USO08029992B2
(12) United States Patent (10) Patent No.: US 8,029,992 B2 Rapko et al. (45) Date of Patent: Oct. 4, 2011
(54) METHODS OF EVALUATING CELLS AND guishes chondrocytes from Synovial cells. Tissue Engineering. Part CELL CULTURES C. Methods, vol. 16, No. 6, pp. 1367-1375, Dec. 2010.* Appendix A of Real-Time PCR Systems: Applied Biosystems (75) Inventors: Stephen M. Rapko, Franklin, MA (US); 7900HT Fast Real-Time PCR System and 7300/7500 Real-Time Stephen J. Duguay, Salem, MA (US) PCR Systems, Chemistry Guide, Applied Biosystems, 2005, Part No. 4348358 Rev. E. pp. A1-A4. Applied Biosystems, "Amplification Efficiency of TaqMan(R) Gene (73) Assignee: Genzyme Corporation, Cambridge, MA Expression Assays”. Application Note TaqMan(R) Gene Expression (US) Assays, Publication 127AP05-03 (2006), printed as pp. 1-6. Applied Biosystems, Taq Man R Gene Expression Assays, ASSay ID (*) Notice: Subject to any disclaimer, the term of this Hs00157103 ml, retrieved online at https://products.ap patent is extended or adjusted under 35 pliedbiosystems.com on Oct. 30, 2006, printed as pp. 1-3. U.S.C. 154(b) by 559 days. Applied Biosystems, Taq Man R Gene Expression Assays, ASSay ID Hs00185803 ml, retrieved online at https://products.ap (21) Appl. No.: 12/098,033 pliedbiosystems.com on Oct. 30, 2006, printed as pp. 1-3. Basad, et al. Treatment of Chondral Defects by Matrix-Guided (22) Filed: Apr. 4, 2008 Autologous Chondrocyte Implantation (MACI), in Cartilage Sur gery and Future Perspectives 50-56 (2003). (65) Prior Publication Data Benya, et al., “Dedifferentiated Chondrocytes Rexpress the Differ entiated Collagen Phenotype When Cultured in Agarose Gels”. Cell, US 2008/02484.81 A1 Oct. 9, 2008 30:215-224 (1982). Binette, et al., “Expression of a Stable Articular Cartilage Phenotype Related U.S. Application Data without Evidence of Hypertrophy by Adult Human Articular Chondrocytes. In Vitro”, Jour: of Orthopaed Research., 16:207-216 (60) Provisional application No. 60/910,574, filed on Apr. (1998). 6, 2007. Brittberg et al., “Treatment of Deep Cartilage Defects in the Knee with Autologous ChondrocyteTransplantation'. N.E.J. of Medicine, (51) Int. Cl. 331;889-895 (1994). CI2O I/68 (2006.01) Brittberg, M., “Autologous Chondrocyte Transplantation'. Clin. CI2O 1/02 (2006.01) Orthopaedics and Related Research, 367S:S147-155 (1999). CI2P 19/34 (2006.01) Buckwalter, et al., “The Effect of Link Protein on Proteoglycan GOIN33/53 (2006.01) Aggregate Structure'. Jour: of Biological Chem., 259(9):5361-5363 (1984). (52) U.S. Cl...... 435/6: 435/29: 435/91.2: 435/7.1 Cherubino, et al., “Autologous chondrocyte implantation using a (58) Field of Classification Search ...... None bilayer collagen membrane: A preliminary report. Jour: of See application file for complete search history. Orthopaedic Surgery, 11(1):10-15 (2003). Galbraith, et al., “Global analysis of cell type-specific gene expres (56) References Cited sion'. Comparative and Functional Genomics, 4:208-215 (2003). U.S. PATENT DOCUMENTS (Continued) 6,150,163 A 11/2000 McPherson et al. 7,169,610 B2 1/2007 Brown 2003/0087259 A1* 5/2003 Clancy et al...... 435/6 Primary Examiner — Jennifer Dunston 2007/0292949 A1 12/2007 Duguay (74) Attorney, Agent, or Firm — Finnegan, Henderson, Farabow, Garrett & Dunner, LLP FOREIGN PATENT DOCUMENTS WO WO 2006/094836 A2 9, 2006 WO WO 2007 149328 12/2007 (57) ABSTRACT OTHER PUBLICATIONS Methods of evaluating the composition of a cell culture (e.g., Pusztai and Hess. Clinical trial design for microarray predictive to distinguish chondrocytes from fibroblasts) and methods for marker discovery and assessment. Annals of Oncology, vol. 15, pp. evaluating the phenotype of an individual cell (e.g., as a 1731-1737, 2004.* chondrocyte) are disclosed. The methods may be used, for Golub et al. Molecular classification of cancer: Class discovery and example, for assessing chondrocyte cultures used for treat class prediction by gene expression monitoring. Science, vol. 286, ment of cartilage defects. In some embodiments, the inven pp. 531-537, Oct. 1999.* tion involves identifying cell culture composition or the iden Tan et al. Classification analysis of the transcriptosome of tity of a cell based on expression level of a fibroblast marker. nonlesional cultured dermal fibroblasts from systemic sclerosis In other embodiments, the invention involves comparing patients with early disease. Arthitis & Rhematism, vol. 52, No. 3, pp. expression levels of at least one chondrocyte marker and at 865-876, Mar. 2005.* Entrez Gene entry for MFAP5 microfibrillar associated protein 5 least one fibroblast marker in a cell culture sample or in an Homo sapiens). Gene ID: 8076, updated on Nov. 1, 2010, printed as individual cell. In illustrative embodiments, the chondrocyte pp. 1-5 on Nov. 9, 2010.* marker is hyaluronan and proteoglycan link protein 1 Entrez Gene entry for HAPLN1 hyaluronan and proteoglycan link (HAPLN1), and the fibroblast marker is microfibrillar asso protein 1 Homo sapiens. Gene ID: 1404, updated on Nov. 1, 2010, ciated protein 5 (MFAP5). printed as pp. 1-6 on Nov. 9, 2010.* Rapko et al. Identification of the chondrocyte lineage using microfibril-associated glycoprotein-2, a novel marker that distin 34 Claims, 18 Drawing Sheets US 8,029.992 B2 Page 2
OTHER PUBLICATIONS Gibson, et al., “Microfibril-associated Glycoprotein-2 (MAGP-2) Is GenBank Accession No. NM 000095, “Homo sapiens cartilage Specifically Associated with Fibrillin-containing Microfibrils but Exhibits More Restricted Patterns of Tissue Localization and Devel oliomeric matrix protein (COMP), mRNA'. Dec. 2003. opmental Expression Than Its Structural Relative MAGP-1”,Jour: of GenBank Accession No. NM 000346, "Homo sapiens SRY (Sex Histochem. & Cytochem., 46(8):871-885 (1998). determining region Y)-box 9 (campomelic dysplasia, autosomal sex Haudenschild, et al., “Differential Expression of Multiple Genes reversal) (SOX9), mRNA'. Jul. 2005. During Articular Chondrocyte Redifferentiation'. The Anatomical GenBank Accession No. NM 000900, “Homo sapiens matrix Gla Record, 263:91-98 (2001). protein (MGP), mRNA'. Feb. 2006. Imabayashi, et al., “Redifferentiation of dedifferentiated GenBank Accession No. NM 00101 1705, "Homo sapiens chondrocytes and chondrogenesis of human bone marrow Stromal leukocyte cell derived chemotaxin 1 (LECT1), transcript variant 2, cells via chondrosphereformation with expression profiling by large mRNA'. Aug. 2006. scale cDNA analysis”. Experimental Cell Research, 288:35-50 GenBank Accession No. NM 001135, "Homo sapiens aggrecan 1, (2003). transcript variant 1, mRNA'. Oct. 2004. Lemaire, et al., “Increased Expression of Type I Collagen Induced by GenBank Accession No. NM 001844. "Homo sapiens collagen, Microfibril-Associated Glycoprotein 2'. Arthritis & Rheumatism, type II, alpha I (COL2A1), transcript variant 1, mRNA'. Apr. 2003. 52: 1812-1823 (2005). GenBank Accession No. NM 001851, “Homo sapiens collagen, Lemaire, et al., “Mutant Fibrillin 1 From tight Skin Mice Increases type IX, alpha 1 (COL9A1), transcript variant 1, mRNA'. Oct. 2006. Extracellular Matrix Incorporation of Microfibril-Associated GenBank Accession No. NM 001854, "Homo sapiens collagen, Glycoprotein 2 and type I Collagen'. Arthritis & Rheumatism, type XI, alpha 1 (COL11A1), transcript variant A, mRNA'. Sep. 50(3):915-926 (2004). 2006. Leung, et al., “Fundametnals of cDNA microarray data analysis'. GenBank Accession No. NM 001884. "Homo sapiens hyaluronan TRENDS in Genetics, 19(11):649-659 (2003). and proteoglycan link protein 1 (HAPLN1)”, Oct. 2005. Linsenmeyer et al., “Monoclonal Antibodies to Connective Tissue GenBank Accession No. NM 003880, “Homo sapiens WNT1 Macromolecules: Type II Collagen'. Biochem. Biophys. Res. Com., inducible signaling pathway protein 3 (WISP3), transcript variant 1, 92(2):440-6 (1980). mRNA'. Aug. 2006. Marlovits, et al., “Early postoperative adherence of matrix-induced GenBank Accession No. NM 005711. "Homo sapiens EGF-like autologous chondrocyte implantation for the treatment of full-thick repeats and discoidin I-like domains 3 (EDIL3), mRNA'. Oct. 2005. ness cartilage defects of the femoral condyle'. Knee Surg. Sports GenBank Accession No. NM 006272, "Homo sapiens S100 cal Traumatol Arthrosc. 12:451-457 (2005). cium binding protein, beta (neural) (S100B), mRN”. Aug. 2005. Rapko, et al., “DNA Methylation Analysis as Novel Tool for Quality GenBank Accession No. NM 006393, "Homo sapiens nebulette Control in Regenerative Medicine”. Tissue Engineering, 13(9):2271 (NEBL), transcript variant 1 mRNA'. Apr. 2008. 2280 (2007). GenBank Accession No. NM 007015, "Homo sapiens leukocyte Reno et al., “Rapid Isolation of Total RNA from Small Samples of cell derived chemotaxin 1 (LECT1), transcript variant 1, mRNA'. Hypocellular, Dense Connective Tissues'. Biotechniques, Apr. 2005. 22(6):1082-1086 (1997). GenBank Accession No. NM 013227. “Homo sapiens aggrecan 1 International Search Report issued in PCT/US2008/059275, mailed (AGC1), transcript variant 2, mRNA'. Oct. 2004. on Aug. 5, 2008. GenBank Accession No. NM 018058, “Homo sapiens cartilage Written Opinion issued in PCT/US2008/059275, mailed on Aug. 5, acidic protein 1 (CRTAC1), mRNA'. Sep. 2005. 2008. GenBank Accession No. NM 033150, “Homo sapiens collagen, Kolettas, E. et al. “Expression of cartilage-specific molecules is type II, alpha 1 (COL2A1), transcript variant 2, mRNA'. Apr. 2003. retained on long-term culture of human articular chondrocytes' J. GenBank Accession No. NM 078485. “Homo sapiens collagen, Cell Science 108:1991-1999 (1995). type IX, alpha 1 (COL9A1), transcript variant2, mRNA'. Oct. 2006. Marlovits, S. etal. “Differential Gene-Expression of Human Articu GenBank Accession No. NM 080629, "Homo sapiens collagen, lar Chondrocytes and Human Fibroblasts in Two- and Three-dimen type XI, alpha 1 (COL11A1), transcript variant B, mRNA'. Sep. sional Cell Culture” FASEB.J. 15(4): A34 (Mar. 7, 2001). 2006. Rapko, S. etal. “Identification of dedifferentiated chondrocytes using GenBank Accession No. NM 080630, “Homo sapiens collagen, gene expression—the disc array Osteoarthritis and Cartilage type XI, alpha 1 (COL11A1), transcript variant C. mRNA'. Sep. 15(Suppl. B): poster P195, p. B137 (Sep. 2007). 2006. Stokes, D.G. et al. "Assessment of the Gene Expression Profile of GenBank Accession No. NM-003480, “Homo sapiens microfibrillar Differentiated and Dedifferentiated Human Fetal Chondrocytes by associated protein (MFAP5), mRNA'. Sep. 2005. Microarray Analysis” Arthritis & Rheumatism 46(2):404-419 (Feb. Gibson, et al., “Further Characterization of Proteins Associated with 2002). Elastic Fiber Microfibrils Including the Molecular Cloning of MAGP-2 (MP25), Jour: of Biol. Chem., 271:1096-1 103 (1996). * cited by examiner U.S. Patent US 8,029,992 B2
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Fig. 16 US 8,029,992 B2 1. 