Exosomes Support the Viability of Mesenchymal Stem Cells in the Storage Condition of 4 oC
Main Article Content
Abstract
Umbilical cord-derived mesenchymal stem cells (UCMSCs) and their exosomes have attracted significant attention due to their various potential for therapeutic applications. In this study, we expanded UCMSCs and evaluated several short-term preservation buffers on the cell viability. The results showed that UCMSCs were split up to the 5th passage (P5), which expressed typical characteristics of mesenchymal stem cells in terms of morphology, marker expression, colony forming, and multilineage differentiation ability. UCMSCs survived up to 48 hours in three preservation buffers. Additionally, three buffers without exosomes were able to maintain a cell survival rate of > 75% at 24 hours for all experiment groups. For the buffers supplied with exosomes, we observed a cell survival rate of > 80% at 24 hours in all experiment groups, and
at 48 hours, the percentage of viable cells was higher than that of the buffer group without exosome supplement, except for the group of 2 x 106 cells/mL in buffer A (Glucose, Human serum albumin, Heparin calcium, Axit ascorbic, Non-essential amino acid) and C (Ringer lactate), and
5 x 106 cells/mL in buffer C. Furthermore, exosomes increased the cell clustering in all three buffer solutions. These results indicate the potential of directly using preservation buffers and cells in vivo and the real application.
Keywords:
Umbilical cord-derived mesenchymal stem cells, exosomes, storage buffer, cell viability.
References
[1] G. Sheng, The Developmental Basis of Mesenchymal Stem/stromal Cells (MSCs), BMC Developmental Biology, Vol. 15, 2015, pp. 44, https://doi.org/10.1186/s12861-015-0094-5.
[2] J. Ma, J. Wu, L. Han, X. Jiang, L. Yan, J. Hao, H. Wang, Comparative Analysis of Mesenchymal Stem Cells Derived from Amniotic Membrane, Umbilical Cord, and Chorionic Plate under Serum-free Condition, Stem Cell Research & Therapy, Vol. 10, No. 1, 2019, pp. 19, https://doi.org/10.1186/s13287-018-1104-x.
[3] T. N. Inoue, H. He, Umbilical Cord-derived Mesenchymal Stem Cells: Their Advantages and Potential Clinical Utility, World Journal Stem Cells, Vol. 6, No. 2, 2014, pp. 195-202, https://doi.org/10.4252/wjsc.v6.i2.195.
[4] J. Bagge, J. N. MacLeod, L. C. Berg, Cellular Proliferation of Equine Bone Marrow- and Adipose Tissue-derived Mesenchymal Stem Cells Decline with Increasing Donor Age, Frontiers in Veterinary Science, Vol. 7, 2020; pp. 602403, https://doi.org/10.3389/fvets.2020.602403.
[5] L. M. Hang, N. T. Lung, H. H. Diem, B. Q. Trung, N. T. N. Ha, M. T. Hien, T. P. Dong, N. D. Tu, N. T. Liem, T. T. T. Uyen, Differential Development of Umbilical Cord-Derived Mesenchymal Stem Cells During Long-Term Maintenance in Fetal Bovine Serum-Supplemented Medium and Xeno- and Serum-Free Culture Medium, Cellular Reprogramming, Vol. 23, No. 6, 2021, pp. 359-369, https://doi.org/10.1089/cell.2021.0050.
[6] M. D. Nicola, C. Carlo-Stella, M. Magni, M. Milanesi, P. D. Longoni, P. Matteucci, S. Grisanti, A. M. Gianni, Human Bone Marrow Stromal Cells Suppress T-Lymphocyte Proliferation Induced by Cellular or Nonspecific Mitogenic Stimuli, Blood, Vol. 99, No. 10, 2002, pp. 3838-43, https://doi.org/10.1182/blood.v99.10.3838.
[7] V. Miceli, M. Bulati, G. Iannolo, G. Zito, A. Gallo, P. G. Conaldi, Therapeutic Properties of Mesenchymal Stromal/Stem Cells: The Need of Cell Priming for Cell-Free Therapies in Regenerative Medicine, International Journal of Molecular Sciences., Vol. 22, No. 2, 2021, https://doi.org/10.3390/ijms22020763.
[8] G. D. Sagaradze, N. A. Basalova, A. Y. Efimenko, V. A. Tkachuk, Mesenchymal Stromal Cells as Critical Contributors to Tissue Regeneration, Frontiers in Cell and Developmental Biology, Vol. 8, 2020, https://doi.org/10.3389/fcell.2020.576176.
