Serum-free cryopreservation solution for regenerative medicine research
Application:
Survival rate after thawing of approximately 1,400 cell types of the cell bank
JCRB was low: Improvement of survival rate could be observed for 4 test cell
lines stored with BambankerTM
Cat. No. BB01, BB02
Data kindly provided by the National Institute of Biomedical Laboratories JCRB cell bank, Dr. Arihiro Ohara.
Methods
The JCRB cell bank handles approximately 1,400 differentcell lines. A low survival rate after thawing frozen celllines (KHYG-1, KAI3, HL60, OVMANA) has let us to testBambankerTM and compare it to the up to then usedpreservation medium for the four cell lines. The growthefficiency after thawing was compared for cells storedwith the currently used commercially available preservationmedium and BambankerTM. The freezing of the cellswere performed with a slow freezing method: The cellswere frozen and stored at -80 °C.
Cell preservation method:
| Preservation medium | Freezing method |
|---|---|
| BambankerTM | Slow method |
| Commercial medium | Slow method |
Comparison method for three cell lines in suspension :
- KHYG-1 (human NK-like cell line)
- KAI3 (human NK cell line)
- HL60 (human leukemia line)
Cryopreservation
- All cultured cells were harvested in the logarithmic growth phase.
- Collection of cells by centrifugation in a centrifugation tube.
- Supernatant discarded after centrifugation.
- Cell precipitate loosen by tapping or light vortexing.
- Addition of 1 ml preservation media to approximately 1 x 106 cells in a storage tube.
- Suspending cells in the preservation medium.
- Storage for 2 weeks at -80 °C.
Thawing and cultivation
- Thawing of frozen cells in an 37 °C water bath.
- Suspension of the cells in 10 ml cell culture medium in a centrifuge tube.
- Supernatant discarded after centrifugation.
- Cell precipitate loosen by tapping or light vortexing.
- Resuspension in culture medium. Sample of cells for cell number measurement.
- Incubation at 37 °C and 5 % CO2 in a 96-well plate.
- Daily determination of viable cell number.
Comparison method for an adherent cell line:
Adherent cell line OVMANA (human cell line derived from ovarian tumour)
Cryopreservation
- Cultured cells were harvested in the logarithmic growth phase.
- Detachment of cells by trypsin treatment.
- Collection of cells by centrifugation in a centrifugation tube.
- Supernatant discarded after centrifugation.
- Addition of 1 ml preservation medium to approximately 1 x 105 cells in a storage tube.
- Mixing of cells with the preservation medium.
- Storage for 2 weeks at -80 °C.
Thawing and cultivation
- Thawing of the frozen cells in an 37 °C water bath.
- Suspension of the cells in 10 ml cell culture medium in a centrifuge tube.
- Supernatant discarded after centrifugation.
- Cell precipitate loosen by tapping or light vortexing.
- Resuspension in culture medium. Sample of cells for cell number measurement.
- Incubation at 37 °C and 5 % CO2 in a 96-well plate.
- Detachment of the cells with trypsin treatment after 7 days of culture for cell number determination.
Results
The results for the above described tests were compared for the both preservation media.
Cell lines in suspension
Adherent cell line
| Bambanker | Commercial product | |
|---|---|---|
| Recovery rate immediately after thawing | 32 % | 39 % |
The survival rate after thawing of the four cell lines (KHYG-1, HL60, KAI3, OVMANA) is with the currently used commercially available product and Bambanker™ very low. However after thawing, all four cell lines cell proliferation was improved with Bambanker™ when compared to the currently used commerial product.