2 METHODS OF EVALUATING CELLS AND other types that may be present in the cell culture. Further CELL CULTURES more, many known fibroblast markers are expressed in both de-differentiated chondrocytes and native cartilage chondro This application claims the benefit of provisional applica cytes, albeit at different levels. Accordingly, the expression tion 60/910,574 filed Apr. 6, 2007, which is incorporated by 5 level of such a fibroblast marker may not necessarily indicate reference in its entirety. whether cells present in the sample are de-differentiated This invention relates to methods of determining the com chondrocytes, fibroblasts, or fibroblast-like cells. position of a cell culture, more particularly, to methods of There is a need for methods of identifying chondrocytes, distinguishing between chondrocytes and fibroblasts. fibroblasts and fibroblast-like cells, particularly, methods Injuries to articular cartilage have poor rates of repair, in 10 part due to the lack of blood Supply in cartilage tissue (Basad applicable to cell culture. et al., In: Hendrich et al., Cartilage Surgery and Future Per In certain aspects, the methods of the invention provide spectives. Thieme Verlag, 49-56 (2003)). Trauma to knee methods of evaluating the composition of a cell culture (e.g., joints can result in, for example, chondral and osteochondral to distinguish chondrocytes from fibroblasts) and methods for lesions, and Such injuries may progress to osteoarthritis (Brit 15 evaluating the phenotype of an individual cell (e.g., as a tberg et al., New England Journal of Medicine, 331(14): chondrocyte). The methods of the invention may be used, for 889-895 (1994)). In severe cases of osteoarthritis, a total knee example, for assessing chondrocyte cultures used for the replacement may be needed. However, the artificial prosthe treatment of cartilage defects. In some embodiments, the ses used in knee replacements have limited lifetimes, thus invention involves identifying cell culture composition or the knee replacements are not optimal remedies, particularly for identity of a cell based on expression level of a fibroblast non-elderly patients (Brittberg et al., Supra). marker. In other embodiments, the invention involves com In some cases, articular cartilage injuries may be repaired paring expression levels of at least one chondrocyte marker by autologous chondrocyte implantation (Brittberg et al., and at least one fibroblast marker in a cell culture sample or in Clin. Orthopaed. Rel. Res., 367S: S147-S155 (1999)). In this an individual cell. In illustrative embodiments, the chondro procedure, chondrocytes are harvested from a patient, 25 cyte marker is hyaluronan and proteoglycan link protein 1 expanded in cell culture to increase the number of chondro (HAPLN1), and the fibroblast marker is microfibrillar asso cytes, and then implanted back into the injury site of the ciated protein 5 (MFAP5). patient. The chondrocytes are covered with a flap of periosteal The invention is based, at least in part, on the identification tissue to seal the chondrocytes into the injury site. Although of MFAP5 as a cell phenotype marker that is highly expressed the cultured chondrocytes have a tendency to de-differentiate 30 in certain non-chondrocytic cell types, such as fibroblasts and in culture, in a successful implant, de-differentiated chondro synoviocytes, while being expressed at significantly lower cytes preserve their re-differentiation potential and will re levels in chondrocytes. The invention is further based, at least differentiate into chondrocytes that produce a hyaline carti in part, on the finding that the expression level ratios of laginous tissue upon implantation. MFAP5 and a chondrocyte marker, such as HAPLN1, is a In a modified technique known as matrix-induced autolo 35 reliable indicator of the cell phenotype in cultures derived gous chondrocyte implantation (MACIR implantation proce from cartilage biopsies. While under some conditions it may dure), cultured chondrocytes are loaded onto a collagen be preferable to use both types of markers (i.e., fibroblast and matrix before they are implanted into the patient (Basadet al., chondrocyte markers) in order to confirm the composition of Supra). In addition, the collagen matrix can be fixed with cell culture or the phenotype of an individual cell, the inven fibrin glue rather than Suturing, making it a simpler Surgical 40 tion also provides embodiments in which determining the technique. normalized expression level of the MFAP5 marker alone may Different techniques and media can be used to culture be sufficient for that purpose. chondrocytes. Examples of serum-free media for chondro In some embodiments, the fibroblast marker is other than cyte culture and methods for isolation and propagation of MFAP5 and is such that its normalized expression levels are chondrocytes are described, for example, in U.S. Pat. Nos. 45 lower in chondrocytes than in fibroblasts. In some embodi 6,150,163 and 7,169,610, and in U.S. Provisional Patent ments, the fibroblast marker is such that its normalized Application No. 60/805.307, which are incorporated herein expression levels are lower in chondrocytes (e.g., primary by reference. and/or passaged chondrocytes) than in fibroblasts and/or syn Fibroblasts or fibroblast-like cells (such as synoviocytes) oviocytes. In some embodiments, the fibroblast marker is may be co-isolated with chondrocytes and, thus, co-propa 50 expressed at least 2-, 5-, 8-,10-fold lower, or less, in chon gated in a cell culture in the course of preparing chondrocyte drocytes than in fibroblasts and/or synoviocytes. implants. Chondrocytes are known to take on a fibroblastic Thus, in one aspect, the invention provides a method of appearance when they de-differentiate in culture (Benya and evaluating the composition of a cell culture (Method 1), e.g., Shaffer, Cell, 30: 215-224 (1982)). Nevertheless, they main cell culture that tentatively contains chondrocytes; and a tain their differentiation potential, i.e., they are able to re 55 method of evaluating the phenotype of an individual cell express a chondrocytic phenotype upon implantation. As a (Method 2). In the embodiments of Method 1, the expression result, it can be difficult to distinguish cultured de-differenti level of a respective marker is determined as the average ated chondrocytes from co-cultured fibroblasts or fibroblast expression level of that marker in a plurality of cells (e.g., like cells based on appearance. culture sample). In the embodiments of Method 1, the com In addition, gene expression patterns in cultured, de-differ 60 position of a cell culture may be evaluated as a whole to entiated chondrocytes are different from those of native car determine whether it contains chondrocytes. In the embodi tilage chondrocytes. For example, many markers that are ments of Method 2, the expression level of a marker is deter highly expressed in native cartilage chondrocytes are mined as the expression level of that marker in the individual expressed at reduced levels in cultured chondrocytes (Binette cell being evaluated. Thus, while Method 1 identifies the et al., J. Orthopaed. Res., 16: 207-216 (1998)). Accordingly, 65 composition of the cell culture, Method 2 identifies the phe expression of such a chondrocyte marker may not necessarily notype of an individual cell, e.g., whether or not the cell is a distinguish a de-differentiated chondrocyte from cells of chondrocyte. US 8,029,992 B2 3 4 In some embodiments, Method 1 comprises: or any other appropriate culture. The method is also useful for a) obtaining a plurality of cells from a cell culture; identifying individual cells derived from any appropriate bio b) determining the average expression level of a fibroblast logical samples in which it is desirable to identify individual marker of the invention in a plurality of cells from the cells, including cartilage samples, synovium samples, fibro cell culture; and blast samples, etc. The fibroblast and chondrocyte markers in c) determining the composition of the culture based on the Method 2 may be chosen and evaluated as described for expression level; Method 1. wherein the expression level below a predetermined thresh In some embodiments, Method 2 comprises old indicates that the cell culture contains chondrocytes. a) determining the expression level of a fibroblast marker Alternatively, the expression level above a predetermined 10 of the invention in the cell; and threshold indicates that the cell culture does not contain chon b) determining the phenotype of the cell based on the drocytes (e.g., the culture does not comprise at least 50%, expression level of the fibroblast maker; 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or wherein the expression level below a predetermined thresh more chondrocytes). old indicates that the cell is a chondrocyte. Alternatively, the In some embodiments, Method 1 involves comparing 15 expression level above a predetermined threshold indicates expression levels of a fibroblast marker (MFAP5 or another that the cell is not a chondrocyte (e.g., a fibroblast or a syn fibroblast marker) and a chondrocyte marker (e.g., HAPLN1) oviocyte). In some embodiments, Method 2 comprises: to a control or to each other. In some embodiments, the a) determining the expression level of a chondrocyte fibroblast marker and the chondrocyte marker are such that marker in the cell; the ratio of their expression levels (chondrocyte marker to b) determining the expression level of a fibroblast marker fibroblast marker) in primary and/or passaged chondrocytes in the cell; and is equal to, or greater than, 5, 10, 20, 30, 50, 75, 100 or more c) evaluating the phenotype of the cell based on the expres times the expression ratio in cultured fibroblasts. sion level of the chondrocyte marker and the expression In particular, in Some embodiments, Method 1 comprises: level of the fibroblast marker. a) obtaining a plurality of cells from a cell culture; 25 In some embodiments, the cell is identified as a chondrocyte b) determining the average expression level of a chondro if the expression level of the chondrocyte marker is above a cyte marker in the plurality of cells; predetermined threshold level, while the expression level of c) determining the average expression level of a fibroblast the fibroblast marker is below a predetermined threshold marker in the plurality of cells; and level. Alternatively, the cell is not a chondrocyte if the expres d) determining the composition of the culture based on the 30 sion level of the chondrocyte marker is below a predeter average expression level of the chondrocyte marker and mined threshold level, while the expression level of the fibro the average expression level of the fibroblast marker. blast marker is above a predetermined threshold level. In In some embodiments, the culture is identified as containing Some embodiments, step c) of evaluating the phenotype of the chondrocytes if the expression level of the chondrocyte cell includes comparing the expression levels of the chondro marker is above a predetermined threshold, while the expres 35 cyte marker and the expression level of the fibroblast marker. sion level of the fibroblast marker is below a predetermined Additional aspects of the invention will be set forth in the threshold. Alternatively, the expression level of the fibroblast description that follows. marker above a predetermined threshold indicates that the cell culture does not contain chondrocytes (e.g., the culture BRIEF DESCRIPTION OF THE FIGURES does not comprise at least 50%, 55%, 60%. 65%, 75%, 80%, 40 85%, 90%, 95%, 98%, 99% or more chondrocytes). FIG. 1 is a flow diagram illustrating stages in an exemplary In some embodiments, the step of determining the culture manufacturing process used for producing cultured chondro composition includes comparing the average expression lev cytes from chondrocyte biopsies. els of the chondrocyte marker and the average expression FIG. 2 depicts HAPLN1 expression levels in several cell level of the fibroblast marker. In some embodiments, the 45 strains as determined by a standard curve method of RT-PCR. markers’ expression levels are compared relative to each Expression levels were normalized to 18S ribosomal RNA. other (thus, the thresholds may be defined, e.g., as a given The expression level in primary chondrocytes (PC) was difference between the expression levels of two markers, or a scaled to 1; other ratios were scaled accordingly. Cell strains ratio thereof). For example, in Some embodiments, a ratio of used are listed in Table 2. a chondrocyte marker (e.g., HAPLN1) expression level to 50 FIG.3 depicts expression levels of MFAP5 in the same cell that of a fibroblast marker (e.g., MFAP5), which is greater strains as shown in FIG. 2, as determined by a standard curve than a predetermined threshold, e.g., 0.25, 0.55, 1, 2, 2.2, 5, method of RT-PCR. Expression levels were normalized to 10, 25, 50 or more, indicates that the cell culture contains 18S ribosomal RNA. The expression level in primary chon chondrocytes. drocytes (PC) was scaled to 1; other ratios were scaled In some embodiments of Method 1, the expression levels of 55 accordingly. chondrocyte and fibroblast markers are determined at the FIG. 4 depicts the ratios of HAPLN1 and MFAP5 expres RNA level, e.g., by a standard curve method of quantitative sion levels from FIGS. 2 and 3. The ratio in primary chon RT-PCR or by a comparative C. method of quantitative RT drocytes (PC) was scaled to 1; other ratios were scaled PCR (which measures the difference in the number of thresh accordingly. old cycles required for the fibroblast marker and the chondro 60 FIG. 5 depicts the ratios of HAPLN1 and MFAP5 expres cyte markers). sion levels in the same strains as shown in FIG. 2. The expres In a related aspect, the invention provides a method of sion levels were determined by a comparative C. method of evaluating the phenotype of an individual cell (Method 2), RT-PCR, and the ratios were calculated as e.g., using flow cytometry or single-cell RT-PCR. The method 2 (CTMEAPs -Cr HAPLN1). is useful for identifying individual cells from a cell culture, 65 FIG. 6 depicts HAPLN1 expression levels in a number of including a cell culture derived from cartilage or synovium, a additional chondrocyte and synoviocyte strains. The expres chondrocyte culture, a fibroblast culture, synoviocyte culture, sion levels were determined by a standard curve method of US 8,029,992 B2 5 6 RT-PCR and normalized to 18S ribosomal RNA. The expres custom-designed primers and probes as described in Example sion level in primary chondrocytes (PC) was scaled to 1; other 6. The HAPLN1:MFAP5 ratios were calculated as ratios were scaled accordingly. Cell strains used are listed in 2 (CTMEAPs -Cr HAPLN1). Table 3. FIG. 16 depicts molar ratios of expression levels of FIG.7 depicts MFAP5 expression levels in the same strains HAPLN1 and MFAP5 in various cell strains, which are listed as shown in FIG. 6. The expression levels were determined by in Table 12, using absolute copy numbers of the markers as a standard curve method of RT-PCR and normalized to 18S determined by an absolute quantitation method. RT-PCR was ribosomal RNA. The expression level in primary chondro performed as described in Example 3, except that 2 ul of cytes (PC) was scaled to 1; other ratios were scaled accord cDNA was used per 13 uL PCR reaction. Standard curves ingly. 