Pre-stimulating stem cell as well as preparation method and application thereof
A technology of stem cells and pre-stimulation, applied in the field of tissue engineering, can solve problems such as restoration and complex microenvironment, and achieve the effects of mild treatment conditions, enhanced secretion function, and enhanced secretion function
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[0032] A method for preparing pre-stimulated stem cells of the present invention comprises the following steps:
[0033] (1) selecting non-target stem cells as auxiliary cells, inducing pyroptotic auxiliary cells, and obtaining pyroptotic cell extracts and / or pyroptotic cells;
[0034] (2) using pyroptotic cell secretions, extracts and / or pyroptotic cells for the cultivation of target stem cells;
[0035] (3) Obtain pre-stimulated stem cells with stress state.
[0036] The principle of the technical solution of the present invention will be described in detail below.
[0037] The steps that the present invention comprises are as follows:
[0038] a. Auxiliary cell culture and induction of pyroptosis:
[0039]In the present invention, non-target stem cells are selected as auxiliary cells, and pyroptosis is induced by physical, chemical or biological methods. Because cell pyroptosis is a series of changes that occur autonomously after cells are stimulated by the outside worl...
Embodiment 1
[0062] Example 1: Preparation of Pre-stimulated Limbal Stem Cells for Rat Tissue Engineering Lamellar Cornea Construction
[0063] 1. Induction of pyroptosis in rat corneal stromal cells
[0064] Rat corneal stromal cells were selected as auxiliary cells, and 1*10 6 cells / cm 2 Cells were seeded in a 10cm diameter plastic culture dish and cultured in an incubator for 24 hours. The culture medium was high-glucose DMEM+10% fetal bovine serum+1% L-glutamine+1% NEAA+1% penicillin-chain Mycin solution (100X)+0.02% EGF+0.1% transferrin+0.1% insulin, the culture condition is 37°C, 5% carbon dioxide. After 24 hours, the culture solution containing 1 mol / ml lipopolysaccharide (LPS) / nigericin mixture was replaced for 48 hours.
[0065] 2. Collection of pyroptotic cell secretions
[0066] Add the above-mentioned LPS / nigericin mixture and cultivate for 48 hours, then collect the culture medium, wash it twice with PBS, blow gently to blow off the cell surface substances and collect the ...
Embodiment 2
[0072] Example 2: Preparation of pre-stimulated human bone marrow mesenchymal stem cells for the treatment of bone defects caused by arthritis
[0073] 1. Induction of pyroptosis in human peripheral blood mononuclear cells
[0074] Human peripheral blood mononuclear cells were selected as auxiliary cells, and 2*10 6 cells / cm 2 Cells were seeded in 10 cm diameter plastic culture dishes and cultured in an incubator for 24 hours. The culture medium was RPMI1640 + 10% fetal bovine serum, and the culture conditions were 37°C and 5% carbon dioxide. After 24h, fresh culture medium was replaced, and 50mJ / cm2 of ultraviolet light was irradiated for 24h.
[0075] 2. Extract pyroptotic bodies
[0076] After 24 hours, culture was continued for 24 hours, the culture medium was collected, washed twice with PBS, blown gently to blow off the cell surface substances and collected PBS. The collected culture solution and PBS were subjected to ultracentrifugation at 80,000 rpm, and the supern...
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