Freezing and Thawing experiment for large volume of Human iPS cell-derived cardiomyocytes


Upon the development of cell-processed products and regenerative medical products, scale-up of cell culture process is considered to be essential. However, there is concern that to achieve scale-up could affect the efficiency of cell culture or other processes. Besides, in case of using aoutologous cells, the cells can’t be used for threatment immediately because it takes long time to cultivate after collection. For this reason, allogenic cells (ES cells, iPS cells), cells donated by donors have been attracting attention recently. By collecting and cultivating a lot of cells from donors beforehand, they can be used for treatment sooner. This time we evaluate cryopreservation of human iPS cell-derived cardiomyocytes for treatment volume based on the success of cryopreservation of undifferentiated iPS cells at preliminary experiment. Also, we used CryoMACS Freezing Bag and ThawSTAR CB the same as preliminary experiment.  

Preliminary experiment


  1. 【Cells】 undifferentiated human iPS cells
  2. 【Volume】 70mL cell suspension
  3. 【Concentration】 5×10⁶cells/mL
  4. 【Freezing medium】 STEM-CELLBANKER®
  5. 【Freezing period】 approx. 1.5 months
  6. 【Bag】 CryoMACS Freezing Bag 250mL
Before freezing

CryoMACS Freezing Bag after thawing with ThawSTAR CB


  1. 【Thawing time】 30 min. (47 × 2 tubes: 90min)
  2. 【Viability after thawing】 95.5%
  3. 【Proliferration rate after thawing】
    Seeded cells: 0.1×10⁶cells, Culture period: 4 days
  • 250 mL bag: 2.55 times
  • Control 1 tube: 2.49 times
  • 47×2mL tubes: 1.85 times
  • 47 tubes

    250 mL bag

    Control (1 tube)


    In large scale of freeezng and thawing, using bag allows us to increase work efficiency and reduce damage to cells.




    In this experiment, cardiomyocytes derivrd from human iPS cells were used, and we froze and thawed the cells twice.

    1. 【Cells】 Cardiomyocytes derived from research iPS cells
      Cells were collected in 15-17 days after starting differentiated.
    2. 【Volume】 20mL cell suspension
    3. 【Concentration】 5×10⁶cells/mL
    4. 【Freezing period】 1-2 weeks at -150C
    5. 【Bag】 CryoMACS Freezing Bag 50mL
      50 mL bag is equivalent to 14 × 2 mL tubes (total liquid volume: 21mL)


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    Cell culture

    1. Working time and post-thawing viability

    * 14 tubes (total liquid volume: 21mL)


    7.5 min.


    6 min.


    60 min.

    ▶Total: 1 hour or more Viability: 94%


    * 50mL bag (Liquid volume: 20mL) 1st experiment


    3.6 min.


    3 min.


    10 min.

    ▶Total: 20 minutes or less Viability Viability: 91%


    *50mL bag (Liquid volume: 20mL) 2nd experiment


    3.6 min.


    3 min.


    10 min.

    ▶Total: 20 minutes or less Viability: 91.5%



    5 Eveluation after culture

    ▶Post-thawing adhesive culture

    14 tubes 50mL bag (1st) 50mL bag (2nd)
    2 days
    6 days

     Adhesiveness of 50 mL bag cells was equivalent to that of 14 tubes. There was no diffrence.



    ▶Motion analysis (SONY SI8000): After 6 days of post-thawing adhesive culture

    14vial 50mL bag
    Pulse rate: 32.3/min Pulse rate: 31.4/min

     Pulse rate of 50mL bag cells was equivalent to that of 14 tubes.. There was no diffrence.

    ▶Ability to generate cardiac spheroids

    Before freezing 14 tubes 50mL bag (1st) 50mL bag (2nd)

    Ability to generate cardiiac spheroids using 50mL bag cells was equivalent to that of 14 tubes.There was no diffrence.

    DATA: proveded by the research fgroup of Dr. Shugo Toyama.
    Department of Cardiovascular Medicine. Keio University School of Medicine

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