CN119060950A

CN119060950A - Cell membrane sheet, decellularized matrix material and preparation method thereof

Info

Publication number: CN119060950A
Application number: CN202410694733.3A
Authority: CN
Inventors: 周琪; 李伟; 郝捷; 吴骏; 顾奇; 王柳; 郭保杰; 魏武妹; 赵喜源
Original assignee: Beijing Institute Of Stem Cell And Regenerative Medicine; Institute of Zoology of CAS
Current assignee: Beijing Institute Of Stem Cell And Regenerative Medicine; Institute of Zoology of CAS
Priority date: 2023-06-02
Filing date: 2024-05-31
Publication date: 2024-12-03

Abstract

The invention provides a cell membrane which expresses immune regulation related factors, nutritional factors and extracellular matrix related proteins in a high mode and expresses pro-inflammatory factors in a low mode and a preparation method of the cell membrane. The invention also provides decellularized matrix materials based on the cell membrane sheets and composite cell membrane sheets based on the same. The invention also provides application of the cell membrane, the composite cell membrane and the decellularized matrix material in regenerative medicine.

Description

Cell patch, acellular matrix material and preparation method thereof

Technical Field

The invention relates to the field of regenerative medicine, and particularly provides a cell membrane which highly expresses immune regulation related factors, nutritional factors and extracellular matrix related proteins and low expresses pro-inflammatory factors, a preparation method thereof, a cell membrane cell material based on the cell membrane cell and a composite cell membrane cell based on the cell membrane cell material, and application of the cell membrane cell, the composite cell membrane cell and the cell membrane cell material in regenerative medicine.

Background

Extracellular matrix is a complex network of macromolecular substances synthesized and secreted by various tissues and cells in the body, which are distributed and aggregated on the cell surface and on intercellular substances. The acellular matrix refers to an extracellular matrix material with cell components removed, and is used as a natural scaffold, so that the acellular matrix material has bioactivity, biocompatibility and non-immunogenicity, can retain cell growth factors, enhances growth, migration, proliferation, differentiation and angiogenesis of seed cells, and is important for regeneration and functional repair of tissues and organs.

The application of acellular matrix in regenerative medicine and tissue engineering is becoming more and more popular, and the acellular matrix can take various forms including membrane, powder and hydrogel, wherein the hydrogel has strong plasticity, can be used in different modes and combinations, such as being used as a coating, a cell carrier, a drug carrier such as a growth factor, a mixed/modified material, biological ink for 3D printing of tissues, and the like, and has wide application prospect. The current research on hydrogels has mostly focused on hydrogels derived from heterogeneous tissues or hydrogels derived from human tissues, the heterogeneous tissues have a risk of carrying pathogens, the human tissues are limited, and the organs have great individual differences. There are also relatively few studies on the preparation of hydrogels from cellular sources.

The decellularized matrix hydrogels can be prepared from cell sheets that form endogenous scaffolds from extracellular matrix secreted by the cells themselves, although some methods of preparing cell sheets have been reported, there remains a need in the art for cell sheets with better bioactivity.

Disclosure of Invention

The inventors provided in previous studies Immune and Matrix Regulating Cells (IMRC) that have mesenchymal stem cell-like properties but at the same time possess unique gene expression profiles that differ from Umbilical Cord Mesenchymal Stem Cells (UCMSCs). The inventors have unexpectedly found that cell membranes which secrete more active factors and extracellular matrix-related proteins and have better mechanical properties can be prepared based on the cells, thereby providing the following invention.

Cell membrane

In a first aspect, the invention provides a cell patch formed by immune and stromal regulatory cells (IMRC) and their secreted extracellular matrix, wherein the extracellular matrix comprises at least COL15A1, COL8A1, EFEMP2, FBLN5, MFAP2, MXRA5, PCOLCE, THBS2, LOXL2, TIMP1, CLEC2D, HPX, BGN, GDF.

In certain embodiments, the amount of the above-described protein factor is increased compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane). In certain embodiments, the amount of the above-described protein factors is each independently increased by at least about 1.2-fold, at least about 1.5-fold, at least about 1.8-fold, at least about 2-fold, at least about 2.5-fold, at least about 3-fold, at least about 3.5-fold, at least about 4-fold, at least about 4.5-fold, at least about 5-fold, at least about 5.5-fold, at least about 6-fold, at least about 6.5-fold, at least about 7-fold, at least about 7.5-fold, at least about 8-fold, at least about 8.5-fold, at least about 9-fold, at least about 9.5-fold, at least about 10-fold, at least about 15-fold, at least about 20-fold, at least about 25-fold, at least about 30-fold, at least about 35-fold, at least about 40-fold, at least about 45-fold, at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold over primary mesenchymal stem cell membrane (e.g.g).

In certain embodiments, the amount of any of the proteins described above is determined by subjecting the decellularized patch (i.e., decellularized matrix) to protein mass spectrometry. In certain embodiments, the cell patch that is tested is a patch that is cultured for 21 days.

In certain embodiments, the cell membrane possesses increased FN expression levels, ELN expression levels, and/or POSTN expression levels as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

In certain embodiments, the cell membrane has an FN expression level of at least about 5-fold, at least about 6-fold, e.g., about 7-fold, as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane). In certain embodiments, the FN expression level is about 5-10 fold, about 5-9 fold, about 5-8 fold, about 6-10 fold, about 6-9 fold, or about 6-8 fold.

In certain embodiments, the cell membrane has an ELN expression level of at least about 50-fold (e.g., at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 110-fold, at least about 120-fold, or at least about 130-fold; e.g., about 131-fold) as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane). In certain embodiments, the ELN expression level is about 100-150 fold, about 100-145 fold, about 100-140 fold, about 100-135 fold, about 110-150 fold, about 110-145 fold, about 110-140 fold, about 110-135 fold, about 120-150 fold, about 120-145 fold, about 120-140 fold, about 120-135 fold, or about 125-135 fold.

In certain embodiments, the cell membrane has a POSTN expression level of at least about 50-fold (e.g., at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 110-fold, at least about 120-fold, or at least about 130-fold; e.g., about 131.84-fold) compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane). In certain embodiments, the POSTN expression level is about 100-150 fold, about 100-145 fold, about 100-140 fold, about 100-135 fold, about 110-150 fold, about 110-145 fold, about 110-140 fold, about 110-135 fold, about 120-150 fold, about 120-145 fold, about 120-140 fold, about 120-135 fold, or about 125-135 fold.

In certain embodiments, the cell membrane has a FN expression level of about 6-8 times, an ELN expression level of about 120-140 times (e.g., about 120-135 times, about 125-135 times), and a POSTN expression level of about 120-140 times (e.g., about 120-135 times, about 125-135 times) as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

In certain embodiments, the cell membrane has a COL1A1 expression level of at least about 8-fold (e.g., at least about 9-fold, e.g., about 9.9-fold), such as about 8-12-fold, about 8-11-fold, about 8-10-fold, about 9-12-fold, about 9-11-fold, about 9-10-fold, as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

In certain embodiments, the cell membrane has a COL1A2 expression level of at least about 10-fold (e.g., at least about 11-fold, at least about 12-fold, e.g., about 12.98-fold), such as about 10-15-fold, about 10-14-fold, about 10-13-fold, about 11-15-fold, about 11-14-fold, about 11-13-fold, as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

In certain embodiments, the cell membrane has a COL4A1 expression level of at least about 5-fold (e.g., at least about 6-fold, e.g., about 6.74-fold), such as about 5-10-fold, about 5-9-fold, about 5-8-fold, about 5-7-fold, about 6-7-fold, as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

In certain embodiments, the cell membrane has a TNC expression level of at least about 18-fold (e.g., at least about 19-fold, at least about 20-fold, or at least about 21-fold, e.g., about 21.13-fold), such as about 18-25-fold, about 18-24-fold, about 18-23-fold, about 18-22-fold, about 19-25-fold, about 19-24-fold, about 19-23-fold, about 19-22-fold, about 20-25-fold, about 20-24-fold, about 20-23-fold, about 20-22-fold, as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

In certain embodiments, the cell membrane has a COL4A2 expression level of at least about 8-fold (e.g., at least about 9-fold, such as about 9.54-fold), such as about 8-fold to about 12-fold, about 8-fold to about 11-fold, about 8-fold to about 10-fold, about 9-fold to about 12-fold, about 9-fold to about 11-fold, about 9-fold to about 10-fold, as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

In certain embodiments, the cell membrane has an LAMC1 expression level of at least about 1.1-fold (e.g., at least about 1.2-fold, e.g., about 1.28-fold), such as about 1.1-1.5-fold, about 1.1-1.4-fold, about 1.1-1.3-fold, about 1.2-1.5-fold, about 1.2-1.4-fold, about 1.2-1.3-fold, as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

In certain embodiments, the cell membrane has a CXCL12 expression level of at least about 1.2-fold (e.g., at least about 1.3-fold, at least about 1.4-fold, e.g., about 1.49-fold), such as about 1.2-1.8-fold, about 1.2-1.7-fold, about 1.2-1.6-fold, about 1.2-1.5-fold, about 1.3-1.8-fold, about 1.3-1.7-fold, about 1.3-1.6-fold, about 1.3-1.5-fold, as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

In certain embodiments, the cell membrane is provided with one or more characteristics selected from the group consisting of (1) COL1A1 expression levels of about 8-12 fold (e.g., about 8-11 fold, about 8-10 fold, about 9-12 fold, about 9-11 fold, about 9-10 fold) compared to primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane); (2) COL1A2 expression levels of about 11 to 15 times (e.g., about 11 to 14 times, about 11 to 13 times), (3) COL4A1 expression levels of about 5 to 8 times (e.g., about 5 to 7 times, about 6 to 7 times), (4) TNC expression levels of about 20 to 24 times (e.g., about 20 to 23 times, about 20 to 22 times), (5) COL4A2 expression levels of about 8 to 12 times (e.g., about 8 to 11 times, about 8 to 10 times, about 9 to 12 times, about 9 to 11 times, about 9 to 10 times), and (6) LAMC1 expression levels of about 1.1 to 1.4 times (e.g., about 1.1 to 1.3 times, about 1.2 to 1.4 times, about 1.2 to 1.3 times), and (7) CXCL12 expression levels of about 1.2 to 1.6 times (e.2 to 1.5 times, about 1.3 to 1.6 times, about 1.3 times).

In certain embodiments, the expression level described in any one of the embodiments above is mRNA level. In certain embodiments, the mRNA expression level is determined by extracting total RNA of the cell membrane and performing RT-PCR or single cell RNA sequencing (scRNA-seq). In certain embodiments, the mRNA expression level is determined by RT-PCR using the primer sequences set forth in SEQ ID NOs: 1-30.

In certain embodiments, the cell patch detected in any of the above embodiments is a patch cultured for 21 days.

In certain embodiments, the cell membrane secretes LIF and exhibits at least about a 300-fold (e.g., at least about 350-fold, at least about 400-fold; e.g., about 437-fold) increase, such as about 300-500-fold, about 300-450-fold, about 350-450-fold, or about 400-450-fold increase, as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane). In certain embodiments, the LIF is at the protein level. In certain embodiments, the secretion change is determined by culturing the cell membrane in culture supernatant (e.g., 21 days of culture). In certain embodiments, the cell patch that is tested is a patch that is cultured for 21 days.

In certain embodiments, the LIF content is at least about 100pg/mL (e.g., at least about 110pg/mL, at least about 120pg/mL, at least about 150pg/mL, at least about 160pg/mL, at least about 180pg/mL, or at least about 190pg/mL; e.g., about 190.19 pg/mL), e.g., about 100-300 pg/mL, about 100-250 pg/mL, about 100-220 pg/mL, about 100-210 pg/mL, about 100-200 pg/mL, about 120-300 pg/mL, about 120-250 pg/mL, about 120-220 pg/mL, about 120-210 pg/mL, about 120-200 pg/mL, about 150-300 pg/mL, about 150-220 pg/mL, about 180-180 pg/mL, about 180-185 pg/mL. In certain embodiments, the secretion amount is determined by culturing the cell membrane in culture supernatant (e.g., 21 days of culture). In certain embodiments, the secretion amount is measured using a Bio-Plex 200 suspension chip system.

In certain embodiments, the cell membrane secretes HGF and exhibits an increase of at least about 1.1 fold (e.g., at least about 1.2 fold, e.g., about 1.25 fold), such as about 1.1-1.5 fold, about 1.1-1.4 fold, about 1.1-1.3 fold, about 1.2-1.5 fold, about 1.2-1.4 fold, about 1.2-1.3 fold, as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane). In certain embodiments, the secretion change is determined by culturing the cell membrane in culture supernatant (e.g., 21 days of culture). In certain embodiments, the cell patch that is tested is a patch that is cultured for 21 days.

In certain embodiments, the HGF content is at least about 100pg/mL (e.g., at least about 110pg/mL; e.g., about 111.86 pg/mL), e.g., about 100 to 120pg/mL, about 105 to 120pg/mL, about 110 to 115pg/mL. In certain embodiments, the secretion amount is determined by culturing the cell membrane in culture supernatant (e.g., 21 days of culture). In certain embodiments, the secretion amount is measured using a Bio-Plex 200 suspension chip system.

In certain embodiments, the proinflammatory factor secreted by the cell membrane is reduced compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

In certain embodiments, the proinflammatory factor is selected from the group consisting of IL6, IL1 alpha, IL1 beta.

