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Cryopreservation Strategy for Tissue Engineering Constructs Consisting

Cryopreservation Strategy for Tissue Engineering Constructs Consisting

CryoLetters 36 (5), 325-335 (2015) © CryoLetters, [email protected]

CRYOPRESERVATION STRATEGY FOR ENGINEERING CONSTRUCTS CONSISTING OF HUMAN MESENHYMAL STEM CELLS AND HYDROGEL Yingnan Wu1,2,#, Feng Wen3,#, Sok Siam Gouk3, Eng Hin Lee1,2 and Lilia L. Kuleshova3,*

1Tissue Engineering Program; 2Department of Orthopaedic Surgery, YLL School of Medicine; 3Low Preservation Unit, National University Medical Institutes, YLL School of Medicine, National University of Singapore, Singapore . #These authors contributed equally. *Corresponding author email: [email protected]

Abstract BACKGROUND: The development of strategy for - constructs, particularly biologically inspired nanoscale materials and hydrogels mimicking the in vivo environment is an active area. A cryopreservation strategy mimicking the in vivo environment for cell- hydrogel constructs may enhance cell proliferation and biological function. OBJECTIVE: To demonstrate the efficacy of vitrification as a platform technology involving and human mesenchymal stem cells (hMSCs). MATERIALS AND METHODS: Microcarriers made from alginate coated with and collagen are used. Conventional and vitrification were compared. The vitrification strategy includes 10 min step-wise exposure to a vitrification (40% v/v EG, 0.6M sucrose) and immersion into liquid . RESULTS: Confocal imaging of live/dead staining of hMSCs cultured on the surface of microcarriers demonstrated that vitrified cells had excellent appearance and prolonged spindle shape morphology. The proliferation ability of post- vitrified cells arbitrated to protein Ki-67 gene expression was not significantly different in comparison to untreated control, while that of post-freezing cells was almost lost. The ability of hMSCs cultured on the surface of microcarriers to proliferate has been not affected by vitrification and it was significantly better after vitrification than after conventional freezing during continuous culture. Collagen II related mRNA expression by 4 weeks post-vitrification and post-freezing showed that ability to differentiate into cartilage was sustained during vitrification and reduced during conventional freezing. No significant difference was found between control and vitrification groups only. CONCLUSION: Vitrification strategy coupled with advances in hMSC-expansion platform that completely preserves the ability of stem cells to proliferate and subsequently differentiate allows not only to reach a critical cell number, but also demonstrate prospects for effective utilization and transportation of cells with their support system, creating demand for novel biodegradable materials. Keywords: vitrification, hydrogel, microcarrier, mesenchymal , chondrogenic differentiation

INTRODUCTION 4,7,8,11,12]. In the present work, we aim to prove the efficacy of vitrification as a platform Ascertaining the superiority of vitrification technology for tissue engineered constructs with for tissue engineering constructs is particularly human mesenchymal stem cells (hMSCs). of intrest [1-12]. Designing of biomaterials with Vitrification has great potential as a part of properties that will not be transformed during tissue engineering approach, yet, it has been interaction with cryoprotective agents, cooling to largely investigated in the application to native cryogenic and subsequent warming tissues and cells [13-29]. There are fundamental is a challenge. Vitrification for cell-biomaterial differences between vitrification and freezing to constructs, including bio-inspired nanoscale cryopreservation [14,19,25]. Vitrification avoids materials and hydrogels mimicking the in vivo formation during cooling and warming inside environment is an active area of our research [1- cells and biomaterial [1-12,25] while freezing

