Improved cryopreservation of cardiomyocyte aggregates differentiated from GMP iPSC in a 3D culture format

improved-cryopreservation-of-cardiomyocyte-aggregates-differentiated-from-gmp-ipsc-in-a-3d-culture-format
Improved cryopreservation of cardiomyocyte aggregates differentiated from GMP iPSC in a 3D culture format

References

  1. van Berlo, J. H. & Molkentin, J. D. An emerging consensus on cardiac regeneration. Nat. Med. 20, 1386–1393. https://doi.org/10.1038/nm.3764 (2014).

    Google Scholar 

  2. Savarese, G. et al. Global burden of heart failure: a comprehensive and updated review of epidemiology. Cardiovasc. Res. 118, 3272–3287. https://doi.org/10.1093/cvr/cvac013 (2023).

    Google Scholar 

  3. Boulet, J., Wanderley, M. R. B. Jr. & Mehra, M. R. Contemporary Left Ventricular Assist Device Therapy as a Bridge or Alternative to Transplantation. Transplantation 108, 1333–1341. https://doi.org/10.1097/TP.0000000000004834 (2024).

    Google Scholar 

  4. Kobold, S. et al. Manually Curated Database on Clinical Studies Involving Cell Products Derived from Human Pluripotent Stem Cells. Stem. Cell Rep. 15, 546–555. https://doi.org/10.1016/j.stemcr.2020.06.014 (2020).

    Google Scholar 

  5. Ilic, D. & Ogilvie, C. Pluripotent Stem Cells in Clinical Setting-New Developments and Overview of Current Status. Stem. Cells 40, 791–801. https://doi.org/10.1093/stmcls/sxac040 (2022).

    Google Scholar 

  6. Jebran, A. F. et al. Engineered heart muscle allografts for heart repair in primates and humans. Nature 639, 503–511. https://doi.org/10.1038/s41586-024-08463-0 (2025).

    Google Scholar 

  7. Laflamme, M. A. et al. Cardiomyocytes derived from human embryonic stem cells in pro-survival factors enhance function of infarcted rat hearts. Nat. Biotechnol. 25, 1015–1024. https://doi.org/10.1038/nbt1327 (2007).

    Google Scholar 

  8. Burridge, P. W. et al. A universal system for highly efficient cardiac differentiation of human induced pluripotent stem cells that eliminates interline variability. PLoS ONE 6, e18293. https://doi.org/10.1371/journal.pone.0018293 (2011).

    Google Scholar 

  9. P. Hofbauer, S.M. Jahnel, S. Mendjan, In vitro models of the human heart, Development 148 (2021). https://doi.org/10.1242/dev.199672.

  10. Zuppinger, C. 3D Cardiac Cell Culture: A Critical Review of Current Technologies and Applications. Front. Cardiovasc. Med. 6, 87. https://doi.org/10.3389/fcvm.2019.00087 (2019).

    Google Scholar 

  11. Hofbauer, P. et al. Cardioids reveal self-organizing principles of human cardiogenesis. Cell 184, 3299–3317. https://doi.org/10.1016/j.cell.2021.04.034 (2021).

    Google Scholar 

  12. Terheyden-Keighley, D. et al. GMP-compliant iPS cell lines show widespread plasticity in a new set of differentiation workflows for cell replacement and cancer immunotherapy. Stem. Cells Transl. Med. 13, 898–911. https://doi.org/10.1093/stcltm/szae047 (2024).

    Google Scholar 

  13. Halloin, C. et al. Continuous WNT Control Enables Advanced hPSC Cardiac Processing and Prognostic Surface Marker Identification in Chemically Defined Suspension Culture. Stem. Cell Rep. 13, 775. https://doi.org/10.1016/j.stemcr.2019.09.001 (2019).

    Google Scholar 

  14. Kriedemann, N. et al. Standardized production of hPSC-derived cardiomyocyte aggregates in stirred spinner flasks. Nat. Protoc. 19, 1911–1939. https://doi.org/10.1038/s41596-024-00976-2 (2024).

    Google Scholar 

  15. M. Uhrig, F. Ezquer, M. Ezquer, Improving Cell Recovery: Freezing and Thawing Optimization of Induced Pluripotent Stem Cells, Cells 11 (2022). https://doi.org/10.3390/cells11050799.

  16. Han, H., Zhan, T., Guo, N., Cui, M. & Xu, Y. Cryopreservation of organoids: Strategies, innovation, and future prospects. Biotechnol. J. https://doi.org/10.1002/biot.202300543 (2024).

    Google Scholar 

  17. Preininger, M. K., Singh, M. & Xu, C. Cryopreservation of Human Pluripotent Stem Cell-Derived Cardiomyocytes: Strategies, Challenges, and Future Directions. Adv. Exp. Med. Biol. 951, 123–135. https://doi.org/10.1007/978-3-319-45457-3_10 (2016).

