WO2023208112A1 - 无动物源性细胞膜片及其制备方法和应用 - Google Patents

无动物源性细胞膜片及其制备方法和应用 Download PDF

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WO2023208112A1
WO2023208112A1 PCT/CN2023/091164 CN2023091164W WO2023208112A1 WO 2023208112 A1 WO2023208112 A1 WO 2023208112A1 CN 2023091164 W CN2023091164 W CN 2023091164W WO 2023208112 A1 WO2023208112 A1 WO 2023208112A1
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mesenchymal stem
cells
medium
culture
stem cells
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PCT/CN2023/091164
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French (fr)
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常德华
靳新
高爽
王娟
刘帅
赵玉菲
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京东方科技集团股份有限公司
京东方再生医学科技有限公司
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Publication of WO2023208112A1 publication Critical patent/WO2023208112A1/zh

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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • A61P37/02—Immunomodulators
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/90—Serum-free medium, which may still contain naturally-sourced components
    • C12N2500/92—Medium free of human- or animal-derived components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/998—Proteins not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin
    • C—CHEMISTRY; METALLURGY
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    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/56—Fibrin; Thrombin

Definitions

  • the present disclosure relates to the fields of tissue engineering and regenerative medicine, and in particular to animal-derived cell membrane sheets and preparation methods and applications thereof.
  • Cell sheets are a more efficient method of stem cell transplantation than previous transplantation methods involving injection of cells in a stem cell suspension.
  • the cell membrane sheet can effectively prevent the loss of stem cells during the transplantation process and improve the efficiency of stem cell transplantation.
  • cell membrane sheets do not use enzymes and analogues for cell digestion during the preparation process, which effectively avoids the destruction of extracellular matrix and reduction of cell function caused by enzyme digestion.
  • Cell membrane sheet transplantation can make stem cells better function in the body. perform its function.
  • the preparation process of mesenchymal stem cell membrane usually involves digesting the subcultured mesenchymal stem cells into single cells with biological enzymes, followed by washing to remove the residue of mesenchymal stem cell culture medium, and then resuspending the cells in membrane-forming medium. Finally, inoculate in a pre-coated temperature-sensitive petri dish. Under the culture conditions of 37°C, the cells grew adherently in the culture dish and proliferated to confluence. After the culture is completed, the culture temperature is lowered, and the cells will automatically detach from the temperature-sensitive surface in sheets, and the cell membrane sheets can be harvested.
  • Cell membrane sheets are sheet-like structures composed of a single layer or multiple layers of cells.
  • the film-forming medium used in the preparation of mesenchymal stem cell membrane sheets and the matrix coated in temperature-sensitive culture dishes remain in the intercellular spaces and on the surface, and are difficult to be completely washed away by external force.
  • the film-forming media used in the membrane preparation methods that have been reported so far are divided into three categories: serum-containing media, commercially available serum-free media, and self-prepared serum-free media.
  • serum can promote the attachment of cells to the surface of temperature-sensitive culture dishes and promote the adhesion between cells.
  • the bovine serum in the serum-containing medium contains heterologous macromolecule sensitizing components bovine Serum albumin, not safe enough for clinical use.
  • Serum-free medium for mesenchymal stem cells on the market and self-prepared serum-free medium for mesenchymal stem cells in the literature contain a variety of exogenous growth factors in order to support cell growth, and the residues of exogenous growth factors may also cause safety issues. risk.
  • the coating matrix currently used in the preparation process of diaphragms is scientific research grade such as fibronectin and laminin. Special reagents, these reagents do not comply with GMP standards during the production process.
  • the film-forming medium used in this method has simple ingredients, including a basic medium and a binder (such as human serum albumin (such as pharmaceutical grade)), which does not contain any animal-derived components (such as serum) and exogenous growth factors. .
  • a binder such as human serum albumin (such as pharmaceutical grade)
  • adhesion factors such as human fibrinogen
  • the mesenchymal stem cell membrane product prepared by the above method of the present disclosure is safer and does not contain animal-derived components (such as serum) and exogenous growth factor residues (such as bovine serum albumin residues that meet drug standards).
  • the film-forming process The medium formula used is simple.
  • the mesenchymal stem cell membrane product of the present disclosure has a certain degree of toughness and can be folded and flattened in appropriate culture media, buffers or preservation solutions. Diaphragm products can still maintain good diaphragm shape after being stored in a specific preservation solution at 4°C for 24 hours.
  • the cell survival rate is as high as over 70%. They can secrete high levels of pro-angiogenic factors and anti-inflammatory factors and inhibit lymphocytes.
  • Proliferation and inflammatory factor secretion can be used clinically for a variety of diseases, including autoimmune system diseases, organ damage diseases, rejection and GVHD during organ transplantation, etc., while maintaining stem cell characteristics and having specific surface markers. It has the ability to induce differentiation in three directions and can be used clinically to repair damaged tissues.
  • the present disclosure relates to the following aspects.