[9] M. D. Kim, S. S Kim., H. Y.Cha, S. H. Jang, D. Y. Chang, W. Kim, H. Suh-Kim, J. H. Lee, Therapeutic Effect of Hepatocyte Growth Factor-Secreting Mesenchymal Stem Cells in a Rat Model of Liver Fibrosis, Experimental & Molecular Medicine, Vol. 46, No. 8, 2014, pp. e110, https://doi.org/10.1038/emm.2014.49.
[10] G. Li, X. A. Zhang, H. Wang, X. Wang, C. L. Meng, C. Y. Chan, D. T. Yew, K. S. Tsang, K. Li, S. N. Tsai, S. M. Ngai, Z. C. Han, M. C. Lin, M. L. He, H. F. Kung, Erratum to: Comparative Proteomic Analysis of Mesenchymal Stem Cells Derived From Human Bone Marrow, Umbilical Cord and Placenta: Implication in The Migration, Advances in Experimental Medicine and Biology, Vol. 720, 2012, https://doi.org/10.1007/978-1-4614-0254-1_18.
[11] R. Pal, M. Hanwate, M. Jan, S. Totey, Phenotypic and Functional Comparison of Optimum Culture Conditions for Upscaling of Bone Marrow-Derived Mesenchymal Stem Cells, Tissue Engineering and Regenerative Medicine, Vol. 3, No. 3, 2009, pp. 163-174, https://doi.org/10.1002/term.143.
[12] H. S. Sohn, J. S. Heo, H. S. Kim, Y. Choi, H. O. Kim, Duration of in Vitro Storage Affects the Key Stem Cell Features of Human Bone Marrow-Derived Mesenchymal Stromal Cells for Clinical Transplantation, Cytotherapy, Vol. 15, No. 4, 2013, pp. 460-466, https://doi.org/10.1016/j.jcyt.2012.10.015.
[13] K. Muraki, M. Hirose, N. Kotobuki, Y. Kato, H. Machida, Y. Takakura, H. Ohgushi, Assessment of Viability and Osteogenic Ability of Human Mesenchymal Stem Cells After Being Stored in Suspension for Clinical Transplantation, Tissue Engineering., Vol. 12, No. 6, 2006, pp. 1711-1719, https://doi.org/10.1089/ten.2006.12.1711.
[14] K. A. Lee, W. Shim, M. J. Paik, S. C. Lee, J. Y. Shin, Y. H. Ahn, K. Park, J. H. Kim, S. Choi, G. Lee, Analysis of Changes in The Viability and Gene Expression Profiles of Human Mesenchymal Stromal Cells over Time. Cytotherapy, Vol. 11, No. 6, 2009, pp. 688-97, https://doi.org/10.3109/14653240902974032.
[15] P. Mazur, Principles of Cryobiology, CRC Press, 2006, pp. 3-56.
[16] J. G. Baust, J. M. Baust, Advances in Biopreservation (1st ed.), CRC Press, 2006.
[17] Y. Zhou, B. Zhao, X. L. Zhang, Y. J. Lu, S. T. Lu, J. Cheng, Y. Fu, L. Lin, N. Y. Zhang, P. X. Li, J. Zhang, J. Zhan, Combined Topical and Systemic Administration with Human Adipose-Derived Mesenchymal Stem Cells (Hadsc) and HADSC-Derived Exosomes Markedly Promoted Cutaneous Wound Healing and Regeneration, Stem Cell Research & Therapy, Vol. 12, No.1, 2021, pp. 257, https://doi.org/10.1186/s13287-021-02287-9.
[18] H. H. Diem, N. D. Tu, N. H. Phuong, N. X. Hung, D. T. X. Phuong, D. V. Duc, D. T. M. Phuong, B. T. H. Hue, T. Q. Mai, V. M. Duc, H. T. M. Nhung, N. T. Liem, T. T. T. Uyen, Differential Wound Healing Capacity of Mesenchymal Stem Cell-Derived Exosomes Originated from Bone Marrow, Adipose Tissue and Umbilical Cord Under Serum- and Xeno-Free Condition, Frontiers in Molecular Biosciences, Vol. 7, 2020, pp. 119, https://doi.org/10.3389/fmolb.2020.00119.
[19] N. T. Huyen, D. H. Hoang, D. M. Chau, N. X. Hung, H. H. Diem, D. X. Hai, T. Q. Trung, N. D. Tu, N. T. Liem, T. T. T. Uyen, Cytokine-primed Umbilical Cord Mesenchymal Stem Cells Enhanced Therapeutic Effects of Extracellular Vesicles on Osteoarthritic Chondrocytes, Front Immunol., Vol. 13, 2022, pp. 1041592,
https://doi.org/10.3389/fimmu.2022.1041592.
