title: Scalable expansion of human induced pluripotent stem cells in the defined xeno-free E8 medium under adherent and suspension culture conditions.
abstract: Large-scale production of human induced pluripotent stem cells (hiPSCs) by robust and economic methods has been one of the major challenges for translational realization of hiPSC technology. Here we demonstrate a scalable culture system for hiPSC expansion using the E8 chemically defined and xeno-free medium under either adherent or suspension conditions. To optimize suspension conditions guided by a computational simulation, we developed a method to efficiently expand hiPSCs as undifferentiated aggregates in spinner flasks. Serial passaging of two different hiPSC lines in the spinner flasks using the E8 medium preserved their normal karyotype and expression of undifferentiated state markers of TRA-1-60, SSEA4, OCT4, and NANOG. The hiPSCs cultured in spinner flasks for more than 10 passages not only could be remained pluripotent as indicated by in vitro and in vivo assays, but also could be efficiently induced toward mesodermal and hematopoietic differentiation. Furthermore, we established a xeno-free protocol of single-cell cryopreservation and recovery for the scalable production of hiPSCs in spinner flasks. This system is the first to enable an efficient scale-up bioprocess in completely xeno-free condition for the expansion and cryopreservation of hiPSCs with the quantity and quality compliant for clinical applications.
authors: Department of Chemical and Biomolecular Engineering, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA; Stem Cell Program, Institute of Cell Engineering, The Johns Hopkins University School of Medicine, Edward D. Miller Research Building, Room 747, 733 N. Broadway, Baltimore, MD 21205, USA; Institute for NanoBioTechnology, The Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
authors: Ying
authors: Y
authors: Wang
authors: Bin-Kuan
authors: BK
authors: Chou
authors: Sarah
authors: S
authors: Dowey
authors: Chaoxia
authors: C
authors: He
authors: Sharon
authors: S
authors: Gerecht
authors: Linzhao
authors: L
authors: Cheng
mesh_terms: D000954:Antigens, Surface / Q000378:metabolism; D002448:Cell Adhesion; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D049109:Cell Proliferation; D002478:Cells, Cultured; D003470:Culture Media / Q000737:chemistry*; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology* / Q000378:metabolism; D007621:Karyotyping; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3 / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D055746:Stage-Specific Embryonic Antigens / Q000378:metabolism
chemical_list: D000954:Antigens, Surface; D003470:Culture Media; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D011509:Proteoglycans; D055746:Stage-Specific Embryonic Antigens; C107064:TRA-1-60 antigen, human; C066584:stage-specific embryonic antigen-4
references: Biotechnol Prog. 2006 May-Jun;22(3):801-10
references: 16739964
references: Stem Cell Res. 2012 May;8(3):388-402
references: 22459095
references: Biotechnol Bioeng. 2004 Jan 5;85(1):34-46
references: 14705010
references: Tissue Eng Part A. 2010 Feb;16(2):405-21
references: 19739936
references: Biotechnol Bioeng. 2005 Sep 20;91(6):688-98
references: 15971228
references: Nat Methods. 2011 May;8(5):424-9
references: 21478862
references: Nat Protoc. 2012 Nov;7(11):2013-21
references: 23080273
references: Biotechnol Appl Biochem. 2012 Mar-Apr;59(2):97-105
references: 23586790
references: Tissue Eng. 2006 Nov;12(11):3233-45
references: 17518637
references: Pharmacol Rev. 2004 Dec;56(4):549-80
references: 15602010
references: Lancet. 2004 Jul 10-16;364(9429):163-71
references: 15246729
references: Am J Physiol Heart Circ Physiol. 2005 Apr;288(4):H1915-24
references: 15576436
references: Nat Protoc. 2013 Jan;8(1):111-30
references: 23257981
references: Stem Cell Res. 2010 May;4(3):165-79
references: 20363202
references: Nature. 2007 Jul 19;448(7151):313-7
references: 17554338
references: Trends Biotechnol. 1991 Dec;9(12):427-37
references: 1367747
references: Tissue Eng Part C Methods. 2012 Nov;18(11):831-51
references: 22559864
references: Stem Cells Dev. 2008 Dec;17(6):1227-41
references: 19006458
references: Cell Res. 2011 Mar;21(3):518-29
references: 21243013
references: Nat Protoc. 2011 May;6(5):572-9
references: 21527915
references: Cell Stem Cell. 2008 May 8;2(5):461-71
references: 18462696
references: Tissue Eng Part C Methods. 2012 Oct;18(10):772-84
references: 22519745
references: J Cell Sci. 2005 Oct 1;118(Pt 19):4495-509
references: 16179608
references: Nat Protoc. 2008;3(5):768-76
references: 18451785
references: Nat Protoc. 2012 Nov;7(11):2029-40
references: 23099485
references: Biotechnol Bioeng. 2007 Sep 1;98(1):282-94
references: 17318906
references: Hum Reprod. 2009 Mar;24(3):580-9
references: 19056770
references: Nat Biotechnol. 2006 Feb;24(2):185-7
references: 16388305
references: Blood. 2005 Sep 1;106(5):1601-3
references: 15914555
references: Nature. 2009 Jun 25;459(7250):1131-5
references: 19440194
references: Blood. 2009 Dec 24;114(27):5473-80
references: 19797525
references: Leuk Lymphoma. 2009 Sep;50(9):1434-41
references: 19603344
references: Biotechnol Bioeng. 1996 Feb 20;49(4):456-66
references: 18623601
references: Blood. 2007 Dec 1;110(12):4111-9
references: 17761519
references: Nat Protoc. 2011 May;6(5):689-700
references: 21527925
references: Nat Biotechnol. 2010 Apr;28(4):361-4
references: 20351691
references: Tissue Eng Part C Methods. 2010 Aug;16(4):573-82
references: 19737071
references: Nat Biotechnol. 2007 Jun;25(6):681-6
references: 17529971
references: Stem Cell Res. 2010 Jul;5(1):51-64
references: 20478754
references: Methods Mol Biol. 2011;767:137-46
references: 21822872
references: Nat Methods. 2012 Mar 25;9(5):509-16
references: 22447133
references: J Biotechnol. 2008 Jan 1;133(1):146-53
references: 17935814
references: Science. 2007 Dec 21;318(5858):1917-20
references: 18029452
references: Biotechnol Bioeng. 2009 Feb 1;102(2):493-507
references: 18767184