325 allows ice outside cells in the medium and with MSCs will involve much greater number of biomaterial as concentration is cells for transplantations. usually low [13,14]. This is an advantage of Culturing stem cells on microcarriers that vitrification over freezing which provides provide suitable biomechanical properties and amorphous solidification, resulting in less biocompatibility is one of reliable methods to mechanical stress to the cells/tissues and cell- increase a number of cells in several folds [40]. carriers. Over the years we have provided The microcarrier system can be applied to scale- evidence that a vitrification strategy is vital for up the hMSCs expansion to multiply amount of successful preservation of engineered constructs cells required in certain clinical applications. [1-4,7,8,10-12,17]. It provides intactness of cell Cryopreservation of cells along with their membrane [12], attachment ability of cells to the culture support system has its own advantages. carrier [8] and cell-cell interactions [1,2,15,16]. Clinical treatments are frequently altered or In a biomaterial context, we have provided delayed. Cryopreservation of cells with their evidence that properly calculated composition of culture support system adds to the flexibility of vitrification and the cooling-warming clinical scheduling and facilitates continuous cycle do not impair the integrity and quality of cell expansion. The detachment and dissociation the materials involved, allowing free migration of MSCs may trigger unwelcome behaviour in and aggregation of cells if necessary [1,2]. It cells, such as differentiation. The re-introduction was also demonstrated by us that vitrification is of hMSCs into their original culture system superior in the maintenance of viability and again with the purpose of perpetual culture metabolic function of cells [1-4,7,8,11,12, and requires special research facilities which proliferation and differentiation potential of 3D becomes a rather challenging task in the clinical clusters of neuronal stem cells [15,16] and swine setting. Finally, cryopreservation of detached mesenchymal stem cells (MSCs) cultured on cells is much less effective compared to that for 2D-, 3D tissue engineered constructs for bone attached cells [12]. A cryopreservation strategy regeneration [7,8]. coupled with a MSC-expansion platform permit In cartilage regeneration, the development not only attaining a critical cell number, but also of effective cryopreservation protocols is a effective utilization and transportation of MSC- useful tool to preserve human MSCs from a hydrogel cultures at any time point or location, variety of sources for future clinical application. creating the demand for novel biodegradable Owing to its intrinsic property of being a materials. A vitrification strategy accompanies nonvascularized tissue, damaged articular the biomaterial platform and is applicable to cartilage severely lacks the capacity to heal or other similar cultures, in particular, a growing regenerate. The research is of high significance. number of novel hydrogel culture systems will Despite recent advances in surgical and non- benefit from a fully developed vitrification surgical interventions, treatment of cartilage strategy. In recent time, it was proven in swine lesions still remains a problem [30]. Cell-based model that combination of a gel scaffold with therapy has shown promising results in treating MSCs can be used successfully for the purpose damaged cartilage [31,32]. Since the 1970s, of new formation of cartilaginous tissue (41). MSCs have been used as chondrocyte progenitor Articular cartilage defects were repaired through cells for cartilage healing [33] but it was not transplantation of MSCs/scaffolds in a primate until the end of 1990s that intense investigation model (42). The collagen scaffold improves the into the regenerative potential of proliferated repair of cartilage (43). Therefore, developing a MSCs [34-36]. MSCs are multipotent cells reliable strategy for preservation of allogeneic present in the bone marrow in low quantity (1 in hMSCs on microcarries involving bioabsorbable 104-105 mononuclear cells), which are capable of materials, serving as permissive substrates for differentiating into chondrocytes, osteocytes, cell growth is an important issue in the field of myocytes, and adipocytes [37]. MSCs have low regenerative medicine. or little immunogenicity when transplanted into another host [38,39]. This advantage makes MSCs more amenable to cell therapy. Treatment

326 MATERIALS AND METHODS was coated on the alginate bead surface followed by conjugation of collagen type I to the chitosan layer (Figure 1.1). All steps were performed at

