Reagents for preparing macrophages by differentiation of human pluripotent stem cells and uses thereof

Abstract

The application discloses a reagent for preparing macrophages by human pluripotent stem cell differentiation and application thereof. Specifically disclosed are a reagent and a method for preparing macrophages, wherein the reagent comprises a culture solution and a 3D micro-scaffold material, and the culture solution is composed of culture solution II, culture solution III, culture solution IV, culture solution V and culture solution VI. The application utilizes the 3D micro-scaffold to develop a chemical composition-determined, animal-derived component-free and low-cost stem cell differentiation culture system which is beneficial to clinical-level stem cell differentiation, and can make human pluripotent stem cells continuously and efficiently differentiate into a large amount of high-purity macrophages. The reagent and the method have the characteristics of short time consumption, high differentiation efficiency, low cost and being more beneficial to large-scale automatic industrial production. The preparation method provided by the application can be used for large-scale production of human macrophages, and the quality is stable and the safety is high, thereby providing a large amount of cell sources for tissue engineering, drug research and development and cell therapy.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, and relates to reagents for preparing macrophages through differentiation of human pluripotent stem cells and their applications, specifically to a method and application for the long-term, continuous, efficient, and large-scale generation of macrophages from human pluripotent stem cells. Background Technology

[0002] Macrophages (Mφ) are white blood cells located in tissues, originating from monocytes, which in turn originate from precursor cells in the bone marrow. Both macrophages and monocytes are phagocytic cells that participate in non-specific defense (innate immunity) and specific defense (cell-mediated immunity) in vertebrates. Macrophages are important innate immune cells in the human body, playing a crucial role in maintaining tissue homeostasis, inflammatory responses, tissue repair and regeneration, infection resistance, and tumor immune regulation. Macrophages participate in the recognition, phagocytosis, and degradation of cellular debris and pathogens. They also play a role in presenting antigens to T cells and inducing other antigen-presenting cells to express co-stimulatory molecules, thereby initiating adaptive immune responses. Macrophages not only play an important role in innate immunity but also in the development and progression of acute and chronic inflammation and tumors. Current research on macrophage reinfusion therapy in liver fibrosis, alveolar effusion disease, diabetic nephropathy, and tumors has made some progress. It is evident that macrophages possess a variety of important physiological functions and are closely related to the occurrence and development of many diseases, thus demonstrating high research value and promising application prospects.

[0003] Currently, macrophage culture mainly employs direct culture or induced culture methods. Direct culture involves directly isolating and culturing macrophages; this method is relatively cumbersome and has a low macrophage recovery rate. Induced culture utilizes exogenous cytokines to induce precursor cells such as stem cells and monocytes into macrophages. Existing macrophage culture methods still suffer from limited proliferation rates, difficulty in obtaining macrophages sustainably, efficiently, stably, and in large quantities over extended periods, and challenges in amplification and gene manipulation, significantly limiting related research and clinical applications. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to obtain macrophages continuously, efficiently, stably, and / or in large quantities. The technical problem to be solved is not limited to the described technical subject matter; other technical subject matter not mentioned herein will be clearly understood by those skilled in the art through the following description.

[0005] To address the aforementioned technical problems, this invention first provides a reagent for preparing macrophages through differentiation of human pluripotent stem cells. The reagent may include a culture medium and a 3D microscaffold material. The culture medium may consist of culture medium II, culture medium III, culture medium IV, culture medium V, and culture medium VI.

[0006] The culture medium II may be any of the following:

[0007] A1) A culture medium containing insulin-free B27 additive and human bone morphogenetic protein 4;

[0008] A2) A culture medium containing insulin-free B27 additive, L-glutamine or its substitute, non-essential amino acids, penicillin, streptomycin, vitamin C and human bone morphogenetic protein 4;

[0009] The non-essential amino acids may be glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine.

[0010] The culture medium III may be a culture medium containing a GSK3 inhibitor, and the culture medium is a liquid prepared by using the culture medium II as a solvent and the GSK3 inhibitor as a solute;

[0011] The culture medium IV can be any of the following:

[0012] B1) A culture medium containing B27 additive with added insulin, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor;

[0013] B2) A culture medium containing B27 additive with added insulin, L-glutamine or its alternative, non-essential amino acids, penicillin, streptomycin, vitamin C, human vascular endothelial growth factor VEGF-165 and human fibroblast growth factor.

[0014] The non-essential amino acids may be glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine.

[0015] The culture medium V may be a culture medium containing human macrophage colony-stimulating factor, and the culture medium is a liquid prepared by using culture medium IV as a solvent and human macrophage colony-stimulating factor as a solute;

[0016] The culture medium VI can be any of the following:

[0017] C1) A culture medium containing B27 additive with added insulin, human interleukin-3 and human macrophage colony-stimulating factor;

[0018] C2) A culture medium containing B27 supplement with added insulin, L-glutamine or its alternative, non-essential amino acids, penicillin, streptomycin, vitamin C, human interleukin-3 and human macrophage colony-stimulating factor;

[0019] The non-essential amino acids may be glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine.

[0020] Further, the culture medium II described in A1) may be a culture medium containing 1%-2% by volume of insulin-free B27 additive and 5-10 ng / ml human bone morphogenetic protein 4.

[0021] Furthermore, the 1%-2% can be 2%, and the 5-10 ng / ml can be 5 ng / ml.

[0022] Further, the culture medium II described in A2) may specifically be a culture medium containing 1%-2% by volume of insulin-free B27 additive, 0.5-1 mM L-glutamine or its substitute, 0.5%-1% by volume of non-essential amino acids, 50-100 U / ml penicillin, 50-100 μg / ml streptomycin, 25-50 ng / ml vitamin C and 5-10 ng / ml human bone morphogenetic protein 4.

[0023] Further, the 1%-2% can be 2%, the 0.5-1 mM can be 1 mM, the 0.5%-1% can be 1%, the 50-100 U / ml can be 100 U / ml, the 50-100 μg / ml can be 100 μg / ml, the 25-50 ng / ml can be 50 ng / ml, and the 5-10 ng / ml can be 5 ng / ml.

[0024] Further, the concentration of glycine in culture medium II can be 750.0 ng / mL; the concentration of L-alanine in culture medium II can be 890 ng / mL; the concentration of L-asparagine in culture medium II can be 1320 ng / mL; the concentration of L-aspartic acid in culture medium II can be 1330 ng / mL; the concentration of L-glutamic acid in culture medium II can be 1470 ng / mL; the concentration of L-proline in culture medium II can be 1150 ng / mL; and the concentration of L-serine in culture medium II can be 1050 ng / mL.

[0025] Furthermore, in the culture medium III, the content of the GSK3 inhibitor in the culture medium may be 1-2 μM or 2 μM.

[0026] Further, the culture medium IV described in B1) may be a culture medium containing 1%-2% by volume of B27 additive with added insulin, 25-50 ng / ml human vascular endothelial growth factor VEGF-165 and 5-10 ng / ml human fibroblast growth factor.

[0027] Furthermore, the 1%-2% can be 2%, the 25-50 ng / ml can be 50 ng / ml, and the 5-10 ng / ml can be 10 ng / ml.

