Method for regulating distance between two species of cells

[0024] Example 1: A method for regulating the distance between hepatocytes and fibroblast cells

[0025] The 15 mg/mL sodium alginate solution and 3.6 mg/ml collagen solution were mixed uniformly in a volume ratio of 1:2, so that the final concentrations of the sodium alginate and collagen solution were 5 mg/mL and 2.4 mg/mL, respectively. Then mix the human fibroblasts with the well-mixed sodium alginate and collagen solution, and adjust the cell density to 2×10 6 cells/mL. 300 μl of the cell suspension was slowly added to a circular glass slide with a diameter of 3 cm placed in a cell culture plate, and placed in a cell culture incubator for 2 hours to gel the collagen. Then add 1mL 100mM CaCl 2 Add the solution to the cell culture plate, calcify for 20 minutes, sodium alginate and Ca 2+ Chelate to form calcium alginate gel, and finally get collagen/calcium alginate gel. Then, 1.5 mg/mL chitosan (molecular weight 65kDa, degree of deacetylation 90%) solution was added, and by controlling the reaction time, gel films with thicknesses of 2 μm, 10 μm, 30 μm and 50 μm can be obtained. Then put it in a cell culture box, use the fibroblast self-cultivation medium for 1 day, then inoculate the hepatocytes isolated from the rat liver on the surface of the gel membrane of different thickness, and conduct co-cultivation. See the preparation process figure 2. Among them, the positive control group is the inoculation of fibroblasts and primary hepatocytes on the surface of the gel membrane, and the two cells are in contact with each other; the negative control group is the sodium alginate and collagen mixture that has been mixed into the fibroblasts. Gel was formed on the transwell membrane, and then the primary hepatocytes were seeded on the slab gel membrane without contact between the two cells. The medium is changed every day, and the medium in the culture plate is collected for the determination of albumin and urea concentration. Using albumin and urea as the indicators to investigate the function of liver cells at different cell distances. See the results of the experiment image 3 The results show that the amount of albumin secretion and the amount of urea synthesis are basically the same when the distance between the two cells is 2μm, 10μm and 30μm; compared with the positive control group, when the distance between the two cells is 2μm, 10μm and 30μm, the white There were no significant differences in protein secretion and urea synthesis; each group was better than the 50μm group. This shows that the maintenance of liver cell function requires mutual contact between liver cells and fibroblasts. In order to maintain the function of liver cells, the distance between the two types of cells must be within 30μm.

[0026] Example 2: A method for regulating the distance between human embryonic stem cells (hES) and fibroblast cells

[0027] First, prepare arginine-glycine-aspartic acid (RGD) modified sodium alginate. Dissolve sodium alginate (molecular weight 430kDa, ratio of guluronic acid to mannuronic acid 1.5) in 2-(N-morpholino)ethanesulfonic acid (MES) buffer containing 0.5M NaCl (pH 6.5 ) To obtain a 1% (W/V) sodium alginate solution. Then, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), N-hydroxysulfosuccinimide (sulfo-NHS) and RGD polypeptide were added, and the reaction was stirred at room temperature for 24 hours. The molar ratio of EDC to sodium alginate is 1:20, the molar ratio of EDC to sulfo-NHS is 2:1, and the mass ratio of RGD to sodium alginate is 1:1000. Then, it is dialyzed and freeze-dried to obtain RGD-modified sodium alginate.

[0028] After that, weigh 5 mg of RGD modified sodium alginate and dissolve it in 100 mL of physiological saline to prepare a sodium alginate solution with a concentration of 0.5 mg/mL. Then mix the mouse fibroblasts with the prepared sodium alginate solution and adjust the cell density to 2×10 6 cells/mL. Slowly add 300μl of the cell suspension onto a circular glass slide with a diameter of 3cm placed in a cell culture plate, let it stand for 5 minutes, and then immerse it in 100mM CaCl 2 In the solution, calcify for 20 minutes to form a flat RGD modified calcium alginate gel. Then, 1.5 mg/mL chitosan (molecular weight 65kDa, degree of deacetylation 90%) solution was added, and by controlling the reaction time, gel films with thicknesses of 2 μm, 30 μm and 50 μm can be obtained. Then put them in a cell culture box, culture them with fibroblasts' own culture medium for 1d, and then inoculate human embryonic stem cells on the surface of gel membranes of different thicknesses for co-cultivation. In the negative control group, the sodium alginate solution mixed with fibroblasts was placed on the transwell membrane to form a gel, and the embryonic stem cells were inoculated on the flat gel membrane. There was no contact between the two cells. The medium was changed every day. After 7 days of culture, the expression of the undifferentiated markers Oct4, SSEA-4, TRA-1-81 and alkaline phosphatase (ALP) of embryonic stem cells was investigated by immunofluorescence. In addition, the embryonic stem cells on the surface of the gel membrane were digested and RNA was collected to detect the expression of the undifferentiated genes Oct4, SSEA-4, TRA-1-81 and alkaline phosphatase (ALP) of embryonic stem cells. Scanning electron microscope to observe whether there is mutual contact between the two kinds of cells. RT-PCR agarose gel electrophoresis results show that when the distance between the two cells is 2μm and 10μm, embryonic stem cells all express Oct4, SSEA-4, TRA-1-81 and alkaline phosphatase (ALP) and the above genes are expressed There is no significant difference in the amount, and the distance between the two kinds of cells is 30μm in the group and the negative control group, embryonic stem cells do not express the above-mentioned undifferentiated genes. The immunofluorescence results showed that when the distance between the two cells was 2μm and 10μm, embryonic stem cells all expressed Oct4, SSEA-4, TRA-1-81 and alkaline phosphatase (ALP) related markers, which were consistent with the gene results. Scanning electron microscopy results show that when the distance between the two cells is 10 μm, there is a pseudopod contact between the two cells; when the distance between the two cells is 30 μm, there is no contact between the two cells. This indicates that the maintenance of the characteristics of human embryonic stem cells requires mutual contact between embryonic stem cells and fibroblasts. In order to maintain the characteristics of embryonic stem cells, the distance between the two cells must be within 10μm.