Tips and tricks for successfully culturing and adapting human induced pluripotent stem cells
2021; Cell Press; Volume: 23; Linguagem: Inglês
10.1016/j.omtm.2021.10.013
ISSN2329-0501
AutoresRocío Castro-Viñuelas, Clara Sanjurjo‐Rodríguez, María Piñeiro-Ramil, Silvia Rodríguez-Fernández, Isidoro López-Baltar, Isaac Fuentes‐Boquete, Francisco J. Blanco, Silvia Díaz‐Prado,
Tópico(s)Biomedical Ethics and Regulation
ResumoReprogramming somatic cells toward pluripotency became possible over a decade ago. Since then, induced pluripotent stem cells (iPSCs) have served as a versatile and powerful tool not only for basic research but also with the long-term goal of using them in human cell transplantation after differentiation. Nonetheless, downstream applications are frequently blurred by the difficulties that researchers have to face when working with iPSCs, such as trouble with clonal selection, in vitro culture and cryopreservation, adaptation to feeder-free conditions, or expansion of the cells. Therefore, in this article we aim to provide other researchers with practical and detailed information to successfully culture and adapt iPSCs. Specifically, we (1) describe the most common problems when in-vitro culturing iPSCs onto feeder cells as well as its possible troubleshooting, and (2) compare different matrices and culture media for adapting the iPSCs to feeder-free conditions. We believe that the troubleshooting and recommendations provided in this article can be of use to other researchers working with iPSCs and who may be experiencing similar issues, hopefully enhancing the appeal of this promising cell source to be used for biomedical investigations, such as tissue engineering or regenerative medicine applications. Reprogramming somatic cells toward pluripotency became possible over a decade ago. Since then, induced pluripotent stem cells (iPSCs) have served as a versatile and powerful tool not only for basic research but also with the long-term goal of using them in human cell transplantation after differentiation. Nonetheless, downstream applications are frequently blurred by the difficulties that researchers have to face when working with iPSCs, such as trouble with clonal selection, in vitro culture and cryopreservation, adaptation to feeder-free conditions, or expansion of the cells. Therefore, in this article we aim to provide other researchers with practical and detailed information to successfully culture and adapt iPSCs. Specifically, we (1) describe the most common problems when in-vitro culturing iPSCs onto feeder cells as well as its possible troubleshooting, and (2) compare different matrices and culture media for adapting the iPSCs to feeder-free conditions. We believe that the troubleshooting and recommendations provided in this article can be of use to other researchers working with iPSCs and who may be experiencing similar issues, hopefully enhancing the appeal of this promising cell source to be used for biomedical investigations, such as tissue engineering or regenerative medicine applications. Induced pluripotent stem cells (iPSCs) have the ability to proliferate indefinitely in culture without reduction of the quality, besides potential of differentiation into any desired cell type.1Rowe R.G. Daley G.Q. Induced pluripotent stem cells in disease modelling and drug discovery.Nat. Rev. Genet. 2019; 20: 377-388Google Scholar Therefore, when first discovered in 2006,2Takahashi K. Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors.Cell. 2006; 126: 663-676Google Scholar these cells were thought to be the “panacea” for biomedical applications. In the case of tissue engineering, iPSCs hold great potential for personalized tissues, which can be used for regenerative medicine and/or in vitro studies to tailor other medical interventions.3Loskill P. Huebsch N. Engineering tissues from induced pluripotent stem cells.Tissue Eng. Part A. 2019; 25: 707-710Google Scholar However, all these advantages and potential applications are blurred by the difficulties that researchers have to face when working with iPSCs, such as trouble with clonal selection, in vitro culture, adaptation, and/or expansion of the cells.4Chen K.G. Mallon B.S. McKay R.D.G. Robey P.G. Human pluripotent stem cell culture: considerations for maintenance, expansion, and therapeutics.Cell Stem Cell. 2014; 14: 13-26Google Scholar iPSCs' recovery after freezing is still an issue5Nishishita N. Muramatsu M. Kawamata S. An effective freezing/thawing method for human pluripotent stem cells cultured in chemically-defined and feeder-free conditions.Am. J. Stem Cells. 