In certain embodiments, IL6 exhibits at least about a 100-fold (e.g., at least about a 110-fold, such as about 114.7-fold) decrease, such as about a 100-120-fold, about a 110-120-fold decrease, as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

In certain embodiments, the IL 1a exhibits at least about a 5-fold (e.g., at least about a 6-fold, such as about a 6.16-fold) decrease, such as about a 5-8-fold, about a 5-7-fold, about a 6-8-fold, about a 6-7-fold decrease, as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

In certain embodiments, the IL1 β exhibits at least about a 6-fold (e.g., at least about 7-fold, e.g., about 7.16-fold) decrease, e.g., about 6-9-fold, about 6-8-fold, about 7-9-fold, about 7-8-fold decrease, as compared to a primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

In certain embodiments, the expression level of the pro-inflammatory factor is mRNA level. Preferably by extracting total RNA from the cell membrane and performing RT-PCR or single cell RNA sequencing (scRNA-seq). In certain embodiments, the mRNA expression level is determined by RT-PCR using the primer sequences set forth in SEQ ID NOs: 1-30. In certain embodiments, the cell patch that is tested is a patch that is cultured for 21 days.

In certain embodiments, the cell membrane of the present invention is a sheet-like structure formed by IMRC cells and their secreted extracellular matrix.

In certain embodiments, the extracellular matrix has a fiber diameter of less than 0.5 μm, such as 0.2 to 0.5 μm,0.2 to 0.4 μm,0.3 to 0.4 μm. In certain embodiments, the extracellular matrix has a fiber density of 12-16 strands/25 μm.

In certain embodiments, the young's modulus of the cell membrane is at least 500Pa, e.g., at least 800Pa, at least 1000Pa, at least 2000Pa, at least 3000Pa, or at least 3500Pa.

In certain embodiments, the cell membrane has a thickness of at least 10 μm, e.g., at least 30 μm, at least 50 μm, at least 60 μm, at least 70 μm, at least 80 μm, at least 90 μm, or at least 100 μm.

In certain embodiments, the cell membrane surface is smooth and planar.

As used herein, the term "about" when referring to a measurable value such as an amount, percentage, etc., is meant to encompass a variation of + -10%, + -5%, or + -1% from the measurable value.

Herein, the term "primary mesenchymal stem cells" refers to mesenchymal stem cells isolated directly from tissues (e.g., adipose tissue, umbilical cord, bone marrow, or cord blood) taken out of the body. Illustratively, primary Umbilical Cord Mesenchymal Stem Cells (UCMSCs) as described herein may be obtained by washing with PBS to remove all residual blood from the umbilical cord, removing arteries and veins, cutting the umbilical cord to a size of about 2 millimeters, transferring umbilical cord fragments to a medium supplemented with 5% KOSR, 1% Ultroser G, 1 XL-glutamine, 1 XNEAA, and 5ng/mL bFGF in αMEM at 37℃in a 5% CO ₂ environment. UCMSCs are passaged when reaching 80% confluence. After 5 cycles of culture, the cells were harvested for identification.

Preparation of cell membranes

In a second aspect, the invention provides a method of preparing a cell membrane comprising culturing IMRC cells in a medium comprising vitamin C or a derivative thereof to obtain the cell membrane.

In certain embodiments, the IMRC cells and their secreted extracellular matrix are lamellar.

In certain embodiments, the cell membrane is formed by the IMRC cells and their secreted extracellular matrix.

In certain embodiments, the method further comprises separating the cell membrane from the culture vessel.

In certain embodiments, the vitamin C derivative is selected from the group consisting of ascorbic acid 2-glucoside, ascorbyl glucoside, or erythorbic acid.

In certain embodiments, IMRC cells are seeded into a culture vessel coated with a matrix.

In certain embodiments, the coating matrix is selected from collagen, gelatin, laminin, fibronectin, or derivatives thereof.

In certain embodiments, the IMRC cells are seeded at a density of 1X 10 ³/cm²～1×10⁶/cm², e.g., 1×10³/cm²～1×10⁵/cm²,1×10³/cm²～5×10⁴/cm²,5×10³/cm²～1×10⁵/cm²,5×10³/cm²～5×10⁴/cm²,, e.g., 2X 10 ⁴/cm².

In certain embodiments, the vitamin C or derivative thereof is present in an amount of 1 to 100. Mu. Mol/L, for example 10 to 100. Mu. Mol/L,10 to 80. Mu. Mol/L,10 to 60. Mu. Mol/L,10 to 50. Mu. Mol/L. In certain embodiments, the vitamin C or derivative thereof is present in an amount of 40. Mu. Mol/L.

In certain embodiments, the medium from which the cell patch is prepared is the same medium as the IMRC cells are cultured.

In certain embodiments, the medium is a basal medium to which one or more serum or serum substitutes, one or more non-essential amino acids, stabilized dipeptide of glutamine or L-alanyl-L-glutamine, and one or more growth factors are added.

In certain embodiments, the one or more serum or serum replacement comprises KOSR and ultraser G.

In certain embodiments, the non-essential amino acid is selected from the group consisting of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, and combinations thereof, e.g., NEAA.

In certain embodiments, the basal medium is selected from the group consisting of alpha-MEM, MEM, DMEM, high-sugar DMEM, KO-DMEM, and any combination thereof.

In certain embodiments, the one or more growth factors comprise bFGF and TGF- β, and optionally further comprise one or more selected from VEGF, EGF, or PDGF.

In certain embodiments, the medium is a basal medium to which 1-5% (v/v) Ultroser G, 5-30% (v/v) KOSR, 1-5mM 1 XL-glutamine or L-alanyl-L-glutamine stabilized dipeptide, 1-100ng/mL bFGF, 1-100ng/mL TGFbeta, and amino acids glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine are added at a concentration of each 0.1-0.5mM, the basal medium being selected from KO-DMEM, KO-DMEM/F12, alpha-MEM, DMEM, DMEM/F12.

In certain embodiments, the medium may further comprise one or more selected from VEGF, EGF, PDGF, e.g., at a concentration of each 1-100 ng/mL.

In certain embodiments, the medium is a basal medium (e.g., alpha-MEM) to which are added 5-30% KOSR, 1-5% Ultroser G, 1-5mM L-glutamine, 0.1-0.5mM NEAA, 1-100 ng/mL bFGF, and 1-100 ng/mL TGF-beta. In certain embodiments, the bFGF and TGF- β are present in an amount of 4 to 100ng/mL, for example 4～90ng/mL,4～80ng/mL,4～60ng/mL,4～50ng/mL,4～40ng/mL,4～30ng/mL,4～20ng/mL,4～10ng/mL,5～90ng/mL,5～80ng/mL,5～60ng/mL,5～50ng/mL,5～40ng/mL,5～30ng/mL,5～20ng/mL,5～10ng/mL. in certain embodiments, the bFGF and TGF- β are present in an amount of 5 to 100ng/mL,4 to 50ng/mL, or 5 to 50ng/mL.

In some embodiments, the medium is a basal medium (e.g., alpha-MEM) to which 5-20% KOSR, 1-5% Ultroser G, 1-5 mM L-glutamine, 0.1-0.5 mM NEAA, 5-100 ng/mL bFGF, and 5-100 ng/mL TGF-beta are added.

In some embodiments, the medium is a basal medium (e.g., alpha-MEM) to which 5-10% KOSR, 1-5% Ultroser G, 1-5 mM L-glutamine, 0.1-0.5 mM NEAA, 5-10 ng/mL bFGF, and 5-10 ng/mL TGF-beta are added.

In certain embodiments, the medium is a basal medium (e.g., alpha-MEM) supplemented with 5% KOSR, 1% Ultroser G, 2mM L-glutamine, 0.1mM NEAA and 5ng/mL, bFGF and 5ng/mL TGF-beta.

In certain embodiments, the culture is for at least 1 day. In certain embodiments, the culture is for at least 7 days, such as at least 14 days, such as 14 days to 28 days, such as 14 days to 21 days. In certain embodiments, the culture is for about 21 days.

In certain embodiments, the cell membranes of the invention can be cultured for a prolonged period of time, e.g., for at least 35 days, at least 42 days, or at least 45 days, while still maintaining a smooth surface. Thus, the method of the invention can provide a membrane with a higher thickness by extending the incubation time, for example, for at least about 45 days, to obtain a smooth surface cell membrane with a thickness of up to 100 μm.

In certain embodiments, the method further comprises pre-culturing the IMRC cells in a medium without added vitamin C or a derivative thereof, e.g., for 1-2 days, followed by culturing with added vitamin C or a derivative thereof.

In a third aspect, the invention also provides a cell membrane prepared by the method of the second aspect. In certain embodiments, the cell membrane obtained by the method of the second aspect is as defined in any embodiment of the first aspect.

IMRC cells

IMRC refers to immune-and matrix-regulatory cells (IMRC), see, e.g. Wu,J.,Song,D.,Li,Z.et al.Immunity-and-matrix-regulatory cells derived from human embryonic stem cells safely and effectively treat mouse lung injury and fibrosis.Cell Res 30,794–809(2020).

In certain embodiments, IMRC cells are characterized by cells that express CD73, CD90, CD105, CD29, HLA-ABC, cells that contain ≡90% (e.g.,. Gtoreq.95%,. Gtoreq.96%,. Gtoreq.97%,. Gtoreq.98%,. Gtoreq.99%, or 100%), and cells that contain ≡1% (e.g.,. Ltoreq.0.5%,. Ltoreq.0.2%,. Ltoreq.0.1%, or ≡0.01%) express CD34, CD45, HLA-DR. In certain embodiments, the IMRC cell is CD34^-/CD45^-/HLA–DR^-/CD90⁺/CD29⁺/CD73⁺/CD105⁺.

In certain embodiments, the mRNA expression level of MMP1 of the IMRC cells is at least about 50-fold that of primary Umbilical Cord Mesenchymal Stem Cells (UCMSCs).

In certain embodiments, the surface marker is preferably detected by immunology. In certain embodiments, the mRNA expression level is preferably determined by RT-PCR.

In certain embodiments, the IMRC cell has an MMP1mRNA expression level that is at least about 50-fold, at least about 60-fold, at least about 70-fold, at least about 80-fold, at least about 90-fold, at least about 100-fold, at least about 150-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, at least about 1000-fold, at least about 2000-fold, at least about 3000-fold, at least about 5000-fold, at least about 8000-fold, at least about 10000-fold, or at least about 12000-fold that of the primary umbilical mesenchymal stem cell. In certain embodiments, the IMRC cells have an MMP1mRNA expression level that is at least about 80-fold, at least about 85-fold, at least about 90-fold, at least about 95-fold, at least about 100-fold that of primary umbilical cord mesenchymal stem cells. In certain embodiments, the IMRC cells have an MMP1mRNA expression level that is at least about 100-fold that of primary umbilical cord mesenchymal stem cells.

In certain embodiments, the IMRC cells have an MMP1 positive rate of no less than 90%, such as no less than 95%, no less than 98%, or no less than 99% via single cell RNA sequencing (scRNA-seq). In certain embodiments, the primary UCMSC has a MMP1 positive rate of no greater than 10%, such as no greater than 5%, no greater than 2%, or no greater than 1%.

In certain embodiments, the IMRC cell exhibits an increase (e.g., at least about a 2-fold increase) in the expression level of at least 1 (e.g., at least 2, at least 3, or all) genes selected from the group consisting of LIF, VEGFA, GREM, CDC20, as compared to the expression level of these genes in the primary UCMSC. In certain embodiments, the expression level is an mRNA level, preferably determined by RT-PCR or RNA-seq.

In certain embodiments, the IMRC cell exhibits a decrease (e.g., at least about a 2-fold decrease) in the expression level of at least 1 (e.g., at least 2, at least 3, or all) genes selected from IL-1B, CXCL, CCL2, CXCL1, as compared to the expression level of these genes in the primary UCMSC. In certain embodiments, the expression level is an mRNA level, preferably determined by RT-PCR or RNA-seq.

In certain embodiments, the IMRC has the ability to undergo triple differentiation (tri-lineage differentiation) to form interstitial tissues such as adipocytes, chondrocytes, and osteoblasts.

In certain embodiments, the IMRC is produced from embryonic stem cells (e.g., human embryonic stem cells) or induced pluripotent stem cells.

Preparation of IMRC cells

Detailed teaching has been provided in the art regarding methods of preparing IMRC cells.

For example, an exemplary method can be seen in Wu,J.,Song,D.,Li,Z.et al.Immunity-and-matrix-regulatory cells derived from human embryonic stem cells safely and effectively treat mouse lung injury and fibrosis.Cell Res 30,794–809(2020). wherein, to generate hEBs, hESCs are incubated with 1mg/mL of a dispersing enzyme (Gibco, 17105-04) at 37℃for 5 minutes to disperse to form small pieces, followed by incubation in KO-DMEM (Gibco, A12861-01) supplemented with 20% KOSR (Gibco, A3020902), 1 XL-glutamine (Gibco, A12860-01), 1 XNEAA (Gibco, 11140050) and 10ng/mL bFGF (R & D systems, minneapolis, MN, USA; 233-FB) to form hEBs. Then hEBs was transferred to a plate coated with glass cellulose and incubated for another 14 days. Outgrowth of hEBs takes place during this time. Grown cells were isolated with Tryple (Gibco, A12859-01) and passaged at a low cell density of 1X 10 ⁴ cells per square centimeter in "IMRCs medium" consisting of alpha-MEM (Gibco, 12561-049) and 5% KOSR, 1% Ultroser G (Pall corporation, new York, NY, USA; 159550-017), 1 XL-glutamine, 1 XNEAA, 5ng/mL bFGF and 5ng/mL TGF-beta (Peprotech, 96-100-21-10). After optionally subjecting the cells to at least one passage after reaching 80% confluence in IMRCs medium, the harvested cells are IMRC cells, which show a fibroblast morphology expressing typical mesenchymal stem cell specific surface markers including CD73, CD90, CD105 and CD29, while being negative for typical hematopoietic markers (CD 45, CD34, HLA-DR).

