Standardized Cryopreservation of Human Primary Cells

Thomas V. Ramos1, Aby J. Mathew2, Maria L. Thompson3, Rolf O. Ehrhardt3

1 HemaCare Corporation, Van Nuys, California, 2 BioLife Solutions, Bothell, Washington, 3 BioCision, Larkspur, California
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Appendix A.3I
DOI:  10.1002/0471143030.cba03is64
Online Posting Date:  September, 2014
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Abstract

Cryopreservation is the use of low temperatures to preserve structurally intact living cells. The cells that survive the thermodynamic journey from the 37°C incubator to the −196°C liquid nitrogen storage tank are free from the influences of time. Thus, cryopreservation is a critical component of cell culture and cell manufacturing protocols. Successful cryopreservation of human cells requires that the cells be derived from patient samples that are collected in a standardized manner, and carefully handled from blood draw through cell isolation. Furthermore, proper equipment must be in place to ensure consistency, reproducibility, and sterility. In addition, the correct choice and amount of cryoprotectant agent must be added at the correct temperature, and a controlled rate of freezing (most commonly 1°C/min) must be applied prior to a standardized method of cryogenic storage. This appendix describes how human primary cells can be frozen for long‐term storage and thawed for growth in a tissue culture vessel. Curr. Protoc. Cell Biol. 64:A.3I.1‐A.3I.8. © 2014 by John Wiley & Sons, Inc.

Keywords: PBMC; primary cells; cryopreservation; cell freezing; CoolCell

     
 
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Table of Contents

  • Basic Protocol 1: Freezing Primary Cells (Purified from a Leukapheresis Collection)
  • Basic Protocol 2: Thawing Primary Cells
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Freezing Primary Cells (Purified from a Leukapheresis Collection)

  Materials
  • Phosphate‐buffered saline (PBS; Life Technologies, cat. no. 14190‐136)
  • Freshly collected leukapheresis material (LeukoPak; HemaCare, cat. no. PB001F‐1)
  • Ficoll (GE Healthcare, cat. no. 17‐1440‐03)
  • CryoStor CS10 cryopreservation solution (BioLife Solutions, cat. no. 210102)
  • Dry ice
  • 5‐, 10‐, 25‐, and 50‐ml serological pipets, sterile (USA Scientific)
  • Pipet aid (Drummond Scientific)
  • 50‐ml conical centrifuge tubes, sterile (USA Scientific)
  • Swinging‐bucket centrifuge set at room temperature (18° to 24°C; Eppendorf, cat. no. 5810)
  • CoolRack 50‐ml thermo‐conductive conical tube module (BioCision)
  • Neubauer hemacytometer or automated cell counter (e.g., ViaCell, MACS Quant)
  • Cryogenic vials, leak‐free (LN 2‐tested), sterile (e.g., Corning, Nunc, TruCool or equivalent)
  • Labels for the cryogenic vials (e.g., Freezerbondz label; Brady, cat. no. THT‐179‐492)
  • Indelible marker, resistant to cold, water, and ethanol
  • CoolRack CFT30 thermo‐conductive cryogenic tube module (BioCision)
  • −20° and −80°C freezer
  • CoolCell cell freezing container (BioCision)
  • Liquid nitrogen (Vapor Phase) freezer
  • NOTE: The use of sterile technique throughout these procedures is critical.

Basic Protocol 2: Thawing Primary Cells

  Materials
  • Frozen cryogenic vial containing cell line (see Basic Protocol 1)
  • Dry ice
  • 70% isopropyl alcohol
  • Thawing medium (see recipe)
  • RPMI 1640 medium (Life Technologies, cat. no. 11875‐119)
  • Fetal bovine serum (FBS; Life Technologies, cat. no. 14037)
  • CoolRack CFT30 thermo‐conductive cryogenic tube module
  • 37°C water bath or Thermal Tray HP platform
  • Biological safety cabinet, certified
  • 15‐m conical tubes with caps
  • Centrifuge (Eppendorf 5810 or equivalent)
  • Tissue culture flasks
  • NOTE: The use of sterile technique throughout these procedures is critical.
  • NOTE: Be sure to properly use personal protective equipment while handling liquid nitrogen and dry ice.
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Figures