10 were prepared from synthetic HAPLN1 and MFAP5 RNA FIG. 8 depicts the ratios of HAPLN1 and MFAP5 expres transcript standards run at 10, 10, and 10 copies/reaction. sion levels from FIGS. 6 and 7. The ratio in primary chon The quantities of HAPLN1 and MFAP5 mRNA copies drocytes (PC) was scaled to 1; other ratios were scaled present in each test sample were determined from these stan accordingly. dard curves. 15 The invention is based, at least in part, on the identification FIG.9 depicts the ratios of HAPLN1 and MFAP5 expres of MFAP5 as a gene that is highly expressed in certain non sion levels in the same strains as shown in FIG. 6. The expres chondrocytic cell types, such as fibroblasts and synoviocytes, sion levels were determined by a comparative C. method of while being expressed at significantly lower levels in chon RT-PCR, and the ratios were calculated as drocytes. Accordingly, in Some embodiments the invention 2 (CTMEAPs -Cr HAPLN1). provides methods of using MFAP5 as a cell phenotype FIG. 10A depicts the ratios of HAPLN1 and MFAP5 marker. MFAP5 is a serine-threonine-rich protein that binds expression levels in the same strains as shown in FIGS. 2 and to fibrillins and was reported to be involved in the stabiliza 6, as well as additional chondrocyte, synoviocyte, and dermal tion of type I procollagen (Lemaire et al., Arthritis & Rheu fibroblast strains identified in Table 4. The expression levels matism, 52(6): 1812-1823 (2005)). The nucleotide and amino were determined by a comparative C. method of RT-PCR 25 acid sequences of human MFAP5 can be found under Gen using custom-designed primers and probes as described in Bank R. Accession No. NM 003480; its nucleotide sequence Example 3. The HAPLN1:MFAP5 ratios were calculated as is also provided as SEQID NO:1. In addition to, or in place of 2 (Czers-Crew). FIG. 10B depicts the ratios of MFAP5, other fibroblast markers can also be used in the HAPLN1 and MFAP5 expression levels in additional cell methods of the invention, as described below. strains identified in Table 5. The expression levels were deter 30 Accordingly, in one aspect, the invention provides a mined using the same methods as described for FIG. 10A. method of evaluating the composition of a cell culture com FIG. 11 shows a comparison between expression level prising chondrocytes (Method 1) and a method of evaluating ratios for HAPLN1 and MFAP5 in monolayer and collagen the phenotype of an individual cell (Method 2). scaffold cultures. Cell strains used are listed in Table 7. In the embodiments concerning Method 1, the expression HAPLN1 and MFAP5 expression levels were determined by 35 level of a respective marker is determined as the average a standard curve method of RT-PCR. Expression levels were expression level of that marker in a plurality of cells. In the normalized to 18S ribosomal RNA. The ratio in monolayer embodiments of Method 1, the composition of a cell culture culture of primary chondrocytes (PC) was scaled to 1; other may be evaluated as a whole to determine whether it contains ratios were scaled accordingly. chondrocytes. In the embodiments of Method 2, the expres FIG. 12 shows a comparison between expression level 40 sion level of a marker is determined as the expression level of ratios for HAPLN1 and MFAP5 in the monolayer and col that marker in an individual cell being evaluated. Thus, while lagen-Scaffold cultures using the same strains as shown in Method 1 identifies the composition of cell culture, Method 2 FIG.11. The expression levels of HAPLN1 and MFAP5 were identifies the phenotype of an individual cell, e.g., whether or determined by a comparative C method of RT-PCR, and the not the cell is a chondrocyte. ratios were calculated as 2 (Carles-Czzuelay). 45 In some embodiments, the fibroblast marker is other than FIG. 13 depicts the change in the expression level ratios for MFAP5 and is such that its normalized expression levels are HAPLN1 and MFAP5 as a function of culture level. Three lower in chondrocytes than in fibroblasts. In some embodi synoviocyte strains were cultured from primary culture (cul ments, the fibroblast marker is such that its normalized ture level 1) through fourth passage (culture level 5), as shown expression levels are lower in chondrocytes (e.g., primary in the figure. The expression levels of HAPLN1 and MFAP5 50 chondrocytes, cultured de-differentiated chondrocytes) than were determined by a comparative C. method of RT-PCR, in fibroblasts (e.g. dermal fibroblasts) and/or synoviocytes. In and the ratios were calculated as 2 (Czarres-Czzuelvi). some embodiments, the fibroblast marker is expressed at least FIGS. 14A and 14B depict the change in the expression 2-, 5-, 8-, 10-fold lower, or less, in chondrocytes than in level ratios for HAPLN1 and MFAP5 as a function of culture fibroblasts and/or synoviocytes. Such additional markers can level. In FIGS. 14A and 14B, chondrocyte strains were 55 be identified using, e.g., gene array analysis, as described in, sampled from cartilage (culture level 0) and then cultured e.g., Leung et al., Trends in Genetics, 19(11): 649-659 (2003). from primary (culture level 1) through second passage (cul In some embodiments, Method 1 comprises determining ture level 3), as shown in the figure. The expression levels the expression level of a fibroblast marker of the invention in were determined by a comparative C. method of RT-PCR a plurality of cells from a cell culture, wherein the expression using custom-designed primers and probes as described in the 60 level below a predetermined threshold indicates that the cell Example 5. The HAPLN1:MFAP5 ratio was calculated as culture contains chondrocytes. Alternatively, the expression 2 (CTMEAPs -Cr HAPLN1). level above a predetermined threshold indicates that the cell FIG. 15 depicts ratios of expression levels of HAPLN1 and culture does not contain chondrocytes (e.g., the culture does MFAP5 in cultures of mixed populations of chondrocytes and not comprise at least 50%, 55%, 60%. 65%, 75%, 80%, 85%, synoviocytes. Three trials were conducted, each with varying 65 90%. 95%, 98%, 99% or more chondrocytes). proportions of the two cell types. The expression levels were In illustrative embodiments, the fibroblast marker is determined by a comparative C. method of RT-PCR using MFAP5, and a higher-than-threshold expression of MFAP5 US 8,029,992 B2 7 8 by the cell culture indicates that the culture contains a sub In some embodiments, Method 1 and Method 2 involve stantial number of non-chondrocytes. In some embodiments, comparing expression levels of a fibroblast marker (MFAP5 or another fibroblast marker) and a chondrocyte marker (e.g., the predetermined threshold level is 1) equal to or less than HAPLN1 or another chondrocyte marker) to a control or to that of MFAP5 expression in pure fibroblast cultures (e.g., 2-, each other. The order in which the expression levels of either 3-, 4-, or 5-fold lower) or 2) equal to or greater than (e.g., 2-, marker are determined can vary. For example, one can first 3-, 4-, or 5-fold greater) the level of MFAP5 expression in determine the expression level of a chondrocyte marker and pure chondrocyte cultures (e.g., primary chondrocytes then determine the expression level of a fibroblast marker, or obtained from cartilage biopsies). For fibroblast markers Vice versa. In some embodiments, the expression levels of other than MFAP5, the predetermined threshold can be analo both types of markers can be determined simultaneously. 10 Examples of some chondrocyte markers useful in the meth gously selected based on the expression levels of the respec ods of the invention, including their GenBankTM accession tive marker in pure fibroblasts and/or chondrocytes. The “pre numbers and SEQID NOS, are provided in Table 1. Thus, in determined” level does not need to be chosen prior to Some embodiments, the chondrocyte marker is chosen from determining marker expression levels and may be chosen HAPLN1, MGP, EDIL3, WISP3, AGC1, COMP, COL2A1, after expression levels are determined, based for example, on 15 COL9A1, COL11A1, LECT1, S100B, CRTAC1, SOX9, and the statistical analysis of the expression results. NEBL. TABLE 1 Examples of chondrocyte markers GenBankTM Marker Name Accession No. SEQID NO Reference hyaluronan and NM 001884 SEQID NO: 2 Buckwalter et al., J. Biol. proteoglycan link Chem., 259(9): 5361-5363 (1984) protein 1 (HAPLN1) matrix Gla protein NM 000900 SEQID NO:3 Monroe et al., Nat Genet., (MGP) 21 (1): 142-4 (1999) GF-like repeats NM OO5711 SEQID NO. 4 Genes Dev., 12(1): 21-33 and discoidin I-like (1998) domains 3 (EDIL3) WNT1 inducible NM OO3880 SEQID NO: 5 Kutz et al., Mol. Cell. Biol., signaling pathway 25(1): 414-21 (2005) protein 3 (WISP3) aggrecan 1 (AGC1) NM 001135 SEQID NO: 6 Roughley et al., Eur. Cell Mater, 11: 1-7 (2006) cartilage oligomeric NM 000095 SEQID NO: 7 Song et al., J. Hum. matrix protein Genet., 48(5): 222-5 (COMP) (2003) type II collagen NM 001844 SEQID NO: 8 Nishimura et al., Hum. (COL2A1) Mutat., 26(1): 36-43 (2005) type IX collagen NM OO1851 SEQID NO: 9 Czarny-Ratajczak et al., (COL9A1) Am. J. Hum. Genet., 69(5):969-80 (2001)) type XI collagen NM 001854 SEQID Poulson et al., J. Med. (COL11A1) NO: 10 Genet., 41 (8): e107 (2004) eukocyte cell NM 007015 SEQID Hiraki et al., Eur. J. derived chemotaxin NO: Biochem., 260(3): 869-78 1 protein (LECT1) (1999) S100 calcium NM OO6272 SEQID Steffansson et al., Nature, binding protein NO: 12 295(5844): 63-4 (1982) beta (S100B) cartilage acidic NM 018058 SEQID Stecket al., Biochem. J., protein 1 NO: 13 353: 169-174 (2001) (CRTAC1) SRY-box 9 protein NM 000346 SEQID Kou and Ikegawa, J. Biol. (SOX9) NO: 14 Chem., 279(49): 50942-8 (2004) nebulette (NEBL) NM OO6393 SEQID Grogan et al., Arth. & NO: 15 Rheum., 56(2): 586-95 (2007)
The plurality of cells from the culture under evaluation Additional chondrocyte markers can be identified using, may be represented by a sample or an aliquot obtained from e.g., gene array analysis, as described in, e.g., Leung et al., that culture. For example, in case of cultures grown on col 60 Trends in Genetics, 19(11): 649-659 (2003). Generally, a lagen matrices, punch sampling can be used as described in chondrocyte marker is a gene or protein whose normalized the Examples. The plurality of cells, typically, will contain at expression levels are higher in chondrocytes (e.g., primary chondrocytes, cultured de-differentiated chondrocytes) than least the number of cells sufficient to conduct a given method in fibroblasts (e.g. dermal fibroblasts) and/or synoviocytes. In of expression analysis, or more. For example, for PCR as few 65 Some embodiments, the chondrocyte marker is expressed at as 10-1,000 cells are usually sufficient, but a lower number least 2, 4, 5, 8, 10, 50, 75, 100 times or greater in chondrocytes can also be used. than in fibroblasts and/or synoviocytes. US 8,029,992 B2 10 In some embodiments, the fibroblast marker and the chon ate culture. The method is also useful for identifying indi drocyte marker are chosen in Such a way that the ratio of their vidual cells derived from any appropriate biological samples expression levels in primary chondrocytes and/or in passaged in which it is desirable to identify individual cells, including chondrocytes is equal to or greater than 5, 10, 20, 30, 50, 75, cartilage samples, synovium samples, fibroblast samples, etc. 100 or more times that in dermal fibroblasts and/or synovio In some embodiments, Method 2 comprises determining the cytes. expression level of a fibroblast marker of the invention in the In particular, in Some embodiments, Method 1 comprises: cell, wherein the expression level below a predetermined a) obtaining a plurality of cells from a cell culture; threshold indicates that the cell is a chondrocyte. Alterna b) determining the average expression level of a chondro tively, the expression level above a predetermined threshold cyte marker in the plurality of cells; 10 c) determining the average expression level of a fibroblast indicates that the cell is not a chondrocyte (e.g., the cell is a marker in the plurality of cells; and fibroblast or a synoviocyte). In some embodiments, Method 2 d) determining the composition of the culture based on the comprises: average expression level of the chondrocyte marker and a) determining the expression level of a chondrocyte the average expression level of the fibroblast marker. 