2015; 4: 38-49Google Scholar,6Ye H. Wang Q. Efficient generation of non-integration and feeder-free induced puripotent stem cells from human peripheral blood cells by Sendai virus.Cell. Physiol. Biochem. 2018; 50: 1318-1331Google Scholar and, in addition, it has been reported that iPSC colonies may disappear and break up into single cells during initial colony morphology-based selection.7Pfannkuche K. Fatima A. Gupta M.K. Dieterich R. Hescheler J. Initial colony morphology-based selection for iPS cells derived from adult fibroblasts is substantially improved by temporary UTF1-based selection.PLoS One. 2010; 5: e9580Google Scholar The use of iPSCs in all downstream applications requires the establishment of protocols that will allow large-scale, cost-effective cultivation of cells, without compromising on their quality.8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar Reprogramming methods and iPSC culture strategies initially involved the use of mouse or human feeder layers, thus coinciding with the protocol established by Thomson for in vitro culturing embryonic stem cells (ESCs).9Thomson J.A. Embryonic stem cell lines derived from human blastocysts.Science. 1998; 282: 1145-1147Google Scholar These feeder cells secrete essential growth factors, extracellular matrix components, and cytokines into the culture media, which support pluripotent cell growth and proliferation.8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar,10Yao S. Chen S. Clark J. Hao E. Beattie G.M. Hayek A. Ding S. Long-term self-renewal and directed differentiation of human embryonic stem cells in chemically defined conditions.Proc. Natl. Acad. Sci. U S A. 2006; 103: 6907-6912Google Scholar,11Sams A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google Scholar Although a robust method, feeder-based systems are labor intensive, hard to scale,11Sams A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google Scholar and can also be a source of animal pathogens and mycoplasma contamination.8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar,11Sams A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google Scholar,12Mannello F. Tonti G.A. Concise review: no breakthroughs for human mesenchymal and embryonic stem cell culture: conditioned medium, feeder layer, or feeder-free; medium with fetal calf serum, human serum, or enriched plasma; serum-free, serum replacement nonconditioned medium, or ad hoc formula? All that glitters is not gold!.Stem Cells. 2007; 25: 1603-1609Google Scholar Moreover, this system does not allow the performance of all the required tests to characterize new-generated iPSC lines, since feeder cells can interfere with molecular and/or flow cytometry experiments.13Skottman H. Hovatta O. Culture conditions for human embryonic stem cells.Reproduction. 2006; 132: 691-698Google Scholar This is why in certain occasions iPSCs have to be “moved” to feeder-free culture systems. But when pluripotent cells are switched from one cell culture condition to another, they need to adapt to the new system to regain homeostasis14Healy L. Ruban L. Culture adaptation and abnormal cultures.in: Atlas of Human Pluripotent Stem Cells in Culture. Springer US, 2014: 167-175Google Scholar; this adaptation process is usually harmful for the cells, especially in the case of newly derived cell lines, often experiencing more differentiation and apoptosis than normal.14Healy L. Ruban L. Culture adaptation and abnormal cultures.in: Atlas of Human Pluripotent Stem Cells in Culture. Springer US, 2014: 167-175Google Scholar, 15Wagner K. Welch D. Feeder-free adaptation, culture and passaging of human IPS cells using complete knockout serum replacement feeder-free medium.J. Vis. Exp. 2010; 41: 2236Google Scholar, 16Stover A.E. Schwartz P.H. Adaptation of human pluripotent stem cells to feeder-free conditions in chemically defined medium with enzymatic single-cell passaging.Methods Mol. Biol. 2011; 767: 137-146Google Scholar Despite feeder-free reprogramming being proven feasible,6Ye H. Wang Q. Efficient generation of non-integration and feeder-free induced puripotent stem cells from human peripheral blood cells by Sendai virus.Cell. Physiol. Biochem. 