Fibroblast and its preparation method and application, cell secretion and its application
A fibroblast, cell secretion technology, applied in the biological field
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[0039] A method for preparing fibroblasts according to one embodiment is obtained, such as figure 1 As shown, the method includes the following steps S110-S130. Cells are among the products that cannot be clearly characterized by structural and / or compositional features alone, and cannot be adequately characterized by other features than the method of preparation. According to Section 4.3, Chapter 10, Part Two of the Examination Guidelines, claims for this type of product are allowed to be characterized by a preparation method.
[0040] S110, providing feeder cells.
[0041] Feeder cells refer to a monolayer of certain cells that have been treated with a mitotic blocking agent. Granulosa cells, fibroblasts, and fallopian tube epithelial cells can be used as feeder cells. After being treated with mitosis blocking agent, the feeder layer cells did not divide and proliferate, but still maintained metabolic activity. The mitotic blocking agent may be mitomycin. Feeder cells c...
Embodiment 1
[0072] Preparation of fibroblasts
[0073] Undifferentiated human embryonic stem cells (hESCs) were seeded on mouse fibroblasts (MEF) fixed with 4% paraformaldehyde, and embryonic stem cells and feeder cells were cultured in induction medium. Wherein the induction medium comprises basal medium and cell growth hormone, the basal medium is DMEM / F12 medium, the volume percentage of fetal calf serum in the induction medium is 5%, and the concentration of adenine in the induction medium is 0.15mmol / L, the concentration of insulin in the induction medium is 5μg / mL, and the concentration of hydrocortisone in the induction medium is 0.5μg / mL. The induction medium was replaced every 24 h. On the 4th to 7th day of the induction culture, BMP-4 was additionally added to the induction medium, and the amount of BMP-4 added was 0.5 nmol / L. After induction of differentiation, fibroblasts derived from embryonic stem cells were obtained. The obtained fibroblasts can be expanded and stored fo...
Embodiment 2
[0075] Preparation of cell secretion fluid from fibroblasts
[0076]The fibroblasts were prepared by the method in Example 1, and after expansion, they were digested with trypsin and passaged onto culture plates pre-plated with type I collagen, and continued to be expanded and passaged. The cell culture medium for cultivating fibroblasts includes basal medium and cell growth hormone, the basal medium is DMEM / F12 medium, the volume percentage of fetal bovine serum in the cell culture medium is 10%, adenine is in the induction medium The concentration of insulin is 0.15mmol / L, and the concentration of insulin in the cell culture medium is 5μg / mL. The medium was changed every 24 hours, the replaced culture medium was collected, and the cell debris in the culture medium was removed by centrifugation, and the supernatant was the cell secretion of fibroblasts. The contents of bFGF, VEGF and TGF-β1 in the cell secretion were detected by ELISA test.
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