Cell cryopreservation kit and application method thereof

A kit and cryopreservation technology, applied in the field of cell cryopreservation kits, can solve the problems of cell damage, low biological safety and compatibility, reduce oxidative stress damage, and improve biological safety and compatibility , The effect of algebraic extension of frozen storage

Active Publication Date: 2022-01-28
北京京蒙细胞生物科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Based on this, it is necessary to provide a cell cryopreservation kit and its application method for the problems that traditional cell cryopreservation methods are likely to cause cell damage and have low biosafety and compatibility.

Method used

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  • Cell cryopreservation kit and application method thereof
  • Cell cryopreservation kit and application method thereof
  • Cell cryopreservation kit and application method thereof

Examples

Experimental program
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Effect test

preparation example Construction

[0062] As an optional embodiment, a small amount of DMSO can be used to provide co-solubilization of the cranberry extract. That is, the first frozen mother liquor preparation method can be as follows:

[0063] Weigh an appropriate amount of bilberry extract under dark conditions and dissolve it in a small amount of DMSO; weigh an appropriate amount of hydroxyethyl starch and dextran 40 and dissolve it in physiological saline as a system stabilizer; Mix with hydroxyethyl starch and dextran 40 solution, and adjust the final concentration to 90g / L~110g / L bilberry extract, 90g / mL~110g / mL hydroxyethyl starch, 180g / L~ The dextran 40 of 220g / L makes the first cryopreservation mother liquor and keeps away from light.

[0064] Before use, the first cryopreservation mother solution and the second cryopreservation mother solution are mixed according to the required ratio to prepare a cell cryopreservation buffer protection system.

[0065] As an optional embodiment, the cell cryoprese...

Embodiment 1

[0098] High biosafety culture of human mesenchymal stem cells:

[0099] According to the clinical trial product standard of mesenchymal stem cells, the large-scale expansion of mesenchymal stem cells is carried out in the GMP workshop.

[0100] Collect umbilical cord, amniotic membrane, placenta, fat and other tissues of healthy people, wash away the residual blood in the blood vessels with phosphate buffer solution (PBS), separate and remove the blood vessel tissues and blood cells, and cut the remaining tissues to about 1-2mm 2 Tissue block; transfer to 1g / L collagenase II, digest at 37°C for 2h, centrifuge at 2000r / min for 10min; remove the lower layer and digest with 2.5g / L trypsin for 20min, centrifuge at 2000r / min for 10min; discard the supernatant, keep the precipitate, Blow into a suspension with PBS, filter with a 100 μm filter, and centrifuge at 2000r / min for 10min to obtain single cells; wash twice with PBS, at 20000 / cm 2 The density was inoculated in serum-free ce...

Embodiment 2

[0129] The steps of Example 2 are the same as those of Example 1, except that in the cryopreservation step of mesenchymal stem cells, DMSO cryopreservation solution is used for cryopreservation.

[0130] Under sterile conditions, prepare the DMSO cryopreservation solution according to the volume ratio of human serum albumin:Dextran40:DMSO: 2:7:1, and add the DMSO cryopreservation solution dropwise to the cells after cryopreservation pretreatment under sterile conditions The cells were resuspended in DMSO to prepare a DMSO frozen cell suspension, and the cell concentration was adjusted to 1×10 6 Cells / ml, divide the cell suspension into cryopreservation tubes, 4mL~4.5ml per tube, tighten the cap to resuspend the cells as before, and mark the tube wall, indicating the cell name, freezing date, etc.; press the following The temperature is lowered in sequence: room temperature → 4°C for 30 minutes → program to warm the box at -80°C overnight → liquid nitrogen.

[0131] like Fig...

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Abstract

The invention provides a cell cryopreservation kit which comprises a first cryopreservation mother solution and a second cryopreservation mother solution, wherein the first cryopreservation mother solution contains a bilberry extract with the concentration of 90 g/L to 110 g/L, hydroxyethyl starch with the concentration of 90 g/mL to 110 g/L and dextran 40 with the concentration of 180 g/L to 220 g/L; and the second cryopreservation mother solution contains proline with the concentration of 90 g/mL to 110 g/mL, human serum albumin with the concentration of 180 g/mL to 220 g/mL and dextran 40 with the concentration of 270 g/mL to 330 g/mL. The bilberry extract and the proline are adopted as main raw materials, oxidative stress damage to cells caused by ice crystal formation in the cell cryopreservation process is effectively reduced, the cell viability, the form, the multiplication capacity and the differentiation potential of the cryopreserved cells can be maintained, the biological activity of the cryopreserved cells is maintained, the cryopreservation algebra is prolonged, and the biological safety and the compatibility are improved.

Description

technical field [0001] The present invention relates to the field of cell and bioengineering, in particular to a cell cryopreservation kit and its application method. Background technique [0002] Stem cells are special cell populations with the potential of self-renewal and multidirectional differentiation, including embryonic stem cells (embryonic stem cells, ES), induced pluripotent stem cells (induced pluripotent stem cells, iPSCs), mesenchymal stem cells (mesenchymal stem cells) , MSCs), etc. Stem cell drug development is the most potential direction of the biomedical industry today. Theoretically, stem cells cultured in vitro can maintain a continuous increase in number while retaining the biological potential to differentiate into other types of cells and organs. This feature makes it possible for unlimited production of stem cell drugs. However, the traditional continuous culture of stem cells usually leads to aging and loss of function. On the other hand, it will a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02C12N5/071
CPCA01N1/0226A01N1/0221A01N1/021C12N5/0602
Inventor 王云虹丁炜柏小丽黄芳蕾万兴中
Owner 北京京蒙细胞生物科技股份有限公司
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