2018; 50: 1318-1331Google Scholar,17Warren L. Wang J. Feeder-free reprogramming of human UNIT 4A.6 fibroblasts with messenger RNA; 24510287.Curr. Protoc. Stem Cell Biol. 2013; 1: 4A.6.1-4A.6.27Google Scholar, 18Steichen C. Si-Tayeb K. Wulkan F. Crestani T. Rosas G. Dariolli R. Pereira A.C. Krieger J.E. Human induced pluripotent stem (hiPS) cells from urine samples: a non-integrative and feeder-free reprogramming strategy.Curr. Protoc. Hum. Genet. 2017; 2017: 21.7.1-21.7.22Google Scholar, 19Park S. Mostoslavsky G. Generation of human induced pluripotent stem cells using a defined, feeder-free reprogramming system.Curr. Protoc. Stem Cell Biol. 2018; 45: e48Google Scholar feeder-based reprogramming methods are still the most common surface on which to develop the reprogramming process.6Ye H. Wang Q. Efficient generation of non-integration and feeder-free induced puripotent stem cells from human peripheral blood cells by Sendai virus.Cell. Physiol. Biochem. 2018; 50: 1318-1331Google Scholar,20Liu G. David B.T. Trawczynski M. Fessler R.G. Advances in pluripotent stem cells: history, mechanisms, technologies, and applications.Stem Cell Rev. Rep. 2020; 16: 3-32Google Scholar In our group we have recently published the successful generation of three iPSC lines derived from human skin fibroblasts.21Castro-Viñuelas R. Sanjurjo-Rodríguez C. Piñeiro-Ramil M. Hermida-Gómez T. Toro-Santos J.D.E. Blanco-García F. Fuentes-Boquete I. Díaz-Prado S. Generation of human induced pluripotent stem cells (iPSc) from hand osteoarthritis patient-derived fibroblasts.Osteoarthritis Cartilage. 2019; 27: S428-S429Google Scholar,22Castro-Viñuelas R. Sanjurjo-Rodríguez C. Piñeiro-Ramil M. Rodríguez-Fernández S. Fuentes-Boquete I. Blanco F.J. Díaz-Prado S.M. Generation of a human control iPS cell line (ESi080-A) from a donor with no rheumatic diseases.Stem Cell Res. 2020; 43: 101683Google Scholar It is worth mentioning that the problems that we had to face during the process of generation and characterization of the cells were numerous, especially regarding the adaptation to feeder-free conditions. There are currently a lot of published combinations of matrices and culture media to replace feeder layers (reviewed in Dakhore et al.,8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar Sams and Powers,8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar, 11Sams A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google Scholar, 13Skottman H. Hovatta O. Culture conditions for human embryonic stem cells.Reproduction. 2006; 132: 691-698Google Scholar, 20Liu G. David B.T. Trawczynski M. Fessler R.G. Advances in pluripotent stem cells: history, mechanisms, technologies, and applications.Stem Cell Rev. Rep. 2020; 16: 3-32Google Scholar, 23Anderson N.C. Chen P.-F. Meganathan K. Saber W.A. Petersen A.J. Bhattacharyya A. Kroll K.L. Sahin M. Cross-IDDRC Human Stem Cell Working GroupBalancing serendipity and reproducibility: pluripotent stem cells as experimental systems for intellectual and developmental disorders.Stem Cell Reports. 2021; 16: 1446-1457Google Scholar, 24Nakamura S. Sugimoto N. Eto K. Ex vivo generation of platelet products from human iPS cells.Inflamm. Regen. 2020; 40: 30Google Scholar, 25Kishimoto K. Shimada A. Shinohara H. Takahashi T. Yamada Y. Higuchi Y. Yoneda N. Suemizu H. Kawai K. Kurotaki Y. et al.Establishment of novel common marmoset embryonic stem cell lines under various conditions.Stem Cell Res. 2021; 53: 102252Google Scholar Skottman and Hovatta,8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar, 11Sams A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google Scholar, 13Skottman H. Hovatta O. Culture conditions for human embryonic stem cells.Reproduction. 