(A))…… … (B))…… … (C) Figure 1.1 Scanning electron microscopy of microcarriers. (A) alginate microcarrier, (B) chitosan-coated alginate microcarrier, (C) collagen-conjugated chitosan/alginate microcarrier.Scale bares are 20µm. room temperature. Microcarriers coated by chitosan and collagen type I were tested for process of introduction to and removal of cryoprotectant as well as cooling to -196°C and warming to 38°C as described in section 2.2 to confirm the integrity. Phase contrast microscopy was used to assess the integrity of microcariers during optimization. Microcarriers can withstand vitrification procedure described in Figure 1.2 without compromising the structural integrity. Seeding of hMSCs on the microcarrier surface hMSCs were harvested from T175 culture flask at P3 and mixed with microcarrier system in a 15mL conical tube and cultured in growth Figure 1.2 Schematic representation of vitrification- media (Gibco Ltd, stem cells expansion warming and dilution for cryopreservation of hMSCs cultured on surface of microcarriers. medium) for 3 days. The cells spontaneously Stem cell culture: hMSC isolation and expansion adhered/ aggregated on the surface of the Adult hMSCs were obtained from whole microcarriers. bone marrow aspirates of consented donors and Cryopreservation of hMSCs on microcarriers culture expanded. Mononuclear cell expansion Vitrification. On day 14 after seeding, medium was low glucose Dulbecco’s Modified hMSCs on microcarriers were transferred to a Eagle’s medium (DMEM, GIBCO, USA) with cell strainer of 40 µm pre-equilibrated with 10% 10% fetal bovine serum (FBS, GIBCO, USA) ethylene glycol (EG), followed by 25% EG (Fig. and 1% antibiotics (50 U/mL penicillin and 50 1.2). All equilibration and dilution solutions mg/mL streptomycin (GIBCO, USA). Adherent were protein or serum-free. The exposure to MSCs were passaged at ~80% confluency and vitrification solution (VS) was done in three the medium was replenished twice a week. The consecutive steps to eliminate likelihood of ice- purity of hMSCs cultures was confirmed. formation inside the carrier (each step for 3 Vitrification protocol was established for minutes). It was established in an earlier study hMSCs seeded on the surface of a microcarriers using porcine MSCs [8] that this duration of system of passage (P) 3. Only P3 cultures were exposure was within the limit of VS used for freezing and control groups. which MSCs could tolerate. Equilibration prior Microcarriers: modified microbead system to cryopreservation was performed at room Microcarriers, modified micro-bead system, temperature. Cell-biomaterial constructs with were engineered using chitosan and collagen as minimum amount of VS were exposed to vapour described in detail [40]. Briefly, alginate beads phase of in a flat container were formed by dropping alginate solution into (15ml) for 20 sec, prior to their immersion into calcium chloride solution. Subsequently chitosan liquid nitrogen. Samaples were rapidly warmed to 38°C. Dilution was achieved by incubating 327 cells in 1M sucrose for 5 min followed by 4 Ethidium homodimer-1 (red: dead) at the consecutive steps from 0.7M sucrose to DMEM excitation/emission of ~495/~515nm. Cell solutions, with 2.5-min intervals. After cooling- proliferation was investigated by performing warming cycle, hMSCs on microcarriers were Picogreen assay in combination with DNA maintained in incubator until time of assessment. quantification. DNA content of samples was Cryopreservatiion by freezing. With the calculated from the standard curve obtained slow cooling method, hMSCs were exposed to using thymus / bacteriophage DNA. 10% (v/v) dimethylsulfoxide (Me2SO) freezing activity was measured by Alamar Blue assay. media in step wise-manner, 2.5% (v/v) in each Cells seeded on microcarriers were incubated in step and 10% FCS and 90% FCS were placed Alamar Blue fluorescent dye for 1.5 h and cryovials (0.7×1.0 cm). Samples were removed absorbance measured at 560/590 nm using from culture and exposed to for FLUOstar Optima - BMG Labtech (Germany). 3 min at room temperature before transferring to Differentiation ability of hMSCs −80°C freezer at 1°C /min in a ‘Frosty boy’ Cells harvested from microcarriers were (Nalgene, USA) apparatus. For thawing, induced to chondrogenic and osteogenic cryovials were immersed in a 37°C water-bath differentiation. Chondrogenic- and osteogenic- until no visible ice was detected. Samples were differentiation have been induced in serum free, then removed from the Me2SO solution and chemically defined media. Chondrogenesis was washed in fresh medium warmed to 37°C, and induced in the presence of a defined medium kept in culture in the growth chamber at 37°C that included 100 nM dexamethasone and 10 with an atmosphere 5% CO2/95% air. ng/ml transforming growth factor–β3 (TGF-β3). Assessment of cell viability and proliferation Osteogenic induced medium consisting of Cell viability and integrity of microcarriers standard medium plus 50mM L-ascorbic asid-2- structure was examined by confocal microscope. phosphate, 10 mM β-glycerophosphate and 100 Microcarriers with hMSCs were sectioned as nM dexamethasone was used. The media was whole structure and stained with live/dead replaced every two days. The cell/microbeads cytotoxicity kit, calcein AM (green: live) and mixtures were cultured in 15ml falcon tubes and

