Stem Cell Freezing

INTRODUCTION

Stem cells are considered pluripotent when they can differentiate into many different kinds of specialized tissue. Heralded as invaluable for new disease therapies, standardized protocols for handling of these pluripotent stem cells need to be ascertained. By establishing best practices, optimal protocols for the cryopreservation and recovery of pluripotent stem cells can be achieved. Recently published papers in Nature Protocols advocate the use of BioCision’s CoolCell® alcohol-free controlled-rate freezing container in the preservation, storage, and recovery of sensitive stem cells.[1][2] The ATCC also now recommends the incorporation of a CoolCell container into their iPSC stem cell handling guide.[3]

Below is a summary of considerations when freezing down pluripotent stem cells:

1) Media used for freezing: Usually contains a cryoprotective agent, such as 10% DMSO. Serum-free protein solutions as well as standard blood-serums are also an option.

2) Dissociation method: E.g trypsinization and mechanical separation have proven to be disadvantageous in the recovery of pluripotent stem cells.  Less harsh dissociation methods such as chelating agents like EDTA show promise.

3) Type of freezing method to use: Choices range from tissue-wrapped vials in Styrofoam boxes, to isopropanol filled containers, to expensive electronically-controlled rate freezers.  The accepted standard freezing rate for stem cells to -80°C is -1°C/minute [see table below for side-by-side comparision].

4) Temperature control is critical: During the entire process from cell handling, addition of freezing media, to freezing and thawing, the cells must be maintained at pre-determined temperatures.

 

COOLCELL® FREEZING CONTAINERS FOR CRYOPRESERVATION

CoolCell alcohol-free freezing containers are the optimal container for freezing human embryonic stem cells (hESCs) to -80°C.  An independent study conducted by Roslin Cellab, showed a 33 percent increase in hESC viable cell count using a CoolCell container (P<0.005) when compared to other bench-top methods three days post-thaw (Figure 1).  As noted by Aidan Courtney, director at Roslin Cellab, “The results of our study show that the CoolCell technology clearly outperforms all other methods and provides excellent efficiency and reproducibility for stem cell cryopreservation and recover.” [4]

 

Fig 1.  After 2 weeks in LN2, 3 vials of hESCs from each method were thawed and cells counted immediately (Day 1) and after 3 days growth (Day 3).  CoolCell method showed 33% increase in cells after 3 days growth.

 

THAWING OF VIALS FROM -80°C TO PROLIFERATIVE CONDITIONS

Studies have shown that the method employed to recover the pluripotent stem cells from frozen stocks is also important. One of the main factors in increasing survival and proliferative capacity of the stem cells is temperature, and how quickly they are brought back from the cryogenic conditions to room temperature.  Using a CoolBox™ ice-free cooling workstation and CoolRack® thermo-conductive cryogenic vial module in the thawing process ensures proper temperature maintenance.

Here are some vital points to consider during stem cell thawing:

1)    Maintain temperature of frozen vials at -80°C by placing them in a CoolRack CF thermo-conductive cryogenic vial module on dry ice when removing them from storage at -80°C or from liquid nitrogen.

2)    Thaw vials to 37°C in a water bath without shaking, agitating, or inverting the vial.  Observe closely and remove when a small piece of ice is still visibly present.  Place the vials immediately in a CoolBox XT containing a CoolRack CF module equilibrated to 4°C.

3)    Gently resuspend cells in room temperature media and place onto tissue culture media equilibrated in 37°C tissue culture incubator.

CoolBox XT with CoolRack CF15 module

 

SUMMARY

When freezing and recovering pluripotent stem cells, keep in mind the temperature at which the various steps take place.  By carefully controlling the temperatures at which the steps take place, recovery of the cultures show marked increase in their number 3 days post-thawing.  Careful control of temperatures during this critical process enhances their recovery.

REFERENCES

[1] Shi Y, Kirwan P, Livesey FJ.  “Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks.”  Nature Protocols. 2012.

[2] Menendez L, Kulik MJ, Page AT, Park SS, Lauderdale JD, Cunningham ML, Dalton S. “Directed differentiation of human pluripotent cells to neural crest stem cells.”  Nature Protocols.  2013.

[3] ATCC iPSC Guide

[4] Press Release: New Data Show BioCision’s CoolCell® Family of Products Outperform Traditional Cryopreservation Methods.  2011.

[5] Shu Z, Kang X, Chen H, Zhou X, Purtteman J, Yadock D, Heimfeld S, and Gao DDevelopment of a Reliable, Low-cost, Controlled Cooling Rate Instrument for the Cryopreservation of Hematopoietic Stem Cells. Cytotherapy. 2010.

 

Quick Protocol:


Freezing Stem Cells with a CoolCell container

  • 1) Resuspend washed cells at 1 X 106 cells in cryopreservative
  • 3) Place cryogenic vials in CoolCell freezing container, ensuring entire device is filled with vials (use CoolCell Filler Vials for empty wells or create your own "dummy" vials to fill empty spaces)
    4) Place CoolCell container in -80℃ freezer
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Protocols:


"Cryopre servation and Thawing of Cells"
Yokoyama WM, Thompson ML, Ehrhardt RO.
Universit y of California School of Medicine, San Francisco, CA
Curr
Protoc Immunol. 2012 Nov;99 Appendix 3:3G
More protocols >

 

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A Win-Win for Stem Cells Researchers >
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Learn more about:

CoolCell alcohol-free cell freezing containers >
CoolBox XT ice-free cooling workstations >
More cell cryopreservation accessories >