15 marker in the cell; In some embodiments, the culture is identified as containing b) determining the expression level of a fibroblast marker chondrocytes if the expression level of the chondrocyte in the cell; and marker is above a predetermined threshold, while the expres c) evaluating the phenotype of the cell based on the expres sion level of the fibroblast marker is below a predetermined sion level of the chondrocyte marker and the expression threshold. Alternatively, the culture does not contain chon level of the fibroblast marker. drocytes (e.g., the culture does not comprise at least 50%, In some embodiments, the cell is identified as a chondrocyte 55%, 60%, 65%, 75%, 80%, 85%, 90%, 95%, 98%, 99% or if the expression level of the chondrocyte marker is above a more chondrocytes) if the expression level of the chondrocyte predetermined threshold level, while the expression level of marker is below a predetermined threshold, while the expres the fibroblast marker is below a predetermined threshold sion level of the fibroblast marker is above a predetermined 25 level. The fibroblast and chondrocyte markers in the embodi threshold level. ments of Method 2 may be chosen and evaluated as described In further embodiments of Method 1, the invention com for Method 1 above. prises a method of evaluating the composition of a cell cul Flow cytometry can be performed using commercially ture, said method comprising: available antibodies or Such antibodies may be prepared as a) obtaining a cartilage biopsy from a mammal; 30 b) isolating cells from the biopsy: described in, e.g., Linsenmeyer et al., Biochem. Biophys. c) culturing cells isolated in step b) in a cell culture; Res. Com., 92(2): 440-6 (1980). d) obtaining a sample of the cell culture; Cells and cultures being evaluated by the methods of this e) determining the expression levels of MFAP5 and invention may be obtained from any biological sample, HAPLN1 in one or more cells from the sample; and 35 including any tissue, cell culture, or other material, that may f) determining the composition of the culture based on the or may not contain chondrocytes. In some embodiments, the expression levels of MFAP5 and HAPLN1. cells or cultures being evaluated are of mammalian, particu In some embodiments, the step of determining the culture larly human, origin. In some embodiments, the cell culture is composition comprises comparing the average expression grown from cells released from a cartilage biopsy. For levels of the chondrocyte marker and the average expression 40 example, in autologous chondrocyte implantation, cartilage level of the fibroblast marker. In some such embodiments, the cells for the procedure are normally cultured from a cartilage cell culture is evaluated as containing chondrocytes when the biopsy of the patient receiving the implant. Carticel(R) autolo ratio of HAPLN1 expression to that of MFAP5 is greater than gous chondrocyte product (Genzyme Corporation, Cam 0.25. In particular embodiments, this ratio indicates that the bridge, Mass.) is an example of a cultured chondrocyte prod cell culture contains at least 50% chondrocytes. 45 uct. In some embodiments of the invention, the cell culture In some embodiments, the markers’ expression levels are comprises a collagen matrix loaded with chondrocytes. Such compared relative to each other (thus, the thresholds may be chondrocytes may be obtained from a cartilage biopsy and defined, e.g., as a given difference between the expression cultured prior to being loaded on the matrix, e.g., as used in levels of two markers or a ratio thereof). For example, in some the MACIR implant product. The method of the invention is embodiments, a ratio of a chondrocyte marker (e.g., 50 useful for identifying, and/or confirming identify of cells HAPLN1) expression level to that of a fibroblast marker (e.g., loaded onto the collagen Support prior to implanting the MFAP5), which is greater than a predetermined threshold, matrix. e.g., 0.25, 0.55, 1, 2, 2.2, 5, 10, 25, 50 or more, indicates that To illustrate an example of the utility of the cell culture the cell culture contains chondrocytes. determination method, reference is made to FIG. 1. This In some embodiments of Method 1, the expression levels of 55 figure illustrates the steps involved in producing a cultured chondrocyte and fibroblast markers are determined at the chondrocyte product for autologous chondrocyte implanta RNA level, e.g., by a standard curve method of quantitative tion, such as using Carticel(R) autologous chondrocytes, or for RT-PCR or by a comparative C. method of quantitative RT producing a cultured chondrocyte product for the MACIR) PCR (which measures the difference in the number of thresh implantation procedure. In step 1, a cartilage biopsy from a old cycles required for the fibroblast marker and the chondro 60 patient undergoing autologous chondrocyte implantation is cyte markers). shipped for processing (step 2). The biopsy material is In a related aspect, the invention provides a method of digested at step 3 to release and harvest chondrocytes from evaluating the phenotype of an individual cell (Method 2), the cartilage. The released cells are plated in tissue culture e.g., using flow cytometry. The method is useful for identify flasks and expanded in primary culture at Step 4, and if nec ing individual cells from a cell culture, including a cell culture 65 essary, Subcultured. Once the cells reach an adequate number, derived from cartilage or synovium, a chondrocyte culture, a they can be, optionally, cryopreserved at step 5 until the fibroblast culture, synoviocyte culture, or any other appropri patient is ready to receive the implant. Once the patient is US 8,029,992 B2 11 12 ready to receive the cells, they are thawed and plated into In illustrative embodiments, the expression levels of the tissue culture flasks and grown to prepare an assembly culture fibroblast and chondrocyte markers are determined using RT (step 6). PCR, either by a standard curve or by a comparative C. For use in an autologous chondrocyte implant, if a suffi method for relative quantification. In some embodiments, cient number of cells are obtained in the assembly culture, the absolute quantitation of marker copy numbers can be deter cells are centrifuged to a cell pellet and resuspended in ship mined by preparing standard curves using known amounts of ping medium, which is the “final product” such as the Carti the markers. The general methods for conducting such assays cel(R) autologous chondrocyte product (step 8). This “final are described, e.g., in Real-Time PCR Systems: Applied Bio product' is subjected to a number of QC tests, including for systems 7900HT Fast Real-Time PCRSystemand 7300/7500 example, a sterility test, a cell viability test, an endotoxin test, 10 Real-Time PCR Systems, Chemistry Guide, Applied Biosys a mycoplasma test, and a culture composition test (step 9"QC tems, 2005, Part No. 4348358 Rev. E. identity” as described herein) to ensure that the cultured cells In the case of comparing two markers using the compara contain a sufficient number of chondrocytes. If the cultured tive C method, the amount of the ratio of expression levels of cells pass all QC tests, they are shipped (step 10) to the patient a fibroblast marker to that of a chondrocyte marker can be 15 calculated as (1+E) (C7-Cz), wherein C, is the number of for implantation (step 11). the fibroblast marker threshold cycles, C, is the number of Alternatively, when the assembly culture from step 6 is to threshold cycles of the chondrocyte marker, assuming that be used in a MACIR implant, the cells are resuspended in efficiency of amplification (E) is the same for both markers culture medium, seeded onto a collagen Scaffold, and cul and the starting amount of both markers is normalized to the tured for 4 days (step 7). At the end of the culture period, the same amount of endogenous control (e.g., as in two duplicate cells are rinsed with shipping medium to produce a final samples). In the case of Es 1, as illustrated in the Examples, product for MACIR implants. This product is also subjected the ratio can be approximated as 2 (C-C). Otherwise, the to the QC tests outlined above. Accordingly, whether the final calculations can be performed as described in Appendix A of product is a Suspension of cultured chondrocytes, such as Real-Time PCR Systems: Applied Biosystems 7900HT Fast Carticel(R) autologous chondrocytes, or the final product is a 25 Real-Time PCR System and 7300/7500 Real-Time PCRSys scaffold-seeded product for MACIR implants, the method of tems, Chemistry Guide, Applied Biosystems, 2005, Part No. the invention is useful as a lot identification assay or lot 4348358 Rev. E. release assay, to confirm the composition of a cell culture as Further embodiments of the invention are illustrated in the containing chondrocytes prior to shipment of the culture. For following examples, which are intended to be exemplary and example, the “QC identity” (step 9) can be performed at any 30 not intended to be limiting on the scope of the invention. step prior to the final product assembly, e.g., before step 4, 5, 6, 7, or 8. EXAMPLES Many methods of determining gene or protein expression levels are known to persons skilled in the art, e.g., as Example 1 described in Sambrook et al. (eds.) Cloning: A Laboratory 35 Manual, 2nd ed., Cold Spring Harbor Laboratory Press, 1989; Expression of HAPLN1 and MFAP5 in Current Protocols in Molecular Biology (Ausubel et al. (eds.) Chondrocytes, Synoviocytes and Fibroblasts New York: John Wiley and Sons, 1998). Examples of such methods include polymerase chain reaction (including abso Cell Isolation and culture—Human chondrocyte cultures lute quantitation by PCR, real time PCR (RT-PCR) and qRT 40 were isolated from cartilage using the method for producing PCR, multiplex or singleplex PCR), single cell PCR, northern Carticel(R) autologous chondrocytes or the Protease method blot assays, nuclease protection assays, in situ hybridization for producing cultured chondrocytes. Using the method for assays, immunohistochemistry assays, immunocytochemis producing Carticel(R) autologous chondrocytes, cartilage tis try assays, electrophoresis assays such as gel or capillary, Sue was trimmed of bone and synovium and Subjected to a Western blot assays, ELISAS, immunoprecipitation assays, 45 first digestion where tissue was enzymatically treated in col chromatography based assays such as HPLC or gel chroma lagenase solution for 18 hours at 37° C. Cells released from tography, mass spectrometry assays, RNase protection the first digestion were plated in tissue culture flasks with fetal assays, flow cytometry assays, DNA methylation assays, and bovine serum (FBS) and gentamicin containing medium histone modification analysis assays. (EGHXX). The cells were then subjected to a second diges In all methods of the invention, expression levels, at the 50 tion where remaining tissue from the first digestion was RNA or at the protein level, can be determined using any treated with a collagenase/trypsin solution for 2.5 hours at 37° Suitable method, including any one of conventional methods. C. Cells released from the second digestion were plated in RNA levels may be determined by, e.g., quantitative RT-PCR tissue culture flasks with EGHXX. Tissue pieces remaining (e.g., TaqManTM RT-PCR or RT-PCR), Northern blotting, or after the second digestion were plated in tissue culture flasks any other method for determining RNA levels, or as described 55 with EGHXX. Using the Protease isolation method, cartilage in the Examples. Protein levels may be determined, e.g., by tissue was trimmed of bone and synovium and Subjected to a using Western blotting, ELISA, flow cytometry, enzymatic first digestion in Pronase E (Sigma-Aldrich Inc., St. Louis, activity assays, or any other method for determining protein Mo.) solution for 1.5 hours at 37°C. The Pronase solution was levels. Expression levels may be scaled and/or normalized per