__ A __ B __ C __ D __ Figure 2.2 Phase contrast microscopy and fluorescent images of hMSCs cultured on the surface of microcarriers. Initially cells were mixed with microcarriers and processed at dynamic culture. hMSCs cultured on surface of microcarriers (stock culture): (A) at the time of vitrification and (B) after continues culture for additional 9 days. Vitrified and subsequently cultured for 9 days hMSCs on the surface of microcarriers (single bead): (C) stained using fluorescein diacetate (FDA) and (D) without staining. Scale bars are 100µm.

A B _____ C ______

Control Vitrification Freezing

D E F

Figure 3. Confocal microscopy images of LIVE/DEAD cytotoxicity test stained hMSCs cultured on the surface of microcarriers (A) untreated control; (B) after vitrification-warming cycle (C) after freezing-thawing cycle. Green indicates live cells and red indicates dead cells. Scale bars are 100µm, 200µm and 100µm on images (A) (B) and (C) respectively.0(D, E, F) show magnified to identical size parts of images (A, B, C) respectively. 328 incubated at 37°C, 5% CO2/95% air. MSCs were ANOVA with Bonfferoni adjustment. Results harvested after 4 weeks induction. were presented as mean ± standard deviation Sulphated glycosaminoglycans (GAG) were (SD). Significant level was set up at 0.05. measured using the Blyscan Assay. Samples were digested with papain digestion buffer for 18 hr at 60°C followed by incubation with RESULTS dimethyl-methylene blue dye. Readings were Activities before and after vitrification of normalized against the DNA content quantitated hMSCs cultured on surface of microcarriers using Hoechst DNA Assay. were evaluated at dynamic culture conditions. Differentiated cells were used to confirm Microcarriers were transparent (Figure 1.1) at their lineages using immunohistochemical and first and became less transparent with cell histological analysis. For histological evaluation, expansion (Figure 2.2A and 2.2B). Calcein sections were stained with Alcian blue (pH 2.5) staining showed cell growth on microcarrier (chondrogenic lineage) with fast red counter- surface (Figure 2.2C). Figure 2.2C showed the staining, von Kossa (osteogenic lineage) and different edge of different surfaces. Thickness of Alizarin-red O staining with fast red counter- cell layer, boundary between layer of cells and staining as well as Hematoxylin and Eosin. For surface of microcarriers can be visualized. the immunohistochemical analaysis, sections During pre-equilibration and especially dilution (chondrogenic) and monolayer culture procedure volume of cells may change (osteogenic) were incubated with Col-II significantly. This can compromise their (chondrogenic) antibody and osteopontin attachment efficacy to the carrier possibly. (osteogenic) antibody before the horseradish However, it can be observed that cells are peroxidase (HRP)-labelled secondary antibody trapped by collagen layer in our collagen and processed with a BenchMark-automated modified microcarrier system irrespectively to staining system. Sections were their viability (Figure 3). Understandably dead counterstained with hematoxylin. cells are not able contribute to proliferation. Total RNA was extracted from cells using Cryopreservation by vitrification did not alter an RNeasy Mini Kit. cDNA synthesis from total pattern of further cells grows (Figure 2.2 D). RNA was performed using iScript cDNA Similarity non-transparency could be observed synthesis system. Real-time PCR analysis was between stock culture on day 9 (Figure 2.2 B) employed to confirm stage specific gene profile and post-vitrification and continues culture for and lineage commitment (collagen type II). additional 9 days (Figure 2.2 D). Statistical analysis Confocal imaging of live/dead staining of All statistical analyses were performed hMSCs cultured on microcarriers demonstrates using SPSS version 15. Independent-samples T that vitrified cells (Figure 3B) had identical cell Tests were carried out for the metabolic viability based on membrane integrity, as those functional and proliferation tests among control, observed for untreated control (Figure 3A). freezing and vitrification groups within the same These results were sustained in continuous day. Difference within the same treatment culture as observed 72 hours after vitrification among different days was tested by one way by confocal microscopy (image not shown). We found that hMSCs survived equally well on the microcarrier system, regardless of whether they were vitrified or cultured as a untreated control (Figure 3D,E). hMSCs had excellent appearance and prolonged spindle shape morphology (Figure 3D,E). Excellent appearance of hMSCs A remained in next 72h indicates that vitrification did not cause adverse effect on stem cells and did not impose non-lethal injury of hMSCs. The developed step-wise exposure to and removal of cryoprotectant procedures did not cause osmotic shock to hMSCs and in combination with brief equilibration steps did not cause toxic impact on C D the cells either. The collagen surface also Figure 2.1 Scanning electron microscopy (SEM) of increased the adhesive ability of hMSCs on the microcarriers. (A) alginate microcarrier; (B) chitosan coated alginate microcarrier; (C, D) collagen type I conjugated 329 chitosan coated alginate microcarrier (SEM). Scale bars are 50 µm. surface. On the other hand, dead (red) cells Although it is known from scientific literature trapped between life cells on the surface of the that human SCs after freezing are able to carrier after freezing-thawing cycle (Figure 3C) eventually recover and proliferate in continuous clearly demonstrate the shortcoming of culture, proliferation ability was not restored to conventional freezing procedure for preservation the same level as in untreated control. Therefore of hMSCs. Diffused pattern of remaining live this comparative study on proliferation and (green) cells post-freezing provide evidence that metabolic function of hMSCs in long-term life cells have been disturbed significantly by culture has been conducted to establish efficacy freezing (Figure 3F). of our vitrification strategy (Figure 5). Ki-67 expression translates cell proliferation It was found that ability of hMSCs cultured shortly after cryopreservation. We have analysed on microcarriers to proliferate as evaluated by its expression for both vitrification and freezing measurement of DNA quantity was significantly (Figure 4). Proliferation ability of post-vitrified better after vitrification than after conventional cells was not significantly different from that of freezing during the entire period of observation untreated control, while that of post-freezing (p<0.05). However the ability of hMSCs to cells decreased dramatically even with 90% FCS proliferate is not affected during vitrification as added. These results imply that after a short compared to non-treated control group (p>0.05) period of post-cryopreservation recovery culture, cell damage induced by vitrification is minimal. as indicated by high hMSCs proliferation post- vitrification. For clinical settings, injections of SCs immediately after cryopreservation and with