  • the present disclosure relates to a method of preparing a cell membrane sheet, the method comprising the following steps:
  • b. Transfer the cells to a temperature-sensitive culture dish coated with a matrix (also known as adhesion factor) and culture it in a film-forming medium to form a film sheet in the culture dish, wherein the film-forming culture
  • the base contains a basal culture medium and a binder (such as human serum albumin) and does not contain animal-derived components (such as serum, such as bovine serum albumin, etc.) and exogenous growth factors; and
  • temperature-sensitive petri dish or “temperature-sensitive petri dish” refers to a surface coated A petri dish with a layer of temperature-sensitive polymer material.
  • the molecular chain stretches differently at different temperatures, thereby showing hydrophilicity or hydrophobicity, so that the hydrophilicity and hydrophobicity of the polymer material can change with the external temperature. Change with change.
  • the surface of the temperature-sensitive culture dish becomes hydrophilic, the adhesion to the cells and the extracellular matrix they secrete becomes poor, and the cells will fall off in layers.
  • the temperature is lowered below the low critical dissolution temperature of the polymer substance, the surface of the temperature-sensitive culture dish becomes hydrophilic, so that the cells will fall off in layers.
  • the cell patch is a stem cell patch, such as a mesenchymal stem cell patch.
  • the matrix is fibronectin, laminin, gelatin, collagen, vitronectin, or human fibrinogen.
  • the matrix is gelatin.
  • the gelatin concentration (w/w) is 0.01-0.5%, such as 0.05-0.2%, such as 0.1%.
  • the matrix is vitronectin.
  • the concentration of vitronectin is 1-20 ⁇ g/mL, such as 5-15 ⁇ g/mL, such as 10 ⁇ g/mL.
  • the matrix is poly-D lysine (PDL).
  • the concentration of PDL is 0.01-0.5 mg/mL, such as 0.05-0.2 mg/mL, such as 0.1 mg/mL. Coating a temperature-sensitive culture dish with the above matrix enables mesenchymal stem cells to attach to the culture dish.
  • the matrix is human fibrinogen.
  • the concentration of human fibrinogen is 0.1-10 mg/mL, such as 0.2-5 mg/mL, such as 1-2.5 mg/mL.
  • the basal medium in the film-forming medium can be selected from the group consisting of DMEM (high sugar), DMEM (low sugar), RPMI1640, ⁇ -MEM, DMEM/F12 and F12.
  • the basal medium in the film-forming medium is ⁇ -MEM.
  • the concentration of human serum albumin in the film-forming medium is 0.1-10%, preferably 0.1-5%, such as 0.1%, 0.5%, 1%, 1.5%, 2% , 2.5%, 3%, 3.5%, 4%, 4.5% or 5%.
  • the film-forming medium further comprises non-essential amino acids (glycine, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, L- proline, L-serine) and/or L-glutamine.
  • non-essential amino acids glycine, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, L- proline, L-serine
  • L-glutamine glycine, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, L- proline, L-serine
  • the concentration of L-glutamine in the film-forming medium used in step b is from 0.5mM to 4mM, preferably about 2mM.
  • the non-essential amino acid glycerol in the film-forming medium used in step b The concentration of each of amino acid, L-alanine, L-aspartic acid, L-asparagine, L-glutamic acid, L-proline and L-serine is 50 ⁇ M to 200 ⁇ M, preferably about 100 ⁇ M.
  • the mesenchymal stem cells are cultured and passaged with a serum-containing medium (eg, a medium containing fetal bovine serum).
  • a serum-containing medium eg, a medium containing fetal bovine serum
  • the mesenchymal stem cells are cultured and passaged using serum-free medium.
  • the serum-free culture system includes the use of a basal medium selected from the group consisting of: RPMI1640, DMEM, ⁇ -MEM, DMEM/F12, and F12 serum-free medium, and the medium is supplemented with a medium selected from the group consisting of: One or more additives: vitamin C, sodium selenate, hydrocortisone, insulin, transferrin, human serum albumin (plant expression), progesterone, putrescine, biotin, sodium pyruvate, ethanolamine, Carnitine, amino acids, vitamins, glutathione, linoleic acid and linolenic acid.
  • the serum-free culture system includes using a commercial culture medium selected from the group consisting of: CTS TM Stem Pro TM MSC SFM, MesenCult TM -ACF Medium, MesenCult TM -ACF Plus Medium, and MesenCult TM - XF medium.
  • a commercial culture medium selected from the group consisting of: CTS TM Stem Pro TM MSC SFM, MesenCult TM -ACF Medium, MesenCult TM -ACF Plus Medium, and MesenCult TM - XF medium.
  • the method further includes the step of washing the cells after step a and before step b.
  • the mesenchymal stem cells can be derived from a tissue selected from the group consisting of: amniotic fluid, amnion, chorion, chorionic villi, decidua, placenta, umbilical cord blood, umbilical cord, adult bone marrow, adult peripheral Blood and adult adipose tissue.
  • the mesenchymal stem cells may be selected from umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, and bone marrow mesenchymal stem cells, as well as mesenchymal stem cells from other sources known in the art.
  • stem cells may be selected from umbilical cord mesenchymal stem cells, placental mesenchymal stem cells, adipose mesenchymal stem cells, and bone marrow mesenchymal stem cells, as well as mesenchymal stem cells from other sources known in the art.
  • stem cells are umbilical cord mesenchymal stem cells.
