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Tissue and/or cell cryopreservation protection solution and its preparation and application

A technology for protecting liquid and tissue, applied in the field of biomedicine, can solve the problems of solute damage, cell damage, mechanical damage and so on

Active Publication Date: 2020-01-24
BIOCELLS BEIJING BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the traditional cryopreservation solution is easy to cause damage to cells, mainly from solute damage caused by intracellular hyperosmotic pressure, and mechanical damage caused by intracellular ice crystals

Method used

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  • Tissue and/or cell cryopreservation protection solution and its preparation and application
  • Tissue and/or cell cryopreservation protection solution and its preparation and application
  • Tissue and/or cell cryopreservation protection solution and its preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Preparation of tissue cryopreservation solution

[0087] Preparation of non-permeable cryoprotectants:

[0088] Human serum albumin HAS (purchased from Solebol, product number: A8230) (10%, w / v): Take 1.3g of HSA and dissolve it with human AB serum to a final volume of 13ml, mix and dissolve.

[0089] Polyvinylpyrrolidone PVP (purchased from Solebol, product number: P8290) (10%, w / v): Dissolve 1 g of PVP with human AB serum to a final volume of 10 ml, mix and dissolve.

[0090] Dextran-40 (purchased from Suleibao, product number: G8570) (10%, w / v): Dissolve 1 g of Dextran-40 with human AB serum to a final volume of 10 ml, mix well and dissolve.

[0091]D-trehalose (purchased from Shanghai Sangong, product number: DB0374) (10%, w / v): Dissolve 1.2 g of D-trehalose in 12 ml of human AB serum, mix well and dissolve.

[0092] Preparation of freezing solution:

[0093] The following are volume ratios: the total parts are 100 parts, and the numbers in parentheses represent ...

Embodiment 2

[0119] Example 2. Observation of cell morphology and calculation of cell viability

[0120] When the P1 generation cells, P2 generation cells and P3 generation cells grow to about 80% of the adherence (the ratio of the cell adhesion area to the culture flask culture area), digest and collect the cells under sterile conditions, suspend the cells with the culture medium, And draw a small amount of suspension, trypan blue staining, counting.

[0121] The cell status of primary cell culture on day 7 is as follows: figure 1 shown. From figure 1 It can be seen from the results in that No. 1-5 cryopreservation solution groups can significantly protect the viability of cells, which shows that the cryopreservation solution of the present application can be used for tissue cryopreservation.

[0122] The results of cell count and viability calculation are shown in Table 1. The formula for calculating cell viability is: viable cell rate (%)=total number of viable cells / (total number o...

Embodiment 3

[0126] Example 3. Determination of cell purity by flow cytometry

[0127] Cells were fixed with 4% paraformaldehyde for 30 min (4° C.), washed 3 times with PBS, and centrifuged at 3500 rpm for 3 min. Then the cells were permeabilized with 0.4% Triton X-100 at room temperature for 10 min, washed 3 times with PBS, and centrifuged at 3500 rpm for 3 min. The cells were resuspended in 200 μl PBS and divided into 2 equal portions, one of which was added with 1 μl of anti-vimentin FITC (Catalog No.: 11-9897, eBioscience), mixed well and placed in an ice bath in the dark for 30 min, and the other was used as a control group. The samples were washed once with PBS, centrifuged at 3500 rpm for 3 min, and then the cell purity was determined by flow cytometry.

[0128] The results of flow cytometry were as figure 2 shown. From figure 2 As can be seen from the results in No. 1-5 cryopreservation solution group, the purity of fibroblasts can reach 100% relative to the fresh tissue grou...

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Abstract

The application provides a tissue and / or cell cryopreservation protective solution as well as preparation and application thereof. The tissue and / or cell cryopreservation protective solution comprisesa permeating protective agent and a non-permeating protective agent, wherein the cryopreservation protective solution further comprises platelet lysates. In addition, the application further providesa method for cryopreserving tissues and / or cells by using the tissue and / or cell cryopreservation protective solution provided by the application, as well as a cryopreserved tissue and / or cell and use of the cryopreserved tissue and / or cell.

Description

[0001] field of invention [0002] The present application generally relates to the field of biomedicine, and in particular, the present application relates to a tissue and / or cell cryopreservation protection solution and a preparation method thereof. [0003] Background of the invention [0004] At present, the cryopreservation of tissues and / or cells mainly includes fast freezing method (ie, vitrification method) and slow freezing method (ie, programmed cooling method). [0005] The rapid cooling method uses a low-temperature preservation solution that is easy to form a vitrified state as the medium, and uses a rapid cooling method to preserve cells or tissues. The vitrification protection solution is mainly composed of a permeable reagent that is easy to form a vitrified state and an impermeable reagent that dehydrates cells, and the reagent concentration is usually very high, and the total concentration usually reaches more than 50%. However, since the high concentration o...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01N1/02A61K8/98A61L27/36A61L27/38A61L27/60A61Q19/00
CPCA01N1/0221A61K8/981A61K8/985A61L27/362A61L27/3804A61L27/60A61Q19/00
Inventor 魏辉韩化敏田雨佳
Owner BIOCELLS BEIJING BIOTECH CO LTD
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