then removed and a second digestion of the cartilage was total amount of RNA or protein in the sample and/or a control, 60 performed in collagenase solution for 18 hours at 37°C. The which may typically be a housekeeping gene such, as beta released cells were then plated in tissue culture flasks with actin or glyceraldehyde-3-phosphate dehydrogenase EGHXX. After isolation, the cell culture methods were the (GAPDH), or 18S ribosomal RNA, etc.). Normalization is same for cells obtained from either isolation method. Primary typically done to account for variability in the amount of cell cultures were re-fed fresh EGHXX every 2 to 4 days. protein, DNA, or RNA input. For example, in the Examples, 65 When the primary culture flasks reached 50% to 80% conflu expression, levels are normalized to 18S ribosomal RNA ence, they were trypsinized to a single cell Suspension, neu using a standard curve. tralized with EGHXX to inactivate trypsin, and a cell count US 8,029,992 B2 13 14 was performed. The resulting cell Suspension was then either Standard Curve Method. The standard curve method was sampled, further expanded by Subculturing, or cryopreserved performed using the Eukaryotic 18S rRNA Endogenous Con for long term storage. The Subculture of the primary culture is trol assay (catalog number 43 19413E, Applied Biosystems referred to as the secondary culture, or first passage. Subse Inc.), in which 18S rRNA is used as an internal control to normalize RT-PCR results to account for input variation. For quent Subcultures are referred to as the second passage, third quantitation of the relative levels of expression of each gene, passage, fourth passage, etc. Subculturing was performed by dilutions of the primary chondrocyte (PC) cDNA were run to plating cells in tissue culture flasks with EGHXX and re generate a standard curve with the 7500 system software. The feeding with fresh EGHXX every 2 to 4 days. When the level of each test sample's expression was determined from subcultures reached 80% to 100% confluence, they were the standard curve, and the resulting mRNA ratios to the trypsinized to a single cell Suspension, neutralized with 10 primary chondrocyte control (PC) were divided by the sam EGHXX to inactivate trypsin, and a cell count was performed. ple's 18S rRNA ratio to PC to normalize for cDNA loading. The resulting cell Suspension was then either sampled, further Comparative C Method—Comparative Canalysis was expanded, or cryopreserved for long term storage. performed to determine the relative gene expression ratios of HAPLN1 to MFAP5 in the various samples from the real time Human synoviocyte cultures (synovium derived cell cul 15 quantitative RT-PCR gene expression analysis raw data gen tures, also known as synovial fibroblasts) S1 and S2 were erated as described above. The comparative Ct method pro obtained from Cell Applications Inc. (San Diego, Calif.) as vides a relative measure of the ratio of HAPLN1 to MFAP5 cryopreserved primary cultured cells. The synoviocytes were which allows for direct comparison between test samples plated in tissue culture flasks with EGHXX medium and without the need for standards, standard curve analysis, or cultured using the method for producing Carticel(R) autolo actual calibrators. This method can be employed in the case of gous chondrocytes as described above. Human dermal fibro the HAPLN1 and MFAP5 assays used in this example blast cultures were purchased from Cell Applications Inc. as because the following four conditions were met: 1) the assay cryopreserved-primary cultured cells. The dermal fibroblasts performance was consistent from run to run; 2) equivalent were cultured using the method for producing Carticel(R) amounts of RNA were run in HAPLN1, MFAP5, and endog autologous chondrocytes as described above. 25 enous control assays; 3) the C value for the endogenous The cell cultures used in this Example are listed in Table 2. control gene, 18S rRNA, was always lower than either the HAPLN1 Cor MFAP5C, when equivalent amounts of RNA TABLE 2 were run in each assay, and thus 18S C was always quanti fiable when either HAPLN1 or MFAP5 were quantifiable: Cell cultures used in RT-PCR Analysis (Example 1 30 and 4) the method used an arbitrarily selected theoretical Cell Culture Cell Type Type of Cell Culture calibrator defined as a theoretical sample containing the ratio of HAPLN1/MFAP5 which yielded a HAPLN1 C, value PC Chondrocyte Primary culture equal to the MFAP5C value when the other three conditions C1 Chondrocyte Second Passage C2 Chondrocyte Second Passage listed above were met. The derivation of the equation used for S1 Synoviocyte Second Passage this comparative C method is as follows. Where the amount S2 Synoviocyte Second Passage 35 of target gene in a sample, normalized to an endogenous F1 Dermal Fibroblast Second Passage control gene and relative to a calibrator is given by: F2 Dermal Fibroblast Second Passage (1+E)(AACT are gene) RNA and cDNA preparation RNA was isolated from cell wherein cultures using the TRI-spin procedure (Reno et al., Biotech 40 niques 22: 1082-6 (1997)). Isolated RNA concentrations E=amplification efficiency were determined spectrophotometrically. For the preparation of cDNA from samples PC, C1, C2, S1, S2, F1, and F2, the wherein First Strand Synthesis Kit (Roche, Indianapolis, Ind.), using random hexamer primers was run according to the manufac 45 AACT.target e.Sample ACT.target -calibrator ACT turer's instructions. The resulting cDNA was stored at -20° iageigerae C. or -80° C. until analysis. Gene expression analysis—Gene expression analysis was and wherein performed using quantitative real time RT-PCR, using either a standard curve method or a comparative C. method. The 50 A Ctargeigerae Ctarget gene Cendogenous control gene real time PCR method was based on the 5' nuclease cleavage of a dual labeled oligo probe to report sequence specific (See Liu, W. and Saint, D. A., Analytical Biochemistry, 302: primer amplification of the target sequence ("TaqManTM' 52-59 (2002); Livak, K. J. ABI Prism 7700 Sequence Detec assay). Expression of genes encoding cartilage link protein tion System, User Bulletin 2, ABI publication 4303859, (HAPLN1) and microfibrillar associated protein 5 (MFAP5) 1997). Then the ratio of HAPLN1 to MFAP5 can be described were assayed using TaqManTM Gene Expression Assays 55 aS Hs00157103 ml and Hs00185803 ml (Applied Biosystems (1+E)(-AACT traptivi)/(1+E)(-AACTAtraps) Inc.), respectively. The real time PCR was prepared with TaqManTM Universal PCR Master Mix, no UNG (catalog which equals: number 432-4018, Applied Biosystems Inc.), appropriate 60 (1+E)(-sample CrHAPLN-sample TaqManTM Gene Expression Assay (Applied Biosystems) C. endogenous control eel-calibrator CT HAPLN1. and sample cDNA were used according to the Universal PCR calibrator CT, endogenous control genel})/(1+E)(- Mix protocol. The amplifications were run on an ABI 7500 sample CTMEAPs-sample Real-Time PCR system (Applied Biosystems Inc.) using the CT, endogenous control genel-calibrator CTMFAPs standard TaqManTM cycling and data collection program for calibrator C. endogenous control gonel}) this configuration. Duplicate 25 LL reactions were run with 65 If the same amount of sample is run in each assay, then the C. up to 5 ng of input cDNA per well. A threshold of 0.1 units of the sample endogenous control gene can be represented by was used for all assays. the term X. If the same amount of calibrator is run in each US 8,029,992 B2 15 16 assay, then the C of the calibrator endogenous control gene method for differentiating between chondrocyte and synovio can be represented by the termy. Substituting these terms, the cyte cultures. The cultures used in this Example are listed in equation derives to: Table 3. (1+E)(-sample Crittaet N1-x-calibrator 5 Cratap, wi-y)})/(1+E)(-(sample TABLE 3 Cratraps-x-(calibrator Czaraps-yl) Cell cultures used in RT-PCR. Analysis (Example 2 which equals: Cell Culture Cell Type Type of Cell Culture (1+E)(x-sample Criter NF-Iy-calibrator 10 PC Chondrocyte Primary culture Crittaptivil)/(1+E)(x-sample Cratrapsi C3 Chondrocyte Second passage Iy-calibrator Crataps) C4 Chondrocyte Second passage C5 Chondrocyte Second passage If the calibrator is defined as a theoretical sample containing C6 Chondrocyte Second passage the ratio of HAPLN1/MFAP5 which yields a Cze, value C7 Chondrocyte Second passage equal to the Czes value when equivalent amounts of cali S3 Synoviocyte Second passage 15 S4 Synoviocyte Second passage brator are run in each assay, then the term Z can be substituted S5 Synoviocyte Second passage for the calibrator Crew and the calibrator Czares. The S6 Synoviocyte Second passage equation then derives to: S7 Synoviocyte Second passage
sample CTMFAPsi-I-Z) 2O Cell isolation and culture Human chondrocyte cell cul tures C3, C4, C5, C6, and C7 were isolated and cultured using which equals: the method for producing Carticel(Rautologous chondrocytes (1+E)(x-sample Crittaptivif-Ey-ZI-fix-sample as described in Example 1. Human synoviocyte cultures (Syn CTMFAP57+Iy-z) ovium derived cell cultures, also known as synovial fibro blasts) were either isolated at Genzyme or obtained from Cell which equals: 25 Applications Inc. (San Diego, Calif.). Strains S4, S6, and S7 (1+E)(x-sample CT HAPLNL-Ex-sample CTMFAPs) were isolated at Genzyme using various procedures. S4 was isolated by Subjecting minced synovium tissue to digestion in which equals: collagenase solution for 3.5 hours at 37°C., followed by a 30 second digestion in trypsin solution for 1 hour at 37° C. Strain (1+E) (sample CTMEAps-Sample CT HAPLN1) S6 was isolated by Subjecting minced synovium tissue to And if E=1 (100% efficiency), then the relative ratio of digestion in a solution containing collagenase and DNase for HAPLN1 to MFAP5 equals: 2 hours at 37° C. Strain S7 was isolated by subjecting minced synovium to the method for producing Carticel(R) autologous 2 (sample CTMEAps-Sample CT HAPLN1) 35 chondrocytes. After isolation, the synovium derived cells The above equations derive to a final formula leaving only were plated in tissue culture flasks with EGHXX medium and two variables, the sample HAPLN1 C and sample MFAP5 cultured using the method for producing Carticel(R) autolo C, as unknowns. This formula applies when samples are gous chondrocytes as described in Example 1. Strains S3 and assayed under the conditions described above, and the theo S5 were obtained from Cell Applications Inc. as cryopre retical calibrator employed is set as described above. 40 served first passage cells. After thawing, the cells from Strains FIG. 2 depicts HAPLN1 expression levels in several cell S3 and S5 were plated in tissue culture flasks with EGHXX strains as determined by a standard curve method of RT-PCR. medium and cultured using the method for producing Carti FIG. 3 depicts expression levels of MFAP5 in the same cell cel(R) autologous chondrocytes as described in Example 1. samples as shown in FIG. 2, as determined by a standard RNA isolation and cDNA preparation RNA preparations curve method of RT-PCR. HAPLN1 was expressed at higher 45 for chondrocyte cell cultures C3, C4, C5, C6, C7 and syn levels in the chondrocyte cell cultures than in the synoviocyte oviocyte cultures S3, S4, S5, S6, S7 were performed as and fibroblast cell cultures. MFAP5 was expressed at higher described in Example 1. The RNA from these samples was levels in synoviocyte and fibroblast cell cultures than in the reverse transcribed into cDNA using the High Capacity chondrocyte cell cultures. cDNA Reverse Transcription Kit (Applied Biosystems, Inc., FIG. 4 depicts the ratios of HAPLN1 and MFAP5 expres 50 Foster City, Calif.) according to the manufacturers instruc sion levels from FIGS. 2 and 3. The ratio in primary chon tions. The PC clNA from Example 1 was used in this drocytes (PC) was scaled to 1; other ratios were scaled Example. The cDNA was stored at -20°C. or -80° C. until accordingly. analysis. FIG.5 depicts the ratios of HAPLN1 and MFAP5 expres Gene expression analysis—Gene expression analysis was sion levels in the same strains as shown in FIG. 2, however, 55 performed using RT-PCR as described in Example 1. the expression levels were determined by a comparative C. FIG. 6 depicts HAPLN1 expression levels in a number of method of RT-PCR. The results of the Cimethod were similar additional chondrocyte and synoviocyte strains. The expres to the results obtained by the standard curve method. sion levels were determined by a standard curve method of RT-PCR and normalized to 18S ribosomal RNA. Example 2 60 FIG.7 depicts MFAP5 expression levels in the same strains as shown in FIG. 6. The expression levels were