  • the mesenchymal stem cells are umbilical cord mesenchymal stem cells, and the method further includes the step of obtaining mesenchymal stem cells from the umbilical cord before step a).
  • obtaining umbilical cord mesenchymal stem cells from the umbilical cord includes the following steps:
  • mesenchymal stem cells grow to 50%-100% confluence, such as 70%-100% confluence or 80%-100% confluence, remove the tissue block to obtain umbilical cord mesenchymal stem cells.
  • the coating time of the temperature-sensitive petri dish is from 1 hour to 7 days, preferably from 1 to 36 hours, and most preferably from 2 to 18 hours. In some embodiments, the coating temperature is 2-37°C, preferably 37°C.
  • cells are plated into a temperature-sensitive culture dish in an inoculation amount as shown below.
  • the number of cells to be inoculated is 1 ⁇ 10 7 to 10 ⁇ 10 7 , such as 1 ⁇ 10 7 , 2 ⁇ 10 7 , 3 ⁇ 10 7 , 4 ⁇ 10 7 , 5 ⁇ 10 7 , 6 ⁇ 10 7 , 7 ⁇ 10 7 , 8 ⁇ 10 7 , 9 ⁇ 10 7 or 10 ⁇ 10 7 pcs.
  • the volume of the culture medium in which the cells are suspended and seeded is 15-30 mL, such as 15, 20, 25 or 30 mL.
  • the number of cells to be seeded is 1 ⁇ 10 6 to 30 ⁇ 10 6 , such as 2 ⁇ 10 6 to 20 ⁇ 10 6 , 5 ⁇ 10 6 to 15 ⁇ 10 6 or 8 ⁇ 10 6 to 12 ⁇ 10 6 , such as 8 ⁇ 10 6 , 9 ⁇ 10 6 , 10 ⁇ 10 6 , 11 ⁇ 10 6 or 12 ⁇ 10 6 .
  • the volume of the culture medium in which the cells are suspended and seeded is 1.5-5 mL, such as 1.5, 2, 2.5, 3, 3.5, 4, 4.5 or 5 mL.
  • the number of cells to be seeded is 3 ⁇ 10 6 to 75 ⁇ 10 6 , such as 5 ⁇ 10 6 to 60 ⁇ 10 6 , 10 ⁇ 10 6 to 50 ⁇ 10 6 , 15 ⁇ 10 6 to 40 ⁇ 10 6 pcs, 20 ⁇ 10 6 to 30 ⁇ 10 6 pcs or 20 ⁇ 10 6 to 25 ⁇ 10 6 pcs, such as 20 ⁇ 10 6 pcs, 20.5 ⁇ 10 6 pcs, 21 ⁇ 10 6 pcs, 21.5 ⁇ 10 6 pcs, 22 ⁇ 10 6 pcs, 22.5 ⁇ 10 6 pcs, 23 ⁇ 10 6 pcs, 23.5 ⁇ 10 6 pcs, 24 ⁇ 10 6 pcs, 24.5 ⁇ 10 6 pcs or 25 ⁇ 10 6 pcs.
  • the volume of culture medium in which cells are suspended and seeded is 3-12.5mL, such as 3, 3.5, 4, 4.5, 5, 5.5, 6, 6, 5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11 , 11.5, 12 or 12.5mL.
  • the mesenchymal stem cells are detached from the temperature-sensitive culture dish by lowering the temperature, thereby forming a mesenchymal stem cell sheet.
  • the culture temperature is about 37°C
  • the mesenchymal stem cells are detached from the temperature-sensitive culture dish by lowering the temperature to 4-32°C.
  • the present disclosure relates to cell membrane sheets prepared by the methods of the present disclosure.
  • the residual amount of bovine serum albumin in the cell membrane sheet obtained by the above preparation method meets the human drug standard, the content is ⁇ 1ng/cm 2 , the residual amount of human serum albumin is 10ng/cm 2 -15000ng/cm 2 , and the residual amount of human fibrinogen is 10ng/cm 2 -15000ng/cm 2 .
  • the residual amount is 0.5ng/cm 2 -5ng/cm 2 .
  • the mesenchymal cell membrane obtained by the above preparation method can secrete a large amount of HGF, VEGF and IL-8 pro-angiogenic function-related factors, secrete a large amount of anti-inflammatory related factors such as IL-6 and IL-8; and has the ability to inhibit lymphocyte Th1 subtype type, inhibiting lymphocyte proliferation and lymphocyte TNF ⁇ secretion.
  • the present disclosure relates to the use of mesenchymal stem cell sheets obtained by the above preparation method for regulating inflammatory responses or treating autoimmune system diseases in subjects, or for preparing Use in drugs that modulate inflammatory responses or treat autoimmune diseases.
  • the autoimmune disease is rheumatoid, allergy, lupus erythematosus, etc.
  • the present disclosure relates to the use of the mesenchymal stem cell sheet obtained by the above preparation method for damage repair of damaged tissue in a subject, or the preparation of a mesenchymal stem cell sheet for repairing damaged tissue in a subject. Use in damage repair drugs.
  • the damaged tissue is tissue of the heart, liver, pancreas, uterus, or other tissue.