[20] K. E. Thane, A. M. Davis, A. M. Hoffman, Improved Methods for Fluorescent Labeling and Detection of Single Extracellular Vesicles Using Nanoparticle Tracking Analysis, Scientific Reports, Vol. 9, No. 1, 2019, pp. 12295,
https://doi.org/10.1038/s41598-019-48181-6.
[21] S. Rowley, W. Bensinger, T. Gooley, C. Buckner, Effect of Cell Concentration on Bone Marrow and Peripheral Blood Stem Cell Cryopreservation, Blood, Vol. 83, No. 9, 1994, pp. 2731-2736,
https://doi.org/10.1182/blood.V83.9.2731.2731.
[22] S. Bahsoun, M. J. Brown, K. Coopman, E. C. Akam, Cryopreservation of Human Bone Marrow Derived Mesenchymal Stem Cells at High Concentration is Feasible, Biopreservation and Biobanking, Vol. 21, No. 5, 2022, pp. 450-457, https://doi.org/10.1089/bio.2022.0017.
[23] X. Sun, J. H. Jung, O. Arvola, M. R. Santoso, R. G. Giffard, P. C. Yang, C. M. Stary, Stem Cell-Derived Exosomes Protect Astrocyte Cultures From in Vitro Ischemia and Decrease Injury as Post-Stroke Intravenous Therapy, Frontiers in Cellular Neuroscience, Vol. 13, 2019, pp. 394, https://doi.org/10.3389/fncel.2019.00394.
[24] G. D. Micó, M. Mittelbrunn, Role of Exosomes in the Protection of Cellular Homeostasis, Cell Adhesion & Migration, Vol. 11, No. 2, 2017, pp. 127-34, https://doi.org/10.1080/19336918.2016.1251000.
[25] J. Cwalinski, R. Staniszewski, E. Baum, T. Jasinski, B. Mackowiak, A. Bręborowicz, Normal Saline May Promote Formation of Peritoneal Adhesions, International Journal of Clinical and Experimental Medicine, Vol. 8, No. 6, 2015, pp. 8828-8834.
[26] L. Li, J. Qin, Q. Feng, H. Tang, R. Liu, L. Xu, Z. Chen, Heparin Promotes Suspension Adaptation Process of CHO-TS28 Cells by Eliminating Cell Aggregation, Molecular Biotechnology, Vol. 47, No. 1, 2011, pp. 9-17, https://doi.org/10.1007/s12033-010-9306-1.
[27] Y. Lan, Q. Jin, H. Xie, C. Yan, Y. Ye, X. Zhao, Z. Chen, Z. Xie, Exosomes Enhance Adhesion and Osteogenic Differentiation of Initial Bone Marrow Stem Cells on Titanium Surfaces, Frontiers in Cell and Developmental Biology, Vol. 8, 2020, pp. 583234, https://doi.org/10.3389/fcell.2020.583234.
[2] J. Ma, J. Wu, L. Han, X. Jiang, L. Yan, J. Hao, H. Wang, Comparative Analysis of Mesenchymal Stem Cells Derived from Amniotic Membrane, Umbilical Cord, and Chorionic Plate under Serum-free Condition, Stem Cell Research & Therapy, Vol. 10, No. 1, 2019, pp. 19, https://doi.org/10.1186/s13287-018-1104-x.
[3] T. N. Inoue, H. He, Umbilical Cord-derived Mesenchymal Stem Cells: Their Advantages and Potential Clinical Utility, World Journal Stem Cells, Vol. 6, No. 2, 2014, pp. 195-202, https://doi.org/10.4252/wjsc.v6.i2.195.
[4] J. Bagge, J. N. MacLeod, L. C. Berg, Cellular Proliferation of Equine Bone Marrow- and Adipose Tissue-derived Mesenchymal Stem Cells Decline with Increasing Donor Age, Frontiers in Veterinary Science, Vol. 7, 2020; pp. 602403, https://doi.org/10.3389/fvets.2020.602403.
[5] L. M. Hang, N. T. Lung, H. H. Diem, B. Q. Trung, N. T. N. Ha, M. T. Hien, T. P. Dong, N. D. Tu, N. T. Liem, T. T. T. Uyen, Differential Development of Umbilical Cord-Derived Mesenchymal Stem Cells During Long-Term Maintenance in Fetal Bovine Serum-Supplemented Medium and Xeno- and Serum-Free Culture Medium, Cellular Reprogramming, Vol. 23, No. 6, 2021, pp. 359-369, https://doi.org/10.1089/cell.2021.0050.