[0028] Further, the culture medium IV described in B2) may specifically be a culture medium containing 1%-2% by volume of B27 additive with added insulin, 0.5-1 mM L-glutamine or its substitute, 0.5%-1% by volume of non-essential amino acids, 50-100 U / ml penicillin, 50-100 μg / ml streptomycin, 25-50 ng / ml vitamin C, 25-50 ng / ml human vascular endothelial growth factor VEGF-165 and 5-10 ng / ml human fibroblast growth factor.

[0029] Further, the 1%-2% can be 2%, the 0.5-1 mM can be 1 mM, the 0.5%-1% can be 1%, the 50-100 U / ml can be 100 U / ml, the 50-100 μg / ml can be 100 μg / ml, the 25-50 ng / ml can be 50 ng / ml, and the 5-10 ng / ml can be 10 ng / ml.

[0030] Further, the concentration of glycine in culture medium IV can be 750.0 ng / mL; the concentration of L-alanine in culture medium IV can be 890 ng / mL; the concentration of L-asparagine in culture medium IV can be 1320 ng / mL; the concentration of L-aspartic acid in culture medium IV can be 1330 ng / mL; the concentration of L-glutamic acid in culture medium IV can be 1470 ng / mL; the concentration of L-proline in culture medium IV can be 1150 ng / mL; and the concentration of L-serine in culture medium IV can be 1050 ng / mL.

[0031] Furthermore, in the culture medium V, the content of the human macrophage colony-stimulating factor in the culture medium can be 50-100 ng / ml or 50 ng / ml.

[0032] Further, the culture VI described in C1) may be a culture medium containing 1%-2% by volume of B27 additive with added insulin, 10-20 ng / ml human interleukin-3 and 50-100 ng / ml human macrophage colony-stimulating factor.

[0033] Furthermore, the 1%-2% can be 2%, the 10-20 ng / ml can be 10 ng / ml, and the 50-100 ng / ml can be 50 ng / ml.

[0034] Further, the culture medium VI described in C2) may specifically be a culture medium containing 1%-2% by volume of B27 additive with added insulin, 0.5-1 mM L-glutamine or its substitute, 0.5%-1% by volume of non-essential amino acids, 50-100 U / ml penicillin, 50-100 μg / ml streptomycin, 25-50 ng / ml vitamin C, 10-20 ng / ml human interleukin-3 and 50-100 ng / ml human macrophage colony-stimulating factor.

[0035] Further, the 1%-2% can be 2%, the 0.5-1 mM can be 1 mM, the 0.5%-1% can be 1%, the 50-100 U / ml can be 100 U / ml, the 50-100 μg / ml can be 50 ng / ml, the 25-50 ng / ml can be 50 ng / ml, the 10-20 ng / ml can be 10 ng / ml, and the 50-100 ng / ml can be 50 ng / ml.

[0036] Further, the concentration of glycine in culture medium VI can be 750.0 ng / mL; the concentration of L-alanine in culture medium VI can be 890 ng / mL; the concentration of L-asparagine in culture medium VI can be 1320 ng / mL; the concentration of L-aspartic acid in culture medium VI can be 1330 ng / mL; the concentration of L-glutamic acid in culture medium VI can be 1470 ng / mL; the concentration of L-proline in culture medium VI can be 1150 ng / mL; and the concentration of L-serine in culture medium VI can be 1050 ng / mL.

[0037] Culture solutions II, III, IV, V and VI can all be prepared using RPMI 1640 medium as the base culture medium.

[0038] In the above reagents, the 3D microscaffold material can be any of the following:

[0039] D1) The 3D microscaffold material can be a porous material containing pores with a diameter of tens of micrometers.

[0040] D2) The 3D microscaffold material can be a porous material with pore sizes of tens of micrometers, prepared from a mixture of gelatin solution and biological cryoprotectant.

[0041] The aperture (diameter) at the tens of micrometer level can be 50-100 micrometers.

[0042] Furthermore, the 50-100 micrometers can be 50 micrometers.

[0043] The gelatin solution may be a deionized aqueous solution containing dissolved gelatin.

[0044] The biological cryoprotectant may be dimethyl sulfoxide, methanol, or glycerol.

[0045] Further, in the mixture described in D2), the gelatin solution can specifically be a deionized gelatin aqueous solution with a mass-to-volume ratio of 3%-6%, and the biological cryoprotectant has a volume fraction of 1%-5%.

[0046] Furthermore, the gelatin solution in the mixture may specifically be a 4% (w / v) deionized gelatin aqueous solution, and the biological cryoprotectant has a volume fraction of 3%.

[0047] D2) The scaffold prepared from the 3D microscaffold material is a gelatin 3D microscaffold (referred to as gelatin microscaffold).

[0048] Furthermore, during the preparation of the gelatin 3D microscaffold, an aqueous solution of glutaraldehyde with a mass-volume ratio of 0.1%-0.4% w / v can be added to promote the formation of the gelatin 3D microscaffold and a 1% sodium borohydride solution (pH=9.7) can be added to remove residual glutaraldehyde in the subsequent 3D microscaffold.

[0049] In the above reagents, the GSK3 inhibitor may be CHIR-99021, SB216763, BIO, or TWS119.

[0050] The reagents mentioned above may further include culture medium I, which may be a stem cell culture medium containing ROCK inhibitors.

[0051] Furthermore, the ROCK inhibitor may be Y27632.

[0052] Furthermore, in the culture medium I, the content of the ROCK inhibitor (such as Y27632) in the culture medium can be 5-10 μM or 5 μM.

[0053] In the above reagents, the stem cell culture medium can be prepared using the ROCK inhibitor (such as Y27632) as a solute and TeSR-E8 culture medium as a solvent.

[0054] The present invention also provides a kit for preparing macrophages, the kit comprising any of the reagents described herein.

[0055] The present invention also provides a method for preparing macrophages, the method comprising the following steps:

[0056] H1) Human pluripotent stem cells were seeded into culture medium I and cultured for 0.5-1.5 days;

[0057] H2) was replaced with culture medium II and cultured for another 0.5-1.5 days;

[0058] H3) was replaced with the culture medium III and cultured for another 1.5-2.5 days;

[0059] H4) Digest and collect the cells from step H3), seed the cells in a 3D microscaffold prepared using the 3D microscaffold material, and culture them in culture medium IV for 2.5-3.5 days;

[0060] Replace H5 with the culture medium V and continue culturing for 2.5-3.5 days;

[0061] H6) was replaced with the culture medium VI and cultured for 20-80 days to obtain the macrophages.

[0062] In the above method, the inoculation density mentioned in step H4) can be 6 × 10⁻⁶. 5 pcs / cm 2 -10×10 5 pcs / cm 2 .

[0063] In the above method, the inoculation density mentioned in step H1) can be 2.0 × 10⁻⁶. 4 pcs / cm 2 -4.0×10 4 pcs / cm 2 .

[0064] The above method further includes step H7), which is: culturing the macrophages collected in step H6) in culture medium VI for 5-10 days, wherein the collection is a batch of macrophages collected every 3-4 days.

