Experiment of scaling up in freezing and thawing of human iPS cells


With the development of cell processing products and regenerative medicine products, scaling up of cell culture processes has been required. However, there are concerns about the disadvantages of scaling up, such as decreased efficiency of culture work. WAKEN B TECH and Miltenyi Biotec collaborating with the research group of Dr. Shugo Tohyama, Department of Cardiovascular Medicine, Keio University School of Medicine conducted a study with 250 mL cryobag as a scaling up to reach the actual cell processing product storage volume, after the success with preliminary experiment using 50 mL cryobag in cell freeze-thaw and confirming cell growth. ThawSTAR CB, a cryobag thawing device and CryoMACS Freezing Bag were used for the study.

Comparison experiment:
47 x 1.5 mL/tube vs max volume of 250 mL cryobag (70 mL)

[Cells] Human iPS cells

[Volume] 70 mL Cell Solution

[Freezing period] approx. 1.5 months

[Freezing medium] STEM-CELLBANKER®

[Concentration] 5×106 cells/mL

47×2 mL tubes* 250 mL bag Control (1 tube)
labeling 20 min. 1 min. 1 min.
freezing 20 min. 7 min. 1 min.
thawing 90 min. 30 min. 13 min.
Total 130 min. 38 min. 15 min.

*Assuming that 47 tubes of 2 mL serum tubes were operated. 5 tubes were frozen and thawed.
Work time was calculated from the actual work time of 5 × 2 mL serum tubes and assuming 2 people would work.
As the number of processed tubes increases, the risk of contamination may increase.

47 tubes 250 mL bag Control
(1 tube)
Number of frozen cells(×106) 352 350 7.5
Thaw Result Viable cells(×106) 205.32 222.60 5.69
Viability 97.5 95.5 97.5
*Recovery rate 58.2 63.6 75.8

*Recovery rate=Viable cells after thawing / number of frozen cells × 100

Increased bag size from 50mL to 250mL
for freezing and thawing of human iPS cells

Succeeded in scaling up!!

Damage from freeze-thaw

Reduced damage to human iPS cells by using bag!!

Recovery rate, proliferation after
thawing and work efficiency

Better with bag compared to tubes!!

Work time

Significantly shortened by using bag!!

Equipment available at WAKEN B TECH, can be adopted to manufacture and manage pharmaceutical products

Cell freezing process

▶CryoMACS Cell freezing bag (Miltenyi Biotec)
A safe and secure freezing bag that has been certified as a medical device.There are sizes from 50 mL to 1000 mL and it is a global standard bag that can be available all over the world.

▶Protective bag sealer
Validatable Vacuum Sealer VMS163B (SEAL Eito)
Validatable vacuum pack sealer. Seal the protective bag of the CryoMACS freezing bag without any problem and implements double packaging of samples.

▶Aluminium canister (Fingallink, Custom Biogenic Systems)
In addition to the Fingallink product developed exclusively for CryoMACS, a versatile aluminium canister (manufactured by CBS) is also available.

▶Programmable freezer FZ-3100 (Strex)
IQOQ applicable, programmable freezer that utilizes a Stirling engine and no liquid nitrogen. This programmable freezer is optimal for airtight manufacturing sites such as CPC.

Cell preservation and transportation process

▶Transporter container
Ice bucket 4L、9L(Biocision)
Bucket with lid for high insulation and cleanliness. Compatible with dry ice and liquid nitrogen.

▶Liquid nitrogen storage container Fusion (MVE Biological Solutions)
IQOQ applicable.
No need to replenish liquid nitrogen after first the fill. It is a storage container with excellent running cost performance.

Cell thawing process

▶Frozen bag thawing device
ThawSTAR CB (BioLife Solutions)
IQOQ applicable device for optimal thawing without using water. A single unit can be used to thaw frozen bags from 50 mL to 750 mL. Thawing time is within 10 minutes maximum.

These products can also be introduced for optimization at the bedside.

* All products in this page are for reserch use except CryoMACS freezing bags.
Please note that they cannot be used for medical purposes.

Please feel free to contact us through a contact page if you have any questions on this page or products.