In addition, the present inventors' prior patent application CN202010998258.0 also teaches in detail a method of preparing a mesenchymal stem cell population.

In certain embodiments, the method comprises the steps of:

(1) Culturing stem cells to form embryoid bodies using a first medium, wherein the first medium is a basal medium to which one or more serum substitutes, one or more non-essential amino acids, stabilized dipeptides of glutamine or L-alanyl-L-glutamine, and bFGF are added;

(2) Culturing the embryoid body using a second medium to induce differentiation into IMRC cells, wherein the second medium is a basal medium supplemented with one or more serum substitutes, one or more non-essential amino acids, stabilized dipeptide of glutamine or L-alanyl-L-glutamine, and one or more growth factors.

In certain embodiments, step (2) comprises attaching the embryoid bodies to a culture vessel and culturing using a second medium.

As used herein, the term "basal medium" refers to any medium capable of supporting cell growth, typically comprising inorganic salts, vitamins, glucose, buffer systems, and essential amino acids, and typically having an osmolality of about 280-330 mOsmol.

In certain embodiments, the stem cell described in step (1) is a totipotent stem cell or a pluripotent stem cell. In certain embodiments, the pluripotent stem cells are selected from embryonic stem cells, haploid stem cells, induced pluripotent stem cells, or adult stem cells. In certain embodiments, the pluripotent stem cells are embryonic stem cells, such as human embryonic stem cells. In certain embodiments, the pluripotent stem cells are induced pluripotent stem cells.

In certain embodiments, the first medium is provided with one or more of the following features:

(i) The total content of the one or more serum substitutes is 3-30% (v/v), such as about 3% (v/v), about 5% (v/v), about 8% (v/v), about 10% (v/v), about 12% (v/v), about 15% (v/v), about 18% (v/v), about 20% (v/v), about 22% (v/v), about 25% (v/v), about 28% (v/v), or about 30% (v/v);

(ii) The one or more non-essential amino acids are each present in an amount of 0.1 to 0.5mM, e.g., about 0.1mM, about 0.2mM, about 0.3mM, about 0.4mM, or about 0.5mM, respectively;

(iii) The stabilized dipeptide of glutamine or L-alanyl-L-glutamine is present in an amount of 1-5mM, such as about 1mM, about 2mM, about 3mM, about 4mM, or about 5mM;

(iv) The bFGF may be provided in an amount of 1-100ng/mL, e.g., 2-100ng/mL,2-50ng/mL,5-100ng/mL,5-50ng/mL, or 5-20ng/mL, e.g., about 1ng/mL, about 2ng/mL, about 3ng/mL, about 5ng/mL, about 8ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, or about 100ng/mL.

(a) The serum replacement is selected from KOSR, MSC serum-free additives, ultroser ^TM G, and any combination thereof, preferably the serum replacement is KnockOut ^TM SR (e.g., thermo: cat. No. 10828028) (hereinafter referred to simply as KOSR);

(b) The non-essential amino acid is selected from the group consisting of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine, and combinations thereof;

(c) The basal medium is selected from KnockOut ^TM DMEM (e.g., gibco: cat No. 10829018) (hereinafter abbreviated as KO-DMEM), knockOut ^TM DMEM/F-12 (e.g., gibco: cat No. 12660-012) (hereinafter abbreviated as KO-DMEM/F12), DMEM, alpha-MEM, F-12, MEM, BME, RPMI 1640, G-MEM, and any combination thereof, preferably, the basal medium is selected from KO-DMEM, KO-DMEM/F12, DMEM/F12, preferably, the basal medium is KO-DMEM.

In certain embodiments, the first medium comprises 3-30% (v/v) KOSR, 1-5mM L-alanyl-L-glutamine stabilized dipeptide, 1-100ng/mL bFGF, and amino acids glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine at a concentration of each 0.1-0.5 mM.

In certain embodiments, the first medium comprises 3-30% (v/v) KOSR, 1-5mM L-alanyl-L-glutamine stabilized dipeptide, 1-20ng/mL bFGF, and amino acids glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine at a concentration of each 0.1-0.5mM, preferably, the basal medium is selected from KO-DMEM, KO-DMEM/F12, DMEM/F12.

In certain embodiments, the first medium comprises 3-30% (v/v) KOSR, 1-5mM L-alanyl-L-glutamine stabilized dipeptide, 5-20ng/mL bFGF, and amino acids glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine at a concentration of each 0.1-0.5mM, preferably, the basal medium is selected from KO-DMEM, KO-DMEM/F12, DMEM/F12.

In certain embodiments, the first medium comprises KOSR in an amount of 5-20% (v/v), stabilized dipeptide L-alanyl-L-glutamine in an amount of 1-5mM, bFGF in an amount of 5-20ng/mL, and amino acids glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine in a concentration of 0.1-0.5mM each, preferably the basal medium is selected from KO-DMEM, KO-DMEM/F12, DMEM/F12.

In certain embodiments, the first medium further comprises β -mercaptoethanol. In certain embodiments, the beta-mercaptoethanol is present in an amount of 0.1-0.5% (v/v), such as about 0.1% (v/v), about 0.2% (v/v), about 0.3% (v/v), about 0.4% (v/v), or about 0.5% (v/v).

In some embodiments, the first medium comprises about 8-12% (v/v) KOSR, about 1-5% (v/v) NEAA (i.e., 0.1-0.5 mM), about 0.5-2.5% (v/v) GlutaMAX (i.e., 1-5 mM), about 5-10 ng/mL bFGF. Preferably also about 0.1% (v/v) beta-mercaptoethanol.

In certain exemplary embodiments, the first medium comprises about 10% (v/v) KOSR, about 1% (v/v) NEAA (i.e., 0.1 mM), about 1% (v/v) GlutaMAX (i.e., 2 mM), about 8ng/mL bFGF. Preferably also about 0.1% (v/v) beta-mercaptoethanol.

In certain embodiments, the second medium is provided with one or more of the following features:

(i) The total content of the one or more serum substitutes is 1-40% (v/v), such as ,1-35％(v/v),1-30％(v/v),2-30％(v/v),5-30％(v/v),1-20％(v/v),2-20％(v/v),5-20％(v/v),1-10％(v/v),2-10％(v/v), or 5-10% (v/v), such as about 1% (v/v), about 2% (v/v), about 3% (v/v), about 5% (v/v), about 8% (v/v), about 10% (v/v), about 12% (v/v), about 15% (v/v), about 18% (v/v), about 20% (v/v), about 22% (v/v), about 25% (v/v), about 28% (v/v), or about 30% (v/v);

(ii) The one or more non-essential amino acids are each present in an amount of 0.1 to 0.5mM, such as 0.1 to 0.2mM, such as about 0.1mM, about 0.2mM, about 0.3mM, about 0.4mM, or about 0.5mM, respectively;

(iii) The stabilized dipeptide of glutamine or L-alanyl-L-glutamine is present in an amount of 1-5mM, such as 1-3mM, such as about 1mM, about 2mM, about 3mM, about 4mM, or about 5mM;

(iv) The one or more growth factors are each present in an amount of 1-100ng/mL, e.g., about 1ng/mL, about 2ng/mL, about 3ng/mL, about 4ng/mL, about 5ng/mL, about 8ng/mL, about 10ng/mL, about 15ng/mL, about 20ng/mL, about 25ng/mL, about 30ng/mL, about 35ng/mL, about 40ng/mL, about 45ng/mL, about 50ng/mL, about 55ng/mL, about 60ng/mL, about 65ng/mL, about 70ng/mL, about 75ng/mL, about 80ng/mL, about 85ng/mL, about 90ng/mL, about 95ng/mL, or about 100ng/mL.

(a) The serum replacement is selected from the group consisting of KOSR, MSC serum-free additives, ultroser ^TM G, and any combination thereof;

(c) The basal medium is selected from KO-DMEM, KO-DMEM/F12, alpha-MEM, DMEM, F, MEM, BME, RPMI 1640, G-MEM and any combination thereof, preferably selected from KO-DMEM, KO-DMEM/F12, alpha-MEM, DMEM, DMEM/F12.

In certain embodiments, the growth factor comprises bFGF and tgfβ. In certain embodiments, the growth factor comprises 1-100ng/mL bFGF and 1-100ng/mL TGF beta.

In certain embodiments, the bFGF content is 4-100 ng/mL, e.g., 4～90ng/mL,4～80ng/mL,4～60ng/mL,4～50ng/mL,4～40ng/mL,4～30ng/mL,4～20ng/mL,4～10ng/mL,5～90ng/mL,5～80ng/mL,5～60ng/mL,5～50ng/mL,5～40ng/mL,5～30ng/mL,5～20ng/mL,5～10ng/mL. in certain embodiments, the bFGF content is 5-100 ng/mL, 10-100 ng/mL.

In some embodiments, the TGF-beta content is 4-100 ng/mL, e.g., 4-90 ng/mL, 4-80 ng/mL, 4-60 ng/mL, 4-50 ng/mL, 4-40 ng/mL, 4-30 ng/mL, 4-20 ng/mL, 4-10 ng/mL.

In certain embodiments, the growth factor comprises 10-100ng/mL bFGF and 4-100ng/mL TGF beta. In certain embodiments, the growth factor further comprises one or more selected from VEGF, EGF, PDGF at a concentration of 1-100 ng/mL.

In certain embodiments, the serum replacement is KOSR, and one selected from MSC serum free additives (e.g., TBD: cat# SC 2013-G-B) and Ultroser ^TM G (e.g., PALL: cat# 159550-017) (hereinafter referred to as Ultroser G). In certain embodiments, the volume ratio of KOSR to MSC serum-free additive or Ultroser G is from 2:1 to 150:1, such as about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, about 10:1, about 20:1, about 50:1, about 80:1, about 100:1, about 120:1, or about 150:1. In certain embodiments, the volume ratio of KOSR to MSC serum-free additive or Ultroser G is 10:1 to 1:2, e.g., about 10:1, about 9:1, about 8:1, about 7:1, about 6:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, or about 1:2.

In certain embodiments, the KOSR is present in an amount of about 1-30% (v/v), about 1-20% (v/v), about 1-10% (v/v), about 2-10% (v/v), or about 5-10% (v/v). In certain embodiments, the MSC is free of serum additives or Ultroser G in an amount of about 1-10% (v/v), or about 1-5% (v/v).

In certain embodiments, the one or more serum or serum replacement comprises KOSR and ultraser G. In certain embodiments, the second medium comprises 1-5% (v/v) Ultroser G, 5-30% (v/v) KOSR.

In certain embodiments, the second medium further comprises ascorbic acid. In certain embodiments, the amount of ascorbic acid is 1-100 μg/mL, such as 1-50 μg/mL,1-20 μg/mL, or 5-20 μg/mL.

In certain embodiments, the second medium is a basal medium comprising 1-5% (v/v) Ultroser G, 5-30% (v/v) (e.g., 4-20% (v/v)) KOSR, 1-5mM L-alanyl-L-glutamine stabilized dipeptide, 1-100ng/mL bFGF, 1-100ng/mL TGFbeta, and amino acids glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine at a concentration of 0.1-0.5mM each, the basal medium being selected from KO-DMEM, KO-DMEM/F12, α -MEM, DMEM, DMEM/F12.

In certain embodiments, the second medium is a basal medium comprising 1-5% (v/v) Ultroser G, 5-30% (v/v) (e.g., 4-20% (v/v)) KOSR, 1-5mM L-alanyl-L-glutamine stabilized dipeptide, 4-100ng/mL bFGF, 4-100ng/mL TGFbeta, and amino acids glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine at a concentration of 0.1-0.5mM each, the basal medium being selected from KO-DMEM, KO-DMEM/F12, α -MEM, DMEM, DMEM/F12.

In certain embodiments, the second medium is a basal medium comprising 1-5% (v/v) Ultroser G, 5-30% (v/v) (e.g., 4-20% (v/v)) KOSR, 1-5mM L-alanyl-L-glutamine stabilized dipeptide, 10-100ng/mL bFGF, 4-100ng/mL TGFbeta, and amino acids glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine at a concentration of 0.1-0.5mM each, the basal medium being selected from KO-DMEM, KO-DMEM/F12, α -MEM, DMEM, DMEM/F12.

In certain embodiments, the second medium may further comprise 1-50 μg/mL ascorbic acid.

In certain embodiments, the second medium may further comprise one or more selected from VEGF, EGF, PDGF, e.g., at a concentration of each 1-100 ng/mL.