Figure 5. Proliferation of hMSCs aftercryopreserved with different methods. Control represents untreated hMSCs cultured on microcarriers as an independent sample; Vitrification represents hMSCs cultured on microcarriers after vitrification; Freezing represents hMSCs cultured on Figure 4. Cell proliferation associated protein Ki-67 gene microcarriers after conventional freezing-thawing cycle. expressions were analyzed by real time PCR on day 3 Significant difference was observed between control and (72h) post-cryopreservation and compared with untreated freezing groups (*), as well as vitrification and freezing control. Control represents untreated hMSCs cultured on groups, marked with hash (#)(p<0.05). For control and microcarriers as an independent sample. For control and vitrification groups, there were no significant differences vitrification groups, there were no statistical significant between control and treatment within the same day of differences between control and treatment (p>0.05). observation (p>0.05) except day 9 marked (**). Statistically significant difference was observed between control and freezing groups, marked with asterisk (*), as well as vitrification and freezing groups, marked with hash except on day 9 where results were significantly (#)(p<0.05). different. Collagen II related mRNA expression full stem cell potential preserved are desirable. by 4 weeks post-freezing, as revealed by real These requirements can be only fulfilled if time PCR, shown that ability to differentiate into human SCs are cryopreserved using vitrification cartilage was lost during freezing, but sustained since results have shown that protein synthesis is through vitrification (Figure 6). It was also suppressed up to 72h post-freezing. Upon found that Collagen I related mRNA, i.e. ability implantation, the microenvironment surrounding to differentiate into bone, was lower after the injection site is unfavorable (acidic) for freezing than after vitrification, but results were hMSCs development due to inflammation. not significantly different (figure not shown).