  • Figure 1 shows the effect of serum as a coating matrix on the film formation of cell membrane sheets.
  • FBS fetal bovine serum.
  • Figure 2 shows an exemplary appearance of a cell membrane sheet.
  • Figure 3 shows the effect of the presence or absence of human fibrinogen as a coating matrix on cell membrane formation in a serum-free system.
  • Figure 4 shows the effect of the presence or absence of human serum albumin as a binder in the film-forming medium on the film formation of cell membrane sheets in a serum-free system.
  • Figure 5 shows the results of IHC staining of cell membrane sheets.
  • Figure 6 shows the results of H&E staining of cell membrane sheets.
  • Figure 7 shows photos of cell membrane sheets from different batches. Samples 1-3 are independent diaphragm samples.
  • Figure 8 shows the results of the residual amount of bovine serum albumin in cell membrane sheets. Samples 1-3 are independent diaphragm samples.
  • Figure 9 shows the results of the residual amount of human serum albumin in cell membrane sheets. Samples 1-3 are independent diaphragm samples.
  • Figure 10 shows the results of the residual amount of human fibrinogen in cell membrane sheets.
  • Samples 1-3 are independent diaphragm samples.
  • Figure 11 shows the secretion of HGF in cell culture medium, membrane patch culture medium, and membrane patch culture medium. The results showed that the secretion of HGF could be detected in all three, and the secretion amount of HGF in the membrane patch culture medium and membrane patch culture medium was significantly higher than that in the cell culture medium. Samples 1-3 are independent diaphragm samples.
  • Figure 12 shows the secretion of VEGF in cell culture medium, membrane patch culture medium, and membrane patch culture medium. The results showed that the secretion of VEGF could be detected in all three, and the amount of VEGF secreted in the patch culture medium was much higher than that in the cell culture medium and the patch culture medium. Samples 1-3 are independent diaphragm samples.
  • Figure 13 shows the secretion of IL-6 in cell culture medium, membrane patch culture medium, and membrane patch culture medium. The results showed that the secretion of IL-6 in the membrane patch culture medium and membrane patch culture medium was significantly higher than that in the cell culture medium. Samples 1-3 are independent diaphragm samples.
  • Figure 14 shows the secretion of IL-8 in cell culture medium, membrane patch culture medium, and membrane patch culture medium. The results showed that the secretion amount of IL-8 in the membrane patch culture medium and membrane patch culture medium was significantly higher than that in the cell culture medium. Samples 1-3 are independent diaphragm samples.
  • Figure 15 shows the inhibitory effect of the diaphragm on the lymphocyte subset Th1.
  • sample 1 had an inhibitory rate of 23.5% for the lymphocyte subpopulation Th1
  • sample 2 had an inhibitory rate of 16.3% for the lymphocyte subpopulation Th1
  • sample 3 had an inhibitory rate of 20.1% for the lymphocyte subpopulation Th1.
  • Samples 1-3 are independent diaphragm samples.
  • Figures 16A-16B show the inhibitory effect of the diaphragm on lymphocyte proliferation.
  • Figure 16A shows that sample 1 has an inhibitory rate of lymphocyte proliferation of 56.2%, sample 2 has an inhibitory rate of lymphocyte proliferation of 57.8%, and sample 3 has an inhibitory rate of lymphocyte proliferation of 67.6%.
  • Figure 16B shows that mesenchymal stem cells in the cell membrane sheet have an obvious inhibitory effect on mononuclear cells.
  • Samples 1-3 are independent diaphragm samples.
  • Figure 17 shows the inhibitory effect of the diaphragm on TNF ⁇ secretion by lymphocytes.
  • the results show that the diaphragm has an inhibitory effect on the secretion of TNF ⁇ by lymphocytes in an inflammatory environment, indicating that the diaphragm has immunomodulatory and anti-inflammatory effects.
  • Samples 1-3 are independent diaphragm samples.
  • Figures 18A-18C show the results of flow cytometry of surface markers on cell membrane sheets of Sample 1, Sample 2, and Sample 3.
  • Figures 19A-19C show the results of three-way induction differentiation of mesenchymal stem cells in the cell membrane sheets of Sample 1, Sample 2, and Sample 3.
  • Figure 19A shows the results of adipogenic differentiation of mesenchymal stem cells in cell sheets
  • Figure 19B shows the results of osteogenic differentiation of mesenchymal stem cells in cell sheets
  • Figure 19C shows the results of chondrogenic differentiation of mesenchymal stem cells in cell sheets. .
  • Figures 20A-20D show photos of cell membrane sheets prepared in a serum-free film-forming system using different coating matrices and film-forming media containing HSA.
  • Figure 20A shows a photo of a cell membrane sheet prepared using gelatin as the coating matrix.
  • Figure 20B shows a photo of a cell membrane sheet prepared using vitronectin XF as the coating matrix.
  • Figure 20C shows a photo of a cell membrane sheet prepared using PDL as the coating matrix. Photos of the prepared cell membrane sheets.
  • Figure 20D shows photos of the cell membrane sheets prepared using recombinant fibronectin as the coating matrix.