[0065] Furthermore, the collection can begin after culturing in culture medium VI for 20 days, with a batch of macrophages collected every 3-4 days. The macrophages collected in step H6 can be the first three batches of cells.

[0066] Furthermore, in step H5), the replacement with culture medium V for continued culture involves transferring the 3D microscaffold with cells obtained after culturing in step H4 to culture medium V for continued culture.

[0067] The step H6) involves replacing the culture medium VI with culture for 20-80 days, which means transferring the 3D microscaffold with cells obtained after step H5) to culture medium VI and culturing for 20-80 days.

[0068] Macrophages continuously emerge from the 3D microscaffold, and newly generated macrophages will crawl out of the 3D microscaffold. A batch of newly generated macrophages is collected every 3-4 days. The first three batches of macrophages generated in the 3D microscaffold exhibit strong amplification capabilities and can be transferred to culture medium VI for further culture and amplification for 5-10 days.

[0069] In one embodiment of the present invention, the method for preparing macrophages includes the following steps:

[0070] (1) Human pluripotent stem cells (H1 cells or CD34-iPSC cells) were seeded in culture medium I and cultured for 1 day;

[0071] (2) Replace with the culture medium II and continue culturing for 2 days;

[0072] (3) Replace with the culture medium III and continue culturing for 2 days;

[0073] (4) Digest and collect the cells from step (3), seed the cells into a 3D microscaffold prepared using the 3D microscaffold material, and culture them in culture medium IV for 3 days;

[0074] (5) Replace with the culture medium V and continue culturing for 3 days;

[0075] (6) Replace with the culture medium VI and culture for 80 days, collecting a batch of macrophages every 3-4 days.

[0076] In one embodiment of the present invention, the 3D microscaffold material is prepared according to the gelatin porous material preparation method in the published paper (S. Jiang, C. Lyu, P. Zhao, W. Li, W. Kong, C. Huang, GM Genin, Y. Du, Cryoprotectant enables structural control of porous scaffolds for exploration of cellular mechano-responsiveness in 3D, Nat Commun 10(1) (2019) 3491.), the specific steps of which are as follows:

[0077] (1) Prepare a gelatin precursor solution by mixing 4% gelatin powder and cryoprotectant of different concentrations (concentration range can be 1%-5%) in deionized water.

[0078] (2) Incubate the premixed solution on ice for 5 minutes.

[0079] (3) Add different concentrations of glutaraldehyde (concentration range of 0.1%-0.4%) and stir the solution for 20 seconds.

[0080] (4) Add 400 μl of solution into a polymethyl methacrylate plastic mold with dimensions of 20 mm × 20 mm × 1 mm. Freeze in a -20°C freezer for 16 h to form a cryogel.

[0081] (5) After cryogelation for 16 hours, the mold containing the cryogel was placed at room temperature to melt the ice crystals formed by the cryoprotectant and then carefully cleaned with deionized water to remove residual reagents to obtain gelatin microscaffolds.

[0082] (6) The obtained gelatin microscaffold was placed in a 1% sodium borohydride solution (pH=9.7) for 10 minutes to remove residual glutaraldehyde, and then washed with deionized water.

[0083] (7) The cleaned gelatin microscaffolds were collected in a container, placed at -20°C for 2 hours, and then freeze-dried for 2 hours.

[0084] (8) Collect gelatin microscaffolds and store them in a low vacuum environment for cell differentiation culture.

[0085] The cryoprotectant mentioned in step (1) may be dimethyl sulfoxide, and the volume fraction of dimethyl sulfoxide may be 3%.

[0086] The concentration of glutaraldehyde in step (3) can be 0.3% (mass-volume ratio).

[0087] The present invention also provides the use of the reagent or the kit in the preparation of macrophages.

[0088] The macrophages prepared in this article can be generated using human pluripotent stem cells as the starting cells.

[0089] The human pluripotent stem cells (PSCs) mentioned in this article can be human embryonic stem cells (ESCs) or human induced pluripotent stem cells (iPSCs).

[0090] The human embryonic stem cells may be commercially available human embryonic stem cell lines, such as human embryonic stem cell line H1. Specifically, human embryonic stem cell line H1 may be derived from the WiCell cell bank in the United States, catalog number: WA01. The human induced pluripotent stem cells may be CD34-iPSCs. These induced pluripotent stem cells (CD34-iPSCs) are obtained by inducing reprogramming of human umbilical cord blood hematopoietic stem cells (CD34-positive cells) using the Sendai virus reprogramming kit (Invitrogen, catalog number: A16517).

[0091] In this article, L-glutamine or its alternative may specifically be Glutamax (Gibco, catalog number: 35050061).

[0092] In this article, the amino acid sequence of human bone morphogenetic protein 4 (BMP4) is SEQ ID No. 1, the amino acid sequence of human vascular endothelial growth factor (VEGF-165) is SEQ ID No. 2, the amino acid sequence of human fibroblast growth factor (bFGF) is SEQ ID No. 3, the amino acid sequence of human interleukin-3 (IL3) is SEQ ID No. 4, and the amino acid sequence of human macrophage colony-stimulating factor (M-CSF) is SEQ ID No. 5.

[0093] In this article, the cell culture conditions can be 37°C and 5% CO2.

[0094] In this article, the culture dishes for cell culture can be coated with Matrigel, and the specific coating conditions can be coating at 37°C for 2 h.

[0095] This invention develops a culture medium additive and a 3D microscaffold material for the continuous, efficient, and large-scale differentiation of human pluripotent stem cells into macrophages. This additive and 3D microscaffold material are free of animal-derived components, have a defined chemical composition, and are low in cost, making them suitable for clinical-grade stem cell differentiation culture systems. This invention also establishes a novel 3D differentiation method for the differentiation of human pluripotent stem cells into macrophages. Compared with existing methods, the 3D differentiation method can generate macrophages efficiently and over a long period, is simple to operate, saves labor costs, significantly improves differentiation efficiency, and reduces differentiation costs. Furthermore, it is more suitable for large-scale, automated, industrial-scale macrophage production at the bioreactor level.

[0096] The preparation method provided by this invention enables the efficient and large-scale production of macrophages over a long period. This invention systematically differentiates human pluripotent stem cells into mesodermal cells, and then gradually induces the generation of a 3D microenvironment capable of continuously and efficiently producing macrophages through the addition of a 3D microscaffold and the cell culture medium of this invention. Compared with traditional stromal cell co-culture, 2D culture, and embryoid culture methods, this method is simple to operate and can obtain high-purity CD14 and CD163 positive macrophages stably and efficiently over a long period. It not only has a defined chemical composition and contains no animal-derived components, greatly improving the safety of cell preparation, but also features short preparation time, high differentiation efficiency, low cost, and is more conducive to large-scale automated industrial production. The preparation method provided by this invention can produce human macrophages on a large scale with stable quality and high safety, providing a large source of cells for tissue engineering, drug development, and cell therapy. Attached Figure Description

[0097] Figure 1 A morphological diagram of human pluripotent stem cells. Figure 1 The medium scale is 250 micrometers.

