DMSO-free cryopreservation of hiPSC-derived cardiomyocytes: low temperature characterization and protocol development
Abstract Background Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have attracted significant interest for use in disease modeling, drug discovery and potential therapeutic applications. However, conventional hiPSC-CM cryopreservation protocols largely use dimethyl sulfoxide...
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2025-06-01
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| Series: | Stem Cell Research & Therapy |
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| Online Access: | https://doi.org/10.1186/s13287-025-04384-5 |
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| author | Akshat S. Mallya Tessa Burrows Jeanne Hsieh Troy Louwagie James R. Dutton Brenda M. Ogle Allison Hubel |
| author_facet | Akshat S. Mallya Tessa Burrows Jeanne Hsieh Troy Louwagie James R. Dutton Brenda M. Ogle Allison Hubel |
| author_sort | Akshat S. Mallya |
| collection | DOAJ |
| description | Abstract Background Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have attracted significant interest for use in disease modeling, drug discovery and potential therapeutic applications. However, conventional hiPSC-CM cryopreservation protocols largely use dimethyl sulfoxide (DMSO) as the cryoprotectant (CPA), which is linked with a loss of post-thaw recovery and function for various cell types and is not ideal for therapeutic protocols. Additionally, the effect of freezing parameters such as cooling rate and nucleation temperature on post-thaw recovery of hiPSC-CMs has not been explored. Methods hiPSC-CMs were generated by Wnt pathway inhibition, followed by sodium l-lactate purification. Subsequently, biophysical characterization of the cells was performed. A differential evolution (DE) algorithm was utilized to determine the optimal composition of a mixture of a sugar, sugar alcohol and amino acid to replace DMSO as the CPA. The hiPSC-CMs were subjected to controlled-rate freezing at different cooling rates and nucleation temperatures. The optimum freezing parameters were identified by post-thaw recoveries and the partitioning ratio obtained from low temperature Raman spectroscopy studies. The post-thaw osmotic behavior of hiPSC-CMs was studied by measuring diameter of cells resuspended in the isotonic culture medium over time. Immunocytochemistry and calcium transient studies were performed to evaluate post-thaw function. Results hiPSC-CMs were found to be slightly larger than hiPSCs and exhibited a large osmotically inactive volume. The best-performing DMSO-free solutions enabled post-thaw recoveries over 90%, which was significantly greater than DMSO (69.4 ± 6.4%). A rapid cooling rate of 5 °C/min and a low nucleation temperature of -8 °C was found to be optimal for hiPSC-CMs. hiPSC-CMs displayed anomalous osmotic behavior post-thaw, dropping sharply in volume after resuspension. Post-thaw function was preserved when hiPSC-CMs were frozen with the best-performing DMSO-free CPA or DMSO and the cells displayed similar cardiac markers pre-freeze and post-thaw. Conclusions It was shown that a CPA cocktail of naturally-occurring osmolytes could effectively replace DMSO for preserving hiPSC-CMs while preserving morphology and function. Understanding the anomalous osmotic behavior and managing the excessive dehydration of hiPSC-CMs could be crucial to improve post-thaw outcomes. Effective DMSO-free cryopreservation would accelerate the development of drug discovery and therapeutic applications of hiPSC-CMs. |
| format | Article |
| id | doaj-art-e581f662b45d4e93805f2ef0f6069b10 |
| institution | Kabale University |
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| language | English |
| publishDate | 2025-06-01 |