[6] M. D. Nicola, C. Carlo-Stella, M. Magni, M. Milanesi, P. D. Longoni, P. Matteucci, S. Grisanti, A. M. Gianni, Human Bone Marrow Stromal Cells Suppress T-Lymphocyte Proliferation Induced by Cellular or Nonspecific Mitogenic Stimuli, Blood, Vol. 99, No. 10, 2002, pp. 3838-43, https://doi.org/10.1182/blood.v99.10.3838.
[7] V. Miceli, M. Bulati, G. Iannolo, G. Zito, A. Gallo, P. G. Conaldi, Therapeutic Properties of Mesenchymal Stromal/Stem Cells: The Need of Cell Priming for Cell-Free Therapies in Regenerative Medicine, International Journal of Molecular Sciences., Vol. 22, No. 2, 2021, https://doi.org/10.3390/ijms22020763.
[8] G. D. Sagaradze, N. A. Basalova, A. Y. Efimenko, V. A. Tkachuk, Mesenchymal Stromal Cells as Critical Contributors to Tissue Regeneration, Frontiers in Cell and Developmental Biology, Vol. 8, 2020, https://doi.org/10.3389/fcell.2020.576176.
[9] M. D. Kim, S. S Kim., H. Y.Cha, S. H. Jang, D. Y. Chang, W. Kim, H. Suh-Kim, J. H. Lee, Therapeutic Effect of Hepatocyte Growth Factor-Secreting Mesenchymal Stem Cells in a Rat Model of Liver Fibrosis, Experimental & Molecular Medicine, Vol. 46, No. 8, 2014, pp. e110, https://doi.org/10.1038/emm.2014.49.
[10] G. Li, X. A. Zhang, H. Wang, X. Wang, C. L. Meng, C. Y. Chan, D. T. Yew, K. S. Tsang, K. Li, S. N. Tsai, S. M. Ngai, Z. C. Han, M. C. Lin, M. L. He, H. F. Kung, Erratum to: Comparative Proteomic Analysis of Mesenchymal Stem Cells Derived From Human Bone Marrow, Umbilical Cord and Placenta: Implication in The Migration, Advances in Experimental Medicine and Biology, Vol. 720, 2012, https://doi.org/10.1007/978-1-4614-0254-1_18.
[11] R. Pal, M. Hanwate, M. Jan, S. Totey, Phenotypic and Functional Comparison of Optimum Culture Conditions for Upscaling of Bone Marrow-Derived Mesenchymal Stem Cells, Tissue Engineering and Regenerative Medicine, Vol. 3, No. 3, 2009, pp. 163-174, https://doi.org/10.1002/term.143.
[12] H. S. Sohn, J. S. Heo, H. S. Kim, Y. Choi, H. O. Kim, Duration of in Vitro Storage Affects the Key Stem Cell Features of Human Bone Marrow-Derived Mesenchymal Stromal Cells for Clinical Transplantation, Cytotherapy, Vol. 15, No. 4, 2013, pp. 460-466, https://doi.org/10.1016/j.jcyt.2012.10.015.
[13] K. Muraki, M. Hirose, N. Kotobuki, Y. Kato, H. Machida, Y. Takakura, H. Ohgushi, Assessment of Viability and Osteogenic Ability of Human Mesenchymal Stem Cells After Being Stored in Suspension for Clinical Transplantation, Tissue Engineering., Vol. 12, No. 6, 2006, pp. 1711-1719, https://doi.org/10.1089/ten.2006.12.1711.
[14] K. A. Lee, W. Shim, M. J. Paik, S. C. Lee, J. Y. Shin, Y. H. Ahn, K. Park, J. H. Kim, S. Choi, G. Lee, Analysis of Changes in The Viability and Gene Expression Profiles of Human Mesenchymal Stromal Cells over Time. Cytotherapy, Vol. 11, No. 6, 2009, pp. 688-97, https://doi.org/10.3109/14653240902974032.
[15] P. Mazur, Principles of Cryobiology, CRC Press, 2006, pp. 3-56.
[16] J. G. Baust, J. M. Baust, Advances in Biopreservation (1st ed.), CRC Press, 2006.
