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Cell viability improves following inhibition of cryopreservation-induced apoptosis

  • Growth, Differentiation, and Senescence
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Summary

A new concept in cryopreservation solution design was developed that focuses on the use of an intracellular-type, hypothermic maintenance medium coupled with additives that inhibit cryopreservation-induced apoptosis. Hypo-Thermosol® (HTS), a hypothermic (4° C) maintenance medium utilized in the long-term storage of cell, tissue, and organ systems, was tested for cryoprotective capability on a renal cell line (Madin-Darby Canine Kidney cells). HTS and HTS derivatives were tested against conventional cell culture medium (Dulbecco's Minimal Essential medium, DME) as the cryoprotectant carrier solution because (1) cells are exposed to an extended state of hypothermia during the freeze-thaw process, and (2) HTS is designed to protect cells exposed to a hypothermic state. Cells separately cryopreserved in either HTS or DME +5% dimethyl sulfoxide (DMSO) yielded equivalent 24-h postthaw survival (∼30%) and 5-d recovery (∼90%). Cells cryopreserved in CryoStor® CS 5, a HTS derivative containing 5% DMSO, yielded ∼75% 24-h postthaw survival and recovery to 100% within 3 d. DNA gel electrophoresis was performed to determine the mechanisms of cell death contributing to cryopreservation failure. Cells preserved in DME (DMSO-free) died primarily through necrosis, whereas cells preserved in either DME +5% DMSO, HTS, or CryoStor® CS 5 died through a combination of apoptosis and necrosis. This observation led to the inclusion of an apoptotic inhibitor designed to improve cryopreservation outcome. MDCK cells cryopreserved in CryoStor® CS 5 supplemented with an apoptotic inhibitor (Caspase I Inhibitor V), hereafter termed CryoStor® CS 5N, resulted in a 24-h postthaw survival and recovery rate exceeding that of any other cryoprotective solution tested (85%). We conclude that: (1) the use HTS (a dextran-based, intracellular-type solution), without DMSO can yield postthaw viability equivalent to that of standard DMSO-based cryopreservation methods, (2) postthaw viability can be significantly increased through the use of an intracellular-type solution in conjunction with DMSO, (3) the use of HTS allows for cryopreservation to be accomplished with reduced levels of cryoprotectants, and (4) the regulation of apoptosis is essential for the improvement of cryopreservation outcome.

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References

  • Adams, R. M.; Wang, M.; Crane, A. M., etal. Effective cryopreservation and long-term storage of primary human hepatocytes with recovery of viability, differentiation, and replicative potential. Cell Transplantation 4: 579–586; 1995.

    PubMed  CAS  Google Scholar 

  • Cheng, Y.; Deshmukh, M.; DeCosta, A., et al. Caspase inhibitor affords neuroprotection with delayed administration in a rat model of neonatal hypoxic-ischemic brain injury. J. Clin. Invest. 101: 1992–1997; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Chesne, C.; Claire, G.; Fauntrel, A., et al. Viability and function in primary culture of adult hepatocytes from various animal species in human beings after eryopreservation. Hepatology 18; 406–414; 1993.

    PubMed  CAS  Google Scholar 

  • Cook, J. R.; Eichelberger, H.; Robert, S., et al., Cold-storage of synthetic human epidermis in HypoThermosol. J. Tissue Eng. 1 (4): 361–377; 1995.

    Article  Google Scholar 

  • Cotter, T. G. Programmed to die—the genetic regulation of cell death and its implications. BIOforum Int. 1: 8–11; 1997.

    Google Scholar 

  • Coundouris, J. A.; Grant, M. H.; Engeset, J., et al., Cryopreservation of human adult hepatocytes for use in drug metabolism and toxicity studies. Xenobiotica 23: 1399–1409; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Evans, G.; Littlewood, T. Apoptosis: a matter of life and cell death. Science 28: 1317–1322; 1998.

    Article  Google Scholar 

  • Fischlein, T.; Schutz, A.; Uhling, A., et al. Integrity and viability of homograft valves. Eur. J. Cardio-Thorac. Surg. 8: 425–430; 1994.

    Article  CAS  Google Scholar 

  • Fisher, R. L.; Hasal, S. J.; Sanuik, J. T., et al. Cold- and cryopreservation of dog liver and kidney slices. Cryobiology 33: 163–171; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Freshney, R. I. Culture of animal cells: a manual of basic techniques, 3rd ed. New York: Wiley, 1994; 287–307.

    Google Scholar 

  • Gao, D. Y.; Mazur, P.; Critser, J. K. Fundamental cryobiology of mammalian spermatozoa. In: Karow, A. M.; Critser, J. K., ed. Reproductive tissue banking, New York: Academic Press; 1997; 263–328.

