Hepatic stem cell preserving solution and applications of hepatic stem cell preserving solution
A technology of liver stem cells and preservation solution, applied in the application, preservation of human or animal body, animal husbandry, etc., can solve the problem of unclear mechanism of liver stem cell transdifferentiation, achieve no toxic side effects, easy storage and transportation, reduce secretory effect
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Embodiment 1
[0018] Embodiment 1, preparation of hepatic stem cell preservation solution
[0019] Human serum albumin injection was purchased from Harbin Shiheng Bioengineering Pharmaceutical Co., Ltd., the product catalog number is (92) Weiyao Zhunzi (Ha Shiheng) S-01, and each liter contains 20 g of effective dose of human serum albumin protein.
[0020] Heparin calcium injection was purchased from Sanofi Winthrop Industrie Company, the product catalog number is J20040119.
[0021] The preparation method of liver stem cell preservation solution I: 5mL human serum albumin injection (containing 0.1g effective dose of human serum albumin), 0.4mL heparin calcium injection, 2.60g sodium chloride, 2.48g sodium gluconate, 1.82 g sodium acetate, 0.18g potassium chloride and 0.15g magnesium chloride were dilute to 100mL with water.
[0022] The preparation method of liver stem cell preservation solution II: 10mL human serum albumin injection (containing 0.2g effective dose of human serum albumi...
Embodiment 2
[0025] Example 2, Cell Expansion of Human Hepatic Stem Cells
[0026] D-hank'S balanced salt buffer solution: the solvent is water, containing the following solutes: 0.8% NaCl, 0.04% KCl, 0.006% Na 2 HPO 4 ·H 2 O and 0.006% KH 2 PO 4 ; % are mass percentages.
[0027] 1. Wash 1 bottle of human liver stem cells twice with 10ml D-hank’S balanced salt buffer.
[0028] 2. Collect the cells and digest them with trypsin at 37°C for 6 minutes (5-7 minutes are acceptable); observe the cells under an inverted microscope during the digestion process, and gently pat the culture bottle with the palm of your hand to ensure that the human hepatic stem cells are completely digested.
[0029] 3. Stop the digestion, then add 10ml of D-hank’S balanced salt buffer solution to each bottle, and repeatedly pipette to suspend the cells to obtain a cell suspension.
[0030] 4. Count the viable cells of the cell suspension, the method is as follows: mix equal volumes of the cell suspension and 0...
Embodiment 3
[0032] Embodiment 3, the application of the hepatic stem cell preservation solution prepared in embodiment 1
[0033] The human hepatic stem cells in Example 2 were resuspended in the four kinds of hepatic stem cell preservation solutions prepared in Example 1, and the proportions were all 6.8×10 5 Human liver stem cell live cells: 1ml of liver stem cell preservation solution, stored at 4°C for 12 hours, then sampled (cell suspension) to measure cell survival rate.
[0034] Cell viability assay method: Mix 0.5ml of cell suspension with 0.5ml of 0.2% trypan blue solution to obtain a mixed solution; draw 10 microliters of the mixed solution and drop it on the edge of the cover slip to make the mixed solution fill the cover slip and counting plate Between; stand still for 1 minute; randomly select several fields of view, count 300 cells in total (count the number of colored cells, that is, the number of dead cells), cell survival rate=(300-number of dead cells) / 300×100%; repeat cou...
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