  • the tissue pieces are removed and the cells are subcultured. Separate cells from culture vessels by trypsinization and cell scraping. Then, the cells are dispersed in the culture medium by stirring, vortexing, etc., and the cells are seeded into the culture container at a density of 500 to 100,000 cells/ cm2 .
  • the cells expand to 70 to 100% confluence, repeat passage. operate. The cultured umbilical cord mesenchymal stem cells grew adherently in a fibrous form with uniform morphology.
  • the temperature-sensitive culture dish Before preparing the umbilical cord mesenchymal stem cell membrane sheet, first use 0.1-5mg/mL human fibrinogen to coat the temperature-sensitive culture dish, which helps the mesenchymal stem cells adhere to the inner surface of the culture dish. The temperature-sensitive petri dish was coated at 37°C for 2 hours, and then the coating solution was discarded.
  • the cells were washed to remove the residue of umbilical cord mesenchymal stem cell culture medium, and then added to membrane-forming medium ( ⁇ -MEM, containing 50 ⁇ M to 200 ⁇ M non-essential amino acids, 0.5 mM to 4 mM L-glutamine, 0.1%-10 % human serum albumin), resuspend the cells, preheat to 37°C, and then inoculate them in a temperature-sensitive culture dish that has been coated with a matrix that is conducive to cell adhesion. The cells grow adherently in the culture dish.
  • membrane-forming medium ⁇ -MEM, containing 50 ⁇ M to 200 ⁇ M non-essential amino acids, 0.5 mM to 4 mM L-glutamine, 0.1%-10 % human serum albumin
  • FIG. 1 shows a photo of the harvested cell membrane. It can be seen that the membrane is gray-white, has a dense structure, and has a smooth surface.
  • the mesenchymal stem cell membrane obtained above was fixed with 4% paraformaldehyde, and then the cell membrane was cut into tissue sections by frozen section method for immunofluorescence staining (Figure 5).
  • the slicing and staining steps are as follows: take out the fixed mesenchymal stem cell membrane and put it into OCT embedding agent for embedding, and then use a freezing microtome to cut into 10 ⁇ m thick slices. Sections were stained with monoclonal antibodies to fibronectin or integrin- ⁇ 1, incubated overnight, and then stained with FITC-labeled fluorescent secondary antibodies. After staining is completed, a mounting medium containing Hoechst 33258 is added dropwise to cover the tissue sections. Finally, a coverslip was used to cover the tissue section and observed under a fluorescence microscope.
  • the cell membrane pieces were made into tissue sections for H&E staining ( Figure 6).
  • the steps for slicing and staining are as follows: The fixed cell membrane pieces are dehydrated and then embedded in paraffin. Then use a microtome to cut into 4-7 ⁇ m thick sections to make paraffin sections. Place the slices in sequence in xylene I for 10 min - xylene II for 10 min - absolute ethanol I for 5 min - absolute ethanol II for 5 min - 95% alcohol for 5 min - 90% alcohol for 5 min - 80% alcohol for 5 min - 70% alcohol for 5 min - and wash with distilled water.
  • the sections were stained with hematoxylin for 3-8 minutes, washed with tap water, differentiated with 1% hydrochloric acid alcohol for a few seconds, rinsed with tap water, returned to blue with 0.6% ammonia, and rinsed with running water. Stain the sections in eosin staining solution for 1-3 minutes. Place the slices in sequence in 95% alcohol I for 5 min - 95% alcohol II for 5 min - absolute ethanol I for 5 min - absolute ethanol for II for 5 min, xylene I for 5 min - xylene II for 5 min to dehydrate and make them transparent. Take the slices out of xylene and dry them slightly with neutral gum. Cover the slide. Observe under a microscope and take photos.
  • the cell membrane sheet is composed of simple cells and extracellular matrix components secreted by the cells, with a thickness of about 10 cells stacked.
  • the extracellular matrix components can be stained by fibronectin and integrin- ⁇ 1.
  • the steps for apoptosis detection are as follows: Digest the umbilical cord mesenchymal stem cell membrane into single cells and resuspend to a cell concentration of 2 to 5 ⁇ 10 5 /ml. Then use the Annexin V/PI apoptosis staining kit and follow the instructions for staining. The steps are as follows: 1. Use Binding Buffer to wash the cells twice; 2. Use 195 ⁇ L Binding Buffer to resuspend the cells and add 5 ⁇ L Annexin V staining solution to it. Incubate in the dark for 30 minutes. 3. Wash the cells twice with Binding Buffer and resuspend the cells in 190 ⁇ L Binding Buffer. Add 10 ⁇ L of PI staining solution to it. 4. Use flow cytometry to detect the cells after staining. Those cells that are negative in Annexin V/PI staining are normal cells that have not apoptotic.
  • the cell culture medium is obtained by replacing the cell culture medium in culture with fresh culture medium ( ⁇ -MEM supplemented with 20IU/mL bFGF and 10% FBS) after subculture and before making the membrane, and incubating it at 37°C with 5% carbon dioxide for 24 hours. liquid.
  • the membrane culture medium is the culture medium obtained after film formation and does not contain cells.
  • the diaphragm attachment culture medium is to clean the finished diaphragm (such as washing 3 times with PBS), attach it to the culture container, and add umbilical cord mesenchymal stem cell culture medium (supplemented with 10% FBS and 20IU/mL bFGF). ⁇ -MEM), culture medium obtained after incubation at 37°C with 5% carbon dioxide for 24 hours.