determined by Expression of HAPLN1 and MFAP5 in Additional a standard curve method of RT-PCR and normalized to 18S Strains of Chondrocytes, Synoviocytes and ribosomal RNA. Fibroblasts FIG. 8 depicts the ratios of HAPLN1 and MFAP5 expres 65 sion levels from FIGS. 6 and 7. The ratio in primary chon Expression levels of HAPLN1 and MFAP5 were deter drocytes (PC) was scaled to 1; other ratios were scaled mined in additional cell cultures to confirm fidelity of the accordingly. US 8,029,992 B2 17 18 FIG.9 depicts the ratios of HAPLN1 and MFAP5 expres TABLE 4-continued sion levels in the same strains as shown in FIG. 6. The expres sion levels were determined by a comparative C. method of First Set of Cell Cultures used in RT-PCR. Analysis (Example 3 RT-PCR, and the ratios were calculated as 2 (CrMraps-Cr trap, w). Cell Culture Cell Type Type of Cell Culture The RT-PCR results in the additional cell strains were F4 Dermal fibroblast Second passage F5 Dermal fibroblast Second passage consistent with the results obtained in Example 1. F6 Dermal fibroblast Second passage Example 3 10 Expression of HAPLN1 and MFAP5 in TABLE 5 Chondrocytes, Synoviocytes and Fibroblasts Using Second Set of Cell Cultures used in RT-PCR. Analysis (Example 3 Custom-Designed Primers and Probes Cell Culture Cell Type Type of Cell Culture 15 Testing of various chondrocyte, synoviocyte, and dermal C21 Chondrocyte Second Passage fibroblast cultures was performed with primers and probes of C22 Chondrocyte Second Passage C23 Chondrocyte Second Passage known oligonucleotide sequences. C24 Chondrocyte Second Passage Cell isolation and culture The cell strains used in this C25 Chondrocyte Second Passage Example are listed in Tables 4 and 5 below. Human chondro C26 Chondrocyte Second Passage cyte cell cultures C1, C2, C3, C4, C5, C6, C7, C8, C26, C28, C27 Chondrocyte Second Passage C28 Chondrocyte Second Passage C30, and C34 were isolated and cultured using the method for C29 Chondrocyte Second Passage producing Carticel(R) autologous chondrocytes as described C30 Chondrocyte Second Passage in Example 1. Human chondrocyte cell cultures C21, C22, C31 Chondrocyte Second Passage C23, C24, C25, C27, C29, C31, C32, and C33 were isolated C32 Chondrocyte Second Passage 25 C33 Chondrocyte Second Passage (using the Protease method) and cultured as described in C34 Chondrocyte Second Passage Example 1. Cell isolation and culture methods for human S14 Synoviocyte Third Passage synoviocyte cultures S1, S2, S3, S4, S5, S6, and S7 were S15 Synoviocyte Third Passage described in Examples 1 and 2. Synoviocyte culture S9 was S16 Synoviocyte Third Passage S17 Synoviocyte Second Passage isolated by Subjecting minced synovium tissue to digestion in 30 S18 Synoviocyte Second Passage a solution containing collagenase and DNase for 2 hours at F8 Dermal fibroblast Second Passage 37° C. Synoviocyte culture S10 was isolated by subjecting F9 Dermal fibroblast Second Passage minced synovium tissue to digestion in collagenase Solution F10 Dermal fibroblast Second Passage for 3.5 hours at 37° C., followed by a second digestion in F11 Dermal fibroblast Second Passage trypsin solution for 1 hour at 37° C. Synoviocyte strains S11, 35 S12, S13, S14, S15, S16, S17, and S18 were obtained from RNA isolation and cDNA preparation RNA preparations Cell Applications Inc. as cryopreserved first passage cells. from chondrocyte strains C1, C2, C3, C4, C5, C6, C7, syn Dermal fibroblast strains F1, F2, F3, F4F5, F6, F8, F9, F10, oviocyte strains S1, S2, S3, S4, S5, S6, S7, and dermal fibro and F11 were obtained from Cell Applications Inc. as cryo blast strains F1, and F2 were described in Examples 1 and 2. preserved primary cultured cells. All cell cultures were cul 40 For preparation of RNA from chondrocyte strain C8, syn tured using the method for producing Carticel(R) autologous oviocyte strains S9, S10, S11, S12, S13, S14, dermal fibro chondrocytes as described in Example 1. blast strains F3, F4, F5, and F6, and all strains listed in Table 5, the RNeasyTM Mini Kit (Qiagen, Valencia, Calif.) RNA TABLE 4 isolation method was used. For the RNeasyTM isolation, 360 First Set of Cell Cultures used in RT-PCR. Analysis (Example 3 45 LL of lysis solution was added to cell pellets containing up to one million cells. The samples were immediately vortexed at Cell Culture Cell Type Type of Cell Culture full speed for 30 seconds, then placed at 37°C. for 5 minutes. C1 Chondrocyte Second passage After incubation, the samples were shaken by hand for 10 C2 Chondrocyte Second passage seconds, followed by another 30 second vortex at full speed. C3 Chondrocyte Second passage 50 The contents of each tube were collected, and the lysate was C4 Chondrocyte Second passage C5 Chondrocyte Second passage run through a QiashredderTM column (Qiagen). Three hun C6 Chondrocyte Second passage dred and fifty uL of the Qiashredded lysate was used in the C7 Chondrocyte Second passage RNeasyTM procedure following the manufacturer's protocol C8 Chondrocyte Second passage for the isolation of RNA from animal cells. The columns were S1 Synoviocyte Second passage 55 S2 Synoviocyte Second passage eluted with a single elution consisting of 30 uL of water. The S3 Synoviocyte Second passage RNA was reverse transcribed into cDNA using the High S4 Synoviocyte Second passage Capacity cDNA Reverse Transcription Kit (Applied Biosys S5 Synoviocyte Second passage tems, Inc., Foster City, Calif.) according to the manufactur S6 Synoviocyte Second passage S7 Synoviocyte Second passage er's instructions. The resulting cDNA was stored at -20°C. or S9 Synoviocyte First passage 60 -80°C. until analysis. S10 Synoviocyte Third passage Gene expression analysis—RT-PCR assays were per S11 Synoviocyte Third passage formed with custom-designed primers and probes specific for S12 Synoviocyte Third passage S13 Synoviocyte Third passage regions of HAPLN1 and MFAP5 mRNAs. The sequence F1 Dermal fibroblast Second passage information for the custom primers and probes is shown in F2 Dermal fibroblast Second passage 65 Table 6. Abbreviations: 6FAM=6-Carboxyfluorescein, F3 Dermal fibroblast Second passage VICTM is a trademark of Applied Biosystems Inc. and is a fluorophore, MGBNFO minor groove binder non-fluores US 8,029,992 B2 19 20 cent quencher. Primers were obtained from Invitrogen Corp. sample was taken for RNA isolation (the “Day 0” or mono (Carlsbad, Calif.). Probes were obtained from Applied Bio layer sample), and then the cells were resuspended in systems Inc. For HAPLN1, the target of the forward primer is EGHXX medium and seeded onto a 20 cm MAIXTMscaffold nucleotides 543 to 570 of the HAPLN1 sequence deposited in (ACI-MAIXTM collagen membrane, CE, Matricel GmbH, a gene sequence database under GENBANKTM Accession 5 D-52134 Herzogenrath, Germany). The cells were allowed to No. NM 001884.2 (SEQID NO:2), the target of the reverse attach for 1 hour at 37°C., then the scaffold was fed additional primer is nucleotides 603 to 622, and the target of the probe is EGHXX and cultured for 4 days. Scaffold cultures containing nucleotides 584 through 601 of the same sequence. For synoviocytes and dermal fibroblasts were also prepared in the MFAP5, the target of the forward primer is nucleotides 301 same manner. After 4 days of scaffold culture, the cultures through 322 of the MFAP5 sequence deposited under GEN 10 were sampled using an 8 mm biopsy punch (the “Day 4” or BANKTM Accession No. NM 003480.2 (SEQID NO:1); the scaffold sample), and RNA isolation was performed. target of the reverse primer is nucleotides 353 through 372, RNAIsolation and cDNA preparation RNA was isolated and the target of the probe is nucleotides 334 through 350 of using the RNA isolation kit RNEASYTM Mini Kit (Qiagen, the same sequence. Real time PCR was performed with TAC 15 Valencia, Calif.). For the RNEASYTM isolation, 360 uL of MANTM Fast Universal PCR Master Mix, no UNG (catalog lysis solution was added to MACIR) implant samples (up to number 4352042, Applied Biosystems Inc.), 900 nM primers, two 8 mm MACIR) implant punches per preparation). The 250 nM probes, and up to 5 ng of sample cDNA, according to samples were immediately vortexed at full speed for 30 sec the TAQMANTM Fast Universal PCRMix protocol. The reac onds, then placed at 37°C. for 5 minutes. After incubation, the tion Volume was 13 Jul and the amplifications were run on an ABI 7500 Real-Time PCR system (Applied Biosystems Inc.) samples were shaken by hand for 10 seconds to unfold the using the default Fast TAQMANTM cycling and data collec membrane followed by another 30 second vortex at full tion program for this configuration. A threshold of 0.1 units speed. The contents of each tube were collected, and the was used for all assays. The expression levels were deter lysate was run through a biopolymer-Shredding system, mined by a comparative C. method of RT-PCR described in QIASHREDDERTM column (Qiagen). Three hundred and Example 1. 25 fifty uI of the Qiashredded lysate was used in the RNE ASYTM procedure following the manufacturer's protocol for TABLE 6 the isolation of RNA from animal cells. The columns were eluted with a single elution consisting of 30 uL of water. Custom Primer and Probe Sequences Preparation of cDNA from the sample RNA was performed 30 using the High Capacity cDNA Reverse Transcription Kit Marker Forward primer Reverse Primer Probe (Applied Biosystems, Inc., Foster City, Calif.) according to HAPLN1 5. TGAAGGATTAG 5 GCCCCAGTCG 5' VIC/ the manufacturer's instructions. The cDNA was stored at AAGATGATACTGTT TGGAAAGTAA 3 TACAAGGTGTGGTA GTG 3." (SEO ID NO:17) TTCC/MGBNFO 3' -20° C. Or -80° C. (SEQ ID NO: 16) (SEQ ID NO:18) Table 7 lists the cell cultures used and configurations used 35 in this Example. MFAP5 5' CGAGGAGACGA 5' AGCGGGATCA 5' 6FAMA TGTGACT CAAG 3." TTCACCAGAT 3 ACATTCACAGAAGA (SEO ID NO:19) (SEO ID NO: 2O) TCC/MGBNFO 3' TABLE 7 (SEQ ID NO:21) Cell cultures used in RT-PCR. Analysis (Example 4 40 FIG. 10A depicts the ratios of HAPLN1 and MFAP5 Culture Code Cell Type Culture Type Configuration expression levels in the same strains as shown in FIGS. 2 and C9 day O Chondrocyte Second passage 5 x 10 cell pellet 6, as well as additional chondrocyte, synoviocyte, and dermal C10 day O Chondrocyte Second passage 5 x 10 cell pellet fibroblast strains from Table 4. FIG. 10B depicts the ratios of C11 day 0 Chondrocyte Second passage 5 x 10 cell pellet 45 C12 day 0 Chondrocyte Second passage 5 x 10 cell pellet HAPLN1 and MFAP5 expression levels in strains from Table C13 day O Chondrocyte Second passage 5 x 10 cell pellet 5. The results obtained with the custom-designed primers and C14 day 0 Chondrocyte Second passage 5 x 10 cell pellet probes were similar to the results described in Examples 1 and C15 day O Chondrocyte Second passage 5 x 10 cell pellet 2. C16 day O Chondrocyte Second passage 5 x 10 cell pellet C17 day O Chondrocyte Second passage 5 x 10 cell pellet C18 day O Chondrocyte Second passage 5 x 10 cell pellet Example 4 50 S7 day O Synoviocyte Third passage 5 x 10 cell pellet F7 day O Dermal fibroblast Second passage 5 x 10 cell pellet Comparison of HAPLN1 and MFAP5 Expression F2 day O Dermal fibroblast Second passage 5 x 10 cell pellet Levels in Chondrocyte, Synoviocyte and Fibroblast C9 day 4 Chondrocyte MACI (R) implant 8 mm punch C10 day 4 Chondrocyte MACI (R) implant 8 mm punch Cultures in Monolayers and Collagen Scaffolds C11 day 4 Chondrocyte MACI (R) implant 8 mm punch 55 C12 day 4 Chondrocyte MACI (R) implant 8 mm punch Expression levels of HAPLN1 and MFAP5 were compared C13 day 4 Chondrocyte MACI (R) implant 8 mm punch in various types of cultures in monolayers and collagen Scaf C14 day 4 Chondrocyte MACI (R) implant 8 mm punch C15 day 4 Chondrocyte MACI (R) implant 8 mm punch folds. C16 day 4 Chondrocyte MACI (R) implant 8 mm punch Cell isolation and culture Chondrocyte cultures C9, C10, C17 day 4 Chondrocyte MACI (R) implant 8 mm punch C11, C12, C13, C14, C15, C16, C17, and C18 were isolated 60 C18 day 4 Chondrocyte MACI (R) implant 8 mm punch using the Protease method as described in Example 1, and S7 day 4 Synoviocyte MACI (R) implant 8 mm punch cultured as described in Example 1. Synoviocyte culture S7 F7 day 4 Dermal fibroblast MACI (R) implant 8 mm punch was isolated and cultured as described in Examples 1 and 2. F2 day 4 Dermal fibroblast MACI (R) implant 8 mm punch Dermal fibroblast cultures F2 and F7 were obtained from Cell Applications Inc. as cryopreserved primary cultured cells and 65 Gene expression analysis—Gene expression analysis of cultured as described in Example 1. Upon completion of the monolayer and MACIR implant cDNA was performed in second passage culture (third passage for culture S7), a the manner outlined above in Example 1. US 8,029,992 B2 21 22 FIG. 11 shows a comparison between expression level consistently high at all culture levels for the chondrocyte ratios for HAPLN1 and MFAP5 in monolayer and collagen samples relative to the synoviocyte samples. scaffold cultures. HAPLN1 and MFAP5 expression levels were determined by a standard curve method of RT-PCR. Example 6 Expression levels were normalized to 18S ribosomal RNA. The ratio in monolayer culture of primary chondrocytes (PC) Expression of HAPLN1 and MFAP5 in Mixed Cell was scaled to 1; other ratios were scaled accordingly. Cultures FIG. 12 shows a comparison between expression level Gene expression analysis was applied to mixed cultures of ratios for HAPLN1 and MFAP5 in the monolayer and col 10 chondrocyte and synoviocyte cells to evaluate sensitivity lagen-Scaffold cultures using the same strains as shown in level of the method in mixed cultures. Cell cultures of human FIG.11. The expression levels of HAPLN1 and MFAP5 were chondrocytes and human synoviocytes were used to prepare determined by a comparative C, method of RT-PCR, and the mixtures of the two cell types at the following proportions: ratios were calculated as 2 (Carles-Czzuelay). 