[0098] Figure 2 This diagram illustrates the morphological changes of pluripotent stem cells during their differentiation into meso-human germ layer cells (H1 cell line). Figure 2 The medium scale is 100 micrometers.

[0099] Figure 3 This is a microscopic image of a 3D microscaffold. Figure 3 The medium scale is 75 micrometers.

[0100] Figure 4 This is an immunofluorescence image showing the distribution of macrophages in a 3D microscaffold. Figure 4 The medium scale is 100 micrometers.

[0101] Figure 5 Microscopic imaging results of macrophages generated in 3D microscaffolds (rows 1 and 2) and Giemsa staining results (row 3). The scale bar for the second row of images is 80 micrometers, and the scale bar for the third row of images is 20 micrometers.

[0102] Figure 6 The results of flow cytometry analysis of CD45, CD11b, CD14 and CD163, surface markers of macrophages differentiated in 3D microscaffolds.

[0103] Figure 7 This is a cumulative curve showing the number of macrophages generated. The horizontal axis represents the collection time of each batch of macrophages differentiated from stem cells, and the vertical axis represents the cumulative collected CD14. + Number of macrophages.

[0104] Figure 8The images show the results of macrophage phagocytic function assays. The left image shows a fluorescent image of macrophages phagocytosing E. coli with green fluorescent protein, and the right image shows the results from flow cytometry. The scale bar in the images is 20 micrometers.

[0105] Figure 9 To detect gene expression results in macrophages after LPS stimulation by real-time quantitative PCR. Detailed Implementation

[0106] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0107] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0108] Unless otherwise specified, the quantitative experiments in the following examples were all repeated three times, and the results were averaged.

[0109] The biological materials and reagents involved in the following embodiments are:

[0110] Human embryonic stem cell line H1 (H1 cells for short): WiCell Cell Bank, USA, ID: WA01.

[0111] Induced pluripotent stem cells CD34-iPSC (CD34-iPSC cells for short): obtained by inducing reprogramming of human umbilical cord blood hematopoietic stem cells (CD34-positive cells) using the Sendai virus reprogramming kit (Invitrogen, catalog number: A16517).

[0112] DMEM culture medium: Gibco, catalog number: 11965092.

[0113] RPMI 1640 basal culture medium (RPMI 1640 medium): Thermo Fisher Scientific, catalog number: 11875093.

[0114] TeSR-E8 culture medium: STEMCELL, catalog number: 05990.

[0115] E8-Y culture medium (i.e., culture medium I) is a stem cell culture medium containing a ROCK inhibitor. Further, the stem cell culture medium can be prepared using the ROCK inhibitor as a solute and TeSR-E8 culture medium as a solvent. In culture medium I, the content of the ROCK inhibitor (such as Y27632) can be 5-10 μM or 5 μM.

[0116] The E8-Y culture medium (i.e., culture medium I) in the following examples is specifically prepared with Y27632 as solute and TeSR-E8 culture medium as solvent, wherein the content of Y27632 in the E8-Y culture medium (i.e., culture medium I) is 5 μM Y27632 in TeSR-E8 culture medium.

[0117] M1 culture medium (i.e., culture medium II) is a culture medium containing insulin-free B27 additive and human bone morphogenetic protein 4. It can be further prepared as RPMI 1640 culture medium containing 1%-2% (v / v) insulin-free B27 additive (B27 minus insulin), 0.5-1 mM L-glutamine substitute (Glutamax), 0.5%-1% (v / v) non-essential amino acids (NEAA), 50-100 U / ml penicillin, 50-100 μg / ml streptomycin, 25-50 ng / ml vitamin C, and 5-10 ng / ml human bone morphogenetic protein 4 (BMP4).

[0118] The M1 culture medium (i.e., culture medium II) in the following examples is specifically an RPMI 1640 culture medium containing 2% (v / v) insulin-free B27 supplement (B27 minus insulin), 1 mM L-glutamine substitute (Glutamax), 1% (v / v) non-essential amino acids (NEAA), 100 U / ml penicillin, 100 μg / ml streptomycin, 50 ng / ml vitamin C, and 5 ng / ml human bone morphogenetic protein 4 (BMP4). The concentrations of each component of the non-essential amino acids in M1 culture medium (culture medium II) are as follows: glycine 750.0 ng / mL; L-alanine 890 ng / mL; L-asparagine 1320 ng / mL; L-aspartic acid 1330 ng / mL; and L-glutamic acid 1470 ng / mL. The concentration of L-proline in culture medium II was 1150 ng / mL; the concentration of L-serine in culture medium II was 1050 ng / mL.

[0119] M2 culture medium (i.e., culture medium III) is a culture medium containing GSK3 inhibitors, that is, a liquid prepared by using culture medium II as a solvent and GSK3 inhibitors as a solute. The concentration of GSK3 inhibitors (such as CHIR-99021) in culture medium III can be 1-2 μM or 2 μM.

[0120] In the following examples, culture medium III specifically refers to RPMI 1640 culture medium containing 2% (v / v) insulin-free B27 supplement (B27minus insulin), 1 mM L-glutamine substitute (Glutamax), 1% (v / v) non-essential amino acids (NEAA), 100 U / ml penicillin, 100 μg / ml streptomycin, 50 ng / ml vitamin C, 5 ng / ml human bone morphogenetic protein 4 (BMP4), and 2 μM GSK3 inhibitor CHIR-99021. The concentrations of each component of the non-essential amino acids in culture medium III are as follows: glycine 750.0 ng / mL; L-alanine 890 ng / mL; L-asparagine 1320 ng / mL; L-aspartic acid 1330 ng / mL; and L-glutamic acid 1470 ng / mL. The concentration of L-proline in culture medium III was 1150 ng / mL; the concentration of L-serine in culture medium III was 1050 ng / mL.

[0121] M3 culture medium (i.e., culture medium IV) is a culture medium containing B27 supplement with added insulin, human vascular endothelial growth factor VEGF-165, and human fibroblast growth factor. It can be further prepared as RPMI 1640 culture medium containing 1%-2% B27 supplement with added insulin (B27 supplement), 0.5-1mM L-glutamine substitute (Glutamax), 0.5%-1% non-essential amino acids (NEAA), 50-100 U / ml penicillin, 50-100 μg / ml streptomycin, 25-50 ng / ml vitamin C, 25-50 ng / ml human vascular endothelial growth factor VEGF-165, and 5-10 ng / ml human fibroblast growth factor (bFGF).

[0122] The M3 culture medium (i.e., culture medium IV) in the following examples is specifically an RPMI 1640 culture medium containing 2% (v / v) of insulin-added B27 supplement, 1 mM L-glutamine substitute (Glutamax), 1% (v / v) of non-essential amino acids, 100 U / ml penicillin, 100 μg / 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 (bFGF). The concentrations of each component of the non-essential amino acids in the M3 culture medium (culture medium IV) are as follows: glycine 750.0 ng / mL; L-alanine 890 ng / mL; L-asparagine 1320 ng / mL; and L-aspartic acid 1330 ng / mL. The concentrations of L-glutamic acid, L-proline, and L-serine in culture medium IV were 1470 ng / mL, 1150 ng / mL, and 1050 ng / mL, respectively.

