Tissue and/or cell cryopreservation protective solution as well as preparation and application thereof
A technology for protecting fluids and tissues, applied in the field of biomedicine, can solve problems such as solute damage, cell destruction, mechanical damage, etc.
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Embodiment 1
[0082] Preparation of tissue cryopreservation solution
[0083] Preparation of non-permeable cryoprotectants:
[0084] 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.
[0085] 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.
[0086] 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.
[0087]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.
[0088] Preparation of freezing solution:
[0089] The following are volume ratios: the total parts are 100 parts, and the numbers in parentheses represent ...
Embodiment 2
[0115] Example 2. Observation of cell morphology and calculation of cell viability
[0116] 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.
[0117] 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.
[0118] 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
[0122] Example 3. Determination of cell purity by flow cytometry
[0123] 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.
[0124] 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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