2006; 132: 691-698Google Scholar, 20Liu G. David B.T. Trawczynski M. Fessler R.G. Advances in pluripotent stem cells: history, mechanisms, technologies, and applications.Stem Cell Rev. Rep. 2020; 16: 3-32Google Scholar, 23Anderson N.C. Chen P.-F. Meganathan K. Saber W.A. Petersen A.J. Bhattacharyya A. Kroll K.L. Sahin M. Cross-IDDRC Human Stem Cell Working GroupBalancing serendipity and reproducibility: pluripotent stem cells as experimental systems for intellectual and developmental disorders.Stem Cell Reports. 2021; 16: 1446-1457Google Scholar, 24Nakamura S. Sugimoto N. Eto K. Ex vivo generation of platelet products from human iPS cells.Inflamm. Regen. 2020; 40: 30Google Scholar, 25Kishimoto K. Shimada A. Shinohara H. Takahashi T. Yamada Y. Higuchi Y. Yoneda N. Suemizu H. Kawai K. Kurotaki Y. et al.Establishment of novel common marmoset embryonic stem cell lines under various conditions.Stem Cell Res. 2021; 53: 102252Google Scholar Liu et al.,8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar, 11Sams A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google Scholar, 13Skottman H. Hovatta O. Culture conditions for human embryonic stem cells.Reproduction. 2006; 132: 691-698Google Scholar, 20Liu G. David B.T. Trawczynski M. Fessler R.G. Advances in pluripotent stem cells: history, mechanisms, technologies, and applications.Stem Cell Rev. Rep. 2020; 16: 3-32Google Scholar, 23Anderson N.C. Chen P.-F. Meganathan K. Saber W.A. Petersen A.J. Bhattacharyya A. Kroll K.L. Sahin M. Cross-IDDRC Human Stem Cell Working GroupBalancing serendipity and reproducibility: pluripotent stem cells as experimental systems for intellectual and developmental disorders.Stem Cell Reports. 2021; 16: 1446-1457Google Scholar, 24Nakamura S. Sugimoto N. Eto K. Ex vivo generation of platelet products from human iPS cells.Inflamm. Regen. 2020; 40: 30Google Scholar, 25Kishimoto K. Shimada A. Shinohara H. Takahashi T. Yamada Y. Higuchi Y. Yoneda N. Suemizu H. Kawai K. Kurotaki Y. et al.Establishment of novel common marmoset embryonic stem cell lines under various conditions.Stem Cell Res. 2021; 53: 102252Google Scholar Anderson et al.,8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar, 11Sams A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google Scholar, 13Skottman H. Hovatta O. Culture conditions for human embryonic stem cells.Reproduction. 2006; 132: 691-698Google Scholar, 20Liu G. David B.T. Trawczynski M. Fessler R.G. Advances in pluripotent stem cells: history, mechanisms, technologies, and applications.Stem Cell Rev. Rep. 2020; 16: 3-32Google Scholar, 23Anderson N.C. Chen P.-F. Meganathan K. Saber W.A. Petersen A.J. Bhattacharyya A. Kroll K.L. Sahin M. Cross-IDDRC Human Stem Cell Working GroupBalancing serendipity and reproducibility: pluripotent stem cells as experimental systems for intellectual and developmental disorders.Stem Cell Reports. 2021; 16: 1446-1457Google Scholar, 24Nakamura S. Sugimoto N. Eto K. Ex vivo generation of platelet products from human iPS cells.Inflamm. Regen. 2020; 40: 30Google Scholar, 25Kishimoto K. Shimada A. Shinohara H. Takahashi T. Yamada Y. Higuchi Y. Yoneda N. Suemizu H. Kawai K. Kurotaki Y. et al.Establishment of novel common marmoset embryonic stem cell lines under various conditions.Stem Cell Res. 2021; 53: 102252Google Scholar Nakamura et al.,8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar, 11Sams A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google Scholar, 13Skottman H. Hovatta O. Culture conditions for human embryonic stem cells.Reproduction. 2006; 132: 691-698Google Scholar, 20Liu G. David B.T. Trawczynski M. Fessler R.G. Advances in pluripotent stem cells: history, mechanisms, technologies, and applications.Stem Cell Rev. Rep. 2020; 16: 3-32Google Scholar, 23Anderson N.C. Chen P.-F. Meganathan K. Saber W.A. Petersen A.J. Bhattacharyya A. Kroll K.L. Sahin M. Cross-IDDRC Human Stem Cell Working GroupBalancing serendipity and reproducibility: pluripotent stem cells as experimental systems for intellectual and developmental disorders.Stem Cell Reports. 