  • cytokine content in the cell culture medium, membrane patch culture medium and membrane patch culture medium.
  • cytokines are hepatocyte growth factor (HGF) (see Figure 11), vascular endothelial growth factor (VEGF) (see Figure 12), interleukin-6 (IL-6) (see Figure 13) and interleukin- 8(IL-8) (see Figure 14).
  • the finished membrane sheet is washed three times with PBS, it is attached to a culture container and then co-cultured with lymphocytes.
  • An experimental group diaphragm + lymphocytes + lymphocyte activator
  • a negative control group diaphragm + lymphocytes
  • a positive control group lymphocytes + lymphocyte activator
  • flow cytometry was used to detect the effect of the diaphragm on Suppressive effects on Th1 lymphocyte subsets. See Figure 15 for the results.
  • Figure 15 shows that the diaphragm has an inhibitory effect on lymphocyte subtype Th1 cells in an inflammatory environment, indicating that the diaphragm has immunomodulatory and anti-inflammatory effects.
  • the finished membrane sheet is washed three times with PBS, it is attached to a culture container and then co-cultured with lymphocytes.
  • Set up experimental groups diaphragm + lymphocytes + lymphocyte stimulating agent
  • negative control group diaphragm + lymphocytes
  • positive control group lymphocytes + lymphocyte stimulating agent
  • the antibody was used to detect the secretion of TNF ⁇ in each group using ELISA, thereby calculating the inhibitory effect of the membrane on the secretion of TNF ⁇ by lymphocytes. See Figure 17 for the results.
  • Figure 17 shows that the diaphragm has an inhibitory effect on the secretion of TNF ⁇ by lymphocytes in an inflammatory environment, indicating that the diaphragm has immunomodulatory and anti-inflammatory effects.
  • the umbilical cord mesenchymal stem cell membrane was digested into single cells and resuspended to a cell concentration of 1 ⁇ 10 6 /ml. Take 200 ⁇ L into the flow tube, and add the corresponding CD73, CD90, CD105, CD11b, CD19, CD34, CD45, and HLA-DR fluorescent staining antibodies to each tube according to the instructions. After incubation for 30 minutes in the dark, wash the cells twice with PBS buffer solution, and then use a flow cytometer to detect the stained cells.
  • the flow cytometry results of cell membrane surface markers are shown in Figure 18A-18C.
  • the cell surface markers in the cell membrane sheet all meet the requirements (CD73, CD90, CD105 positive rate >95%, CD11b, CD19, CD34, CD45, HLA -DR negative rate ⁇ 2%). It shows that the cells contained in the cell membrane sheet prepared by the present disclosure have stem cell-specific surface markers.
  • the cells in the membrane have the ability to induce differentiation in three directions
  • Example 5 Other coating matrices and film-forming media
  • cell membrane sheets were prepared according to the preparation process as described above. The results are as follows in Table 1.
  • Figures 20A-20D respectively show gelatin (concentration 0.1% w/w) coating + film-forming medium containing HSA ( Figure 20A), vitronectin XF (concentration 10 ⁇ g/mL) coating + film-forming containing HSA Culture medium ( Figure 20B), PDL (concentration 0.1 mg/mL) coating + film-forming medium containing HSA ( Figure 20C) and recombinant fibronectin (concentration 10 ⁇ g/mL) coating + film-forming medium containing HSA (Fig. 20D) Photograph of the prepared diaphragm.
  • Gelatin, vitronectin XF and PDL have lower costs and are conducive to large-scale production and application.

