CN-115851580-B - Reagent for preparing macrophage by human pluripotent stem cell differentiation and application thereof

CN115851580BCN 115851580 BCN115851580 BCN 115851580BCN-115851580-B

Abstract

The invention discloses a reagent for preparing macrophages by differentiating human pluripotent stem cells and application thereof. Specifically disclosed are reagents and methods for preparing macrophages, the reagents comprising a culture broth consisting of culture broth II, culture broth III, culture broth IV, culture broth V, and culture broth VI, and a 3D micro-stent material. The invention develops a stem cell differentiation culture system which has definite chemical components, does not contain animal source components and has low cost by utilizing the 3D micro-scaffold, is favorable for clinical grade stem cells, and can lead human pluripotent stem cells to rapidly, continuously and efficiently differentiate a large number of high-purity macrophages. The reagent and the method have the characteristics of short time consumption, high differentiation efficiency, lower cost, more contribution to large-scale automatic industrialized production and the like. The preparation method provided by the invention can be used for producing human macrophages on a large scale, has stable quality and high safety, and provides a large amount of cell sources for tissue engineering, drug development and cell treatment.

Inventors

NA JIE
WANG PEILIANG
CHEN XIA
ZHANG YAXUAN

Assignees

清华大学

Dates

Publication Date: 20260512
Application Date: 20220711

Claims (4)

1. A method of preparing macrophages, the method comprising the steps of: h1 Inoculating the human pluripotent stem cells into the culture solution I for culturing for 0.5-1.5 days; H2 Changing into culture solution II, and culturing for 0.5-1.5 days; H3 Changing into culture solution III for continuous culture for 1.5-2.5 days; h4 Digesting and collecting the cells in the step H3), inoculating the cells into a 3D micro-stent prepared by a 3D micro-stent material, and culturing for 2.5-3.5 days by adopting a culture solution IV; h5 Changing into culture solution V, and culturing for 2.5-3.5 days; h6 Culturing for 20-80 days by changing into culture solution VI, and collecting the macrophage; The culture solution I is stem cell culture solution containing a ROCK inhibitor; The culture solution II is RPMI 1640 culture solution containing 2% of insulin-free B27 additive, 1mM L-glutamine substitute, 1% of nonessential amino acid, 100U/mL penicillin, 100 mug/mL streptomycin, 50 ng/mL vitamin C and 5 ng/mL human bone morphogenetic protein 4, wherein the nonessential amino acid consists of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine, the concentration of glycine in the culture solution II is 750.0 ng/mL, the concentration of L-alanine in the culture solution II is 890 ng/mL, the concentration of L-asparagine in the culture solution II is 1320 ng/mL, the concentration of L-aspartic acid in the culture solution II is 1330 ng/mL, the concentration of L-glutamic acid in the culture solution II is 1470 ng/mL, the concentration of L-proline in the culture solution II is 1150 ng/mL, and the concentration of L-serine in the culture solution ng/mL; the culture solution III is a culture solution containing a GSK3 inhibitor, and is prepared by taking the culture solution II as a solvent and taking the GSK3 inhibitor as a solute, wherein the GSK3 inhibitor is CHIR-99021, and the content of the CHIR-99021 in the culture solution III is 2 mu M; The culture solution IV is RPMI 1640 culture solution containing 2% of B27 additive added with insulin, 1mM L-glutamine substitute, 1% of nonessential amino acid, 100U/mL penicillin, 100 mug/mL streptomycin, 50 ng/mL vitamin C, 50 ng/mL human vascular endothelial growth factor VEGF-165 and 10 ng/mL human fibroblast growth factor, wherein the nonessential amino acid consists of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine, the concentration of glycine in the culture solution IV is 750.0 ng/mL, the concentration of L-alanine in the culture solution IV is 890 ng/mL, the concentration of L-asparagine in the culture solution IV is 1320 ng/mL, the concentration of L-aspartic acid in the culture solution IV is ng/mL, the concentration of L-glutamic acid in the culture solution IV is 1470 ng/mL, the concentration of L-proline in the culture solution IV is 1150, and the concentration of L-serine in the culture solution is 1050/mL; the culture solution V is a culture solution containing human macrophage colony stimulating factor, and is prepared by taking the culture solution IV as a solvent and taking the human macrophage colony stimulating factor as a solute, wherein the content of the human macrophage colony stimulating factor in the culture solution V is 50 ng/ml; The culture solution VI is RPMI 1640 culture solution containing 2% of B27 additive added with insulin, 1mM L-glutamine substitute, 1% of optional amino acid, 100U/mL penicillin, 100 ug/mL streptomycin, 50 ng/mL vitamin C,10 ng/mL human interleukin-3 and 50 ng/mL human macrophage colony stimulating factor, wherein the optional amino acid consists of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline and L-serine, the concentration of glycine in the culture solution VI is 750.0 ng/mL, the concentration of L-alanine in the culture solution VI is 890 ng/mL, the concentration of L-asparagine in the culture solution VI is 1320 ng/mL, the concentration of L-aspartic acid in the culture solution VI is 1330 ng/mL, the concentration of L-glutamic acid in the culture solution VI is 1470 ng/mL, the concentration of L-proline in the culture solution VI is 1150/L-glutamic acid, the concentration of L-proline and the L-serine concentration in the culture solution VI is 1050-98/mL; the 3D micro-stent material is a porous material prepared from a gelatin solution and a biological cryoprotectant mixed solution, wherein the pore size of the porous material is 50-100 microns, the gelatin solution in the mixed solution is gelatin deionized water solution with the mass volume ratio of 4%, and the biological cryoprotectant is DMSO with the volume ratio of 3%.
2. The method of claim 1, wherein the stem cell culture fluid is formulated with the ROCK inhibitor as a solute and the TeSR-E8 culture fluid as a solvent.
3. The method according to claim 1, wherein the inoculation density in step H4) is 6 x 10 5 /cm 2 -10×10 5 /cm 2 .
4. The method according to any one of claims 1 to 3, further comprising step H7), wherein the macrophages collected in step H6) are cultured in the culture broth VI for 5 to 10 days, and a batch of macrophages is collected every 3 to 4 days.