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| spelling | doaj-art-e581f662b45d4e93805f2ef0f6069b102025-08-20T03:45:11ZengBMCStem Cell Research & Therapy1757-65122025-06-0116111610.1186/s13287-025-04384-5DMSO-free cryopreservation of hiPSC-derived cardiomyocytes: low temperature characterization and protocol developmentAkshat S. Mallya0Tessa Burrows1Jeanne Hsieh2Troy Louwagie3James R. Dutton4Brenda M. Ogle5Allison Hubel6Department of Mechanical Engineering, University of MinnesotaDepartment of Biomedical Engineering, University of MinnesotaDepartment of Biomedical Engineering, University of MinnesotaDepartment of Mechanical Engineering, University of MinnesotaDepartment of Genetics, Cell Biology and Development, University of MinnesotaDepartment of Biomedical Engineering, University of MinnesotaDepartment of Mechanical Engineering, University of MinnesotaAbstract Background Human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have attracted significant interest for use in disease modeling, drug discovery and potential therapeutic applications. However, conventional hiPSC-CM cryopreservation protocols largely use dimethyl sulfoxide (DMSO) as the cryoprotectant (CPA), which is linked with a loss of post-thaw recovery and function for various cell types and is not ideal for therapeutic protocols. Additionally, the effect of freezing parameters such as cooling rate and nucleation temperature on post-thaw recovery of hiPSC-CMs has not been explored. Methods hiPSC-CMs were generated by Wnt pathway inhibition, followed by sodium l-lactate purification. Subsequently, biophysical characterization of the cells was performed. A differential evolution (DE) algorithm was utilized to determine the optimal composition of a mixture of a sugar, sugar alcohol and amino acid to replace DMSO as the CPA. The hiPSC-CMs were subjected to controlled-rate freezing at different cooling rates and nucleation temperatures. The optimum freezing parameters were identified by post-thaw recoveries and the partitioning ratio obtained from low temperature Raman spectroscopy studies. The post-thaw osmotic behavior of hiPSC-CMs was studied by measuring diameter of cells resuspended in the isotonic culture medium over time. Immunocytochemistry and calcium transient studies were performed to evaluate post-thaw function. Results hiPSC-CMs were found to be slightly larger than hiPSCs and exhibited a large osmotically inactive volume. The best-performing DMSO-free solutions enabled post-thaw recoveries over 90%, which was significantly greater than DMSO (69.4 ± 6.4%). A rapid cooling rate of 5 °C/min and a low nucleation temperature of -8 °C was found to be optimal for hiPSC-CMs. hiPSC-CMs displayed anomalous osmotic behavior post-thaw, dropping sharply in volume after resuspension. Post-thaw function was preserved when hiPSC-CMs were frozen with the best-performing DMSO-free CPA or DMSO and the cells displayed similar cardiac markers pre-freeze and post-thaw. Conclusions It was shown that a CPA cocktail of naturally-occurring osmolytes could effectively replace DMSO for preserving hiPSC-CMs while preserving morphology and function. Understanding the anomalous osmotic behavior and managing the excessive dehydration of hiPSC-CMs could be crucial to improve post-thaw outcomes. Effective DMSO-free cryopreservation would accelerate the development of drug discovery and therapeutic applications of hiPSC-CMs.https://doi.org/10.1186/s13287-025-04384-5CardiomyocytesCryopreservationDMSO-freeRaman spectroscopyNADESProtocol development |
| spellingShingle | Akshat S. Mallya Tessa Burrows Jeanne Hsieh Troy Louwagie James R. Dutton Brenda M. Ogle Allison Hubel DMSO-free cryopreservation of hiPSC-derived cardiomyocytes: low temperature characterization and protocol development Stem Cell Research & Therapy Cardiomyocytes Cryopreservation DMSO-free Raman spectroscopy NADES Protocol development |
| title | DMSO-free cryopreservation of hiPSC-derived cardiomyocytes: low temperature characterization and protocol development |
| title_full | DMSO-free cryopreservation of hiPSC-derived cardiomyocytes: low temperature characterization and protocol development |
| title_fullStr | DMSO-free cryopreservation of hiPSC-derived cardiomyocytes: low temperature characterization and protocol development |
| title_full_unstemmed | DMSO-free cryopreservation of hiPSC-derived cardiomyocytes: low temperature characterization and protocol development |
| title_short | DMSO-free cryopreservation of hiPSC-derived cardiomyocytes: low temperature characterization and protocol development |
| title_sort | dmso free cryopreservation of hipsc derived cardiomyocytes low temperature characterization and protocol development |
| topic | Cardiomyocytes Cryopreservation DMSO-free Raman spectroscopy NADES Protocol development |
| url | https://doi.org/10.1186/s13287-025-04384-5 |
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