2021; 16: 1446-1457Google Scholar, 24Nakamura S. Sugimoto N. Eto K. Ex vivo generation of platelet products from human iPS cells.Inflamm. Regen. 2020; 40: 30Google Scholar, 25Kishimoto K. Shimada A. Shinohara H. Takahashi T. Yamada Y. Higuchi Y. Yoneda N. Suemizu H. Kawai K. Kurotaki Y. et al.Establishment of novel common marmoset embryonic stem cell lines under various conditions.Stem Cell Res. 2021; 53: 102252Google Scholar Kishimoto et al.8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar, 11Sams A. Powers M.J. Feeder-free substrates for pluripotent stem cell culture.Methods Mol. Biol. 2013; 997: 73-89Google Scholar, 13Skottman H. Hovatta O. Culture conditions for human embryonic stem cells.Reproduction. 2006; 132: 691-698Google Scholar, 20Liu G. David B.T. Trawczynski M. Fessler R.G. Advances in pluripotent stem cells: history, mechanisms, technologies, and applications.Stem Cell Rev. Rep. 2020; 16: 3-32Google Scholar, 23Anderson N.C. Chen P.-F. Meganathan K. Saber W.A. Petersen A.J. Bhattacharyya A. Kroll K.L. Sahin M. Cross-IDDRC Human Stem Cell Working GroupBalancing serendipity and reproducibility: pluripotent stem cells as experimental systems for intellectual and developmental disorders.Stem Cell Reports. 2021; 16: 1446-1457Google Scholar, 24Nakamura S. Sugimoto N. Eto K. Ex vivo generation of platelet products from human iPS cells.Inflamm. Regen. 2020; 40: 30Google Scholar, 25Kishimoto K. Shimada A. Shinohara H. Takahashi T. Yamada Y. Higuchi Y. Yoneda N. Suemizu H. Kawai K. Kurotaki Y. et al.Establishment of novel common marmoset embryonic stem cell lines under various conditions.Stem Cell Res. 2021; 53: 102252Google Scholar). But when consulting the bibliography for troubleshooting, we could just find depthless explained methodology. It is widely known that generation and in vitro culture of iPSCs is a slow and tricky process,8Dakhore S. Nayer B. Hasegawa K. Human pluripotent stem cell culture: current status, challenges, and advancement.Stem Cells Int. 2018; 2018: 7396905Google Scholar,26Ohnuki M. Takahashi K. Present and future challenges of induced pluripotent stem cells.Philos. Trans. R. Soc. Lond. B Biol. Sci. 2015; 370: 20140367Google Scholar but the literature rarely explains the difficulties and problems found during the process of establishing new iPSC lines and daily working with them. Therefore, in this methodological article we aim to fill in this gap of knowledge by deeply describing the protocols we established for the complex process of generating iPSC lines. Special attention is paid to (1) which are the most common problems when in-vitro-culturing iPSCs onto feeder cells as well as its possible troubleshooting, and (2) comparing different matrices and culture media for adapting the iPSCs to feeder-free conditions. We strongly believe that reporting this kind of problems and suggesting possible ways to have them solved will contribute to help other researchers that might have similar issues when working with iPSCs. •Basic fibroblast growth factor (bFGF) (13256029, Gibco Thermo Fisher Scientific)•Bovine serum albumin (BSA) 9048-46-8, Sigma)•Dimethyl sulfoxide (DMSO) (67-68-5, Sigma-Aldrich Química SA)•Dulbecco's modified Eagle's medium (DMEM) (12-604F, Lonza)•DMEM knockout without L-glutamine (KO-DMEM); (10829018, Gibco Thermo Fisher Scientific)•DPBS with calcium and magnesium (14040133, Gibco Thermo Fisher Scientific)•Fluorescence-activated cell sorting buffer (Becton Dickinson)•Fetal bovine serum (FBS) (10082147, Gibco Thermo Fisher Scientific)•Gentle cell dissociation reagent (07174, STEMCELL Technologies)•Geltrex hESC-qualified, ready-to-use, reduced growth factor basement membrane matrix (A1569601, Gibco)•GlutaMAX 100X (35050061, Gibco Thermo Fisher Scientific)•Human foreskin fibroblasts, HFF-1 cell line (CRL2429, ATCC)•Iscove’s modified Dulbecco medium (IMDM) (12440053; Gibco Thermo Fisher Scientific)•Knockout serum replacement (10828028, Gibco Thermo Fisher Scientific)•2-Mercaptoethanol (50 mM; 31350010, Gibco Thermo Fisher Scientific)•Matrigel basement membrane matrix (354234, Corning)•Non-essential amino acids 100X (11140050, Gibco Thermo Fisher Scientific)•Phosphate-buffered saline tablets (BR0014G, Oxoid)•Phosphate-buffered saline with Ca2+ and Mg (14287, Thermo Fisher Scientific)•Penicillin/streptomycin (P/S) (15140122, Gibco Thermo Fisher Scientific)•Propidium iodide (PI) (P4170, Sigma-Aldrich)•rh-Laminin-521 521 (A29249; Thermo Fisher Scientific)•RNAse (500 μg/mL; Roche)•RevitaCell 100X (A2644501; Gibco)•ROCK inhibitor Y-27632 (04-0012, Stemgent)•Saline serum (154 mmol/L Na and 154 mmol/L Cl–, Fresenius Kabi)•StemFlex basal medium (A3349301, Gibco Thermo Fisher Scientific)•StemFlex Supplement 10X (A3349201, Gibco Thermo Fisher Scientific)•Trypan blue staining (T8154-20 ML, Sigma)•TrypLE Select Enzyme (1X), no phenol red (12563011, Gibco Thermo Fisher Scientific)•Versene solution (15040066, Gibco)•0.05% trypsin-EDTA (25300054, Gibco)•0.25% trypsin-EDTA (25200056, Gibco) •0.22-μm filter (GSWP14250, Millipore)•15- and 50-mL conical tubes•175 μm “stripper” tips (Gynétics)•Cryovials, 1.5 mL•FACScalibur flow cytometer (Becton Dickinson) and CellQuest software (Becton Dickinson)•CoolCell freezing container•Neubauer chamber•Polypropylene conical tubes (Corning Science)•Real-time DS-FI2 photomicrograph camera•Sight DS-L3 digital control monitor•SMZ-745T stereomicroscope•Stripper micropipette (Origio Midatlantic Devices)•100- and 150-mm tissue culture dishes•Six-well tissue culture plates •5% DMEM medium: DMEM supplemented with 5% FBS and 1% P/S. Store at 4°C.•bFGF: 100 μg/mL stock solution of bFGF in 2% filter sterilized BSA. Divide into usage size aliquots (50 μL). Store at −20°C.•CRYOSTEM hPSC freezing medium (Cellseco Biological Industries).•Freezing medium: 90% FBS and 10% DMSO. The use of freshly made freezing medium is recommended. Alternatively, freezing medium can be prepared and stored at 4°C in darkness up to 1 month.•hES medium: KO-DMEM containing 20% knockout serum replacement, 1% non-essential amino acids, 1% GlutaMAX 100X, 1% P/S, 0.1 mM β-mercaptoethanol (0.5 mL of a 50 mM stock solution), and 100 μg/mL bFGF (all from Gibco). Store at 4°C and use within 1 month. Do not pre-warm the whole bottle.•HFF medium: IMDM supplemented with 10% FBS and 1% P/S. Store at 4°C.•Matrigel solution: thaw Matrigel basement membrane matrix slowly at 4°C overnight on ice. While working on ice inside a laminar flow hood, Matrigel solution is prepared at 1:40 dilution in P/S and KO-DMEM. Cool the pipette by aspirating cold KO-DMEM up and down 10–20 times. Using a cold pipette, aseptically transfer Matrigel to KO-DMEM. Invert the solution several times to obtain homogeneous Matrigel solution. Do not shake again and store at 4°C.•rh-Laminin-521: thaw rh-laminin-521 between 2°C and 8°C, mix gently by inversion 3–5 times, divide into usage size aliquots (300 μL recommended) in polypropylene tubes and store at −20°C until expiration date. Avoid extended exposure of protein to ambient temperatures.•ROCK inhibitor Y-27632: reconstitute in DMSO to prepare a stock solution of 10 mM and divide into aliquots of 10 μL. Store aliquots at −20°C up to 6 months.•StemFlex complete medium: thaw the frozen StemFlex Supplement 10X at room temperature (RT) for ∼2 h or overnight at 4°C. Mix the thawed supplement by gently inverting 3–5 times. Aseptically transfer 50 mL of StemFlex Supplement 10X to the bottle of StemFlex basal medium. Gently invert the bottle several times to obtain an homogeneous complete medium. Aliquot the medium and store at −20°C for up to 6 months. To avoid multiple freeze-thaw cycles, we recommend preparing 25- and 50-mL aliquots and use as needed. Before use, warm complete medium required for that day at RT until it is no longer cool to the touch.