Chondrocyte culture with high tissue regeneration ability
A chondrocyte and tissue regeneration technology, applied in tissue culture, cell culture support/coating, cell culture active agent, etc., can solve the problems of fibrocartilage formation, inability to obtain sufficient therapeutic effect, death, etc., and achieve viability high effect
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[0057] The method for preparing chondrocyte cultures with high tissue regeneration ability of the present invention includes the step of culturing cell groups isolated from cartilage tissue with thermoreversible polymers. "Cartilage tissue" in the present disclosure refers to tissue derived from cartilage. Cartilage includes, but is not limited to, articular cartilage, epiphyseal plate, costal cartilage, tracheal cartilage, laryngeal cartilage, sacroiliac joint, jaw joint, sternoclavicular joint, intervertebral disc, pubic symphysis, articular meniscus, articular disc, external auditory canal, Eustachian tube, ear Contour cartilage and epiglottis cartilage, etc. Cartilage tissue can be derived from any organism. Human cartilage tissue is preferable because of low rejection reaction when bone tissue culture is transplanted to human damaged cartilage. It may be an autologous tissue or an allogeneic tissue, but autologous tissue is preferred because of low rejection when admini...
preparation example 1
[0172] 42.0 g of N-isopropylacrylamide and 4.0 g of n-butyl methacrylate were dissolved in 592 g of ethanol. An aqueous solution in which 11.5 g of polyethylene glycol dimethacrylate (PD E6000, manufactured by NOF Co., Ltd.) was dissolved in 65.1 g of water was added thereto, and it heated to 70 degreeC in nitrogen stream. While maintaining 70°C under nitrogen flow, add 0.4 mL of N,N,N',N'-tetramethylethylenediamine (TEMED) and 4 mL of 10% ammonium persulfate (APS) aqueous solution and stir for 30 minutes to make it reaction. Further, 0.4 mL of TEMED and 4 mL of 10% APS aqueous solution were added four times at intervals of 30 minutes to complete the polymerization reaction. After cooling the reaction liquid to below 5°C, add 5 L of cooled distilled water at 5°C for dilution, and concentrate to 2 L at 5°C using an ultrafiltration membrane with a molecular weight cut-off of 100,000.
[0173] 4 L of cooled distilled water was added to this concentrated solution for dilution, a...
Embodiment 1
[0184]
[0185] 1 g of TGP produced in Preparation Example 1 was dissolved in 9 mL of DMEM at 4°C to prepare a 10% TGP solution, and then cells derived from cartilage tissue were dispersed and injected into 6-well plates. After standing at room temperature until gelatinous, add 7-8 ml of antibiotics (gentamycin (50 μg / ml), amphotericin (0.25 μg / ml), penicillin (100 Units / ml) / streptomycin (100 μg / ml) and L-ascorbic acid (5 mg / ml)) were cultured in a 5% carbon dioxide incubator (ESPEC BNA-111). The culture medium was changed every week and cultured for 20 weeks.
[0186]
[0187] (1) Phase contrast microscope observation
[0188] In Comparative Example 1, normal chondrocytes confirmed to be spherical at the beginning of culture gradually began to appear elongated fibroblast-like cells as the culture progressed, and gradually began to degenerate or die from about 12 days to about 20 days. Complete denaturation or death begins at about 4 weeks. For example, sample 1059 die...
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