Vitrification has become an important alternative to standard slow programmable freezing methods for cryopreservation of primate ES cell lines including Human iPS cells because of the higher survival rates of cells after thawing. However, the vitrification requires an ultra-rapid freezing protocol, usually less than 15 seconds between making cell suspensions and freezing in liquid nitrogen.
Cell Reservoir One (Vitrify) is a novel serum-free cell culture freezing medium for vitrification method, which contains a water-soluble glycoprotein SERICIN isolated from the silkworm cocoon as a major constituent. It provides high survival rates of primate cells, such as Monkey ES cells and Human iPS cells even with a longer freezing protocol; up to 60 second from the cell collection to freezing in liquid nitrogen.

*Cell Reservoir One (Vitrify) is produced in corporation with SEIREN. (Patent pending)

Application 1

Comparison of survival rate of Hunan iPS cells (201B7 cell line*)

*Takahashi, K. et al. Cell, 2007 Nov 30;131(5):861-872

Freezing protocol: 60 seconds

Human iPS cells were cryopreserved for more than 2 weeks in Cell Reservoir One (Vitrify) or DAP213. Viability was detected using Alkaline Phosphatase 4 days after thawing. Cell Reservoir One (Vitrify) showed high survival rate, while most of cells in DAP213 were dead.

Freezing protocol: 15 seconds

Human iPS cells were cryopreserved for more than 2 weeks in Cell Reservoir One (Vitrify), DAP213 or Company A’s product. Viability was detected using Alkaline Phosphatase 4 days after thawing. Cell Reservoir One (Vitrify) showed the highest viability.

Data courtesy of a customer

Conclusion

Cell Reservoir One (Vitrify) showed high viability with both 15 and 60 seconds of freezing protocol. With 60 seconds protocol, the survival rate of cells in Cell Reservoir One (Vitrify) was significantly higher than other freezing media.

	Freezing Medium	The Number of Colony
		Vitrification Method		Slow Freezing Method
		60 Seconds	15 Seconds
A	Cell Reservoir One (Vitrify)	672	563	-
B	DAP213	37	479	-
C	Company A	-	-	172

Application 2

Comparison of recovery ratio in primate stem cells

Cell Reservoir One (Vitrify) showed higher recovery ratio of Human iPS cells and Common Marmoset ES cells compared to DAP213.

Recovery ratio of Human iPS cells (253G1 cell line*) after vitrification

*Nakagawa, M. et al. Nat Biotechnol, 2008 Jan;26(1):101-106

Data courtesy of Fukui University

Recovery ratio of common marmoset ES cells (CMES 40 cell line) after vitrification

*Sasaki, E. et al. Stem Cells, 2005 Oct; 23(9):1304-1313

Application 3

Comparison of recovery ratio of Human iPS cells (253G1 cell line) with competitors’

Human iPS cells were cryopreserved for 2 hours with 15 seconds freezing protocol in each company’s freezing medium. Recovery ratio was evaluated 4 days after thawing. Cell Reservoir One (Vitrify) showed the highest recovery ratio.

Data courtesy of Fukui University

*Recovery ratio (%) =
100 x the number of colonies after vitrification and thawing / the number of colonies at general passage

• Cell Reservoir One, Vitrify (PDF 79 KB)

• Cell Reservoir One, Vitrify (PDF 1.9 MB)