•Vitronectin (VTN-N) recombinant human protein, truncated (0.5 mg/mL). Thaw the vial of vitronectin at RT and prepare 60-μL aliquots of vitronectin in polypropylene tubes. Freeze the aliquots at −80°C or use immediately. •For the manual picking of iPSC colonies, equip a laminar flow hood with a stereomicroscope coupled to a real-time camera that allows for visualizing the iPSC colonies while working inside the laminar flow hood (Figure 1A). Note: all of these protocols must be performed in a sterile manner. All reagents, except for BSA, are suitable for cell culture and there is no need of filter sterilization. Note: optimization and testing of the protocols included in this manuscript has been performed using three iPSC lines previously generated in our laboratory: N1-FiPS4F#7, MOA1-FiPS4F#7, and MOA2-FiPS4F#17.19Park S. Mostoslavsky G. Generation of human induced pluripotent stem cells using a defined, feeder-free reprogramming system.Curr. Protoc. Stem Cell Biol. 2018; 45: e48Google Scholar,22Castro-Viñuelas R. Sanjurjo-Rodríguez C. Piñeiro-Ramil M. Rodríguez-Fernández S. Fuentes-Boquete I. Blanco F.J. Díaz-Prado S.M. Generation of a human control iPS cell line (ESi080-A) from a donor with no rheumatic diseases.Stem Cell Res. 2020; 43: 101683Google Scholar Note: generating a stock of feeder cells is necessary for performing cell reprogramming and sub-culturing of iPSC colonies. The protocol we recommend here is based on mitotically inactivating the foreskin fibroblast cell line HFF-1 with γ-irradiation, but other methods for arresting cell cycle such as mitomycin C can be used.27Llames S. García-Pérez E. Meana Á. Larcher F. Río M. Feeder layer cell actions and applications.Tissue Eng. B Rev. 2015; 21: 345-353Google Scholar 1.Before performing the irradiation process, culture the HFF-1 cell line in IMDM supplemented with 10% FBS and 1% P/S (HFF medium), at 37°C in a humidified atmosphere with 5% CO2.2.When cell confluence reaches 90%–100%, perform subcultures for cell expansion until sufficient numbers of cells (approximately 6–10, 150-cm2 culture dishes at 100% confluence) are obtained. Note: do not over expand the HFF-1 before irradiation. Too many cells will result in too many frozen cryovials. We do not recommend using feeder cells that have been stored for longer than 3 months in liquid nitrogen since they tend to deteriorate. This makes the iPSC colonies more likely to break up into single cells and disappear in culture.3.On the day of irradiation, remove the HFF medium from the plates and wash the cells twice with PBS.4.Incubate the HFF-1 cell line with 0.25% trypsin-EDTA for 2–3 min at 37°C.5.To verify that cells are not still adhered to the culture dish, visualize the plates through an inverted microscope.6.When HFF-1 cells are detached from the culture dishes, inactivate the trypsin-EDTA with a double volume of 5% DMEM.7.Collect cell suspension in a 50-mL conical tube and centrifuge for 7 min at 400 × g to obtain a cell pellet.8.Resuspend the cell pellet in 50 mL of warm HFF medium.9.Immediately irradiate the HFF-1-containing tube at 75 Gy. In this protocol we used a Varian Unique linear accelerator (Centro Oncológico de Galicia, Spain). Note: if performing this protocol for the first time, and due to possible differences in the accelerators used, we recommend verifying that 75 Gy irradiation inhibits HFF-1 mitosis by flow cytometry as described below. Here, the cell cycle is evaluated by flow cytometry and using PI.1.Transfer 2 × 105 cells to polypropylene conical tubes and centrifuge at 400 × g for 5 min.2.Wash the pelleted cells twice with saline serum.3.Discard the supernatant and resuspend pelleted cells in a solution composed of 350 μL PI (stock concentration 1.0 mg/mL) and 1.5 μL RNAse (stock concentration 500 μg/mL) per tube.4.Incubate the tubes in the dark for 15 min and then centrifuge for 5 min at 400 × g.5.Resuspend the pellets in fluorescence-activated cell sorting buffer to a final volume of 200 μL.6.Analyze the cells in a FACScalibur flow cytometer using the CellQuest software. Note: non-irradiated HFF-1 cells must be also analyzed as negative control. 1.Count the irradiated HFF-1 cells using trypan blue staining and a Neubauer chamber.2.Centrifuge the cell suspension for 7 min at 400 × g.3.While centrifuging, prepare the necessary amount of f
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