330 followed by collagen type I [46]. It was demonstrated that collagen type I hydrogel , which has been used frequently in clinic for matrix-based chondrocyte transplantation, supports ex vivo pre-differentiation or in situ differentiation of hMSCs for articular cartilage repair [47, 48]. Chitosan/collagen scaffolds are Figure 6. Collagen type II related mRNA also extensively used as the scaffold for cartilage expression 4 weeks post–freezing and post- tissue engineering [41, 49]. Transplantation of vitrification with matching untreated control. chitosan/ collagen type I scaffold with MSCs Control represents untreated hMSCs cultured on stimulates healing of cartilage defects in swine microcarriers; Vitrification represents hMSCs in vivo model [41]. Despite the stress on cell-cell cultured on microcarriers after vitrification. Freezing represents hMSCs cultured on contact during cryopreservation, hMSCs are microcarriers after conventional freezing-thawing capable of undergoing chondrogenesis and cycle. For control and vitrification groups, there ostrogenesis (results are not shown) in our 3D were no significant difference (p>0.05). Statistical system (Figure 7). Developed vitrification difference was observed between control and strategy which is applicable to other similar freezing groups, marked with asterisk (*), as well as systems accompanies biomaterial platform, vitrification and freezing groups, marked with hash particularly growing a number of novel hydrogel (#)(p<0.05). cultures. The promising start strongly supports Chondrogenic and osteogenic abilities of the notion that vitrification concept of hMSCs on microcarriers were not affected by cryopreserving TECs has great prospect for vitrification as compared to non-treated control regenerative medicine, supporting demand for group. The ability to undergo chondrogenic development of new bio-absorbable materials differentiation has confirmed that vitrification is enduring low temperatures. viable approach for cryopreservation of hMSCs We strongly advocate vitrification based on for articular cartilage regeneration (Figure 7). results obtained here and during decades of our research as well as in addition to the DISCUSSION understanding gained in the area of native cartilage cryopreservation through the consistent Human MSCs complex culture generated efforts of Pegg’s group, including original using tissue engineering approach could be liquidus tracking method [18] and by McGann effectively preserved by vitrification. Generally, and colleagues [19-22]. Chondrocyte recovery cryopreservation of systems described here is a of 56% after vitrification of tissue engineered challenge for at least two reasons. First, 3D cartilage has been reported so far [9]. The geometry, although favouring cell growth and cartilage was grown in the bioreactor for up to transplantation requirements might not suit the 28 d prior to vitrification [9]. The chondrocyte requirements for an effective cryopreservation in recovery of 75% after vitrification of intact terms of thermal distribution. Second, stem cells human artificial cartilage has been achieved attached on the surface of constructs are [20]. Furthermore, it was demonstrated that it is vulnerable during cryopreservation as they can possible to reach even slightly higher cell be easily detached during the procedure. We viability in thin tissue segments [24]. Yet, were able to overcome both these challenges freezing approach, considered as a classical using vitrification. approach, is usually the first assayed when it With regard to the efficacy of 3D system in comes to cryopreservation. The present study general, alginate carrier as a supporting platform indicates an inefficiency of conventional was selected in this study. Alginates have been freezing procedure for preservation of hMSCs used as popular hydrogel in tissue engineering (Figure 3C, Figure 4). Freezing allows ice due to their suitable bio-compatibility properties formation, thus it results in rupture of the carrier [44]. Alginates as natural polysaccharides itself, which leads to nearly half of tissue promote minimum protein adsorption; therefore, engineered capsules becoming broken [50]. MSCs are unable to specifically interact with Furthermore, MSCs attached to surfers with alginates’ surface [45]. To create favorable different substrate properties are vulnerable to surface for cell attachment, highly viscous freezing [51]. Freezing in 10% Me2SO when chitosan was coated on the alginate bead applied to cryopreservation of embryonic stem

