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( 12 ) United States Patent US010214714B2 (12 ) United States Patent ( 10 ) Patent No. : US 10 , 214 ,714 B2 Maria de Peppo (45 ) Date of Patent: Feb . 26 , 2019 ( 54 ) PERFUSION BIOREACTOR ( 56 ) References Cited (71 ) Applicant: NEW YORK STEM CELL U . S . PATENT DOCUMENTS FOUNDATION , INC ., New York , NY 5 ,899 ,939 A 5 / 1999 Boyce et al . ( US ) 6 , 143 , 293 A 11 / 2000 Weiss 6 ,472 , 202 B1 10 / 2002 Banes (72 ) Inventor : Giuseppe Maria de Peppo , New York , 8 , 398 , 714 B2 3 /2013 Boiangiu NY (US ) 8 , 895 ,046 B2 11 /2014 Xuenong et al. 8 , 926 ,699 B2 1 / 2015 Burkinshaw 2003 /0100107 Al 5 /2003 Peschle ( 73 ) Assignee : New York Stem Cell Foundation , Inc. , 2006 /0257447 AL 11/ 2006 Hinds et al . New York , NY (US ) (Continued ) ( * ) Notice : Subject to any disclaimer , the term of this patent is extended or adjusted under 35 FOREIGN PATENT DOCUMENTS U . S . C . 154 (b ) by 417 days . WO WO 1996 / 040002 Al 12 / 1996 WO WO 1999 /048541 A1 9 / 1999 (21 ) Appl. No. : 14 /959 ,950 ( Continued ) ( 22 ) Filed : Dec . 4 , 2015 OTHER PUBLICATIONS (65 ) Prior Publication Data International Search Report dated Aug. 22 , 2016 , regarding PCT/ US 2016 /0194593 A1 Jul. 7 , 2016 US2016 /025601 . Related U . S . Application Data (Continued ) (63 ) Continuation -in - part of application No . Primary Examiner — Gautam Prakash PCT /US2014 / 072579 , filed on Dec . 29 , 2014 . (74 ) Attorney , Agent, or Firm — DLA Piper LLP (US ) ( 60 ) Provisional application No . 62/ 087 ,614 , filed on Dec . 4 , 2014 , provisional application No . 61 /921 , 915 , filed on Dec . 30 , 2013 . (57 ) ABSTRACT In some embodiments the present invention provides per (51 ) Int . Cl. fusion bioreactors and cell culture scaffolds suitable for the C12M 1 /00 ( 2006 .01 ) preparation of tissue grafts , such as bone tissue grafts . In C12M 1 / 12 ( 2006 . 01 ) some embodiments , the perfusion bioreactors comprise a C12M 3 /00 ( 2006 .01 ) graft chamber and / or a graft chamber insert configured to (52 ) U . S . CI. hold a tissue graft having a certain shape and / or certain CPC .. .. .. C12M 29 / 10 ( 2013 .01 ) ; C12M 21/ 08 dimensions, and / or to allow culture of such tissue grafts (2013 . 01 ) ; C12M 23 /46 ( 2013. 01 ) ; C12M under press - fit direct perfusion conditions . In some embodi 25 / 14 (2013 .01 ) ments , the perfusion bioreactors comprise an equilibration ( 58 ) Field of Classification Search CPC .. .. C12M 29 / 10 ; C12M 21 /08 , C12M 23 / 46 ; chamber . C12M 25 / 14 See application file for complete search history . 25 Claims, 21 Drawing Sheets US 10 ,214 ,714 B2 Page 2 ( 56 ) References Cited Bioreactors” ; Methods Mol Biol. , Nov. 27 , 2013 , vol. 1202, pp . 173 - 184 . U . S . PATENT DOCUMENTS Grayson at al. : “ Effects of Initial Seeding Density and Fluid Perfu 2008 / 0033548 A1 2 / 2008 Xuenong et al . sion Rate on Formation of Tissue - Engineered Bone ” ; Tissue Eng 2010 / 0249931 A1 9 / 2010 Laurencin et al. Part A , Jul. 11 , 2008 , vol. 14 , pp . 1809 - 1820 . 2010 /0303911 A1 12 / 2010 Sheardown et al. International Search Report dated Feb . 16 , 2016 , regarding PCT/ 2011/ 0151400 AL 6 / 2011 Boiangiu et al. US2015 /064076 . 