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Shanghai Youbai Biotechnology Co., Ltd

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Clinical grade frozen storage solution

NegotiableUpdate on 05/06

Model

Nature of the Manufacturer: Producers

Product Category

Place of Origin

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Overview

Collect the cells to be cryopreserved using a stripping solution and count the number of cells.

Product Details

CP-1 cell cryopreservation solution (clinical grade cryopreservation solution)

Obtained qualification certification for materials used in Japanese regenerative medicine and other products

1. The product has been promoted and applied since 1992

2. Manufacturing in GMP equipment in Japan

3. Conducted non clinical experiments

5. Can be frozen and stored in a -80 ℃ freezer

6. Does not contain ingredients of animal origin

7. Ingredient openness and transparency

8. Possess a Japanese Raw Material Registration Certificate (MF)

Application Examples

Freezing methods for suspended cells and adherent cells

II. Method of using CP-1HG for cryopreservation/thawing of hematopoietic stem cells

III。 Using CP-1HG mesenchymal stem cell cryopreservation/thawing method

Preparation of Cryopreservation Liquid

·1. CP-1 high-grade, 25% HSA, cool the medium to 4 ℃.

·2. Add 6.8 mL of CP-1 advanced, 3.2 mL of 25% HSA, and 10.0 mL of culture medium into a centrifuge tube and mix evenly.

·Before use, please store at 4 ℃.

Cell cryopreservation

·1. Collect the cells to be frozen using a stripping solution and count the number of cells.

·2. Centrifuge to remove the supernatant.

·3. Add CP-1 high-grade/25% HSA/culture medium that has been cooled to 4 ° C in advance (preparation of cryopreservation solute-2), and suspend evenly to 1 × 106 cells/mL.

·4. Place 1 mL each into a freezing tube, transfer to a freezing container, and slowly freeze at -80 ° C.

·5. Move to liquid nitrogen or a deep freezer at -150 ℃ the next day.

Thawing cells

·1. Prepare 9 mL of culture medium in a centrifuge tube beforehand.

·2. Remove the frozen cryotube from liquid nitrogen and rapidly melt it in a 37 ° C warm water bath.

·3. Add all thawed cells to the culture medium and centrifuge.

·4. Remove the supernatant and add an appropriate amount of culture medium.

·5. Cultivate under optimal conditions for each cell type used.

IV Evaluation Results (Cryopreservation of Mesenchymal Stem Cells)

V used CP-1HG for cryopreservation of iPS/ES cells and evaluation

　　Ⅰ．Freezing methods for suspended cells and adherent cells

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Preparation of Cryopreservation Liquid

·1. CP-1 high-grade, 25% HSA, cool the medium to 4 ℃.

·2. Add 6.8 mL of CP-1 advanced, 3.2 mL of 25% HSA, and 10.0 mL of culture medium into a centrifuge tube and mix evenly.

·Before use, please store at 4 ℃.

Cell cryopreservation

·1. Collect the cells to be frozen using a stripping solution and count the number of cells.

·2. Centrifuge to remove the supernatant.

·3. Pre add CP-1 high-grade/25% HSA/culture medium that has been cooled to 4 ° C (preparation of cryopreservation solute-2),

Suspend evenly to 1 × 106 cells/mL.

·4. Place 1 mL each into a freezing tube, transfer to a freezing container, and slowly freeze at -80 ° C.

·5. Move to liquid nitrogen or a deep freezer at -150 ℃ the next day.

Thawing cells

·1. Prepare 9 mL of culture medium in a centrifuge tube beforehand.

·2. Remove the frozen cryotube from liquid nitrogen and rapidly melt it in a 37 ° C warm water bath.

·3. Add all thawed cells to the culture medium and centrifuge.

·4. Remove the supernatant and add an appropriate amount of culture medium.

·5. Cultivate under optimal conditions for each cell type used.

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Can human mesenchymal stem cells (hMSCs), which are expected to be used in regenerative medicine in the future, be cryopreserved in CP-1 high-grade and stably transported for a long time through inexpensive and convenient dry ice?

[Method] Human adipose derived mesenchymal stem cells (MSC-AT) or human bone marrow-derived mesenchymal stem cells (MSC-BM) were frozen in CP-1 High Grade or control (A in the figure below) (1 x 106 cells/mL) and stored in a liquid nitrogen tank. After storage in liquid nitrogen tanks, the adhesion rate to blood vessels should be confirmed under each condition of assumed dry ice transportation (-80 ° C · 7 days, -60 ° C · 3 days, 60 ° C · 7 days), immediate survival rate after melting, and the following day of cultivation (adhesion rate=number of live cells/number of inoculated live cells).

[Results] Under all conditions, the survival rate after melting was above 90%, and no difference was observed (Figure B). However, in frozen hMSCs as a control, many dead cells that did not adhere to the culture box were observed on the second day after thawing and stored at -60 ° C, but almost no dead cells were observed in hMSCs frozen in CP-1, and almost no dead cells were observed. Connect cells (B and C in the figure below).

[Conclusion] Due to the temperature of dry ice itself being about -80 ° C, we believe that hMSCs frozen in CP-1 High Grade can be stabilized and transported for a long time using dry ice.