331 consistent with results of current study, demonstrating superiority of vitrification compared to freezing approaches [15]. Indeed, neurospheres remain intact and cells in intact neurospheres remained viable after vitrification with no difference in proliferation and ability to differentiate [15, 16]. Cryopreservation of SCs as 3D clusters seems to beneficial, drawing a parallel with current results. For MSCs in slow cooling serum is required; however there is no need of serum during vitrification as it involves rapid cooling. Figure 7. Chondrogenic differentiation capability of hMSCs cultured on the surface of microcarriers after a 4- week It is also considered as main advantage of induction. (A) and (B) as in Fig. 3. Scale bars are 100µm vitrification strategy since immersing directly into liquid nitrogen the specimens pre-treated cells is associated with high differentiation rates with cryoprotectants is cost-effective and less upon thawing, concluding that it leads to a loss time-consuming. It is rather rare to find in of the marker Oct-4, a characteristic marker of applied studies supplementary experiments the stem cell phenotype [26]. The majority of which verified vitrification state of biological human ESCs express an apoptotic pathway material studied. It our early studies, we have shortly after freezing. This, together with its demonstrated that through gradual adjustments known adverse reactions when employed as a survival rate of SCs close to 100%, resulting in cryoprotectant for haematopoietic cell therapy vitrification formulation employed here [15,16]. [52], plus its cellular toxicity, has led to attempts Similar trials were conducted on encapsulated to cryopreserve MSCs with other cells [1]. Using swine MSCs 2D- and 3D cryoprotectants either on their own or in engineered constructs as model system; we have combination with Me2SO [53,54]. As a result, proved that our vitrification strategy and effect of seven controlled rate freezing programs formulations are capable to achieve ice-free state and different ratios of Me2SO to non-penetrating during cooling-warming cycle [7,8]. Results of cryoprotectant on viability of rat MSCs have this study indicate that amorphous solidification been studied [54]. Apparently freezing- of hMSCs growing in alginate-chitosan beads associated of stem cells occurs from hours has been achieved (Fig.3B). We found how the to days after thawing [54]. In sharp contrast to texture of nanofibrous scaffold, geometry and control, thawed suspension of hMSCs can be the pore size have to be linked to size of held for a maximum of 2 hours before losing cryoprotectant molecules and the duration of their viability below permissible limits for exposure. Vitreous state was attainable transplantation [55]. Taking into consideration throughout the whole scaffold preventing ice- acidity of Me2SO at the high concentration formation during the cooling-warming cycle required for vitrification, we hypothesize that its [1,2,7,8], if concentration of VS and duration of high acidity may impair the chemical property procedure were properly evaluated. Our study of hydrogels and subsequently affect viability was designed in line with our previous reports and proliferation rate of hMSCs. Results of this [1,2], confirming that exposed to VS fragile study indicate that strategy based on this hydrogel constructs tolerate thermal gradients assumption is valid (Fig. 6). Freezing of hMSCs during rapid cooling and warming. No broken on microcarriers resulted in limited success, microcarriers were found after step-wise irrespectively Me2SO used alone or in introduced to VS, cooling to the temperature of combination with non-penetrating protectants liquid nitrogen and warming to 38°C and [56], this emphasize focus on vitrification in removal off cryoprotectant during pre-testing. context of current study. Therefore, it was assured that neither exposure The establishment of reliable protocols for to cryoprotectants, nor thermal gradients, vitrification of 3D cultures of MSCs will greatly particularly exposure to liquid nitrogen did not facilitate the progress of hMSCs research in the impair integrity of microcarries fabricated in this application to regenerative medicine as it is cost study. Later observation and results of our and time effective. Similarly, our results on previous studies are important outcomes from a neuronal SC 3D clusters (neurospheres) biomaterial point of view, as the fibrin –