In certain embodiments, the method comprises the steps of:

(1) Culturing stem cells to form embryoid bodies using a first medium, the stem cells being embryonic stem cells or induced pluripotent stem cells, wherein the stem cells are cultured to form embryoid bodies using the first medium, wherein the first medium is a basal medium to which one or more serum substitutes, one or more non-essential amino acids, stabilized dipeptides of glutamine or L-alanyl-L-glutamine, and bFGF are added;

(2) Culturing the embryoid body to induce differentiation into mesenchymal stem cells using a second medium, wherein the second medium is a basal medium to which 1-5% (v/v) Ultroser G, 5-30% (v/v) (e.g., 4-20% (v/v)) KOSR, 1-5mM L-alanyl-L-glutamine stabilized dipeptide, 10-100ng/mL bFGF, 4-100ng/mL TGFbeta, 1-50 μg/mL ascorbic acid, and amino acids of glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine are added at concentrations of 0.1-0.5mM each, the basal medium being selected from KO-DMEM, KO-DMEM/F12, alpha-MEM, DMEM, DMEM/F12.

In certain embodiments, the method comprises the steps of:

(1) Culturing stem cells to form embryoid bodies using a first medium that is a basal medium to which 3-30% (v/v) (e.g., 5-20% (v/v)) KOSR, 1-5mM of stabilized dipeptide of L-alanyl-L-glutamine, 1-100ng/mL (e.g., 5-20ng/mL,5-10 ng/mL), and amino acids glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine are added at a concentration of each 0.1-0.5mM, wherein the basal medium is selected from KO-DMEM, KO-DMEM/F12, DMEM/F12;

In certain embodiments, the first medium and the second medium both comprise components that are cell therapy grade (CTS grade). In certain embodiments, the first medium and the second medium comprise basal medium (e.g., KO-DMEM), serum replacement (e.g., KOSR), and stabilized dipeptide of L-alanyl-L-glutamine are cell therapy grade (CTS grade).

In certain embodiments, step (1) comprises culturing the pluripotent stem cells in a low adsorption cell culture dish.

As used herein, the term "low adsorption cell culture dish" refers to a culture dish having a coating on the surface that prevents adsorption of proteins on the surface of the dish, thereby minimizing the adhesion of a monolayer of cells to the culture vessel. Such cell culture dishes are well known to those of skill in the art, including, but not limited to, corning's low attachment dish (cat No. 3262).

In certain embodiments, the duration of the culturing in step (1) is 3-14 days, for example about 3 days, about 4 days, about 5 days, about 7 days, about 10 days, or about 14 days.

In certain embodiments, the duration of the culturing in step (1) is 4-7 days, for example about 5 days.

In certain embodiments, step (2) comprises inoculating the embryoid body of step (1) to the culture vessel at a density of about 1 embryoid body/cm ².

In certain embodiments, step (2) comprises culturing the embryoid body in a culture dish coated with gelatin, type I collagen, type IV collagen, vitronectin, fibronectin, or polylysine.

In certain embodiments, step (2) comprises culturing the embryoid bodies in vitronectin-coated culture dishes.

In certain embodiments, the duration of the culturing in step (2) is from 10 to 21 days, for example, about 10 days, about 14 days, or about 21 days.

In certain embodiments, the duration of the culturing in step (2) is from 10 to 14 days, e.g., about 14 days.

In certain embodiments, step (2) comprises replacing fresh second medium every day or every 1-7 days (e.g., every 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, or 7 days). In certain embodiments, step (2) comprises discarding spent medium (THE SPENT MEDIA) every day or every 1-7 days (e.g., every 1,2,3, 4, 5, 6, or 7 days) and replacing fresh second medium.

In certain embodiments, in steps (1) - (2), the culture conditions are 37 ℃ 5% CO ₂. In certain embodiments, in steps (1) - (2), the culturing is performed in an incubator at 37 ℃ with 5% CO ₂.

In certain embodiments, the cells attached to the culture vessel in step (2) are IMRC cells of the P0 generation. In certain embodiments, when the confluence of the cells attached to the culture vessel described in step (2) is not less than about 80% (e.g., not less than about 85%, not less than about 90%, or not less than about 95%), the cells may be separated from the culture vessel, thereby obtaining P0 generation IMRC cells.

Thus, in certain embodiments, the method further comprises (3) isolating the cells attached to the culture vessel in step (2), thereby obtaining IMRC cells.

In certain embodiments, the method further comprises passaging the IMRC cells of step (3).

In certain embodiments, the cells are passaged when the cell confluency is greater than or equal to about 80% (e.g., greater than or equal to about 85%, greater than or equal to about 90%, or greater than or equal to about 95%).

In certain embodiments, the IMRC cells are passaged for 1, 2,3, 4, or 5 passages.

Methods for passaging cells are well known to those skilled in the art. For example, the method may include separating the cells from the culture vessel and uniformly dispersing the cells in the culture medium, followed by seeding in the culture vessel. And adding a proper amount of culture medium, replacing a proper amount of fresh culture medium at intervals (for example, every 1-5 days) according to the growth state of the cells, and repeating the passage operation when the cells grow to 70-100% confluence. The algebra increases by 1 for each passage operation of the cells.

In certain embodiments, the passaging comprises passaging at a cell density of about 5 x 10 ³-5×10⁴/cm² (e.g., about 5 x 10 ³/cm², about 1 x 10 ⁴/cm², about 2 x 10 ⁴/cm², about 3 x 10 ⁴/cm², about 4 x 10 ⁴/cm², or about 5 x 10 ⁴/cm²).

In certain embodiments, the passaging comprises seeding the cells into the second medium for culturing.

In certain embodiments, the isolating comprises disrupting the attachment of the IMRC cells to the culture vessel by (i) contacting the culture with one or more enzymes selected from trypsin or an analog thereof, collagenase, dispase, papain, a mixture of collagenase and dispase, and a mixture of collagenase and trypsin or an analog thereof, (ii) mechanically isolating using cell scraping or the like, or (iii) contacting the culture with EDTA or EGTA.

In certain embodiments, the isolating comprises disrupting the attachment of the IMRC cells to the culture vessel by enzymatic digestion.

In certain embodiments, the enzyme is trypsin (e.g., gibco: cat. Number 25200072).

Optionally, after culturing to obtain IMRC cells, the cell growth curve may be assayed by MTT method, WST method, DNA content assay, ATP assay, etc., to evaluate the growth activity of the umbilical cord IMRC cells. Alternatively, isolated cultured IMRC cells can be identified by flow cytometry for detection of cell surface markers, three-way differentiation assays, and PCR for detection of cell expression genes.

In certain embodiments, the medium used to culture IMRC cells is the second medium.

Acellular matrix materials

In a fourth aspect, the present invention provides a decellularized matrix material obtained from the cell patch of the first aspect or after decellularization of the cell patch of the third aspect.

In certain embodiments, the decellularization treatment includes physical methods (e.g., direct effects of temperature, pressure, electroporation, and force), chemical methods (e.g., acids and bases, detergents, hypotonic and hypertonic solutions, alcohols and solvents), biological methods (e.g., enzymes, nucleases, and chelators), or any combination thereof.

In certain embodiments, the decellularized matrix material is a decellularized membrane sheet that is a direct product obtained from the decellularized treatment of the cell membrane sheet of the first aspect or the cell membrane sheet of the third aspect.

In certain embodiments, the decellularized matrix material is a decellularized hydrogel that is the product of the decellularization process and digestion of the cell membrane of the first aspect or the cell membrane of the third aspect.

In certain embodiments, the digestion treatment comprises the use of an acid (e.g., acetic acid or hydrochloric acid) and/or a digestive enzyme (e.g., pepsin or trypsin).

Preparation of acellular matrix materials

In a fifth aspect, the invention provides a method of preparing a decellularized matrix material comprising:

(1) Providing a cell patch of the first aspect or a cell patch of the third aspect;

(2) And (3) performing decellularization treatment on the cell membrane.

Decellularization treatments are well known to those of skill in the art and can be seen, for example Kim BS,Das S,Jang J,Cho DW.Decellularized Extracellular Matrix-based Bioinks for Engineering Tissue-and Organ-specific Microenvironments.Chem Rev.2020;120(19):10608-10661. in certain embodiments, including physical methods (e.g., direct effects of temperature, pressure, electroporation, and force), chemical methods (e.g., acids and bases, detergents, hypotonic and hypertonic solutions, alcohols and solvents), biological methods (e.g., enzymes, nucleases, and chelators), or any combination thereof.

In certain embodiments, the decellularization treatment includes the use of surfactants (e.g., nonionic or ionic surfactants such as Triton-X100, sodium Dodecyl Sulfate (SDS), sodium Deoxycholate (SD)).

In certain embodiments, the decellularization treatment comprises freeze thawing, e.g., multiple freeze-thaw cycles.

In certain embodiments, the decellularization treatment includes using 1% Triton-X100, such as 1% Triton-X100 and 20mM NH ₃·H₂ O.

In certain embodiments, the decellularization treatment comprises using 0.1% sds.

In certain embodiments, the product obtained in step (2) is a decellularized membrane.

In certain embodiments, the decellularized matrix material is a hydrogel and the method further comprises subjecting the decellularized product to a digestion process.

Digestion treatments are well known to those skilled in the art and can be found, for example, in Kim BS,Das S,Jang J,Cho DW.Decellularized Extracellular Matrix-based Bioinks for Engineering Tissue-and Organ-specific Microenvironments.Chem Rev.2020;120(19):10608-10661.

In certain embodiments, the digestion treatment comprises the use of acetic acid and pepsin. Illustratively, 0.5M acetic acid and 10mg pepsin may be used.

In certain embodiments, the digestion treatment is further followed by dialysis, lyophilization, and redissolution steps. In certain embodiments, the dissolving comprises using a PBS solution (e.g., pH 7-9, e.g., pH 8).

Composite cell membrane and preparation thereof

In a sixth aspect, the invention provides a composite cell patch comprising IMRC cells and at least one cell selected from the group consisting of fibroblasts, keratinocytes, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, cardiac fibroblasts, vascular wall cells (e.g., smooth muscle cells, pericytes), corneal endothelial cells, retinal pigment epithelial cells.

In certain embodiments, the composite cell patch comprises at least two cell patches stacked, wherein at least one cell patch is a cell patch according to the first aspect or a cell patch according to the third aspect.

In certain embodiments, at least one of the cell sheets of the stack is a cell sheet formed from cells selected from the group consisting of fibroblasts, keratinocytes, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, myocardial fibroblasts, vascular wall cells (e.g., smooth muscle cells and pericytes), corneal endothelial cells, retinal pigment epithelial cells.

In certain embodiments, the laminated cell membranes comprise an adhesive between the layers.

In certain embodiments, the adhesive is made of a material that is biocompatible and degradable in vivo.

In certain embodiments, the adhesive is the decellularized matrix material of the fourth aspect.

In certain embodiments, the binder comprises an artificially synthesized biomaterial selected from the group consisting of polyethylene glycol, polyethylene glycol derivatives, polylactic acid alcohol copolymers, polyanhydrides, polyacrylamides, polyamino acids, polyethylene oxides, polyesters, polymethyl methacrylates, polycarbonates, polyurethanes, polycaprolactone, polyhydroxyalkanoates, polysiloxanes, polyethylene, polyvinyl chloride, polytetrafluoroethylene, polystyrene, polypropylene, N- (3-aminopropyl) methacrylamide hydrochloride (APMA), N- [ tris (hydroxymethyl) methyl ] acrylamide (THMA), maleic anhydride graft copolymers, polyacrylamides, polyacetals, polypyrroles, or any combination thereof. The preferred synthetic material is N- (3-aminopropyl) methacrylamide hydrochloride (APMA), N- [ tris (hydroxymethyl) methyl ] acrylamide (THMA).

In certain embodiments, the adhesive comprises a natural biological material selected from the group consisting of natural proteins, collagen and collagen derivatives, gelatin and gelatin derivatives, agar and agar derivatives, proteoglycans, alginates and their alginate derivatives, matrigel, propolis, cellulose and cellulose derivatives, chitin and chitin derivatives, fibroin and its derivatives, laminin and its derivatives, fibronectin and its derivatives, sodium hyaluronate and hyaluronic acid derivatives, agarose and its derivatives, dextran and its derivatives, sucrose and sucrose derivatives, starch, chitosan and chitosan derivatives, or any combination thereof, preferably gelatin and gelatin derivatives, chitosan and chitosan derivatives.

In another aspect, the invention provides a method of preparing a composite cell patch comprising:

(1) Providing a plurality of cell patches, wherein at least one of the cell patches is according to the first aspect or the cell patches is according to the third aspect;

(2) Disposing an adhesive on one of said cell sheets and then overlaying another of said cell sheets thereon;

(3) Repeating the step (2);

(4) A laminated product having a predetermined number of sheets is obtained.

In certain embodiments, wherein at least one cell patch is a cell patch formed from cells selected from the group consisting of fibroblasts, keratinocytes, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, cardiac fibroblasts, vascular wall cells (e.g., smooth muscle cells, pericytes), corneal endothelial cells, retinal pigment epithelial cells.

In certain embodiments, the adhesive is made of a material that is biocompatible and degradable in the human body.

In a seventh aspect, the invention provides a composite cell patch comprising at least two layers of cell patches stacked, wherein each layer of cell patches stacked comprises an adhesive therebetween, the adhesive being selected from the decellularized matrix materials of the fourth aspect.