2012 /0035742 AL 2 /2012 Vunjak -Novakovic et al . International Search Report dated Apr. 6 , 2015 regarding PCT/ 2012 /0209403 A1 8 / 2012 Morrison et al. US2014 /072579 . 2013 / 0017232 Al 1 / 2013 Varghese Chen , F . et al. : “ Anchoring Dental Implant in Tissue - Engineered 2013 / 0030547 A1 1 / 2013 Burkinshaw Bone Using Composite Scaffold A Preliminary Study in Nude Mouse 2013 / 0030548 Al 1 / 2013 Ling Model " ; Journal of Oral and Maxillofacial Surgery , 2005 , 63 , 2013 / 0274892 Al 10 / 2013 Lelkes et al . 586 - 591. 2013 /0344114 Al 12 / 2013 Chang et al. International Search Report dated Jun . 22 , 2017, regarding PCT/ 2014 /0030762 A11 / 2014 De Plano et al. US2017 /025390 . 2014 / 0147419 A1 5 / 2014 Novakovic et al. Yu , X et al. : “ Bioreactor - based bone tissue engineering : The 2015 /0289889 Al 10 / 2015 Altschuler et al. influence of dynamic flow on osteoblast phenotypic expression and matrix mineralization ” Proceedings of the National Academy of FOREIGN PATENT DOCUMENTS Science, vol. 101 ( 31) ; p . 11203 - 11208 . Extended European Search Report dated Jul. 7 , 2017 , regarding EP WO WO 2006 / 109137 A1 10 / 2006 14 87 7521 . 6 . WO WO 2015 / 103149 AL 7 / 2015 Eldesoni, Karam et al. : “ High Calcium Bioglass Enhances Differ entiation and Survival of Endothelial Progenitor Cells , Inducing OTHER PUBLICATIONS Early Vascularization in Critical Size Bone Defects” ; PLOS One , Nov. 2013 , vol. 8 , No. 11 , e79058 . De Peppo et al .: “ Cultivation of Human Bone - Like Tissue from Japanese Office Action dated Aug . 23 , 2018 , regarding JP 2016 Pluripotent Stem Cell - Derived Osteogenic Progenitors in Perfusion 543600 . U . S . Patent Feb . 26 , 2019 Sheet 1 of 21 US 10 ,214 ,714 B2 ** * * * * ** * * * ** * * * * * * * * * * * * * * * * * leU . S . monoPatent Feb . 26 ,- 2019 - Sheeto 2 of 21. US 10 . ,214 . ,714 B2 2 ** *** * * * * * * 2 U . S . Patent Feb . 26 , 2019 Sheet 3 of 21 US 10 ,214 ,714 B2 wo B . U . S . Patent Feb . 26 , 2019 Sheet 4 of 21 US 10 ,214 ,714 B2 DO XXX U . S . Patent Feb . 26 , 2019 Sheet 5 of 21 US 10 ,214 ,714 B2 *** * * ** * www U . S . Patent Feb . 26 , 2019 Sheet 6 of 21 US 10 ,214 ,714 B2 W8 atent Feb . 26 , 2019 Sheet 7 of 21 US 10 ,214 ,714 B2 LAXXU ** ** ** * ** * * * * * WWW atent Feb . 26 , 2019 Sheet 8 of 21 US 10 ,214 ,714 B2 atent Feb . 26 , 2019 Sheet 9 of 21 US 10 ,214 ,714 B2 W Et KEE 1 U . S . Patent Feb . 26 , 2019 Sheet 10 of 21 US 10 ,214 ,714 B2 atent Feb . 26 , 2019 Sheet 11 of 21 US 10 ,214 , 714 B2 www 13 ] > atent Feb . 26 , 2019 Sheet 12 of 21 US 10 ,214 ,714 B2 atent Feb . 26 , 2019 Sheet 13 of 21 US 10 ,214 ,714 B2 *** :44 ? wwwwwwwwww ? XXXXXX 3 atent Feb . 26 , 2019 Sheet 14 of 21 US 10 ,214 ,714 B2 w FIG.14* ** U . S . Patent Feb . 26 , 2019 Sheet 15 of 21 US 10 ,214 ,714 B2 . FIG.14(cont) atent Feb . 26 , 2019 Sheet 16 of 21 US 10 ,214 ,714 B2 FIG . 15A . icie w FIGISB W postos w U . S . Patent Feb . 26 , 2019 Sheet 17 of 21 US 10 ,214 ,714 B2 pa U . S . Patent Feb . 26 , 2019 Sheet 18 of 21 US 10 ,214 ,714 B2 WW XXXX . U . S . Patent Feb . 26 , 2019 Sheet 19 of 21 US 10 ,214 ,714 B2 w WWWW atent Feb . 26 , 2019 Sheet 20 of 21 US 10 ,214 ,714 B2 atent Feb . 