Videos

Literature Cited

Literature Cited
   Baust, J.M. 2002. Molecular mechanisms of cellular demise associated with cryopreservation failure. Cell Preserv. Technol. 1:17‐31.
   Baust, J.M. 2005. Advances in media for cryopreservation and hypothermic storage. BioProcess Int. 4:S46‐S54.
   Baust, J.M. , Van Buskirk, R.G. , and Baust, J.G. 2002. Gene activation of the apoptotic caspase cascade following cryogenic storage. Cell Preserv. Technol. 1:63‐80.
   Clarke, D.M. , Yadock, D.J. , Nicoud, I.B. , Mathew, A.J. , and Heimfeld S. 2009. Improved post‐thaw recovery of peripheral blood stem/progenitor cells using a novel intracellular‐like cryopreservation solution. Cytotherapy 11:472‐479.
   Cosentino, L.M , Corwin, W. , Baust, J.M. , Diaz‐Mayoral, N. , Cooley, H. , Shao, W. , van Buskirk, R. , and Baust, J.G. 2007. Preliminary report: Evaluation of storage conditions and cryococktails during peripheral blood mononuclear cell cryopreservation. Cell Preserv. Technol. 5:189‐204.
   Nicoud, I.B. , Clarke, D.M. , Taber, G. , Stolowski, K.M. , Roberge, S.E. , Song, M.K. , Mathew, A.J. , and Reems, J.A. 2012. Cryopreservation of umbilical cord blood with a novel freezing solution that mimics intracellular ionic composition. Transfusion 52:2055‐2062.
   Fritsch, G. , Witt, V. , Matthes, S. , Dworzak, M. , Artwohl, M. , Clarke, D. , and Mathew, A.J. 2011. Improved post‐thaw stability validation of peripheral blood cell products utilizing the intracellular‐like CryoStor cryopreservation solution, and preliminary results of clinical application. Biol. Blood Marrow Transplant. 17:S210‐S211.
   Gülen, D. , Maas, S. , Julius, H. , Warkentin, P. , Britton, H. , Younos, I. , Senesac, J. , Pirruccello, S.M. , and Talmadge J.E. 2012. Cryopreservation of adenovirus‐transfected dendritic cells (DCs) for clinical use. Int. Immunopharmacol. 13:61‐68.
   Mazur, P. 1984. Freezing of living cells: Mechanisms and implications. Am. J. Physiol. 247:C125‐C142.
   Mazur, P. , Leibo, S.P. , and Chu, E.H.Y. 1972. A two‐factor hypothesis of freezing injury: Evidence from Chinese hamster tissue‐culture cells. Exp. Cell Res. 71:345‐55.
   Mullen, S.F. and Critser, J.K. 2007. The science of cryobiology. Cancer Treatment Res. 138:83‐109.
   Pegg, D.E. 2007. Principles of cryopreservation. Methods Mol. Biol. 368:39‐57.
   Putnam, A.L. , Safinia, N. , Medvec, A. , Laszkowska, M. , Wray, M. , Mintz, M.A. , Trotta, E. , Szot, G.L. , Liu, W. , Lares, A. , Lee, K. , Laing, A. , Lechler, R.I. , Riley, J.L. , Bluestone, J.A. , Lombardi, G. , and Tang, Q. 2013. Clinical grade manufacturing of human alloantigen‐reactive regulatory T cells for use in transplantation. Am. J. Transplant. 13:3010‐3020.
   Tannenbaum, S.E. , Turetsky, T.T. , Singer, O. , Aizenman, E. , Kirshberg, S. , Ilouz, N. , Gil, Y. , Berman‐Zaken, Y. , Perlman, T.S. , Geva, N. , Levy, O. , Arbell, D. , Simon, A. , Ben‐Meir, A. , Shufaro, Y. , Laufer, N. , and Reubinoff, B.E. 2012. Derivation of xeno‐free and GMP‐grade human embryonic stem cells—Platforms for future clinical applications. PLoS One 7:e35325.
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