In certain embodiments, the at least two cell patches are each independently formed from cells selected from the group consisting of fibroblasts, keratinocytes, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, cardiac fibroblasts, vascular wall cells (e.g., smooth muscle cells, pericytes), corneal endothelium, retinal pigment epithelial cells.

In certain embodiments, the composite cell patch comprises a fibroblast patch and a keratinocyte patch.

In another aspect, a method of preparing a composite cell patch is provided, comprising:

(1) Providing a plurality of cell patches;

(2) Disposing an adhesive on one of said cell sheets and then overlying the other of said cell sheets, wherein said adhesive is selected from the decellularized matrix material of the fourth aspect;

(3) Repeating the step (2);

(4) A laminated product having a predetermined number of sheets is obtained.

In certain embodiments, each of the plurality of cell patches is independently formed from a cell selected from the group consisting of fibroblasts, keratinocytes, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, cardiac fibroblasts, vascular wall cells (e.g., smooth muscle cells, pericytes), corneal endothelium, retinal pigment epithelial cells.

In certain embodiments, the plurality of cell patches comprises a fibroblast patch and a keratinocyte patch.

In another aspect, the use of the decellularized matrix material of the invention as an adhesive is provided. Preferably, the adhesive is used to prepare cell membranes. Preferably, the adhesive is used to prepare a composite cell patch according to the sixth or seventh aspect.

Cell membrane subjected to induced differentiation and preparation

In an eighth aspect, the present invention provides an induced differentiation cell membrane obtained from the cell membrane of the first aspect or the cell membrane of the third aspect. Techniques for inducing differentiation of stem cells are well known to those skilled in the art, and those skilled in the art can select appropriate conditions for inducing differentiation according to the type of differentiated cell of interest, thereby obtaining a corresponding cell sheet induced to differentiation.

In certain embodiments, the cell patch that is induced to differentiate is an ectodermal tissue patch (e.g., neural crest cell patch, neural cell patch, retinal pigment epithelial cell patch, keratinocyte patch).

In certain embodiments, the cell patch that is induced to differentiate is a mesodermal tissue patch (e.g., chondrocyte patch, bone cell patch, adipocyte patch, cardiomyocyte patch, muscle cell patch, vascular endothelial cell patch, renal cell patch).

In certain embodiments, the induced differentiated cell membrane is an endodermal tissue membrane (liver cell membrane, islet beta cell membrane, intestinal cell membrane, gastric cell membrane).

In certain embodiments, the cell membrane that is induced to differentiate is a chondrocyte membrane.

In another aspect, there is provided a method of preparing the above-described induced differentiated cell membrane, comprising:

(1) Providing a cell patch according to the first aspect or a cell patch according to the third aspect;

(2) The cell membrane is cultured under conditions that induce differentiation, for example in a medium supplemented with an induction factor.

In certain embodiments, the method comprises culturing IMRC cells using the method of the second aspect, and replacing the medium with the medium supplemented with the inducing factor and continuing the culturing (e.g., for at least 7 days, at least 14 days) on days 5-10 (e.g., 5-9, 5-8, 6-10, 6-9, 6-8, e.g., 7) of the culturing.

Medical application

In another aspect, a pharmaceutical composition is provided comprising a cell membrane according to the first or third aspect, a decellularized matrix material according to the fourth aspect, or a composite cell membrane according to the sixth or seventh aspect, or an induced differentiated cell membrane according to the eighth aspect, and a pharmaceutically acceptable carrier and/or excipient.

In another aspect, there is provided the use of a cell patch according to the first aspect or a cell patch according to the third aspect, a decellularized matrix material according to the fourth aspect, or a composite cell patch according to the sixth or seventh aspect, or an induced differentiated cell patch according to the eighth aspect, or a pharmaceutical composition as described herein, in the manufacture of a medicament for the regeneration or repair of a tissue or organ.

In another aspect, there is provided the use of a cell patch according to the first aspect or a cell patch according to the third aspect, a decellularized matrix material according to the fourth aspect, or a composite cell patch according to the sixth or seventh aspect, or an induced differentiated cell patch according to the eighth aspect, or a pharmaceutical composition as described herein, in the manufacture of a medicament for the treatment of a tissue or organ injury.

In certain embodiments, the tissue or organ damage includes, corneal damage (e.g., corneal alkali burn), skin damage (e.g., skin burn, skin scald, diabetic foot), heart disease (e.g., myocardial damage), skeletal damage (e.g., skull damage, bone defect), endometrial damage (e.g., uterine cavity adhesion), meniscus damage, and/or muscle damage, teeth Zhou Sunshang (e.g., periodontitis), esophagus (e.g., esophageal mucosa damage), liver damage (e.g., cirrhosis), pancreatic disease (e.g., diabetes, pancreatitis), lung disease (e.g., lung damage, lung fibrosis).

In certain embodiments, the cell membrane is differentiated by osteogenesis or chondrogenesis.

In another aspect, there is provided the use of a cell patch according to the first aspect or a cell patch according to the third aspect, a decellularized matrix material according to the fourth aspect, or a composite cell patch according to the sixth or seventh aspect, or an induced differentiated cell patch according to the eighth aspect, or a pharmaceutical composition as described herein, in the manufacture of a medicament for cell, tissue or organ transplantation. In certain embodiments, the decellularized hydrogels described herein are used to prepare a drug for cell, tissue or organ transplantation as an excipient in the drug.

In another aspect, there is provided the use of the acellular matrix material of the invention as a pharmaceutically acceptable carrier and/or excipient in a medicament. In certain embodiments, the drug comprises a nucleic acid drug, a small molecule drug, a protein drug, and/or a cell, tissue, or organ transplant drug.

In another aspect, there is provided the use of the decellularized matrix material of the invention for cell culture (e.g., culture of pluripotent stem cells, differentiation of pluripotent stem cells), organoid culture, or 3D printing. In certain embodiments, the decellularized matrix material is used as an ink for 3D printing. In certain embodiments, the decellularized matrix material is used as a coating matrix for cell culture or organoid culture.

In another aspect, there is provided a method of culturing a cell or organoid in vitro comprising culturing in the presence of the decellularized matrix material of the fourth aspect. In certain embodiments, the decellularized matrix material is a hydrogel. In certain embodiments, the method comprises culturing on a surface coated with the hydrogel.

In another aspect, there is provided a method of 3D printing comprising using the decellularized matrix material of the fourth aspect as an ink. Preferably, the decellularized matrix material is a hydrogel.

Advantageous effects

The invention provides a cell patch formed by IMRC cells and extracellular matrixes and active factors secreted by the IMRC cells, compared with mesenchymal stem cell patches, the IMRC cell patch has the advantages that the IMRC cell patch expresses immune regulation related factors, nutritional factors and extracellular matrix related proteins, the secreted active factors are more in variety, pro-inflammatory factors are expressed in a low mode, the IMRC cell patch has stronger immune regulation capability, better mechanical properties and more compact and uniform fibers. The IMRC cell membrane provided by the invention has important application value in the field of regenerative medicine.

Embodiments of the present invention will be described in detail below with reference to the accompanying drawings and examples, but it will be understood by those skilled in the art that the following drawings and examples are only for illustrating the present invention and are not to be construed as limiting the scope of the present invention. Various objects and advantageous aspects of the present invention will become apparent to those skilled in the art from the following detailed description of the preferred embodiments and the accompanying drawings.

Drawings

FIG. 1 is a photomicrograph of cell patches on different days of culture.

FIG. 2. Light-mapping of Day21 cell membranes.

FIG. 3 shows a schematic diagram of cell membrane transfer. Transfer of M cell membranes can be achieved with PVDF membranes.

FIG. 4 is a photograph of a long-term cultured cell pellet and a HE staining chart.

FIG. 5 automated large-scale culture of cell patches. Automated culture and culture observation was performed on cell patches using CELLKEEPER instrument.

FIG. 6 schematic representation of cell patch culture and M-cell hydrogel preparation.

FIG. 7 is a photograph of the cell membrane taken after decellularization.

FIG. 8. Identification of the temperature sensitivity of IMRCs cell hydrogels.

FIG. 9 identification of viscosity and modulus of IMRCs cell hydrogels.

FIG. 10 purification of IMRCs cell hydrogels.

FIG. 11.IMRCs cell hydrogel (10 mg/ml) electron microscopy.

Figure 12 in vivo temperature sensitivity verification of imrcs cell hydrogels.

FIG. 13 yield measurement of cell patch hydrogels. Cell patches were subjected to treatments such as decellularization to prepare hydrogels, and the yields were calculated.

FIG. 14 shows extracellular matrix synthesis of cell membrane and detection of expression of genes related to cell proliferation.

FIG. 15 detection of expression of genes involved in inflammation and immune regulation of cell membranes.

FIG. 16 cell patch transcriptome sequencing PCA analysis.

FIG. 17 cell patch transcriptome sequencing GO analysis.

FIG. 18 detection of extracellular matrix and inflammation-related genes expression of cell patches.

FIG. 19 is a schematic diagram of a mechanical property test of cell membranes.

FIG. 20 is a graph showing mechanical properties of cell membranes.

FIG. 21 cell membrane tensile test stress strain.

FIG. 22 mechanical properties test of cell membranes.

FIG. 23 Young's modulus of cell membranes.

FIG. 24 is an electron micrograph of the cell membrane after decellularization.

FIG. 25 immunofluorescence identification of extracellular matrix components of cell patches. .

FIG. 26 immunofluorescent identification of extracellular matrix components of cells.

FIG. 27 shows the measurement of factor secretion from cell membranes using a Bio-Plex 200 suspension chip system, (A) the measurement of immune-related factors, trophic factors, and pro-inflammatory factors in cell membranes, (B) the expression of pro-inflammatory factors, and (C) the expression of immune-related and trophic factors.

FIGS. 28A-28B protein mass spectrometry identification after cell patch decellularization.

FIG. 29 mass spectrometry of cell patches.

FIG. 30 identification of components in cell membranes.

FIG. 31.IMRC cell hydrogels promote proliferation of IMRC cells. Light-mirror photographs of different days of imrc cell plating on hydrogel. B. Compared with the common culture plate, the IMRC cell hydrogel can obviously promote the proliferation of IMRC cells. Identification of surface Marker of imrc cells after hydrogel culture.

FIG. 32.IMRC cell hydrogels promote migration of GFP-IMRC cells.

FIG. 33.IMRC cell hydrogels can encapsulate GFP-IMRC cells, promoting proliferation and expansion of GFP-IMRC cells.

Figure 34 imrc cell hydrogels can promote in vitro vascularization of HUVECs.

FIG. 35 treatment of rat corneal alkali burn with non-decellularized IMRC cell membrane. A. Schematic drawing of rat cornea alkali burn modeling. Photo-glasses of rat corneas at different days after IMRC cell patch treatment. C. Scoring of rat cornea clarity. D-F rat cornea HE staining and immunohistochemical results.

Fig. 36 is a schematic representation of intrauterine adhesion treatment of rats by imrc hydrogel in vivo implantation.

Fig. 37. Intrauterine adhesion implant pictures of imrc hydrogel implant treated rats.

FIG. 38 photomicrographs of IMRC hydrogel in vivo grafts for treatment of rat uterine adhesion, at the time of harvest.

Figure 39 analysis of uterine tissue sections after imrc hydrogel treatment of intrauterine adhesion in rats.

Sequence information

Examples

The invention will now be described with reference to the following examples, which are intended to illustrate the invention, but not to limit it.

Those skilled in the art will appreciate that the examples describe the application by way of example and are not intended to limit the scope of the application as claimed. The experimental methods in the examples are all conventional methods unless otherwise specified. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1 preparation of IMRC cells

1.1 Embryoid Body (EB) production

Human embryonic stem cells (CB 0019, national stem cell resource library) were cultured in a low-attached petri dish (Corning: cat No. 3262) using EB broth, and placed in a 37℃incubator to form EB beads.

EB culture broth was prepared by adding 10% (v/v) KOSR, 1% (v/v) NEAA (i.e., 0.1 mM), 1% (v/v) GlutaMAX (i.e., 2 mM), 8ng/mL bFGF and 0.1% (v/v) beta-mercaptoethanol to KO-DMEM.

1.2 Wall-attached culture of EB balls

The EB spheres obtained in step 1.1 were cultured in vitronectin coated 6-well plates using IMRC culture broth. The liquid is changed every day until about 14 days, and IMRC subculture is carried out. Subculture operations may be repeated, for example, for the fifth generation for subsequent operations or frozen using Cryostor CS a 10.

IMRC medium was prepared by adding 1% (v/v) Ultroser G, 5% (v/v) KOSR, 1% (v/v) NEAA, 1% (v/v) GlutaMAX, 5. Mu.g/mL ascorbic acid, 5ng/mL bFGF, and 5ng/mL TGF beta to alpha-MEM.

Example 2 preparation of cell Membrane

IMRC cells were passaged after reaching approximately 80% confluence in IMRC medium. To prepare cell patches, a total of 2×10 ⁵ IMRC cells cultured to P5 passages were seeded onto collagen I peptide coated cell culture surface (BD Biosciences, 356270) at a cell density of 2×10 ⁴/cm². All cultures were maintained in humidified incubator (Thermo FISHER SCIENTIFIC, waltham, MA, USA) at 37 ℃, 5% carbon dioxide and atmospheric oxygen. IMRC medium consisted of alpha-MEM supplemented with 5% KOSR, 1% Ultroser G, 1 XL-glutamine, 1 XNEAA and 5ng/mL, bFGF and 5ng/mL TGF-beta. On days 3 to 21, 40. Mu. Mol/L of vitamin C (Sigma-Aldrich, st Louis, MO, USA) was added to the medium.