[0123] M4 culture medium (i.e., culture medium V) is a culture medium containing human macrophage colony-stimulating factor (HMP), which is a liquid prepared using culture medium IV as the solvent and HMP as the solute. The concentration of HMP in culture medium V can be 50-100 ng / ml or 50 ng / ml.

[0124] The M4 culture medium (i.e., culture medium V) in the following examples is specifically an RPMI 1640 culture medium containing 2% (v / v) of insulin-added B27 supplement, 1 mM L-glutamine substitute (Glutamax), 1% (v / v) of non-essential amino acids, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 ng / ml vitamin C, 50 ng / ml human vascular endothelial growth factor VEGF-165, 10 ng / ml human fibroblast growth factor (bFGF), and 50 ng / ml human macrophage colony-stimulating factor (M-CSF). The concentrations of each component of the non-essential amino acids in the M4 culture medium (culture medium V) are as follows: glycine 750.0 ng / mL; L-alanine 890 ng / mL; L-asparagine 1320 ng / mL. The concentrations of L-aspartic acid, L-glutamic acid, L-proline, and L-serine in culture medium V were 1330 ng / mL, 1470 ng / mL, 1150 ng / mL, and 1050 ng / mL, respectively.

[0125] M5 culture medium (i.e., culture medium VI) is a culture medium containing B27 supplement with added insulin, human interleukin-3, and human macrophage colony-stimulating factor. It can be further prepared as RPMI 1640 culture medium containing 1%-2% B27 supplement with added insulin (B27 supplement), 0.5-1 mM L-glutamine substitute (Glutamax), 0.5%-1% non-essential amino acids (NEAA), 50-100 U / ml penicillin, 50-100 ug / ml streptomycin, 25-50 ng / ml vitamin C, 10-20 ng / ml human interleukin-3 (IL3), and 50-100 ng / ml human macrophage colony-stimulating factor (M-CSF).

[0126] The M5 culture medium (i.e., culture medium VI) in the following examples is specifically an RPMI 1640 culture medium containing 2% (v / v) of insulin-added B27 supplement, 1 mM L-glutamine substitute (Glutamax), 1% (v / v) of non-essential amino acids (NEAA), 100 U / ml penicillin, 100 ug / ml streptomycin, 50 ng / ml vitamin C, 10 ng / ml human interleukin-3 (IL3), and 50 ng / ml human macrophage colony-stimulating factor (M-CSF). The concentrations of each component of the non-essential amino acids in the M5 culture medium (culture medium VI) are as follows: glycine 750.0 ng / mL; L-alanine 890 ng / mL; L-asparagine 1320 ng / mL; and L-aspartic acid 1330 ng / mL. The concentrations of L-glutamic acid in culture medium VI were 1470 ng / mL; the concentrations of L-proline in culture medium VI were 1150 ng / mL; and the concentrations of L-serine in culture medium VI were 1050 ng / mL.

[0127] Non-essential amino acids (NEAA, 100×): Gibco, Product No.: 11140050

[0128] B27 supplement with added insulin: Gibco, catalog number: 17504-044.

[0129] Insulin-free B27 supplement (B27 minus insulin): Gibco, catalog number: A1895601.

[0130] L-Glutamine substitute (Glutamax): Gibco, catalog number: 35050061.

[0131] Vitamin C: Sigma Aldrich, product number: A4403.

[0132] ROCK inhibitor Y27632 (CAS 146986-50-7): TargetMol, catalog number: T1870. The structural formula of Y27632 is as follows:

[0133]

[0134] Human bone morphogenetic protein 4 (BMP4): Peprotech, catalog number: 120-05; the amino acid sequence of human bone morphogenetic protein 4 is SEQ ID No. 1.

[0135] Human vascular endothelial growth factor (VEGF-165): Suzhou Nearshore Protein Technology Co., Ltd., catalog number: C083; the amino acid sequence of angiogenesis factor (VEGF-165) is SEQ ID No.2.

[0136] Human fibroblast growth factor (bFGF): Suzhou Nearshore Protein Technology Co., Ltd., product number: C779; the amino acid sequence of human fibroblast growth factor (bFGF) is SEQ ID No. 3.

[0137] Human interleukin-3 (IL3): Suzhou Nearshore Protein Technology Co., Ltd., catalog number: CX90; the amino acid sequence of human interleukin-3 (IL3) is SEQ ID No.4.

[0138] Human macrophage colony-stimulating factor (M-CSF): Suzhou Nearshore Protein Technology Co., Ltd., catalog number: C417; the amino acid sequence of human macrophage colony-stimulating factor (M-CSF) is SEQ ID No. 5.

[0139] GSK3 inhibitor CHIR-99021: Tocris Biosciences, catalog number: 4423 / 10. The structural formula of CHIR-99021 is as follows:

[0140]

[0141] Accutase, a cell digestion solution: Shanghai Yisheng Biotechnology Co., Ltd., product number: 40506ES60.

[0142] Matrigel: BD Biosciences, catalog number: 356231.

[0143] FITC-labeled CD45 antibody: Miltenyi Pharmaceuticals, catalog number: 304005.

[0144] APC-labeled CD14 antibody: Biolegend, catalog number: 325607.

[0145] PerCP-Cy5.5 labeled CD11b antibody: Biolegend, catalog number: 301417.

[0146] PE-labeled CD163 antibody: Biolegend, catalog number: 326505.

[0147] Anti-human CD163-APC antibody: Biolegend, catalog number: 326509.

[0148] Lipopolysaccharide (LPS): Sigma-Aldrich, product number: L2880-10MG.

[0149] Rapid Wright-Giemsa stain solution: Shanghai Yisheng Biotechnology Co., Ltd., product number: 60529ES02.

[0150] Trizol® RNA Extraction Buffer: Invitrogen: Catalog No.: 15596026.

[0151] 5×All-In-One RT MasterMix Reverse Transcription Kit: abm, catalog number: G490.

[0152] GoTaq® qPCR master mix Real-time PCR Kit: Promega, Catalog No.: A6001.

[0153] Cell counting chamber: Countstar, catalog number: 12-0005-50.

[0154] Dimethyl sulfoxide: MP Biomedicals, catalog number: 196055.

[0155] Fetal bovine serum: Biological Industries, catalog number: 04-001-1ACS.

[0156] Glutaraldehyde: Shanghai Maclean Biochemical Technology Co., Ltd., Product No.: G849973-500ml.

[0157] Sodium borohydride: Sigma-Aldrich, part number: 480886-25G.

[0158] Example 1: Preparation of macrophages from human pluripotent stem cell differentiation

[0159] In this embodiment, two types of human pluripotent stem cells were used: H1 cells and CD34-iPSC cells. The specific steps for differentiating macrophages using human pluripotent stem cells are as follows:

[0160] 1. Seed human pluripotent stem cells into 12-well plates (1.5 × 10⁶ cells per well). 5 (100 cells) were cultured in TeSR-E8 medium at 37°C until the cell confluence reached 70%-80%.