[17] Y. Zhou, B. Zhao, X. L. Zhang, Y. J. Lu, S. T. Lu, J. Cheng, Y. Fu, L. Lin, N. Y. Zhang, P. X. Li, J. Zhang, J. Zhan, Combined Topical and Systemic Administration with Human Adipose-Derived Mesenchymal Stem Cells (Hadsc) and HADSC-Derived Exosomes Markedly Promoted Cutaneous Wound Healing and Regeneration, Stem Cell Research & Therapy, Vol. 12, No.1, 2021, pp. 257, https://doi.org/10.1186/s13287-021-02287-9.
[18] H. H. Diem, N. D. Tu, N. H. Phuong, N. X. Hung, D. T. X. Phuong, D. V. Duc, D. T. M. Phuong, B. T. H. Hue, T. Q. Mai, V. M. Duc, H. T. M. Nhung, N. T. Liem, T. T. T. Uyen, Differential Wound Healing Capacity of Mesenchymal Stem Cell-Derived Exosomes Originated from Bone Marrow, Adipose Tissue and Umbilical Cord Under Serum- and Xeno-Free Condition, Frontiers in Molecular Biosciences, Vol. 7, 2020, pp. 119, https://doi.org/10.3389/fmolb.2020.00119.
[19] N. T. Huyen, D. H. Hoang, D. M. Chau, N. X. Hung, H. H. Diem, D. X. Hai, T. Q. Trung, N. D. Tu, N. T. Liem, T. T. T. Uyen, Cytokine-primed Umbilical Cord Mesenchymal Stem Cells Enhanced Therapeutic Effects of Extracellular Vesicles on Osteoarthritic Chondrocytes, Front Immunol., Vol. 13, 2022, pp. 1041592,
https://doi.org/10.3389/fimmu.2022.1041592.
[20] K. E. Thane, A. M. Davis, A. M. Hoffman, Improved Methods for Fluorescent Labeling and Detection of Single Extracellular Vesicles Using Nanoparticle Tracking Analysis, Scientific Reports, Vol. 9, No. 1, 2019, pp. 12295,
https://doi.org/10.1038/s41598-019-48181-6.
[21] S. Rowley, W. Bensinger, T. Gooley, C. Buckner, Effect of Cell Concentration on Bone Marrow and Peripheral Blood Stem Cell Cryopreservation, Blood, Vol. 83, No. 9, 1994, pp. 2731-2736,
https://doi.org/10.1182/blood.V83.9.2731.2731.
[22] S. Bahsoun, M. J. Brown, K. Coopman, E. C. Akam, Cryopreservation of Human Bone Marrow Derived Mesenchymal Stem Cells at High Concentration is Feasible, Biopreservation and Biobanking, Vol. 21, No. 5, 2022, pp. 450-457, https://doi.org/10.1089/bio.2022.0017.
[23] X. Sun, J. H. Jung, O. Arvola, M. R. Santoso, R. G. Giffard, P. C. Yang, C. M. Stary, Stem Cell-Derived Exosomes Protect Astrocyte Cultures From in Vitro Ischemia and Decrease Injury as Post-Stroke Intravenous Therapy, Frontiers in Cellular Neuroscience, Vol. 13, 2019, pp. 394, https://doi.org/10.3389/fncel.2019.00394.
[24] G. D. Micó, M. Mittelbrunn, Role of Exosomes in the Protection of Cellular Homeostasis, Cell Adhesion & Migration, Vol. 11, No. 2, 2017, pp. 127-34, https://doi.org/10.1080/19336918.2016.1251000.
[25] J. Cwalinski, R. Staniszewski, E. Baum, T. Jasinski, B. Mackowiak, A. Bręborowicz, Normal Saline May Promote Formation of Peritoneal Adhesions, International Journal of Clinical and Experimental Medicine, Vol. 8, No. 6, 2015, pp. 8828-8834.
[26] L. Li, J. Qin, Q. Feng, H. Tang, R. Liu, L. Xu, Z. Chen, Heparin Promotes Suspension Adaptation Process of CHO-TS28 Cells by Eliminating Cell Aggregation, Molecular Biotechnology, Vol. 47, No. 1, 2011, pp. 9-17, https://doi.org/10.1007/s12033-010-9306-1.
[27] Y. Lan, Q. Jin, H. Xie, C. Yan, Y. Ye, X. Zhao, Z. Chen, Z. Xie, Exosomes Enhance Adhesion and Osteogenic Differentiation of Initial Bone Marrow Stem Cells on Titanium Surfaces, Frontiers in Cell and Developmental Biology, Vol. 8, 2020, pp. 583234, https://doi.org/10.3389/fcell.2020.583234.