1) 0% chondrocytes/100% synoviocytes; The results obtained in scaffold cultures were similar to 15 2) 25% chondrocytes/75% synoviocytes; those obtained in monolayer cultures. 3) 50% chondrocytes/50% synoviocytes; 4) 75% chondrocytes/25% synoviocytes; and 5) 100% chondrocytes/0% synoviocytes. Example 5 Cell isolation and culture Chondrocyte strains C5, C6, and C8 were isolated and cultured as described in Examples 1 and 2. Synoviocyte cultures S6, S7, and S9 were isolated Expression of HAPLN1 and MFAP5 as a Function and cultured as described in Examples 1, 2, and 3. For Mixing of the Passage Number Experiment 1, second passage cultures of chondrocyte strain 6 (C6) and synoviocyte strain 6 (S6) were used. For Mixing The ratio of HAPLN1 to MFAP5 at various culture levels 25 Experiment 2, first passage cultures of chondrocyte strain 8 was investigated. (C8) and synoviocyte strain 7 (S7) were used. For Mixing Cell isolation and culture Chondrocyte cultures C19. Experiment 3, first passage cultures of chondrocyte strain 5 C20, C31, C32, and C33 were isolated using the Protease (C5) and synoviocyte strain 9 (S9) were used. method as described in Example 1, and cultured as described RNA isolation and cDNA preparation RNA and cDNA 30 were prepared using the RNeasyTM Mini Kit (Qiagen) and the in Example 1. Synoviocyte cultures S6 and S7 were isolated High Capacity cDNA Reverse Transcription Kit (Applied and cultured as described in Examples 1 and 2. Synoviocyte Biosystems Inc.) as described in Example 3. culture S8 was isolated by Subjecting minced synovium tissue Gene expression analysis—Gene expression analysis was to digestion in a solution containing collagenase and DNase performed as described in Example 3. for 2 hours at 37° C. Cell culture of S8 was performed as 35 The expression levels were determined by a comparative described in Example 1. For chondrocyte cultures C19, C20, C method of RT-PCR. The HAPLN1:MFAP5 ratios were C31, C32, and C33, samples of cartilage derived cells (la calculated as 2 (Ctes-Czzuelay). The results of the beled “0” in FIGS. 14A and 14B), primary cultured cells mixing experiments are provided in FIG. 15, which shows the (labeled “1” in FIGS. 14A and 14B), first passage cells (la ratios of expression levels of HAPLN1 and MFAP5 in beled “2 in FIGS. 14A and 14B), and second passage cells 40 samples consisting of mixed populations of chondrocytes and (labeled “3” in FIGS. 14A and 14B) were taken. For synovio synoviocytes. In the cultures that contained 75% or less chon cyte culture S7, samples of primary cultured (labeled “1” in drocytes and 25% or more synoviocytes, the HAPLN1: FIG. 13), first passage (labeled “2 in FIG. 13), second pas MFAP5 expression level ratios were about 1 or lower. Higher sage (labeled '3' in FIG. 13), third passage (labeled “4” in ratios corresponded to higher proportions of chondrocytes in FIG. 13), and fourth passage (labeled “5” in FIG. 13) cells 45 the tested samples. The ability of the assay to discern mixtures were taken. For synoviocyte cultures S6 and S8, samples of of cell cultures showed that, on average, Samples composed first passage (labeled “2 on FIG. 13), second passage (la of at least 67% chondrocytes with the balance consisting of beled “3” on FIG. 13), third passage (labeled “4” on FIG. 13), synovial fibroblasts to produce a positive Craces-C, and fourth passage (labeled “5” on FIG. 13) cells were taken. HAPLN1. Contamination by other cell types, such as dermal 50 fibroblasts, can also be detected with this assay. RNA isolation and cDNA preparation RNA and cDNA were prepared using the RNeasyTM Mini Kit (Qiagen) and the Example 7 High Capacity cDNA Reverse Transcription Kit (Applied Biosystems Inc.) as described in Example 3. Analysis of Relationship Between Assay Response Gene expression analysis—Gene expression analysis of 55 and Molecular Ratio of Markers the synoviocyte samples was performed as described in Example 1. Gene expression analysis of the chondrocyte Synthetic RNA transcripts of HAPLN1 and MFAP5 were samples was performed as described in Example 3. employed to determine the relationship between the assay response and the molecular ratio of the markers in test FIGS. 13, 14A, and 14B depict the change in the expres 60 samples. First, primary PCR (Platinum PCR Supermix, Invit Sion level ratios for HAPLN1 and MFAP5 as a function of the rogen catalog number 11306-016) was performed on human passage number. The expression levels were determined by a cDNA using the primers listed in Table 8. For HAPLN1, these comparative C. method of RT-PCR. The HAPLN1:MFAP5 primers amplified nucleotides from positions 256 to 1171 of ratios were calculated as 2 (Carles-Czzuelay). the HAPLN1 gene (Accession No. NM 001884.2). For The ratios of HAPLN1 to MFAP5 were consistently low at 65 MFAP5, these primers amplified nucleotides from positions all culture levels for the synoviocyte samples relative to the 32 to 728 of the MFAP5 gene (Accession No. chondrocyte samples. Theratios of HAPLN1 to MFAP5 were NM 003480.2). PCR products were analyzed on 1.5% tris US 8,029,992 B2 23 24 acetate EDTA (TAE) agarose gels using a 100 base pair TABLE 9 molecular size ladder (100 bp PCRMolecular Ruler, BioRad catalog number 170-8206) for reference and SYBR Green I Expected Transcript Sizes and Conversion Factors (Invitrogen catalog number S-7563) gel staining. The result Expected ing primary amplicons were gel purified with 4% native tris transcript size Molecular weight borate EDTA (TBE) polyacrylamide (PAGE) gels and used as Transcript (bases) (Daltons) Copiesing a template for secondary PCR with the primers listed in Table HAPLN1 917 3.145 x 10 g/mole 1.914 x 10°/ng 8. Gel-purified primary amplicon template for the secondary MFAPS 698 2.394 x 10 g/mole 2.515 x 10°/ng MFAP5 amplification was loaded at 0.56 ng per 200 uL of secondary reaction. Gel-purified primary amplicon template 10 for the secondary HAPLN1 amplification was loaded at 2.5 ng per 900 uL of secondary reaction. Secondary amplicons TABLE 10 were purified from native TBE PAGE gels as described and used as templates for in vitro transcription with an Ambion Assay Response Versus Molecular Ratio of Markers MegaScript T7 Kit. Up to 1.3 ug of secondary amplicon 15 Exact molecular template was used per 20 uL transcription reaction. The Copies of Copies of ratio of Assay response, resulting transcripts were gel purified with 6% TBE-Urea HAPLN1 MFAPS HAPLN1:MFAPS HAPLN1:MFAPS (TBU) polyacrylamide gels, resuspended in 0.1 mM EDTA, 1 x 108 1 x 108 1 O.470 and quantified spectrophotometrically with readings per 1 x 107 1 x 107 1 O448 1 x 10 1 x 10 1 O423 formed in duplicate using a conversion factor of 1 go unit 1 x 10 1 x 10 1 O.452 corresponding to a concentration of 40 ng/L RNA. The gel 1 x 10' 1 x 10' 1 O.443 purified transcripts were analyzed on 6% TBUgels to assess 1 x 10 1 x 10 1 O.478 purity. After determination of purity by PAGE analysis and Average = 0.452 quantitation by spectrophotometry, the number of transcript copies per LL was determined using the conversion factors 25 listed in Table 9. These conversion factors assume an average TABLE 11 base molecular weight of 343 Daltons. Avagadro's constant was considered to be 6.02x10°/mole. It was also assumed Exact Molecular Ratio of Markers at Various Assay Responses that the first base transcribed was the +1 G from the T7 Assay Response, Exact molecular ratio of promoter, followed by the target sequence. The transcripts 30 were diluted in yeast RNA carrier buffer (20 ng/uL solution of HAPLN1:MFAPS HAPLN1:MFAPS Yeast RNA (Ambion catalog number AM7120G) in nuclease 500 1106 100 2212 free water) at concentrations ranging from 103 to 108 copies/ 5 11.06 uL. The dilutions were then tested using the RT-PCR proce 1 2.212 dure given in Example 3 of the patent application, except that 35 O.2 O.4425 2 uL of cDNA was used per 13 ul. PCR reaction. The ratio of O.O1 O.O2212 HAPLN1:MFAP5 was then calculated for each dilution using O.OO2 O.OO4425 the comparative Ct method as described in Example 4 of the patent application. The assay response using the copy number standards was 40 Example 8 compared to the known molecular ratio. These results are shown in Table 10. With the relationship between the assay Absolute Quantitation Analysis of Chondrocyte and response and the molecular ratio determined, the exact Fibroblast Markers molecular ratios at various assay responses were calculated. These results, shown in Table 11, indicate that when the 45 Gene expression analysis using an absolute quantitation comparative Ct determined acceptance boundary equals 1 method was performed. Table 12 lists the cell cultures used in (for example, where HAPLN1:MFAP5=1), this corresponds this Example. The various cultures were isolated and cultured to an exact molecular ratio of HAPLN1:MFAP5 of 2.212. as discussed in the Examples above. RNA from the cell cul tures was isolated using the RNEASYTM Kit as discussed in TABLE 8 50 Example 3. RT-PCR was performed on the cell cultures as described in Example 3, except that 2 ul of cDNA was used Primers Used for Amplification of per 13 uL PCR reaction. In vitro transcribed RNA standards Copy Number Standards for HAPLN1 and MFAP5 (prepared as described in Example Forward Primer Reverse Primer 7) were diluted to yield cDNA with final concentrations of Forward Primer (Secondary (Primary and 55 5x10,5x10, and 5x10 copies peruL. Standard curves were Marker (Primary PCR) PCR) Secondary PCR) generated by graphing the Ctresults from the standards on the HAPLN1, 5 GCCAAGGTGTT 5 TAATACGACTC 5 CTCTGAAGGAG y-axis, Versus the logarithm of the number of copies per TTCACACAG 3 '' ACTATAGGGGCCAA TAGACACCA 3" reaction (10, 10, and 10) on the x-axis. A linear trendline (SEO ID NO: 22) GGTGTTTTCACACA (SEO ID NO :24) was fitted to the data, and the quantities of HAPLN1 and G 3." 60 MFAP5 mRNA copies present in each test sample were deter (SEQ ID NO:23) mined mathematically. This method of quantitation has been MFAPs is CCTAGCCTGGC is TAATACGACTC is CCATTGGGTCT previously described, e.g., in Real-Time PCR Systems: TTTCTTGCTC 3 ACTATAGGGCCTAG CTGCAAATCC 3 Applied Biosystems 7900HT Fast Real-Time PCR System (SEO ID NO:25) CCTGGCTTTCTTGC (SEO ID NO:27 TC 3' and 7300/7500 Real-Time PCR Systems, Chemistry Guide, (SEQ ID NO: 26) 65 Applied Biosystems, 2005, Part No. 4348358 Rev. E. The molar ratio of HAPLN1:MFAP5 was then calculated for each sample. FIG. 16 depicts molar ratios of HAPLN1:MFAP5 in US 8,029,992 B2 25 26 various cell cultures. These results indicate that the molar TABLE 12-continued ratio of HAPLN1:MFAP5 in chondrocytes is high relative to synoviocytes and dermal fibroblasts. Cell cultures used in Absolute Quantitation Analysis (Example 8 Cell Culture Cell Type Type of Cell Culture TABLE 12 5 S16 Synoviocyte Third Passage Cell cultures used in Absolute Quantitation Analysis (Example 8 F1 Dermal Fibroblast Second Passage F2 Dermal Fibroblast Second Passage Cell Culture Cell Type Type of Cell Culture C26 Chondrocyte Second Passage 10 All publications and patent documents cited herein are C27 Chondrocyte Second Passage C28 Chondrocytey Second Passage9. incorporated- 0 by reference in their entirety. To the extent the S14 Synoviocyte Third Passage material incorporated by reference contradicts or is inconsis S15 Synoviocyte Third Passage tent with the present specification, the present specification will Supersede any such material.
