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Cell Reservoir One (Vitrify)

Serum-Free Cell Freezing Medium

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)

Features

  • High viability with a longer freezing protocol (up to 60 seconds)
  • Low toxicity to cells (DMSO and acetamide free)

Application 1 Comparison of survival rate of Human iPS cells (201B7 cell line*)

*Takahashi, K. et al. Cell, 2007 Nov 30; 131(5):861-872
  • Freezing protocol: 60 seconds

    Hunan 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 all cells in DAP213 were dead.

  • Freezing protocol: 15 seconds

    Hunan 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.

  • Conclusion

      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

    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.

    Data courtesy of a customer

Application 2 Comparison of recovery ratio in primate stem cells

Cell Reservoir One (Vitrify) showed higher recovery ratio of human iPS 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

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

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.

*Recovery ratio (%) =

100 x the number of colonies after vitrification and thawing
The number of colonies at general passage

Data courtesy of Fukui University

Application 4 Comparison of recovery ratio in human iPS cells (253G1 cell line) and examination of their undifferentiated state

Cell Reservoir One (Vitrify) showed higher recovery ratio compared to DAP213. The time of freezing protocols (15 seconds or 60 seconds) did not affect the recovery rate of cells in Cell Reservoir One (Vitrify). Expression of undifferentiated marker gene was also confirmed.

  • Recovery ratio

    *Recovery ratio (%) =
    100 x the number of colonies after vitrification and thawing
    The number of colonies at general passage
  • Alkaline phosphatase activity


    A: Unfrozen
    B: FBS/10%DMSO (Frozen by slow freezing method)
    C: DAP213 (Vitrification method, 15 seconds freezing protocol)
    D: Cell Reservoir One (Vitrify)
         (Vitrification method, 15 seconds freezing protocol)

  • Expression of undifferentiated marker gene

    Data courtesy of Fukui University

Protocol

  • Freezing protocol

    1. Prepare tweezers and liquid nitrogen near a clean bench.
    2. Detach primate ES/iPS cells with dissociation solution (0.25% trypsin/collagenase IV solution), and carefully collect the solution not to break down the cell colonies.
    3. Aliquot the cell colonies into centrifuge tubes, and centrifuge them to remove as much supernatant as possible.
      *If Cell Reservoir One (Vitrify) is diluted by the remaining supernatant, the viability may decrease.
    4. Add 200ul of Cell Reservoir One (Vitrify), and carefully mix by pipetting 4-5 times not to break down colonies. Transfer them quickly into a cryopreservation tube, and tighten a cap.
    5. Immerse 2/3 height of the tube into liquid nitrogen for 10 seconds using tweezers, and then immerse it completely. For the successful cryopreservation, (4)-(5) process should be performed within 60 seconds.
    6. Transfer the tube into a liquid nitrogen storage tank.
  • Thawing protocol

    1. Pre-warm 10 ml of cell culture medium in a centrifugation tube at 37C.
    2. Take out the cryopreservation tube containing frozen cells from the liquid nitrogen storage tank, and transfer it to a clean bench leaving it immersed in liquid nitrogen.
    3. Take out the tube from liquid nitrogen Open a cap and discard liquid nitrogen in the tube by turning it upside down.
    4. Thaw the cell quickly by adding more than 800ul of the pre-warmed cell culture medium to the tube and pipetting a few times.
      *The larger volume of medium is used, the quicker the sample thaws. Add suitable volume of medium depending on the tube size.
    5. Transfer the cell suspension (4) to the centrifugation tube (1).
      *Operate (3)-(5) as quickly as possible.
    6. Wash the cryopreservation tube with cell culture medium, and transfer the medium to the centrifugation tube (5)
    7. Remove as much supernatant as possible after centrifugation, and seed cells in fresh medium.

Note

  • Pretest the target cell before use.
  • Nacalai Tesque, Inc. takes no responsibility for any damages or losses arising from use of this product.

Ordering Information

Product Cat.No. Storage Quantity Price(US$)  
Cell Reservoir One (Vitrify) 11325-62 4°C 25 ml 135.00 Buy

SERICIN, a major constituent of Cell Reservoir One, is produced by SEIREN.