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Abstract

本公开提供了一种无动物源性(例如无血清)细胞膜片例如间充质干细胞膜片及其制备方法。本公开还提供了该无动物源性(例如无血清)细胞膜片例如间充质干细胞膜片在受损组织的损伤修复中的用途。

Description

无动物源性细胞膜片及其制备方法和应用 发明领域
本公开涉及组织工程学与再生医学领域，尤其涉及无动物源性细胞膜片及其制备方法和应用。
技术背景
与以往的涉及干细胞悬液注射细胞的移植方法相比，细胞膜片是一种更高效的干细胞移植方式。细胞膜片能够有效地避免干细胞在移植过程中的流失，提高干细胞移植效率。此外，细胞膜片在制备过程中不使用酶及类似物进行细胞消化，这有效地避免了酶消化所带来的细胞外基质的破坏和细胞功能减低，细胞膜片移植能使干细胞在体内更好的发挥其功能。
间充质干细胞膜片制备过程通常涉及将传代培养的间充质干细胞用生物酶消化成单细胞，之后经过清洗以除去间充质干细胞培养基的残留，然后用成膜培养基将细胞重悬后，接种在事先包被好的温度敏感培养皿中。在37℃的培养条件下，细胞在培养皿中贴壁生长，增殖汇合。培养结束后降低培养温度，细胞呈片状自动从温敏表面脱离，即可收获细胞膜片。
细胞膜片是单层或者多层细胞组成的片状结构。间充质干细胞膜片制备过程中使用的成膜培养基和包被在温度敏感培养皿中的基质残留在细胞间隙及表面，通过外力不易被完全清洗掉。目前已经报道的膜片制备方法中使用的成膜培养基分为三类：含血清培养基、市售无血清培养基和自配无血清培养基。传统血清成膜体系中，血清可以促使细胞在温度敏感培养皿的表面贴附并可以促进细胞与细胞之间的黏连，但含血清培养基中的牛血清含有异源大分子致敏成分牛血清白蛋白，对于临床用途不够安全。无血清培养体系中因缺乏血清，导致细胞不易贴壁，无法形成膜片而呈现破碎状态，或在起膜过程中因膜片韧性不足容易发生破碎(图1)。市面上的间充质干细胞无血清培养基和文献中的自配间充质干细胞无血清培养基为了支持细胞生长，都含有多种外源生长因子，而外源生长因子的残留也会有安全风险。
此外，目前膜片制备过程中包被基质为纤连蛋白、层粘连蛋白等科研级 别试剂，这些试剂在生产过程中不符合GMP标准。
因此，亟需开发一种使用更安全的成膜培养基和/或更安全的包被基质的制备形态良好、具有韧性的间充质干细胞膜片的方法，从而获得安全性更高、更易于保存和使用的间充质干细胞膜片。
发明内容
为了解决以上技术问题，本公开提供了一种间充质干细胞膜片产品的生产方法。该方法使用的成膜培养基成分简单，包括基础培养基和粘合剂(例如人血清白蛋白(例如药用级别))，其中不含任何动物源性成分(例如血清)和外源生长因子。此外，在细胞膜片制备过程中，可以使用贴壁因子(例如人纤维蛋白原)作为包被基质。
通过本公开的上述方法制备的间充质干细胞膜片产品更安全，不含有动物源性成分(例如血清)和外源生长因子残留(例如牛血清白蛋白残留量符合药物标准)，成膜过程所用培养基配方简单。此外，本公开的间充质干细胞膜片产品具有一定的韧性，可以在适当的培养基、缓冲液或保存液等中进行折叠和展平。膜片产品在4℃保存在特定的保存液中24小时后仍能够维持良好的膜片形态，细胞存活率高达70％以上，能够分泌高水平的促血管生成因子和抗炎因子，抑制淋巴细胞增殖和炎性因子分泌，能够在临床上用于多种疾病，包括自身免疫系统疾病、器官损伤类疾病，器官移植过程中的排异反应和GVHD等，同时能够维持干细胞特性，具有特定表面标志物，具有三向诱导分化能力，能够在临床上用于受损组织的损伤修复。
相应的，本公开涉及以下方面。
在第一个方面，本公开涉及一种制备细胞膜片的方法，所述方法包括以下步骤：
a.培养和传代细胞；
b.将所述细胞转移到用基质(又称为贴壁因子)包被的温敏培养皿中，在成膜培养基中培养，从而在培养皿中形成膜片，其中所述成膜培养基包含基础培养基和粘合剂(例如人血清白蛋白)且不包含动物源性成分(例如血清，例如牛血清白蛋白等)和外源生长因子；和
c.通过降低温度使细胞从所述温敏培养皿脱离。
本文所用的术语“温度敏感培养皿”或“温敏培养皿”是指表面涂覆了 一层温度敏感性高分子物质的培养皿，该高分子物质在不同温度下分子链段的伸展情况不同，从而表现出亲水性或疏水性，使得该高分子物质的亲疏水性能够随外部温度变化而变化。当温敏培养皿表面呈现亲水性时，与细胞及其分泌的细胞外基质粘合性变差，细胞将成层状脱落。在具体应用中，当将温度降低至该高分子物质的低临界溶解温度之下，该温敏培养皿表面呈现亲水性，从而使得细胞将成层状脱落。
在一些实施方案中，所述细胞膜片是干细胞膜片，例如间充质干细胞膜片。
在一些实施方案中，所述基质为纤连蛋白(fibronectin)、层粘连蛋白(laminin)、明胶、胶原、玻连蛋白或人纤维蛋白原。在一些实施方案中，所述基质为明胶。在一些实施方案中，明胶的浓度(w/w)为0.01-0.5％，例如0.05-0.2％，例如0.1％。在一些实施方案中，所述基质为玻连蛋白。在一些实施方案中，玻连蛋白的浓度为1-20μg/mL，例如5-15μg/mL，例如10μg/mL。在一些实施方案中，所述基质为多聚D赖氨酸(PDL)。在一些实施方案中，PDL的浓度为0.01-0.5mg/mL，例如0.05-0.2mg/mL，例如0.1mg/mL。用以上基质包被温敏培养皿使得间充质干细胞能够附着于所述培养皿。