26 , 2019 Sheet 21 of 21 US 10 ,214 ,714 B2 US 10 , 214 , 714 B2 PERFUSION BIOREACTOR However, static cultures are not optimal to grow centimeter sized bone grafts for clinical translation due to poor nutrient CROSS -REFERENCE TO RELATED supply and removal of metabolic waste . Under these con APPLICATIONS ditions, in fact, mass transport occurs only via diffusion , 5 which is not sufficient to support cell survival and prolif This application claims the benefit of priority of U . S . eration inside the core of large cell / scaffold constructs , Provisional Patent Application No. 62 /087 ,614 , filed Dec . 4 , resulting in necrosis and poor tissue formation . In addition , 2014 , and also claims the benefit of priority of International cell proliferation and matrix synthesis at the construct Patent Application No . PCT/ US2014 / 072579 , filed Dec . 29 . periphery over the culture period further impede medium 2014 , which claims the benefit of priority of U . S . Provi- 10 diffusion and contribute to the formation of a nutrient sional Patent Application No . 62 /087 , 614 , filed Dec . 4 , gradient that drive cell migration towards the substitute 2014 , and U . S . Provisional Patent Application No. 61/ 921 , borders (Goldstein , A . S . , Juarez , T . M ., et al. Effect of 915 , filed Dec . 30 , 2013 , the contents of each of which are convection on osteoblastic cell growth and function in hereby incorporated by reference in their entireties . biodegradable polymer foam scaffolds . Biomaterials 2001 , 15 22 ( 11 ) , 1279 - 1288 ) . On top of this , culture in static condi COPYRIGHT AND INCORPORATION BY tions does not allow provision of those biophysical stimuli REFERENCE that are critical for functional regeneration ( Yeatts , A . B ., Fisher , J . P . Bone tissue engineering bioreactors : dynamic A portion of the disclosure of this patent document culture and the influence of shear stress . Bone 2011 , 48 ( 2 ) , contains material that is subject to copyright protection . The 20 171 - 181 ; Klein -Nulend , J ., Bakker , A . D ., et al. Mecha copyright owner has no objection to the facsimile reproduc nosensation and transduction in osteocytes . Bone 2013 , tion by anyone of the patent document or the patent disclo 54 ( 2 ) , 182 - 190 ) . Advances in bioreactor systems over the sure as it appears in the Patent and Trademark Office patent last two decades have opened new opportunities in the field file or records, but otherwise reserves all copyright rights of bone engineering as they allow to nurture the develop whatsoever. 25 ment of bone tissue by providing an appropriate physiologi cal environment with stimulatory biochemical and biophysi BACKGROUND INFORMATION cal signals (Salter , E ., Goh , B . , et al. Bone tissue engineering bioreactors : a role in the clinic ? Tissue Eng Part B Rev 2012 , Field of the Invention 18 ( 1 ) , 62 - 75 ) . The present invention relates generally to cell culture , and 30 Bioreactors were initially developed to allow the high more particularly to a bioreactor and method of use thereof . mass culture of cells used for applications in diverse areas , Background of the Invention including fermentation , wastewater treatment and purifica The human skeleton consists of 206 distinct bones , which tion , food processing and drug production (Martin , I .
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