Description

Reagent for preparing macrophage by human pluripotent stem cell differentiation and application thereof Technical Field The invention belongs to the technical field of cell culture, relates to a reagent for preparing macrophages by differentiating human pluripotent stem cells and application thereof, and in particular relates to a method for continuously and efficiently culturing human pluripotent stem cells for a long time and generating macrophages in a large quantity and application thereof. Background Macrophages (macrophages, mphi) are white blood cells located in tissue and are derived from monocytes, which in turn are derived from precursor cells in bone marrow. Macrophages and monocytes are phagocytes and are involved in nonspecific defense (innate immunity) and specific defense (cellular immunity) in vertebrates. Macrophages are important natural immune cells of a human body, and have important application values in the aspects of maintaining tissue homeostasis, inflammatory reaction, tissue repair and regeneration, resisting infection, regulating and controlling tumor immunity and the like. Macrophages are involved in the recognition, phagocytosis and degradation of cell debris and pathogens, and also play a role in presenting antigens to T cells and inducing expression of costimulatory molecules by other antigen presenting cells, thereby initiating an adaptive immune response. Macrophages play an important role in innate immunity and also play a vital regulatory role in the development and progression of acute and chronic inflammation and tumors. At present, macrophage feedback treatment is utilized to achieve certain progress in related researches on liver fibrosis, alveolar deposition, diabetic nephropathy and tumors. It can be seen that macrophages have a variety of important physiological functions, are closely related to the occurrence and development of a plurality of diseases, and have higher research value and good application prospects. At present, a direct culture method or an induction culture method is mainly adopted for the macrophage culture. The direct culture method is to directly separate macrophages for culture, and the method has complex operation and low macrophage recovery rate. The induction culture method is a culture method for inducing precursor cells such as stem cells and monocytes into macrophages by using exogenous cytokines. The existing macrophage culturing method still has the problems of limited proliferation times, difficulty in continuous high-efficiency, stable and large-scale acquisition for a long time, difficulty in amplification and gene manipulation, and great limitation on related research and clinical application. Disclosure of Invention The technical problem to be solved by the present invention is how to continue to obtain macrophages with high efficiency, stability and/or mass. The technical problems to be solved are not limited to the described technical subject matter, and other technical subject matter not mentioned herein will be clearly understood by those skilled in the art from the following description. In order to solve the above technical problems, the present invention firstly provides a reagent for preparing macrophages by differentiation of human pluripotent stem cells, which may include a culture solution and a 3D micro-scaffold material, the culture solution may be composed of a culture solution II, a culture solution III, a culture solution IV, a culture solution V and a culture solution VI, The culture solution II can be any one of the following: a1 A culture broth comprising an insulin-free B27 additive and human bone morphogenetic protein 4; a2 A culture broth comprising insulin-free B27 supplement, L-glutamine or a substitute thereof, a non-essential amino acid, penicillin, streptomycin, vitamin C, and human bone morphogenic protein 4; Wherein the non-essential amino acid may be glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine; The culture solution III can be a culture solution containing a GSK3 inhibitor, and the culture solution is prepared by taking the culture solution II as a solvent and taking the GSK3 inhibitor as a solute; the culture solution IV can be any one of the following: B1 A culture medium containing an insulin-added B27 additive, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor; b2 A culture broth containing insulin-added B27 additive, L-glutamine or its substitute, nonessential amino acids, penicillin, streptomycin, vitamin C, human vascular endothelial growth factor VEGF-165, and human fibroblast growth factor; Wherein the non-essential amino acid may be glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine; the culture solution V can be a culture solution containing human macrophage colony stimulating factor, and the culture solution is prepared by taking the culture solution IV as a solvent and