As a control, a membrane of UCMSC cells was prepared using primary Umbilical Cord Mesenchymal Stem Cells (UCMSCs), wherein UCMSC medium consisting of α -MEM supplemented with 5% KOSR, 1% Ultroser G, 1 xl-glutamine, 1 xneaa and 5ng/mL bFGF was used. The medium was changed daily and 40. Mu. Mol/L of vitamin C (Sigma-Aldrich, st Louis, MO, USA) was added to the medium on days 3 to 21.

The photomicrographs of the cell patch on different days of culture are shown in FIG. 1, and the IMRC cell wall-attached growth can be seen to be fibrous and uniform in morphology. On day 21, the optical diagram of the whole cell membrane is shown in fig. 2, the IMRC cell membrane has compact structure, smooth and flat surface, and compared with UCMSC cell membrane, the integrity of the cell membrane is poor, obvious breakage, wrinkles, cracks and unevenness are generated, and in addition, the IMRC cell membrane is thicker than the UCMSC cell membrane.

In addition, transfer of M cell membranes can be achieved with PVDF membranes. The transfer schematic is shown in fig. 3. Specifically, the method comprises the steps of (1) discarding a culture medium of IMRCs cell membranes (IMRC-CSs), (2) cutting the PVDF membrane into the same area as a 6-pore plate, (3) transferring the PVDF membrane onto the IMRC-CSs of the discarded culture medium by using tweezers, exhausting air to enable the PVDF membrane to be attached to the cell membranes, (4) clamping one section of the PVDF membrane by using the tweezers, slightly uncovering the section of the PVDF membrane, wherein the section of the PVDF membrane is attached to the IMRC-CSs integrally, and the section of the PVDF membrane can be transferred, (5) transferring the section to a target area, and dropwise adding PBS buffer solution on the PVDF membrane, and separating the membrane from the IMRC-CSs to finish the transfer of the cell membranes.

Furthermore, the inventors have unexpectedly found that IMRC cell patches can be cultured for a long time to obtain patches up to 100 μm in thickness, as shown in fig. 4. In contrast, the UCMSC cell membrane had developed significant breakage, wrinkles, cracks at 21 days of culture, whereby the UCMSC cell membrane could not obtain a higher thickness cell membrane by means of long-term culture.

In addition, cell patches can be prepared at cell densities ranging from 1×10 ³/cm² to 1×10 ⁶/cm². Cell membranes can be prepared with vitamin C concentrations of 1-100. Mu. Mol/L, or cell membranes can be prepared by replacing Vc derivatives, for example, L-ascorbic acid-phosphoate with a concentration of 10. Mu. Mol/L can be used on days 1-2 of culture, or L-ascorbic acid-phosphoate with a concentration of 10. Mu. Mol/L can be used on days 3-21. Cell membranes can also be prepared by coating the surface of the culture dish with collagen and collagen derivatives, gelatin and gelatin derivatives, laminin and derivatives thereof, fibronectin and derivatives thereof.

In addition, IMRC cells were also subjected to automated culture using CELLKEEPER apparatus to prepare cell patches, the results of which are shown in fig. 5.

In the following examples, the cell patches detected were patches at 21 days of culture unless otherwise specified.

EXAMPLE 3 preparation of acellular matrix Material

The cell membrane can be subjected to decellularization treatment to obtain a decellularized membrane, and further digestion treatment can be performed to obtain the decellularized hydrogel. The preparation schematic is shown in fig. 6.

Decellularization treatment cell patches were decellularized with 1% Triton-x 100+20mM NH ₃H₂ O or 0.1% SDS at 37℃for 15min. The PBS was washed 2 times, 15 min/time. DNA was removed, and 100IU/ml DNase was added and the mixture was treated for 2 hours. The PBS was washed 2 times, 15 min/time.

DNA residual detection A small portion of cell membrane before and after decellularization was taken as sample, DNA was extracted using DNA extraction kit (DNeasy Blood and Tissue Kit, qiagen)), and the DNA residual concentration was detected and read on Nanodrop.

The preparation of hydrogel comprises cutting cell membrane after decellularization, dissolving with acetic acid and pepsin under stirring for 48 hr, dialyzing for 48 hr, and lyophilizing. Weigh and dissolve with PBS solution at ph=8 to prepare 10mg/ml hydrogel.

The photograph of the cell patch after decellularization is shown in FIG. 7. IMRC cell patches were smoother after decellularization, with more dead cell aggregates compared to UCMSC cell patches.

The results of the IMRC cell hydrogel temperature sensitivity assay are shown in fig. 8. Removing cells from IMRC cell membrane with Triton-X100, dissolving with acetic acid and pepsin, dialyzing, lyophilizing, and dissolving, and placing at 37deg.C under different pH values to identify gel. The results show that the IMRC cell hydrogel can be well gelled at the pH of 7-9, which shows that the IMRC cell hydrogel has good temperature sensitivity.

Characterization of rheological mechanical properties the IMRCs hydrogels were rheologically characterized using an MCR 302 (Anton Paar, austria) parallel plate rheometer. By measuring IMRCs storage (G ') and loss modulus (G ' ') of the hydrogel pre-gel, a temperature ramp oscillation test was performed to evaluate the gelation kinetics. The neutralized IMRCs hydrogel pre-gel samples were loaded into Peltier plates, respectively, with a volume of 200. Mu.L. After determining its linear viscoelastic region by amplitude sweep, we used 1% of the amplitude as the set amplitude for the temperature sweep. For a temperature sweep (1%, 0.5%,0.25% IMRCs hydrogel), the oscillation frequency was 1s ^-1, the temperature range was 0 ℃ to 40 ℃, and the slope was 0.5 ℃ min ^-1. Statistical data were obtained by Origin 2021b and plotted, including power law equation fitting.

The results of the identification of the viscosity and modulus of IMRC cell hydrogels are shown in fig. 9. The viscosity of the IMRC cell hydrogel increases sequentially with increasing concentration, and M-ECM (10 mg/mL) begins to solidify at 23.7 ℃ at 1%, which proves that the M cell hydrogel has good temperature sensitivity, and the modulus gradually increases with increasing concentration.

The results of purification of IMRC cell hydrogels are shown in fig. 10. The IMRC cell hydrogels were treated with different concentrations of NaCl and centrifuged. And B, identifying the protein content of the IMRC cell hydrogel after the treatment of NaCl at different concentrations, wherein the solubility of the M cell hydrogel is maximized when the treatment of 900mM NaCl is carried out along with the increase of the NaCl concentration. After NaCl treatment, the IMRC cell hydrogel still has good temperature sensitivity and can be gelled.

Scanning electron microscopy of IMRC cell hydrogels (10 mg/ml) is shown in FIG. 11. The IMRC cell hydrogel is a porous network structure after gel formation.

The temperature sensitivity verification in IMRC cell hydrogels is shown in figure 12. A. Injecting 100ul of IMRC-gel under the skin of SD rat, taking out after 10min, and B. Taking out the IMRC-gel to be colloid, which shows that the IMRC-gel is an injectable hydrogel with temperature sensitivity.

Yield of cell membrane hydrogel the results of the yield test of IMRC cell hydrogel are shown in fig. 13 as significantly better than UCMSC membrane.

Example 4 characterization of Gene expression in cell sheets

Total RNA is extracted from the prepared cell membrane, and RNA-SEQ transcriptome sequencing or Real-time PCR quantification is performed to identify the gene expression characteristics, wherein the primers are shown as SEQ ID NOs 1-30.

As shown in FIG. 14, the RNA-seq data shows that the genes upregulated by IMRC cell membrane compared with UCMSC cell membrane are accumulated to the pathways related to extracellular matrix synthesis and cell proliferation (A), the genes upregulated by IMRC compared with UCMSC include ELN, FN1, COL1A2, TNC, POSTN, COL A1, COL4A2 (B), and mRNA quantification results show that the expression of extracellular matrix related genes COL1A1, COL1A2, FN1, ELN, COL4A1, COL4A2, LAMC1, TNC, POSTN is significantly higher than UCMSC cell membrane (C). In conclusion, the extracellular matrix secretion of IMRC cell membranes was significantly higher than that of UCMSCs.

As shown in FIG. 15, the RNA-seq data shows that the genes of which the UCMSC cell membrane is up-regulated compared with the IMRC cell membrane are accumulated to the inflammation related pathway (A), the genes of which the IMRC is down-regulated compared with the UCMSC comprise IL1B, CCL and IL1A, IL6 (B), and the mRNA quantitative result shows that the expression quantity of the genes related to inflammation and immune regulation is obviously different between the IMRC cell membrane and the UCMSC cell membrane (C). In conclusion, the inflammatory gene expression of the IMRC cell membrane is lower, the immune regulation gene expression is higher, and the IMRC cell membrane has stronger immune regulation capacity than UCMSC.

The mRNA quantitative results of representative genes are shown in the following table.

TABLE 1 mRNA quantification results of representative genes

Gene	IMRC cell membrane	UCMSC cell membrane	Multiple of change
				FN	10.79	1.528	7.06
ELN	11139	84.81	131.34
				POSTN	342.3	2.597	131.81
COL1A1	9.824	0.9870	9.95
				COL1A2	12.85	0.9895	12.99
COL4A1	4.925	0.7303	6.74
				TNC	48.93	2.315	21.14
COL4A2	7.243	0.7589	9.54
				LAMC1	0.7340	0.5696	1.29
CXCL12	1.453	0.9706	1.50
				IL6	0.03095	3.551	0.01
IL1α	0.07279	0.4485	0.16
				IL1β	0.0006423	0.004602	0.14

Transcriptome sequencing PCA analysis of cell patches as shown in fig. 16, IMRC cell patches were significantly different from the transcriptome of UCMSC cell patches.

Transcriptome sequencing GO analysis of cell membrane as shown in fig. 17, IMRC cell membrane clusters genes with higher expression than UCMSC into extracellular matrix assembly and vascular development-related signaling pathways at day21, and UCMSC clusters genes with higher expression than IMRCs into inflammation-related signaling pathways at day 21.

The expression of extracellular matrix and inflammation-related genes in cell membrane as shown in fig. 18, IMRC cell membrane expressed extracellular matrix-related genes including FN1, COL1A2, TNC, POSTN, ECM2, ELN, COL6A3, COL6A2, and low expression pro-inflammatory-related genes including IL1A, IL1B, IL6, compared to UCMSC.

Example 5 determination of mechanical Properties of cell Membrane

AFM tensile test:

AFM (Nanowizard, bruker, U.S.) was used to test the elastic modulus of cell membranes, a method that has been widely used for biological material and cell property characterization. AFM measurements were performed using silicon nitride cantilevers with nominal spring constants of 0.12N/m (NP-O10, nanoworld, U.S.) and 30 μm diameter silicon beads (Thermo). Each sample was measured at 5 points and 8 force curves were collected at each end. The application of mechanical and Hertz models can be used for data fitting and calculation with spherical indenters involved in indentation. The data of the load portion of the load-displacement curve is used to determine the Young's modulus using a fit of all data points from the contact point to the point of maximum load.

Uniaxial tensile test:

IMRC and UCMSC cell membrane samples were the samples tested. The type of test equipment used was an Instron 5943 tensile tester (Instron, canton, MA, USA). Clamps were used to secure the ends of the sample. We measured the width W and thickness T of the cell patch samples. Before starting the experimental measurement, we measured the initial length L of the sample between the clamps. The load was applied at a rate of 10mm/min until the sample ruptured. We recorded the force F and the change in sample length al during the experiment. We then obtained the stress σ=f/a during the experiment, where a is the cross-sectional area of the sample. The calculation formula of the battery plate is as follows. A=wt. The strain is calculated as follows. epsilon=Δl/L ₀. The initial linear slope of the stress-strain curve was used to calculate the tensile elastic modulus (young's modulus, E) (from 10% to 30% strain). All of these factors can be combined into one equation of F:

Mechanical properties of cell membrane the schematic diagram of the mechanical properties of the cell membrane is shown in FIG. 19, and IMRC cell membrane and UCMSC cell membrane were subjected to instron and AFM at day 21.

Cell membrane mechanical properties test chart as shown in fig. 20, IMRC cell membrane, UCMSC cell membrane were subjected to instron tensile test at day 21. The IMRC cell membrane can withstand greater tensile forces.

The stress strain results of the cell membrane tensile test are shown in fig. 21. The results of the mechanical properties of the cell membrane are shown in FIG. 22. The results show that when instron tests are performed on day21, the IMRC cell membranes have significant differences in UTF, UTS, elastic modulus and UCMSC, indicating that the IMRC cell membranes have stronger mechanical properties compared with UCMSC cell membranes.

The results of the Young's modulus test of the cell membrane are shown in FIG. 23, and the IMRC cell membrane has a larger Young's modulus than the UCMSC cell membrane, indicating that the IMRC cell membrane has stronger mechanical properties.

The electron micrograph of the cell membrane after decellularization is shown in FIG. 24, and the fiber diameter and fiber density of the IMRC cell membrane are more uniform and greater than those of UCMSC.