SEQUENCE LISTING
<16 Os NUMBER OF SEO ID NOS: 27
<21 Os SEQ ID NO 1 &211s LENGTH: 29 OO &212s. TYPE: DNA <213> ORGANISM: Homo sapiens
<4 OOs SEQUENCE: 1 atticoagcct cattgtaa.ca cacattctac goctagoctd gotttcttgc tict c cct cat 60 ct cattgttt cagoggaggc caaatctgaa gtCctitt CC a gggagtggct Ctgttcatct 12O tatt cqc cag ccaaagtagg aacagcgitaa gaggagagag acac attcag cagccalaagg 18O act cqgtgga aagag cagaa caccataga C aatatgtc.gc ticttgggacc caaggtgctg 24 O
ctgtttcttg ctgcatt cat catcaccitct gactggatac ccct gggggt caatagt caa 3 OO cgaggagacg atgtgactica agcgacticca galaa cattca cagaagatcc taatctggtg 360
aatgatcc.cg ctacagatga aa cagttittg gctgttittgg citgatattgc acct tccaca 42O gatgacttgg cct coct cag taaaaaaat accactgcag agtgctggga tigagaaattit 48O
acctgcacaa ggctic tact c tdtgcatcgg ccggittaaac aatgcattca toagittatgc 54 O
ttcaccagtt tacgacgitat gtacatcgt.c aacaaggaga totgct citcg to ttgttctgt 6 OO
aaggaac acg aagctatgaa agatgagctt to cqtcaga tiggctggtct gcc.ccctagg 660 agact cogtic gotccaatta citt.ccgacitt cotc cct gtgaaaatgtgga tittgcagaga 72O Cccaatggit C titgat catt gaaaaagagg aaagaagaala aaatgt atgg gtgagaggaa 78O
ggaggat.ctic cttctitctcc aaccattgac agctaac cct taga cagtat ttcttaaacc 84 O
aatcc titttg caatgtc.cag ctitttacccc tact citctac tttitt caccc aaactgataa 9 OO catttat ct c attittctago acttaaaata caaagtictat attattgcat aattittgctg 96.O
cittct caata toatagacac agitgaataga tigatgacitat atggcttata tacaaacatt O2O ctatotacaa tittcaaggga gactaaactt taggctaata at ctitt act a ttgaatctgt O8O
ctgatataga tottagggitt galagaagcta totttgttcta tttgggctaa ccatagaatt 14 O
to atttattt to ct cacaat attitt cotag accaact coc catcattcac gtgttcct ct 2 OO ttact cittac tittaactatt ttgctggctt gcc.cgaaaat ttgcctggca agtic titcc tt 26 O atalagacaca to atggtaag titttgtagt c ct gtaagatt ctdcaacaca gt caagaatt 32O
atacaatcct actagoaata tataagg acc caaaatgtct tctgctaagc ticagaggctg 38O
gggctaaagc atgaggact a tigc.ca.gctat agaacttgga ct cataattic gctato caat 4 4 O
ttitt catgca gttgtctagt cqggaagitaa ggttggaaac taagttct cat ttactgattic 5 OO
US 8,029,992 B2 73 74 - Continued <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: HAPLN1 forward primer <4 OOs, SEQUENCE: 16 tgaaggatta gaagatgata ctgttgttg 28
<210s, SEQ ID NO 17 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: HAPLN1 reverse primer <4 OOs, SEQUENCE: 17 gccc.cagtic taaagtaa
<210s, SEQ ID NO 18 &211s LENGTH: 18 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: HAPLN1 probe sequence
<4 OOs, SEQUENCE: 18 taca aggtgt gg tatt Co 18
<210s, SEQ ID NO 19 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: MFAP5 forward primer sequence
<4 OOs, SEQUENCE: 19 cgaggagacg atgtgactica ag 22
<210s, SEQ ID NO 2 O &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: MFAP5 reverse primer sequence
<4 OOs, SEQUENCE: 2O agcgggat.ca tt Caccagat
<210s, SEQ ID NO 21 &211s LENGTH: 17 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: MFAP5 probe sequence <4 OOs, SEQUENCE: 21 acatt cacag aagatcc 17
<210s, SEQ ID NO 22 &211s LENGTH: 2O &212s. TYPE: DNA <213> ORGANISM; artificial 22 Os. FEATURE: <223> OTHER INFORMATION: Primer sequence <4 OOs, SEQUENCE: 22 gccalaggtgt titt cacacag US 8,029,992 B2 75 76 - Continued
SEQ ID NO 23 LENGTH: 4 O TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: Primer sequence
< 4 OOs SEQUENCE: 23 taatacgact Cactataggg gccaaggtgt titt cacacag 4 O
SEQ ID NO 24 LENGTH: 2O TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: Primer sequence
< 4 OOs SEQUENCE: 24 Ctctgaagca gtag acacca
SEO ID NO 25 LENGTH: 21 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: Primer sequence
< 4 OOs SEQUENCE: 25 cctagoctogg ctittcttgct c 21
SEQ ID NO 26 LENGTH: 41 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: Primer sequence
< 4 OOs SEQUENCE: 26 taatacgact cactataggg cctagoctogg ctittcttgct c 41
SEO ID NO 27 LENGTH: 21 TYPE: DNA ORGANISM: Artificial FEATURE: OTHER INFORMATION: Primer sequence <4 OOs, SEQUENCE: 27 c cattgggtc. tctgcaaatc c 21
The invention claimed is: plurality of cells; and, in step d), determining the composition 1. A method of evaluating the composition of a cell culture, of the culture based on the average expression level of the said method comprising: 55 chondrocyte marker and the average expression level of a) obtaining a culture of cells for expansion of chondro MFAP5. cytes; b) Rising a plurality of cells from the cell culture; 3. The method of claim 2, wherein MFAP5 and the chon c) determining the average expression level of microfibril drocyte marker are such that the ratio of their expression lar associated protein 5 (MFAP5) in the plurality of cells; levels (chondrocyte marker to MFAP5) in chondrocytes is and 60 equal to or greater than 5 times the ratio in dermal fibroblasts d) determining the composition of the culture based on the or synoviocytes. average expression level of MFAP5; 4. The method of claim 2, wherein the chondrocyte marker wherein the average expression level of MFAP5 below a is selected from the group consisting of hyaluronan and pro predetermined threshold indicates that the cell culture com teoglycan link protein 1 (HAPLN1), matrix Gla protein prises chondrocytes. 65 (MGP), GF-like repeats and discoidin I-like domains 3 2. The method of claim 1, further comprising determining (EDIL3), WNT1 inducible signaling pathway protein 3 the average expression level of a chondrocyte marker in the (WISP3), aggrecan 1 (AGC1), cartilage oligomeric matrix US 8,029,992 B2 77 78 protein (COMP), type II collagen, alpha 1 (COL2A1), type d) determining the expression levels of MFAP5 and IX collagen, alpha 1 (COL9A1), type XI collagen, alpha 1 HAPLN1 in a plurality of cells obtained from the cell (COL11A1), leukocyte cell derived chemotaxin 1 protein culture; and (LECT 1), S100 calcium binding protein beta (S100B), carti e) determining the composition of the culture based on the lage acidic protein 1 (CRTAC1), SRY-box9 protein (SOX9), 5 expression levels of MFAP5 and HAPLN1. and nebulette (NEBL). 21. The method of claim 20, wherein the expression levels 5. The method of claim 2, wherein the chondrocyte marker of MFAP5 and HAPLN1 are determined at the RNA level. is HAPLN1. 22. The method of claim 21, wherein the expression levels 6. The method of claim 2, wherein the expression levels of are determined using PCR. MFAP5 and the chondrocyte marker are determined at the 10 RNA level. 23. The method of claim 22, wherein the expression levels 7. The method of claim 6, wherein the expression levels are are determined using comparative C, PCR method. determined using PCR. 24. The method of claim 20, wherein an expression level 8. The method of claim 7, wherein the expression levels are ratio of HAPLN1 to MFAP5 greater than 0.25, as determined determined using comparative CPCR method. 15 using comparative C, PCR method, indicates that the cell 9. The method of claim 2, wherein an expression level ratio culture contains chondrocytes. of the chondrocyte marker to MFAP5 of greater than 0.25, as 25. The method of claim 20, wherein a molar ratio of determined using comparative CPCR method, indicates that HAPLN1 to MFAP5 greater than 0.55 indicates that the cell the cell culture contains chondrocytes. culture contains chondrocytes. 10. The method of claim 2, wherein a molar ratio of the 26. A method of evaluating the composition of a cell cul chondrocyte marker to MFAP5 of greater than 0.55 indicates ture comprising: that the cell culture contains chondrocytes. a) obtaining a cartilage biopsy from a mammal; 11. The method of claim 1, wherein the cell culture com b) isolating cells from the biopsy: prises cells obtained from a cartilage biopsy. c) culturing the isolated cells in a cell culture to produce 12. The method of claim 11, wherein the cartilage biopsy is 25 chondrocytes; taken from a knee joint. d) determining the expression levels of MFAP5 and a chon 13. The method of claim 11, wherein, following the evalu drocyte marker in a plurality of cells obtained from the ation of the cell culture, the cells from the cell culture are cell culture; and administered to a patient in need thereof. e) determining the presence of chondrocytes in the cell 14. The method of claim 13, wherein the cells from the 30 culture based on the expression levels of MFAP5 and the culture are used for autologous chondrocyte implantation. chondrocyte marker. 15. A method of evaluating the composition of a cell cul 27. The method of claim 26, wherein the chondrocyte ture, the method comprising: marker is selected from the group consisting of hyaluronan a) obtaining a culture of cells for expansion of chondro and proteoglycan link protein 1 (HAPLN1), matrix Gla pro cytes; 35 tein (MGP), GF-like repeats and discoidin I-like domains 3 b) determining the expression level of MFAP5 in the cell (EDIL3), WNT1 inducible signaling pathway protein 3 culture; and (WISP3), aggrecan 1 (AGC1), cartilage oligomeric matrix c) determining the composition of the cell culture based on protein (COMP), type II collagen, alpha 1 (COL2A1), type the expression level of MFAP5. IX collagen, alpha 1 (COL9A1), type XI collagen, alpha 1 16. The method of claim 15, further comprising determin 40 (COL11A1), leukocyte cell derived chemotaxin 1 protein ing the expression level of a chondrocyte marker in the cell (LECT 1), S100 calcium binding protein beta (S100B), carti culture; and, in step c), determining the composition of the lage acidic protein 1 (CRTAC1), SRY-box9 protein (SOX9), cell culture based on the expression levels of the chondrocyte and nebulette (NEBL). marker and MFAP5. 28. The method of claim 26, wherein the chondrocyte 17. The method of claim 16, wherein the chondrocyte 45 marker is HAPLN1. marker is selected from the group consisting of hyaluronan 29. A method of evaluating the composition of a cell cul and proteoglycan link protein 1 (HAPLN1), matrix Gla pro ture to confirm the presence of chondrocytes comprising: tein (MGP), GF-like repeats and discoidin I-like domains 3 a) obtaining a plurality of cells from a cell culture compris (EDIL3), WNT1 inducible signaling pathway protein 3 ing cells derived from cartilage or synovium; and (WISP3), aggrecan 1 (AGC1), cartilage oligomeric matrix 50 b) determining the average expression level of microfibril protein (COMP), type II collagen, alpha 1 (COL2A1), type lar associated protein 5 (MFAP5) in the plurality of cells; IX collagen, alpha 1 (COL9A1), type XI collagen, alpha 1 wherein the average expression level of MFAP5 below a (COL11A1), leukocyte cell derived chemotaxin 1 protein predetermined threshold indicates that the cell culture com (LECT 1), S100 calcium binding protein beta (S100B), carti prises chondrocytes. lage acidic protein 1 (CRTAC1), SRY-box9 protein (SOX9), 55 30. A method of evaluating the composition of a chondro and nebulette (NEBL). cyte cell culture to identify the presence of fibroblasts and/or 18. The method of claim 16, wherein the chondrocyte synoviocytes comprising: marker is HAPLN1. a) obtaining a plurality of cells from a chondrocyte cell 19. The method of claim 16, wherein the expression levels culture; and of the chondrocyte marker and MFAP5 are determined at the 60 b) determining the average expression level of microfibril RNA level. lar associated protein 5 (MFAP5) in the plurality of cells; 20. A method of evaluating the composition of a cell cul wherein an average expression level of MFAP5 above a pre ture, said method comprising: determined threshold indicates that the cell culture comprises a) obtaining a cartilage biopsy from a mammal; fibroblasts and/or synoviocytes. b) isolating cells from the biopsy: 65 31. A method of evaluating the composition of a cell cul c) culturing the isolated cells in a cell culture to produce ture to determine the ratio of chondrocytes to fibroblasts chondrocytes; and/or synoviocytes comprising: US 8,029,992 B2 79 80 a) performing a cell count of a chondrocyte cell culture 33. The method of claim 32, wherein the chondrocyte b) obtaining a plurality of cells from the cell culture; and marker is selected from the group consisting of hyaluronan c) determining the average expression level of microfibril and proteoglycan link protein 1 (HAPLN1), matrix Gla pro lar associated protein 5 (MFAP5) in the plurality of cells; tein (MGP), GF-like repeats and discoidin I-like domains 3 wherein an average expression level of MFAP5 above a pre (EDIL3), WNT1 inducible signaling pathway protein 3 determined threshold indicates that the cell culture comprises (WISP3), aggrecan 1 (AGC1), cartilage oliqomeric matrix more fibroblasts and/or synoviocytes than chondrocytes and protein (COMP), type II collagen, alpha 1 (COL2A1), type an average expression level of MFAP5 below a predetermined IX collagen, alpha 1 (COL9A1), type XI collagen, alpha 1 threshold indicates that the cell culture comprises more chon (COL11A1), leukocyte cell derived chemotaxin 1 protein 10 (LECT 1), S100 calcium binding protein beta (S100B), carti drocytes than fibroblasts and/or synoviocytes. lage acidic protein 1 (CRTAC1), SRY-box9 protein (SOX9), 32. The method of claim 31, further comprising determin and nebulette (NEBL). ing the expression level of a chondrocyte marker in the plu 34. The method of claim 32, wherein the chondrocyte rality of cells; and determining the ratio of chondrocytes to marker is HAPLN1. fibroblasts and/or synoviocytes based on the expression lev els of the chondrocyte marker and MFAP5. UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 8,029,992 B2 Page 1 of 1 APPLICATIONNO. : 12/098033 DATED : October 4, 2011 INVENTOR(S) : Stephen Rapko et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below:
In claim 31, column 79, line 1, “culture should read --culture:--. In claim 33, column 80, line 6, “oliqomeric should read --oligomeric--.
Signed and Sealed this Third Day of January, 2012
David J. Kappos Director of the United States Patent and Trademark Office