    Google Scholar 

  • Green, D. R.; Reed, J. C. Apoptosis: mitochondria and apoptosis. Science 281: 1309–1316; 1998.

    Article  PubMed  CAS  Google Scholar 

  • Hollister, W. R.; Mathew, A. J.; Baust, J. G., et al. The effects of freezing on cell viability and mechanisms of cell death in an in vitro human prostate cancer cell line. Mol. Urol. 2 (1): 13–18; 1998.

    Google Scholar 

  • Kerr, J. F. R.; Wyllie, A. H., Currie, A. R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26: 239–247; 1972.

    PubMed  CAS  Google Scholar 

  • Langer, R.; Vacanti, J. P. Tissite engineering. Science 260: 920–926; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Mathew, A.; Baust, J. G.; Van Buskirk, R. G. Optimization of Hypo Thermosol® for the hypothermic storage of cardiomyocytes—Addition of EDTA. In Vitro Toxicol. 10 (4): 407–415; 1997.

    CAS  Google Scholar 

  • Nagle, W. A.; Soloff, B. L.; Moss, A. J., et al. Cultured Chinese hamster cells undergo apoptosis after exposure to cold but nonfreezing temperatures. Cryobiology 27: 439–451; 1990.

    Article  PubMed  CAS  Google Scholar 

  • Neronov, A. J.; Bratanov, M. B.; Tsenov, I. A. Cryopreservation of myeloma cells, hybridoma cells, and lymphocytes with different cryoprotectants. Cryobiology 29 (2): 296–299; 1992.

    Article  PubMed  CAS  Google Scholar 

  • Parks, J. E. Hypothermia and mammalian gameters. In: Karow, A. M.; Critser, J. K., ed. Reproductive tissue banking, New York: Academic Press; 1997; 229–261.

    Google Scholar 

  • Paynter, S.; Cooper, A.; Thomas, N., et al. Cryopreservation of multi-cellular embryos and reproductive tissues. In: Karrow, A. M.; Crister, J. K., ed. Reproductive Tissue Banking, New York: Academic Press; 1997; 359–393.

    Google Scholar 

  • Polge, C.; Smith, A. U.; Parkes, A. S. Revival of spermatozoa after vitrification and dehydration at low temperatures. Nature 164: 666; 1949.

    PubMed  CAS  Google Scholar 

  • Rowe, A. W. Cryopreservation of blood—an historical perspective. Infusionsther. Transfusionsmed. 22: 36–40; 1995.

    Google Scholar 

  • Taylor, M. J.; Bailes, J. E.; Elrifai, A. M., et al. A new solution for life without blood: asanguineous flow-flow perfusion of a whole-body perfusate during 3 hour cardiac arrest and prolonged hypothermia. Circulation 91 (2): 431–444; 1995.

    PubMed  CAS  Google Scholar 

  • Taylor, M. J.; Elrafai A. M.; Bailes, J. E. Hypothermia in relation to the acceptable limits of ischemia for bloodless surgery. Adv. Low Temp. Biol. 3: 1–64; 1996.

    Google Scholar 

  • Thormberry, N. A.; Lazenbnik, Y. Caspeses: enemies within. Science 281: 1312–1316; 1998.

    Article  Google Scholar 

  • Van Buskirk, R. G.; Rauch, J.; Robert, S., et al. Alamar Blue—assesment of hypothermic storage of normal human epidermal keratinocytes (NHEK) using Alamar Blue. In Vitro Toxicol. 9 (3): 297–303; 1996.

    Google Scholar 

  • Voytik-Harbin, S. L.; Brightman, A. O.; Waisner, B., et al. Application and evaluation of the alamarBlue® assay for cell growth and survival of fibroblasts. In Vitro Cell. Dev Biol. 34 (3): 239–246; 1998.

    CAS  Google Scholar 

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Authors and Affiliations

  1. Institute of Biomedical Technology, State University of New York, 13902, Binghamton, New York

    John M. Baust, Robert Van Buskirk & John G. Baust

  2. BioLife Solutions, Inc., 20850, Rockville, Maryland

    Robert Van Buskirk & John G. Baust

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  1. John M. Baust
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  2. Robert Van Buskirk
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  3. John G. Baust
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Baust, J.M., Van Buskirk, R. & Baust, J.G. Cell viability improves following inhibition of cryopreservation-induced apoptosis. In Vitro Cell.Dev.Biol.-Animal 36, 262–270 (2000). https://doi.org/10.1290/1071-2690(2000)036<0262:CVIFIO>2.0.CO;2

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  • DOI: https://doi.org/10.1290/1071-2690(2000)036<0262:CVIFIO>2.0.CO;2

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