Example 6 protein secretion assay of cell membrane

Immunofluorescent staining of cell patches followed by observation and data collection under a confocal laser microscope using antibodies comprising ：collagenⅠ(Abcam,Cambridge,MA,USA;Ab34710),1:150rabbit anti-collagenⅣ(Abcam,Cambridge,MA,USA;Ab6586),1:150rabbit anti-laminin(GeneTex,Irvine,CA,USA;GTX27463),and 1:150rabbit anti-fibronectin(GeneTex,Irvine,CA,USA;GTX112794).

The results of immunofluorescence assays of cell membranes and cells are shown in FIGS. 25 and 26, respectively, in which the extracellular matrix component of IMRC cell membranes is expressed higher than UCMSC.

Factor secretion assays were performed on cell membranes. Culture supernatants of IMRC cell membranes, UCMSC cell membranes at day21 were harvested and assayed for factor secretion using a Bio-Plex 200 suspension chip system. The results are shown in FIG. 27, wherein the LIF content of IMRC cell membranes was 190.19pg/mL, which showed 437-fold increase over UCMSC cell membranes, and the HGF content was 111.86pg/mL, which showed 1.25-fold increase over UCMSC cell membranes, after 21 days of culture. Compared with UCMSC cell membrane, IMRCs cell membrane immune regulation related factors and trophic factors are expressed more and proinflammatory factors are expressed less. Compared with UCMSCs cell membrane, IMRCs cell membrane is more beneficial to clinical application and has better treatment effect.

LC-MS identification is carried out on matrigel after cell membrane decellularization, in short, SDS-PAGE is carried out on matrigel serving as a sample, corresponding gel is obtained after SDS-PAGE gel electrophoresis experiments, corresponding band areas are cut off after dyeing, LC-MS mass spectrum identification is carried out after treatment, mass spectrum analysis (DDA mode) is carried out, and each scanning cycle has one MS full scan and comprises the following 15 MS/MS scans. (HCD collision energy: 28; four-bar screening window 1.6Da; dynamic exclusion time: 35s; data alignment reference UniProt-Homo database (pancreatin; maximum missed cut site: 2; fixed modification: carbamidomethyl (C); variable modification Oxidation (M), dioxidation (W), DEAMIDATED (NQ), actyl (Protein N-MS tolerance: + -10 ppm MS/MS tolerance: + -0.6 Da; protein score C.I. > 95%) was defined as identification success).

As shown in FIGS. 28A-28B, the identification results of the protein mass spectrum after cell membrane decellularization show that the extracellular matrix related proteins in IMRCs are more in variety and the factors are more in variety. Proteins with increased extracellular matrix content compared to UCMSCs cell membrane, IMRCs include COL15A1, COL8A1, EFEMP2, FBLN5, MFAP2, MXRA5, PCOLCE, THBS2, LOXL2, TIMP1, CLEC2D, HPX, BGN, GDF.

As shown in FIG. 29, the mass spectrometry analysis results after cell membrane decellularization show that the extracellular matrix type abundance after IMRC membrane decellularization is higher than that of UCMSC membrane. The major components in IMRCs, UCMSCs cell membranes are FN1 and COL1A1, and FN1 ranks highest among IMRCs cell membranes.

The protein content, the Collagen and the GAG content of IMRCs and UCMSCs after cell membrane decellularization are identified, the BCA experimental method (using thermo 23252) is used for the total protein content, the Sircol dye reagent is used for the Collagen content measurement, and the Blyscan dye reagent is used for the GAG content measurement. The results are shown in FIG. 30, which shows that IMRCs cells have higher extracellular matrix content than UCMSCs.

EXAMPLE 7 hydrogel for promoting proliferation and migration of IMRC cells

The experimental method comprises the following steps:

(1) Plate IMRC hydrogel (M-Mgel) or filtered hydrogel (M-Filtration of the material filtered with 0.22 filter) was plated at a concentration of 60. Mu.g/mL, 1mL per well of 6-well plate, and placed in a 37℃incubator for 30min. (2) Cell inoculation, namely inoculating IMRCs into a common pore plate or an IMRC hydrogel coated pore, culturing for 3 days, and changing the liquid every day, wherein the cell density is 1x10 ⁴/cm². (3) Cell digestion count, and identification of surface markers.

Results:

as shown in FIG. 31, the coating of IMRC hydrogel significantly promoted proliferation of IMRC cells, while the expression of the cell surface Marker was unchanged, compared to the conventional culture plate. The above data indicate that IMRC hydrogels can promote proliferation of IMRC cells and maintain the cell properties unchanged.

As shown in FIG. 32, the migration of GFP-IMRC cells was significantly promoted after coating the IMRC hydrogel, compared to the common plates and the coated Collagen group.

As shown in FIG. 33, the IMRC hydrogel can encapsulate GFP-IMRC cells, promoting proliferation and expansion of GFP-M cells.

EXAMPLE 8 use of hydrogels for promoting in vitro vascularization of HUVECs

The experimental method comprises the following steps:

(1) mu.L of IMRC hydrogel (5-10 mg/mL) or 100. Mu.L of Collagen (5-10 mg/mL) was added to a 96-well plate, and the mixture was allowed to stand in an incubator for 15min. (2) The fluorescently labeled HUVECs were inoculated into normal plates, coated Collagen plates, coated IMRC hydrogel plates. (3) Under the living cell workstation, the setup program performs a photographing record every half an hour for a total of 24 hours.

Results:

As shown in FIG. 34, HUVEC cells were seeded on a common plate, a coated Collagen plate, and a coated IMRC hydrogel plate, and as a result, it was found that the IMRC hydrogel can promote in vitro vascularization of HUVECs.

EXAMPLE 9 cell patch for treatment of corneal alkali burn

The rat cornea alkali burn molding is to select 7-8 week old male SD rat (Vetong Lihua company) and the rat is anesthetized and molded by inhalation of isoflurane. After immersing a filter paper with a diameter of 7mm in 1M NaOH for 30s, excess NaOH was sucked off on the dried filter paper, and then transferred to the right cornea of a rat for 30s, and then rinsed with 50mL of physiological saline. IMRCs or UCMSCs cell patches were placed into the damaged cornea and the eyelids were then sutured with 5-0 silk threads. Photographing and observing at Day3, day7 and Day21, and taking materials for pathological section analysis of the samples.

As shown in fig. 35, IMRC patch treatment reduced macrophage infiltration at day3 and significantly lower CD31, α -SMA expression in cornea at day21 than in the model group. The scores of the IMRC diaphragm treatment groups are obviously different from those of the NaOH group, and the IMRC diaphragm can be used for well treating rat cornea alkali burn.

EXAMPLE 10 hydrogel for treatment of intrauterine adhesions

And (3) molding the intrauterine adhesion of the rat, and establishing a model of the intrauterine adhesion of the rat by injecting absolute ethyl alcohol into the uterus. Female SD rats (Vetong Liwa) of 7-8 weeks old were selected, all rats were anesthetized by intraperitoneal injection of 2% pentobarbital sodium (0.3 ml/100 g), the abdomen was shaved, and uterine horns were exposed through abdominal incisions. The proximal and distal portions of the uterus were then gently clamped with two vascular clamps, after which 95% ethanol was injected into the right uterine cavity for 5 minutes. The damaged portion was washed three times with physiological saline to further remove the remaining ethanol, and then the vascular clamp was loosened. After one week of animal modeling, animals of the experimental group were again operated on, and the proximal and distal portions of the damaged uterus were gently clamped with two vascular clamps while physiological saline, collagen, and IMRCs hydrogels were injected into the uterus. After three estrus cycles following hydrogel injection, rats were euthanized, uterine tissue excised and sectioned for downstream analysis.

Fig. 36 shows a schematic representation of intrauterine adhesion treatment of rats by IMRC hydrogel implantation.

After implantation IMRCs of the hydrogel, as shown in fig. 37, a significant gel formation is achieved in the uterine cavity. As shown in fig. 38, IMRCs hydrogel treatment groups had no obvious blisters, and alcohol and collagen groups had obvious blisters, indicating that IMRCs hydrogel had a better treatment effect on intrauterine adhesion. As shown in FIG. 39, after the treatment of the IMRC hydrogel, the shape and structure of the rat uterus are complete, the endometrium, the number of blood vessels and the number of glands are remarkably different from those of a model group and a collagen group, which shows that the IMRC hydrogel has remarkable treatment effect on the rat uterine cavity adhesion, the proportion of the rat uterine fibrosis is remarkably reduced, which shows that the IMRC hydrogel can improve the fibrosis condition of the rat uterine cavity adhesion and has good treatment effect.

EXAMPLE 11 preparation of chondrocyte membrane

After the IMRC cell membrane forming sheet is induced for 7 days, the cell forming sheet is replaced by chondroblast induced differentiation liquid, membrane forming induced culture is continued for 14 days, liquid is replaced every day, cells in the cell sheet are differentiated into chondrocytes, the chondrocyte sheet is obtained after harvesting on the 21 st day, and in-vitro property identification and in-vivo function verification are carried out. The chondrocyte membrane induced differentiation liquid is prepared according to the specification of a human mesenchymal stem cell function identification kit (human MESENCHYMAL STEM CELL functional identification kit, cat# SC 006).

While specific embodiments of the invention have been described in detail, it will be appreciated by those skilled in the art that various modifications and alternatives to those details could be developed in light of the overall teachings of the disclosure and that such modifications would be within the scope of the invention. The full scope of the invention is given by the appended claims together with any equivalents thereof.

Claims

1. A cell membrane sheet formed by immune and matrix regulatory cells (IMRC) and extracellular matrix secreted by the cells, wherein the extracellular matrix at least comprises: COL15A1, COL8A1, EFEMP2, FBLN5, MFAP2, MXRA5, PCOLCE, THBS2, LOXL2, TIMP1, CLEC2D, HPX, BGN, GDF15;

Preferably, the content of any of the above proteins is increased compared to that of primary mesenchymal stem cell membrane sheets (such as UCMSC cell membrane sheets);

Preferably, the content of any of the above proteins is independently increased by at least about 1.2 times, at least about 1.5 times, at least about 1.8 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 4.5 times, at least about 5 times, at least about 5.5 times, at least about 6 times, at least about 6.5 times, at least about 7 times, at least about 7.5 times, at least about 8 times, at least about 8.5 times, at least about 9 times, at least about 9.5 times, at least about 10 times, at least about 15 times, at least about 20 times, at least about 25 times, at least about 30 times, at least about 35 times, at least about 40 times, at least about 45 times, at least about 50 times, at least about 60 times, at least about 70 times, at least about 80 times, at least about 90 times, at least about 100 times compared to the primary mesenchymal stem cell membrane (e.g., UCMSC cell membrane).

2. The cell sheet of claim 1, wherein, compared with a primary mesenchymal stem cell sheet (e.g., a UCMSC cell sheet), the cell sheet has an FN expression level of at least about 5 times (e.g., about 6-8 times), an ELN expression level of at least about 100 times (e.g., about 120-140 times), and/or an POSTN expression level of at least about 100 times (e.g., about 120-140 times);

Preferably, the expression level is the mRNA level.

3. The cell membrane of claim 2, which, compared with a primary mesenchymal stem cell membrane (e.g., a UCMSC cell membrane), further has one or more characteristics selected from the following: COL1A1 expression level is at least about 8 times (e.g., about 8 to 12 times); COL1A2 expression level is at least about 11 times (e.g., about 11 to 15 times); COL4A1 expression level is at least about 5 times (e.g., about 5 to 8 times); TNC expression level is at least about 20 times (e.g., about 20 to 24 times); COL4A2 expression level is at least about 8 times (e.g., about 8 to 12 times); LAMC1 expression level is at least about 1.1 times (e.g., about 1.1 to 1.4 times); and/or, CXCL12 expression level is at least about 1.2 times (e.g., about 1.2 to 1.6 times);

Preferably, the expression level is the mRNA level.

4. The cell sheet according to any one of claims 1 to 3, wherein the cell sheet secretes LIF and shows an increase of at least about 300 times (e.g., about 350 to 450 times) compared to a primary mesenchymal stem cell sheet (e.g., a UCMSC cell sheet);

Preferably, the LIF content is at least about 100 pg/mL (e.g., about 180-200 pg/mL, about 185-195 pg/mL);

Preferably, the cell membrane sheet secretes HGF and shows an increase of at least about 1.1 times (e.g., about 1.2-1.4 times) compared to a primary mesenchymal stem cell membrane sheet (e.g., a UCMSC cell membrane sheet);

Preferably, the HGF content is at least about 100 pg/mL (eg, about 110-120 pg/mL, about 110-115 pg/mL).

5. The cell sheet according to any one of claims 1 to 4, wherein the pro-inflammatory factors secreted by the cell sheet are reduced compared with those of a primary mesenchymal stem cell sheet (e.g., a UCMSC cell sheet);

Preferably, the pro-inflammatory factor is selected from IL6, IL1α, IL1β;

Preferably, IL6 shows at least about 100-fold (eg, about 110-120-fold) reduction compared to a primary mesenchymal stem cell sheet (eg, a UCMSC cell sheet);

Preferably, IL1α shows at least about 5-fold (eg, about 5-7-fold) reduction compared to a primary mesenchymal stem cell sheet (eg, a UCMSC cell sheet);

Preferably, IL1β shows at least about 6-fold (eg, about 6-8-fold) reduction compared to a primary mesenchymal stem cell sheet (eg, a UCMSC cell sheet);

Preferably, the expression level is the mRNA level.