332 alginatre and chitosan- alginatre are hydrogels; and swine MSCs combined with results reported hence they have a very high water content and here using this particular EG-based vitrification breakability at cryogenic temperatures [1,2]. solution proved its efficacy in cryopreservation These results play an important role in of stem cells [7,8,15,16]. Vitrification procedure expanding research on hydrogels, mimicking the could be effectively adjusted to cooling/warming in vivo environment and serving as permissive rates employed by addition of polymers to VS substrates for cell growth and proliferation. The used here as was reported by us previously [11]. vitrification strategy used open the possibility of We have demonstrated that negligible increase cryopreservation of hydrogel-based microfluidic of molarity of such VSs allowed effective devices loaded with hMSCs contributing to preservation of cells adhered on large surfaces basic research linked to orthopedics and open [11]. Lately, such vitrification strategy also the prospect of commercialization of 3-D becomes increasingly attractive with work on microfludic cell culture chips for needs of basic adhered SCs [58-60]. research. Similar applicability of cryopreserved Cryopreservation maintains critical cell structured neural cell cultures seeded onto a lab- number and enhances the prospect of scaling up on-chip can be seen in basic research linked to chondrogenic cultures for clinical application. studies of neurodegenerative diseases. Reported here is a cryopreservation strategy for The composition of vitrification solution is hMSCs growing on the surface of a micocarrier a critical factor in preservation process. Ideally, which maintains the integrity and quality of the vitrification solutions should not only protect carrier (Figure 1) while preserving the viability cells against intra- and extra-cellular ice (Figure 3) and the ability of stem cells to formation but also be non-toxic [1-4, 25] proliferate and differentiate (Figure 5, 6, 7). As Vitrification solutions can be made up of the incorporation of hMSCs into hydrogel penetrating and non-penetrating cryoprotectants biomaterials has great potential for regenerative [1-4,18,25]. Introduction of EG as principal medicine, an efficient vitrification protocol will cryoprotectant was the first choice during lay a foundation for hydrogel-based hMSC optimization of composition of final vitrification constructs. solution [10,15,24, 29,30] due its low molecular weight (62.07). In the last few years this topic Acknowledgment: The work was supported evolved again in connection to vitrification of by NUHS Bridging Funds 02/FY13, Singapore. induced pluripotent stem cells (iPS). Several studies on vitrification advocated use of mixture REFERENCES of penetrating cryoprotectants (Me2SO, acetamide, propylene glycol) suggested in 1. Kuleshova LL, Wang XW, Wu YN, Zhou Y vitrification protocols. Recent studies on & Yu H (2004) CryoLetters 25, 241-254. vitrification of iPS demonstrated that EG is still 2. Wu YN, Yu H, Chang S, Magalhães R & a superior cryoprotector [29,30] which is Kuleshova L (2007) Tissue Engineering 13, consistent with our early study [10]. It is a rather 649-58 challenging task to find an accurate vitrification 3. Kuleshova LL, Gouk SS & Hutmacher DW solution for the cryopreservation of any sensitive (2007) Biomaterials 28, 1585-1596. cells and procedures should be carefully well- 4. Kuleshova LL, Hutmacher DW. (2008). In: thought-out for optimum results [29,30]. Tissue Engineering (C. van Blitterswijk, We published the first report on birth of a senior ed.), Elsevier, London, UK, pp.363-40. healthy baby from human oocytes cryopreserved 5. Dahl SL, Chen Z, Solan AK Brockbank KG, with a vitrification solution comprised of 40% Niklason LE, Song YC. (2006) Tissue Eng. EG and 0.6M sucrose in 1999 [19, 57]. 12, 291-300. Subsequently, the same vitrification solution 6. Song YC, Chen ZZ, Mukherjee N, Lightfoot comprised of 40% EG and 0.6M sucrose was FG, Taylor MJ, Brockbank KG, Sambanis A. developed and implemented effectively for (2005) Transplant Proc. 37,253-5. preservation of variety of cell-containing tissue 7. Bhakta, G., Lee K.H., Magalhaes R., Wen F., engineered constructs, and for a number of Gouk S.S., Hutmacher D.W. and Kuleshova species [1-8,10-17]. This vitrification solution L.L. (2009) Biomaterials, 30, 336-343. appears to be optimal due to the correct ratio of 8. Wen, F., R. Magalhães, S. Gouk, G. Bhakta, K. penetrating to non-penetrating cryoprotectant Lee, D. Hutmacher and L. Kuleshova. (2009) (2:1). Our previous success in preserving rodent Tissue Eng., Part C: Methods 15,105-114.

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