    Google Scholar 

  18. Xu, C. et al. Efficient generation and cryopreservation of cardiomyocytes derived from human embryonic stem cells. Regen. Med. 6, 53–66. https://doi.org/10.2217/rme.10.91 (2011).

    Google Scholar 

  19. van den Brink, L. et al. Cryopreservation of human pluripotent stem cell-derived cardiomyocytes is not detrimental to their molecular and functional properties. Stem. Cell Res. https://doi.org/10.1016/j.scr.2019.101698 (2020).

    Google Scholar 

  20. Miller, D. C., Genehr, C., Telugu, N. S., Kurths, S. & Diecke, S. Simple Workflow and Comparison of Media for hPSC-Cardiomyocyte Cryopreservation and Recovery. Curr Protoc Stem Cell Biol 55, e125. https://doi.org/10.1002/cpsc.125 (2020).

    Google Scholar 

  21. R.G.C. Maas, S. Lee, M. Harakalova, C.J.B. Snijders Blok, W.R. Goodyer, J. Hjortnaes, P. Doevendans, L.W. Van Laake, J. van der Velden, F.W. Asselbergs, J.C. Wu, J.P.G. Sluijter, S.M. Wu, J.W. Buikema, Massive expansion and cryopreservation of functional human induced pluripotent stem cell-derived cardiomyocytes, STAR Protoc 2 (2021) 100334. https://doi.org/10.1016/j.xpro.2021.100334.

  22. Zhang, J. Z. et al. Effects of Cryopreservation on Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes for Assessing Drug Safety Response Profiles. Stem. Cell Rep. 16, 168–181. https://doi.org/10.1016/j.stemcr.2020.11.010 (2021).

    Google Scholar 

  23. Prondzynski, M. et al. Efficient and reproducible generation of human iPSC-derived cardiomyocytes and cardiac organoids in stirred suspension systems. Nat. Commun. 15, 5929. https://doi.org/10.1038/s41467-024-50224-0 (2024).

    Google Scholar 

  24. Murray, K. A. & Gibson, M. I. Chemical approaches to cryopreservation. Nat. Rev. Chem. 6, 579–593. https://doi.org/10.1038/s41570-022-00407-4 (2022).

    Google Scholar 

  25. Whaley, D. et al. Cryopreservation: An Overview of Principles and Cell-Specific Considerations. Cell Transpl. 30, 963689721999617. https://doi.org/10.1177/0963689721999617 (2021).

    Google Scholar 

  26. Yamatoya, K. et al. Cryopreservation of undifferentiated and differentiated human neuronal cells. Regen. Ther. 19, 58–68. https://doi.org/10.1016/j.reth.2021.12.007 (2022).

    Google Scholar 

  27. Mandumpal, J. B., Kreck, C. A. & Mancera, R. L. A molecular mechanism of solvent cryoprotection in aqueous DMSO solutions. Phys. Chem. Chem. Phys. 13, 3839–3842. https://doi.org/10.1039/c0cp02326d (2011).

    Google Scholar 

  28. Verheijen, M. et al. DMSO induces drastic changes in human cellular processes and epigenetic landscape in vitro. Sci. Rep. 9, 4641. https://doi.org/10.1038/s41598-019-40660-0 (2019).

    Google Scholar 

  29. R.G.C. Maas, T. Beekink, N. Chirico, C.J.B. Snijders Blok, I. Dokter, V. Sampaio-Pinto, A. van Mil, P.A. Doevendans, J.W. Buikema, J.P.G. Sluijter, F. Stillitano, Generation, High-Throughput Screening, and Biobanking of Human-Induced Pluripotent Stem Cell-Derived Cardiac Spheroids, J. Vis. Exp. https://doi.org/10.3791/64365. (2023).

  30. Janssen, J. et al. Hypothermic and cryogenic preservation of cardiac tissue-engineered constructs. Biomater. Sci. 12, 3866–3881. https://doi.org/10.1039/d3bm01908j (2024).

    Google Scholar 

  31. Meneghel, J., Kilbride, P. & Morris, G. J. Cryopreservation as a Key Element in the Successful Delivery of Cell-Based Therapies-A Review. Front. Med. (Lausanne) https://doi.org/10.3389/fmed.2020.592242 (2020).

    Google Scholar 

  32. Xie, J. et al. Principles and Protocols For Post-Cryopreservation Quality Evaluation of Stem Cells in Novel Biomedicine. Front. Pharmacol. https://doi.org/10.3389/fphar.2022.907943 (2022).

    Google Scholar 

  33. Murray, K. A. & Gibson, M. I. Post-Thaw Culture and Measurement of Total Cell Recovery Is Crucial in the Evaluation of New Macromolecular Cryoprotectants. Biomacromol 21, 2864–2873. https://doi.org/10.1021/acs.biomac.0c00591 (2020).