6. The cell membrane sheet according to any one of claims 1 to 5, wherein the fiber diameter of the extracellular matrix is less than 0.5 μm, such as 0.2-0.5 μm, 0.2-0.4 μm, 0.3-0.4 μm; and/or the fiber density is 12-16 fibers/25 ^μm2 .

7. The cell membrane sheet according to any one of claims 1 to 6, wherein the Young's modulus of the cell membrane sheet is at least 500 Pa, such as at least 800 Pa, at least 1000 Pa, at least 2000 Pa, at least 3000 Pa or at least 3500 Pa.

8. The cell membrane sheet of any one of claims 1-7, wherein the thickness of the cell membrane sheet is at least 10 μm, such as at least 30 μm, at least 50 μm, at least 60 μm, at least 70 μm, at least 80 μm, at least 90 μm, or at least 100 μm.

9. A method for preparing a cell membrane sheet, comprising: culturing IMRC cells in a culture medium containing vitamin C or a derivative thereof (eg, ascorbic acid 2-glucoside, ascorbyl glucoside or isoascorbic acid) to obtain a cell membrane sheet.

10. The method of claim 9, wherein the IMRC cells are seeded in a culture container coated with a matrix;

Preferably, the coating matrix is selected from collagen, gelatin, laminin, fibronectin, or derivatives thereof.

11. The method of claim 9 or 10, wherein the seeding density of the IMRC cells is 1×10 ³ /cm ² to 1×10 ⁶ /cm ² ; for example, 1×10 ³ /cm ² to 1×10 ⁵ /cm ² , 1×10 ³ /cm ² to 5×10 ⁴ /cm ² , 5×10 ³ /cm ² to 1×10 ⁵ /cm ² , 5×10 ³ /cm ² to 5×10 ⁴ /cm ² , for example 2×10 ⁴ /cm ² .

12. The method according to any one of claims 9 to 11, wherein the content of vitamin C or its derivatives is 1-100 μmol/L, such as 10-100 μmol/L, 10-80 μmol/L, 10-60 μmol/L, 10-50 μmol/L.

13. The method of any one of claims 9 to 12, wherein the culture medium is a basal medium supplemented with the following substances: one or more serum or serum replacements, one or more non-essential amino acids, glutamine or a stabilized dipeptide of L-alanyl-L-glutamine, and one or more growth factors;

Preferably, the one or more sera or serum replacements comprise KOSR and Ultroser G;

Preferably, the non-essential amino acid is selected from glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine and combinations thereof; for example, NEAA;

Preferably, the basal culture medium is selected from α-MEM, MEM, DMEM, high-glucose DMEM, KO-DMEM, and any combination thereof;

Preferably, the one or more growth factors include bFGF and TGF-β; optionally, further include one or more selected from VEGF, EGF, or PDGF.

14. The method of claim 13, wherein the culture medium is a basal medium supplemented with the following substances: 1-5% (v/v) Ultroser G, 5-30% (v/v) KOSR, 1-5 mM stabilized dipeptide of L-alanyl-L-glutamine, 1-100 ng/mL bFGF, 1-100 ng/mL TGFβ, and the following amino acids at a concentration of 0.1-0.5 mM each: glycine, L-alanine, L-asparagine, L-aspartic acid, L-glutamic acid, L-proline, L-serine;

Preferably, the basal culture medium is selected from KO-DMEM, KO-DMEM/F12, α-MEM, DMEM, DMEM/F12.

15. The method according to any one of claims 9 to 14, wherein the culture is performed for at least 1 day;

For example, the culture is for at least 7 days, at least 14 days, such as 14 to 28 days or 14 to 21 days.

16. The method of any one of claims 9 to 15, wherein the culturing is for at least 35 days, such as at least 42 days, such as 45 days.

17. A cell membrane sheet prepared by the method according to any one of claims 9 to 16.

18. A decellularized matrix material obtained by decellularizing the cell membrane sheet according to any one of claims 1 to 8 or the cell membrane sheet according to claim 17.

19. The decellularized matrix material according to claim 18, wherein the decellularized matrix material is a membrane sheet obtained by decellularizing the cell membrane sheet according to any one of claims 1 to 8 or the cell membrane sheet according to claim 17;

Preferably, the decellularization treatment comprises physical methods (e.g., direct effects of temperature, pressure, electroporation and force), chemical methods (e.g., acids and alkalis, detergents, hypotonic and hypertonic solutions, alcohols and solvents), biological methods (such as enzymes, nucleases and chelators) or any combination thereof.

20. The decellularized matrix material according to claim 19, wherein the decellularized matrix material is a hydrogel obtained by decellularizing and digesting the cell membrane sheet according to any one of claims 1 to 8 or the cell membrane sheet according to claim 17;

Preferably, the decellularization treatment comprises physical methods (e.g., direct effects of temperature, pressure, electroporation and force), chemical methods (e.g., acids and alkalis, detergents, hypotonic and hypertonic solutions, alcohols and solvents), biological methods (e.g., enzymes, nucleases and chelating agents) or any combination thereof;

Preferably, the digestion treatment comprises the use of an acid (eg acetic acid or hydrochloric acid) and/or a digestive enzyme (eg pepsin or trypsin).

21. A method for preparing a cell-free matrix material, comprising:

(1) Providing the cell membrane sheet according to any one of claims 1 to 8 or the cell membrane sheet according to claim 17;

(2) performing a decellularization treatment on the cell membrane sheet;

Preferably, the decellularization treatment comprises using a surfactant (e.g., a nonionic surfactant or an ionic surfactant, such as Triton-X 100, sodium dodecyl sulfate (SDS), sodium deoxycholate (SD));

Preferably, the decellularization treatment comprises freeze-thawing;

Preferably, the decellularization treatment comprises using 1% Triton-X 100, such as 1% Triton-X 100 and 20 mM NH ₃ ·H ₂ O;

Preferably, the decellularization treatment comprises using 0.1% SDS;

Preferably, the product obtained in step (2) is a decellularized membrane sheet.

22. The method of claim 21, wherein the decellularized matrix material is a hydrogel, and the method further comprises subjecting the decellularized product to a digestion treatment;

Preferably, the digestion treatment comprises the use of an acid (e.g. acetic acid or hydrochloric acid) and/or a digestive enzyme (e.g. pepsin or trypsin);

Preferably, the digestion treatment comprises using acetic acid and pepsin;

Preferably, the digestion treatment further includes the steps of dialysis, freeze-drying and dissolution.

23. A composite cell sheet comprising IMRC cells and at least one cell selected from the group consisting of fibroblasts, keratinocytes, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, cardiac fibroblasts, vascular wall cells (e.g., smooth muscle cells, pericytes), corneal endothelium, and retinal pigment epithelial cells.

24. The composite cell membrane sheet of claim 23, comprising at least two stacked cell membrane sheets, wherein at least one cell membrane sheet is the cell membrane sheet of any one of claims 1 to 8 or the cell membrane sheet of claim 17;

Preferably, at least one layer of the cell membrane is a cell membrane formed by cells selected from the group consisting of fibroblasts, keratinocytes, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, cardiac fibroblasts, vascular wall cells (e.g., smooth muscle cells and pericytes), corneal endothelium, and retinal pigment epithelial cells;

Preferably, adhesive is included between each layer of the stacked cell membrane sheets;

Preferably, the adhesive is made of a biocompatible material that can be degraded in the body;

Preferably, the adhesive is the acellular matrix material according to any one of claims 18-20.

25. A method for preparing a composite cell membrane sheet, comprising:

(1) providing a plurality of cell membrane sheets, at least one of which is the cell membrane sheet according to any one of claims 1 to 8 or the cell membrane sheet according to claim 17;

(2) disposing an adhesive on one of the cell membrane sheets, and then laminating another cell membrane sheet thereon;

(3) Repeating step (2);

(4) obtaining a stacked product having a predetermined number of sheets;

Preferably, at least one of the cell membrane sheets is a cell membrane sheet formed by cells selected from the group consisting of fibroblasts, keratinocytes, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, cardiac fibroblasts, vascular wall cells (e.g., smooth muscle cells, pericytes), corneal endothelium, and retinal pigment epithelial cells;

Preferably, the adhesive is made of a material that is biocompatible and degradable in the human body;

26. A composite cell membrane sheet, comprising at least two stacked cell membrane sheets, wherein an adhesive is contained between each of the stacked cell membrane sheets, and the adhesive is selected from the decellularized matrix material according to any one of claims 18 to 20;

Preferably, the at least two layers of cell membranes are each independently formed by cells selected from the following: fibroblasts, keratinocytes, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, cardiac fibroblasts, vascular wall cells (e.g., smooth muscle cells, pericytes), corneal endothelium, retinal pigment epithelial cells;

Preferably, the composite cell membrane sheet comprises a fibroblast membrane sheet and a keratinocyte membrane sheet.

27. A method for preparing a composite cell membrane sheet, comprising:

(1) Providing multiple cell membrane sheets;

(2) disposing an adhesive on one of the cell membrane sheets, and then stacking another cell membrane sheet thereon; wherein the adhesive is selected from the decellularized matrix material according to any one of claims 18 to 20;

(3) Repeating step (2);

(4) obtaining a stacked product having a predetermined number of sheets;

Preferably, the plurality of cell membrane sheets are each independently formed by cells selected from the following: fibroblasts, keratinocytes, cardiomyocytes, vascular endothelial cells, vascular smooth muscle cells, cardiac fibroblasts, vascular wall cells (e.g., smooth muscle cells, pericytes), corneal endothelium, retinal pigment epithelial cells;

Preferably, the plurality of cell membrane sheets include fibroblast membrane sheets and keratinocyte membrane sheets.

28. Use of the decellularized matrix material according to any one of claims 18 to 20 as an adhesive;

Preferably, the adhesive is used to prepare a cell membrane sheet;

Preferably, the cell membrane sheet is a composite cell membrane sheet as described in claim 23, 24 or 26.

29. A cell sheet induced to differentiate, which is obtained by inducing differentiation of the cell sheet according to any one of claims 1 to 8 or the cell sheet according to claim 17;

Preferably, the induced differentiated cell membrane is selected from ectoderm tissue membrane (e.g., neural crest cell membrane, nerve cell membrane, retinal pigment epithelial cell membrane, keratinocyte membrane), mesoderm tissue membrane (e.g., chondrocyte membrane, osteocyte membrane, adipocyte membrane, myocardial cell membrane, muscle cell membrane, vascular endothelial cell membrane, kidney cell membrane), endoderm tissue membrane (hepatocyte membrane, pancreatic β cell membrane, intestinal cell membrane, gastric cell membrane);

Preferably, the cell sheet induced to differentiate is a chondrocyte sheet.

30. A pharmaceutical composition comprising the cell membrane sheet according to any one of claims 1 to 8 or the cell membrane sheet according to claim 17, the decellularized matrix material according to any one of claims 18 to 20, or the composite cell membrane sheet according to claim 23, 24 or 26, or the cell membrane sheet according to claim 29, and a pharmaceutically acceptable carrier and/or excipient.

31. Use of the cell membrane sheet according to any one of claims 1 to 8, the cell membrane sheet according to claim 17, the decellularized matrix material according to any one of claims 18 to 20, the composite cell membrane sheet according to claim 23, 24 or 26, the cell membrane sheet according to claim 29, or the pharmaceutical composition according to claim 30 in the preparation of a drug for regeneration or repair of tissues or organs.

32. Use of the cell membrane sheet according to any one of claims 1 to 8, the cell membrane sheet according to claim 17, the decellularized matrix material according to any one of claims 18 to 20, the composite cell membrane sheet according to claim 23, 24 or 26, the cell membrane sheet according to claim 29, or the pharmaceutical composition according to claim 30 in the preparation of a drug for treating tissue or organ damage;

Preferably, the tissue or organ damage includes corneal damage (e.g. corneal alkali burn), skin damage (e.g. skin burn, skin scald, diabetic foot), heart disease (e.g. myocardial damage), bone damage (e.g. skull damage, bone defect), endometrial damage (e.g. intrauterine adhesion), meniscus damage, and/or muscle damage, periodontal damage (e.g. periodontitis), esophagus (e.g. esophageal mucosal damage), liver damage (e.g. cirrhosis), pancreatic disease (e.g. diabetes, pancreatitis), lung disease (e.g. lung damage, pulmonary fibrosis);

Preferably, the cell sheet is induced to differentiate into osteoblasts or chondrocytes.

33. Use of the cell membrane sheet described in any one of claims 1 to 8 or the cell membrane sheet described in claim 17, the decellularized matrix material described in any one of claims 18 to 20, the composite cell membrane sheet described in claim 23, 24 or 26, or the cell membrane sheet described in claim 29, or the pharmaceutical composition described in claim 30 in the preparation of a medicament for cell, tissue or organ transplantation.

34. Use of the decellularized matrix material according to any one of claims 18 to 20 as a pharmaceutically acceptable carrier and/or excipient in a drug;

Preferably, the drug comprises a nucleic acid drug, a small molecule drug, a protein drug, and/or a cell, tissue or organ transplant drug.

35. Use of the decellularized matrix material according to any one of claims 18 to 20 for cell culture (e.g., culture of pluripotent stem cells, differentiation of pluripotent stem cells), organoid culture, or 3D printing;

Preferably, the acellular matrix material is used as ink for 3D printing;

Preferably, the decellularized matrix material is used as a coating matrix for cell culture or organoid culture.