[0161] 2. After completing step 1, remove the 12-well plate, aspirate the culture supernatant, and wash twice with RPMI 1640 medium preheated to 37°C. At this point, the morphological images of H1 cells and CD34-iPSC cells are shown below. Figure 1 (Scale bar 250 micrometers).

[0162] 3. After completing step 2, take the 12-well plate, add 1 ml of cell digestion solution Accutase to each well, incubate at 37°C for 3-5 minutes, then terminate the digestion with 1 ml of RPMI 1640 basal culture medium, and collect the cells by centrifugation.

[0163] 4. Seed the cells collected in step 3 into culture dishes (the culture dishes have been coated with Matrigel at 37°C for 2 hours), at a seeding density of 2.0 × 10⁶ cells / mL. 4 pcs / cm 2 -4.0×10 4 pcs / cm 2 The culture was carried out in E8-Y medium (i.e., culture medium I) at 37℃ in a 5% CO2 incubator for 1 day.

[0164] 5. After completing step 4, take the culture dish, discard the culture supernatant, replace it with M1 culture medium (i.e., culture medium II), and culture it in a 37°C, 5% CO2 incubator for 1 day. Human pluripotent stem cells are induced to differentiate into mesodermal cells through steps 4 and 5.

[0165] 6. After completing step 5, remove the culture dish, discard the culture supernatant, and replace it with M2 culture medium (i.e., M1 culture medium containing 2 μM GSK3 inhibitor CHIR-99021), and incubate at 37°C and 5% CO2 for 2 days.

[0166] 7. After completing step 6, take the culture dish, discard the culture supernatant, add 1 ml of Accutase cell digestion solution to digest until single cells are formed, then add 1 ml of RPMI 1640 basal culture medium to stop digestion, and centrifuge at 300 g for 3 minutes to collect the cells.

[0167] 8. Seed the cells collected in step 7 into 3D microscaffolds at a seeding density of 6 × 10⁻⁶. 5 pcs / cm 2 -10×10 5 pcs / cm 2 M3 culture medium (i.e., culture medium IV) was used and cultured in a 37℃, 5% CO2 incubator for 3 days (with some of the culture medium replaced with fresh M3 culture medium (i.e., culture medium IV) each day).

[0168] 9. After completing step 8, replace the culture medium with M4 culture medium (i.e., culture medium V) and incubate at 37℃ in a 5% CO2 incubator for 3 days (replacing part of it with fresh M4 culture medium each day).

[0169] 10. After completing step 9, transfer the cell-bearing 3D microscaffold to M5 culture medium (i.e., culture medium VI) and culture at 37°C in a 5% CO2 incubator for 20-80 days. Macrophages will continuously generate within the 3D microscaffold, and newly generated macrophages will emerge from it. Collect a batch of newly generated macrophages emerging from the 3D microscaffold every 3-4 days. The first three batches of macrophages generated in the 3D microscaffold have a strong amplification capacity; these three batches can be transferred to M5 culture medium and cultured for further expansion for 5-10 days.

[0170] In step 10, the cell-laden 3D microscaffolds are transferred to M5 culture medium (i.e., culture medium VI) and cultured in a 37°C, 5% CO2 incubator. Every 3-4 days, a batch of cells that have migrated from the 3D microscaffolds is collected. The first three batches (first, second, and third batches) show strong cell proliferation ability; these three batches are transferred to M5 culture medium and cultured for another 5-10 days. After 5-10 days of culture, when the cells no longer proliferate, they are digested with acutase for 5 minutes and collected. The fourth and fifth batches, as well as subsequent batches, have weaker cell proliferation ability from the 3D microscaffolds and do not require further expansion culture; they are collected directly.

[0171] In the above steps, the 3D microscaffold is a gelatin porous microscaffold. The steps for preparing the 3D microscaffold using the 3D microscaffold material described in this article are as follows:

[0172] (1) Prepare a gelatin precursor solution by mixing 4% gelatin powder and 3% dimethyl sulfoxide in deionized water.

[0173] (2) Incubate the premixed solution on ice for 5 minutes.

[0174] (3) Add 0.3% glutaraldehyde and stir the solution for 20 seconds.

[0175] (4) Add 400 μl of solution into a polymethyl methacrylate plastic mold with dimensions of 20 mm × 20 mm × 1 mm. Freeze in a -20°C freezer for 16 h to form a cryogel.

[0176] (5) After cryogelation for 16 hours, the mold containing the cryogel was placed at room temperature to melt the ice crystals formed by dimethyl sulfoxide and then carefully washed with deionized water to remove residual reagents, thus obtaining gelatin microscaffolds.

[0177] (6) The obtained gelatin microscaffold was placed in a 1% sodium borohydride solution (pH=9.7) for 10 minutes to remove residual glutaraldehyde, and then washed with deionized water.

[0178] (7) The cleaned gelatin microscaffolds were collected in a container, placed at -20°C for 2 hours, and then freeze-dried for 2 hours.

[0179] (8) Collect gelatin microscaffolds and store them in a low vacuum environment for cell differentiation culture.

[0180] During the above culture process, human pluripotent stem cells are first induced to differentiate into mesodermal cells (…). Figure 1 and Figure 2 The resulting mesodermal cells were then collected and seeded into 3D microscaffolds. Figure 3 Through gradual induction and differentiation, a macrophage-generating microenvironment is formed within a 3D microscaffold. Figure 4 Furthermore, macrophages are continuously generated from the 3D microscaffold, and a batch can be collected every 3-4 days. Microscopic observation and Giemsa staining images show that the cells contain many vacuolar structures of varying sizes, with an elongated spindle shape and an adherent state. Figure 5 Therefore, the cells generated by the 3D microscaffold have a standard macrophage morphology.

[0181] Example 2: Detection of cells during the differentiation of pluripotent stem cells into macrophages

[0182] I. Immunofluorescence detection of macrophages in 3D microscaffolds

[0183] 1. Take the 3D microscaffold for generating macrophages from step 10 in Example 1, fix it with 4% paraformaldehyde at room temperature for 10 minutes, then remove the 4% paraformaldehyde and wash it 3 times with PBS buffer.

[0184] 2. After completing step 1, add PBS buffer containing 5% (volume fraction) BSA and block at room temperature for 1 h.

[0185] 3. After completing step 2, add anti-human CD163-APC antibody (volume ratio 1:100), incubate at 4℃ for 16 h, and then wash 3 times with PBST (PBS buffer containing 0.1% Tween-20).

[0186] 4. After completing step 3, add DAPI (volume ratio 1:1000), incubate at room temperature for 10 minutes, and then wash three times with PBST buffer. Observe the cell staining under a laser confocal microscope.

[0187] Immunofluorescence results of macrophages in 3D microscaffolds are shown in [Figure number missing]. Figure 4 The results showed that macrophages differentiated from human pluripotent stem cells formed a unique 3D tissue microenvironment that promoted macrophage generation within the 3D microscaffold.

[0188] II. Giemsa staining assay for macrophages

[0189] The experimental cells were macrophages prepared by differentiation of human pluripotent stem cell H1 cells.