    Google Scholar 

  34. Burridge, P. W. et al. Chemically defined generation of human cardiomyocytes. Nat. Methods 11, 855–860. https://doi.org/10.1038/nmeth.2999 (2014).

    Google Scholar 

  35. P.W. Burridge, A. Holmstrom, J.C. Wu, Chemically Defined Culture and Cardiomyocyte Differentiation of Human Pluripotent Stem Cells, Curr. Protoc. Hum. Genet. 87 (2015) 21 23 21–21 23 15. https://doi.org/10.1002/0471142905.hg2103s87.

  36. Kempf, H., Kropp, C., Olmer, R., Martin, U. & Zweigerdt, R. Cardiac differentiation of human pluripotent stem cells in scalable suspension culture. Nat. Protoc. 10, 1345–1361. https://doi.org/10.1038/nprot.2015.089 (2015).

    Google Scholar 

  37. Kriedemann, N. et al. Protein-free media for cardiac differentiation of hPSCs in 2000 mL suspension culture. Stem. Cell Res. Ther. 15, 213. https://doi.org/10.1186/s13287-024-03826-w (2024).

    Google Scholar 

  38. Xu, X. et al. The roles of apoptotic pathways in the low recovery rate after cryopreservation of dissociated human embryonic stem cells. Biotechnol. Prog. 26, 827–837. https://doi.org/10.1002/btpr.368 (2010).

    Google Scholar 

  39. Kim, Y. Y. et al. Cryopreservation of human embryonic stem cells derived-cardiomyocytes induced by BMP2 in serum-free condition. Reprod. Sci. 18, 252–260. https://doi.org/10.1177/1933719110385130 (2011).

    Google Scholar 

  40. Chen, Y. et al. A versatile polypharmacology platform promotes cytoprotection and viability of human pluripotent and differentiated cells. Nat. Methods 18, 528–541. https://doi.org/10.1038/s41592-021-01126-2 (2021).

    Google Scholar 

  41. I. Gruh, A. Martens, S. Cebotari, A. Schrod, A. Haase, C. Halloin, W. Triebert, T. Goecke, M. Arar, K. Hoeffler, P. Frank, K. Lampe, A. Moussavi, V. Fricke, N. Kriedemann, M. Szepes, K. Mätz-Rensing, J. Eiringhaus, A.-L.d. Vries, I. Barnekow, C.S. Ferrel, S. Hohmann, M. Witte, T. Kohrn, J. Teske, V. Lupanov, A. Franke, M. Kühnel, D. Jonigk, S. Boretius, C. Veltmann, D. Duncker, A. Hilfiker, A. Haverich, R. Zweigerdt, U. Martin, 2024 Cell therapy with human iPSC-derived cardiomyocyte aggregates leads to efficient engraftment and functional recovery after myocardial infarction in non-human primates, Biorxiv. https://doi.org/10.1101/2023.12.31.573775. (2024).

  42. Kobayashi, H. et al. Regeneration of Nonhuman Primate Hearts With Human Induced Pluripotent Stem Cell-Derived Cardiac Spheroids. Circulation 150, 611–621. https://doi.org/10.1161/CIRCULATIONAHA.123.064876 (2024).

    Google Scholar 

  43. Liedtke, S., Korschgen, L., Korn, J., Duppers, A. & Kogler, G. GMP-grade CD34(+) selection from HLA-homozygous licensed cord blood units and short-term expansion under European ATMP regulations. Vox Sang. 116, 123–135. https://doi.org/10.1111/vox.12978 (2021).

    Google Scholar 

  44. Okita, K. et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem. Cells 31, 458–466. https://doi.org/10.1002/stem.1293 (2013).

    Google Scholar 

  45. Nicoletti, I., Migliorati, G., Pagliacci, M. C., Grignani, F. & Riccardi, C. A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J. Immunol. Methods 139, 271–279. https://doi.org/10.1016/0022-1759(91)90198-o (1991).

    Google Scholar 

  46. Riccardi, C. & Nicoletti, I. Analysis of apoptosis by propidium iodide staining and flow cytometry. Nat. Protoc. 1, 1458–1461. https://doi.org/10.1038/nprot.2006.238 (2006).

    Google Scholar 

  47. Sala, L. et al. MUSCLEMOTION: A Versatile Open Software Tool to Quantify Cardiomyocyte and Cardiac Muscle Contraction In Vitro and In Vivo. Circ. Res. 122, e5–e16. https://doi.org/10.1161/CIRCRESAHA.117.312067 (2018).

    Google Scholar 

  48. van Meer, B. J. et al. Quantification of Muscle Contraction In Vitro and In Vivo Using MUSCLEMOTION Software: From Stem Cell-Derived Cardiomyocytes to Zebrafish and Human Hearts. Curr. Protoc. Hum. Genet. https://doi.org/10.1002/cphg.67 (2018).

    Google Scholar 

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