[0190] The experimental cells were prepared as follows: In step 10, the cell-laden 3D microscaffolds were transferred to M5 culture medium (i.e., culture medium VI) and cultured in a 37°C, 5% CO2 incubator. Every 3-4 days, a batch of cells that had migrated from the 3D microscaffolds was collected. The first three batches (first, second, and third batches) showed strong cell proliferation ability; these cells were then transferred to M5 culture medium and cultured for another 5-10 days. After 5-10 days of culture, when the cells stopped proliferating, they were digested with acutase for 5 minutes and collected. The collected cells were then subjected to Moussa staining. The fourth and fifth batches showed weaker cell proliferation ability; these two batches were directly subjected to Moussa staining after collection without further expansion culture.

[0191] 1. Adjust the concentration of experimental cells to 1×10⁻⁶. 7 Take 10 μL and drop it onto a glass slide. Keep the pusher slide at a 30-degree angle to the glass slide and place it directly in front of the droplet. Move it slightly back to contact the droplet. You will see the droplet spread along the lower edge of the pusher slide. Then slide it smoothly forward along the plane of the glass slide at a uniform speed to create a uniform cell sap film.

[0192] 2. Draw lines on both ends of the liquid film with a crayon and place it flat on the staining rack. The liquid film must be completely dry before staining; otherwise, the liquid film will easily fall off during staining.

[0193] 3. Add 400 μl of rapid Wright-Giemsa stain solution to quickly cover the liquid film and stain for about 1 minute.

[0194] 4. Do not discard reagent one. Directly add 1 ml of reagent two, gently shake the slide or blow air into the blood smear with a bulb syringe to mix the staining solution thoroughly, and stain for 5-8 minutes.

[0195] 5. Wash with water for 10 minutes, air dry, observe and photograph under a microscope.

[0196] Giemsa staining results are shown in Figure 5 The third line shows that the staining results show that the cells turn purple after staining. The stained cells are relatively large and contain many hollow structures in the cytoplasm, exhibiting typical macrophage morphological characteristics.

[0197] III. Flow Cytometry Detection of Macrophage Surface Markers

[0198] The experimental cells were macrophages prepared by differentiation of human pluripotent stem cell H1 cells.

[0199] The experimental cells were prepared as follows: In step 10, the 3D microscaffolds containing cells were transferred to M5 culture medium (i.e., culture medium VI) and cultured in a 37°C, 5% CO2 incubator. A batch was collected every 3-4 days, and five batches of cells were collected (first batch, second batch, third batch, fourth batch, and fifth batch). The cells from the first three batches (first batch, second batch, and third batch) were transferred to M5 culture medium and cultured for expansion for 5-10 days. After expansion, the cells were used for flow cytometry to detect macrophage surface markers. The fourth and fifth batches of cells had weak expansion capacity, and these two batches of cells were directly used for flow cytometry to detect macrophage surface markers.

[0200] 1. Digest the experimental cells with acutase for 5 minutes, collect the cells, and centrifuge at 300 g for 3 minutes. Resuspend the cells in PBS buffer containing 5% (v / v) fetal bovine serum to obtain a cell suspension (containing 1×10⁻⁶ cells / mL). 5 (cells).

[0201] 2. Add FITC-labeled CD45 antibody, Percp-Cy5.5-labeled CD11b antibody, APC-labeled CD14 antibody, and PE-labeled CD163 antibody to the cell suspension from step 1, and incubate at room temperature in the dark for 10 minutes. Then wash twice with PBS buffer containing 5% (v / v) fetal bovine serum, and collect the cells by centrifugation.

[0202] 3. After completing step 2, resuspend the cells in 300 μl of PBS buffer containing 5% (volume fraction) fetal bovine serum and detect them using flow cytometry.

[0203] Cell detection results obtained from H1 cell differentiation are as follows: Figure 6 As shown in the figure. The results indicate that, when cells differentiated using culture medium M4 were collected five times consecutively every 3-4 days from day 15 onwards, more than 80% of the cells co-expressed typical macrophage surface markers such as CD45, CD11b, CD14, and CD163. This demonstrates that the 3D macrophage differentiation system established using the differentiation medium and 3D microscaffold developed in this method can stably generate a high proportion of macrophages over a long period of time and in multiple batches.

[0204] IV. Plotting the cumulative curve of macrophage generation

[0205] 1. Record the number of human pluripotent stem cells collected in step 4 of Example 1 and the collection time point, the number of mesodermal cells differentiated after step 6 and the collection time point, the number of mesodermal cells seeded onto the 3D microscaffold in step 8, and the number of macrophages continuously generated in step 10 and the collection time point. The cell count method is as follows: after thoroughly mixing the cell suspension obtained each time, take 20 μl and add it to a cell counting chamber. Calculate the cell density using a Countstar cell counter and multiply it by the corresponding volume of cell suspension to obtain the total number of cells.

[0206] 2. Calculate the number of mesodermal cells generated per million pluripotent stem cells. The formula is: Number of mesodermal cells generated per million pluripotent stem cells = (Number of differentiated mesodermal cells ÷ Number of initially differentiated pluripotent stem cells) × 10 6 .

[0207] 3. Calculate the number of macrophages generated per million human pluripotent stem cells. The formula is: Number of macrophages generated per million human pluripotent stem cells = (Number of macrophages generated per batch per 3D microscaffold ÷ Number of mesodermal cells seeded into each 3D microscaffold) × (Number of differentiated mesodermal cells ÷ Number of initially differentiated human pluripotent stem cells) × 10 6 .

[0208] 4. Plot the cumulative curve of the number of macrophages continuously generated from the differentiation of one million human pluripotent stem cells. The horizontal axis represents the collection time of each batch of macrophages generated from stem cell differentiation, and the vertical axis represents the total number of macrophages collected cumulatively.

[0209] The results are as follows Figure 7 As shown, 1×10 6 Human pluripotent stem cells began differentiation, and by day 23, a total of 4.6 × 10⁶ cells had been generated. 9 Macrophages. A total of 5.5 × 10⁻⁶ macrophages were generated by day 47. 9 Macrophages. This demonstrates that the 3D macrophage differentiation system established using the differentiation medium and 3D microscaffold developed in this method can efficiently generate large quantities of macrophages over a long period and in multiple batches.

[0210] V. Macrophage Phagocytosis Experiment

[0211] The experimental cells were macrophages obtained from the first batch of cells collected in step 10 and cultured for 10 days.

[0212] 1. The macrophages obtained in step 10 of Example 1 are processed at a ratio of 1×10⁻⁶. 5 pcs / cm 2 The cells were seeded at a density in a cell culture plate and cultured overnight to allow them to adhere to the plate.

[0213] 2. On the second day, E. coli with GFP fluorescent labeling was inoculated onto the cells at a concentration of 1:5 (E. coli:cells) and incubated for 2-4 hours; mesenchymal stem cells were used as a negative control.

[0214] 3. After 2-4 hours, aspirate the culture medium, gently wash three times with PBS, then gently scrape the cells off with a pipette tip, pipette them into a single-cell suspension, and detect the phagocytic ratio by flow cytometry.

[0215] The results are as follows Figure 8 As shown, macrophages can engulf GFP-labeled E. coli into their cells. Flow cytometry analysis showed that 98% of macrophages engulfed GFP-labeled E. coli after two hours of incubation, while the mesenchymal cells of the negative control did not have the ability to engulf fungi. This demonstrates that the differentiated macrophages have the ability to engulf pathogenic microorganisms, which is consistent with the functional characteristics of macrophages.

[0216] VI. LPS-stimulated macrophage experiment

[0217] The experimental cells were macrophages obtained from the first batch of cells collected in step 10 and cultured for 10 days.

[0218] 1. The macrophages obtained in step 10 of Example 1 are processed at a ratio of 2 × 10⁻⁶. 5 pcs / cm 2 The cells were seeded at a density in a cell culture plate and cultured overnight to allow them to adhere to the plate.

[0219] 2. On the second day, add medium containing 10% non-inactivated fetal bovine serum and 25 ng / ml RPMI 1640, and incubate for 48 hours.

[0220] 3. On the fourth day, cells were stimulated for 48 hours with 10 ng / ml of lipopolysaccharide (LPS). The negative control was not given LPS.

[0221] 4. After completing step 3, aspirate the supernatant, rinse twice with PBS, and then aspirate the PBS.

[0222] 5. After completing step 4, add 1 ml of TRIzol® RNA extraction solution to extract total RNA from the cells.

[0223] 6. After completing step 5, reverse transcription is performed using the 5×All-In-One RT MasterMix Reverse Transcription Kit. Follow the instructions to reverse transcribe and synthesize the first-strand cDNA.

[0224] 7. After completing step 6, using the above cDNA as a template, perform a Q-PCR reaction using the Promega kit (GoTaq® qPCR mastermix real-time PCR kit). The PCR reaction system is shown in Table 1:

[0225]

[0226] The reaction was performed on a CFX 96 (BIO-RAD) real-time PCR instrument under the following conditions:

[0227] 94℃ for 5 min; 94℃ for 30 s;

[0228] 60℃ for 30 seconds, 72℃ for 30 seconds, repeat 40 cycles;

[0229] 72℃ for 10 min.

[0230] The qRT-PCR primer sequences used in this experiment are shown in Table 2.

[0231]

[0232] The results are as follows Figure 9 As shown, after LPS stimulation, macrophages in Example 1 showed significantly upregulation of the expression of inflammation-related cytokines and chemokines such as TNFα, IL1b, IL8, IL10 and CCL2 compared with the control group, exhibiting a response similar to that of macrophages in response to LPS stimulation. This demonstrates that macrophages can upregulate inflammation-related factors in response to inflammatory responses after LPS stimulation.

[0233] The above results indicate that macrophages can be differentiated in large quantities and for a long time using M1, M2, M3, M4, and M5 culture media and 3D microscaffold materials.

[0234] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims.

Claims

1. A method for preparing macrophages, characterized in that, The method includes the following steps: H1) Human pluripotent stem cells were seeded into culture medium I and cultured for 0.5-1.5 days; Replace H2 with culture medium II and continue culturing for 0.5-1.5 days; Replace H3 with culture medium III and continue culturing for 1.5-2.5 days; H4) Digest and collect the cells from step H3), and seed the cells into a 3D microscaffold made of 3D microscaffold material, and culture them in culture medium IV for 2.5-3.5 days; Replace H5 with culture medium V and continue culturing for 2.5-3.5 days; H6) was replaced with culture medium VI and cultured for 20-80 days, and the macrophages were collected. The culture medium I is a stem cell culture medium containing ROCK inhibitors; Culture medium II is an RPMI 1640 culture medium containing 2% (v / v) insulin-free B27 additive, 1 mM L-glutamine substitute, 1% (v / v) non-essential amino acids, 100 U / ml penicillin, 100 μg / ml streptomycin, 50 ng / ml vitamin C, and 5 ng / ml human bone morphogenetic protein 4. The non-essential amino acids consist of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. The concentrations of glycine, L-alanine, L-glutamic acid, L-proline, and L-serine in culture medium II are as follows: glycine 750.0 ng / mL; L-alanine 890 ng / mL; L-asparagine 1320 ng / mL; L-aspartic acid 1330 ng / mL; L-glutamic acid 1470 ng / mL; and L-proline 1150 ng / mL. ng / mL; the concentration of L-serine in culture medium II was 1050 ng / mL; The culture medium III is a culture medium containing a GSK3 inhibitor. The culture medium is a liquid prepared by using the culture medium II as a solvent and the GSK3 inhibitor as a solute. The GSK3 inhibitor is CHIR-99021. The concentration of CHIR-99021 in the culture medium III is 2 μM. Culture medium IV was an RPMI 1640 culture medium containing 2% (v / v) insulin-added B27 supplement, 1 mM L-glutamine substitute, 1% (v / v) non-essential amino acids, 100 U / ml penicillin, 100 μg / 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. The non-essential amino acids consisted of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. The concentrations of glycine, L-alanine, L-asparagine, L-glutamic acid, L-proline, and L-serine in culture medium IV were: 750.0 ng / mL; L-alanine: 890 ng / mL; L-asparagine: 1320 ng / mL; L-aspartic acid: 1330 ng / mL; and L-glutamic acid: 1470 ng / mL. The concentration of L-proline in culture medium IV was 1150 ng / mL; the concentration of L-serine in culture medium IV was 1050 ng / mL. The culture medium V is a culture medium containing human macrophage colony-stimulating factor (HMP). The culture medium is a liquid prepared by using culture medium IV as a solvent and HMP as a solute. The concentration of HMP in culture medium V is 50 ng / ml. Culture medium VI is an RPMI 1640 culture medium containing 2% (v / v) of insulin-added B27 supplement, 1 mM L-glutamine substitute, 1% (v / v) of non-essential amino acids, 100 U / 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. The non-essential amino acids consist of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, and L-serine. The concentrations of glycine, L-alanine, L-asparagine, L-glutamic acid, L-proline, and L-serine in culture medium VI are as follows: 750.0 ng / mL; 890 ng / mL; 1320 ng / mL; 1330 ng / mL; and 1470 ng / mL. ng / mL; the concentration of L-proline in culture medium VI was 1150 ng / mL; the concentration of L-serine in culture medium VI was 1050 ng / mL; The 3D microscaffold material is a porous material prepared from a mixture of gelatin solution and a biological cryoprotectant; the pore size of the porous material is 50-100 micrometers, the gelatin solution in the mixture is a deionized gelatin aqueous solution with a mass-volume ratio of 4%, and the biological cryoprotectant is DMSO with a volume fraction of 3%.

2. The method according to claim 1, characterized in that, The stem cell culture medium is prepared using the ROCK inhibitor as a solute and TeSR-E8 culture medium as a solvent.

3. The method according to claim 1, characterized in that, The inoculation density described in step H4) is 6 × 10⁻⁶. 5 pcs / cm 2 -10×10 5 pcs / cm 2 .

4. The method according to any one of claims 1-3, characterized in that, The method further includes step H7), which is: culturing the macrophages collected in step H6) in culture medium VI for 5-10 days, wherein the collection is carried out by collecting a batch of macrophages every 3-4 days.

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