在优选的实施方案中，所述基质为人纤维蛋白原。在一些实施方案中，所述人纤维蛋白原的浓度为0.1-10mg/mL，例如0.2-5mg/mL，例如1-2.5mg/mL。
在一些实施方案中，在步骤b中，成膜培养基中的基础培养基可以选自DMEM(高糖)、DMEM(低糖)、RPMI1640、α-MEM、DMEM/F12和F12。在一个优选的实施方案中，成膜培养基中的基础培养基为α-MEM。
在一些实施方案中，在步骤b中，成膜培养基中的人血清白蛋白的浓度为0.1-10％，优选0.1-5％，例如0.1％、0.5％、1％、1.5％、2％、2.5％、3％、3.5％、4％、4.5％或5％。
在一些实施方案中，在步骤b中，成膜培养基还包含非必需氨基酸(甘氨酸、L-丙氨酸、L-天冬氨酸、L-天门冬酰胺、L-谷氨酸、L-脯氨酸、L-丝氨酸)和/或L-谷氨酰胺。
在一些实施方案中，在步骤b中使用的成膜培养基中的L-谷氨酰胺的浓度为0.5mM至4mM，优选为约2mM。
在一些实施方案中，在步骤b中使用的成膜培养基中的非必需氨基酸甘 氨酸、L-丙氨酸、L-天冬氨酸、L-天门冬酰胺、L-谷氨酸、L-脯氨酸和L-丝氨酸各自的浓度为50μM至200μM，优选为约100μM。
在一些实施方案中，在步骤a中，用含血清培养基(例如含有胎牛血清的培养基)培养和传代所述间充质干细胞。
在一些实施方案中，在步骤a中，用无血清培养基培养和传代所述间充质干细胞。在一些实施方案中，所述无血清培养体系包括使用选自以下的基础培养基：RPMI1640、DMEM、α-MEM、DMEM/F12和F12无血清培养基，并且所述培养基补充有选自以下的一种或多种添加剂：维生素C、硒酸钠、氢化可的松、胰岛素、转铁蛋白、人血清白蛋白(植物表达)、孕酮、腐胺、生物素、丙酮酸钠、乙醇胺、肉毒碱、氨基酸、维生素、谷胱甘肽、亚油酸和亚麻酸。
在另一些实施方案中，所述无血清培养体系包括使用选自以下的商业化培养基：CTSTM Stem ProTM MSC SFM、MesenCultTM-ACF培养基、MesenCultTM-ACF Plus培养基和MesenCultTM-XF培养基。
在一些实施方案中，所述方法在步骤a之后步骤b之前还包括对细胞进行清洗的步骤。
在上述方法的一些实施方案中，所述间充质干细胞可以来源于选自以下的组织：羊水、羊膜、绒毛膜、绒毛膜绒毛、蜕膜、胎盘、脐带血、脐带、成人骨髓、成人外周血和成人脂肪组织。
在一些实施方案中，所述间充质干细胞可以选自脐带间充质干细胞、胎盘间充质干细胞、脂肪间充质干细胞和骨髓间充质干细胞，以及本领域已知的其它来源的间充质干细胞。在优选的实施方案中，所述间充质干细胞是脐带间充质干细胞。
在一些实施方案中，所述间充质干细胞是脐带间充质干细胞，并且所述方法在步骤a)之前还包括从脐带获得间充质干细胞的步骤。
在一些实施方案中，从脐带获得脐带间充质干细胞包括以下步骤：
i.从脐带分离华通氏胶；
ii.将华通氏胶剪碎成小组织块，并在无血清培养体系中培养所述组织块足够的时间，使得间充质干细胞从组织块爬出；和
iii.待所述间充质干细胞生长至50％-100％汇合，例如70％-100％汇合或80％-100％汇合时，移除组织块，从而获得脐带间充质干细胞。
在一些实施方案中，所述温敏培养皿的包被时间为1小时至7天，优选1-36小时，最优选2-18小时。在一些实施方案中，所述包被温度为2-37℃，优选37℃。
在一些实施方案中，将细胞接种到温敏培养皿的接种量如下所示。
当接种到100mm培养皿中时，接种细胞数目为1×107至10×107个，例如1×107、2×107、3×107、4×107、5×107、6×107、7×107、8×107、9×107或10×107个。悬浮接种细胞的培养液的体积为15-30mL，例如15、20、25或30mL。
当接种到35mm培养皿中时，接种细胞数目为1×106至30×106个，例如2×106至20×106个、5×106至15×106个或8×106至12×106、例如8×106个、9×106个、10×106个、11×106个或12×106个。悬浮接种细胞的培养液的体积为1.5-5mL，例如1.5、2、2.5、3、3.5、4、4.5或5mL。
当接种到60mm培养皿中时，接种细胞数目为3×106至75×106个，例如5×106至60×106个、10×106至50×106个、15×106至40×106个，20×106至30×106个或20×106至25×106个，例如20×106个、20.5×106个、21×106个、21.5×106个、22×106个、22.5×106个、23×106个、23.5×106个、24×106个、24.5×106个或25×106个。悬浮接种细胞的培养液的体积为3-12.5mL，例如3、3.5、4、4.5、5、5.5、6、6,5、7、7.5、8、8.5、9、9.5、10、10.5、11、11.5、12或12.5mL。
在本公开的方法中，通过降低温度使得间充质干细胞从温敏培养皿脱离，从而形成间充质干细胞膜片。例如，在培养温度为约37℃的情况下，通过将温度降低至4-32℃从而使间充质干细胞从所述温敏培养皿脱离。
在第二个方面，本公开涉及通过本公开的方法制备的细胞膜片。
上述制备方法获得的细胞膜片中的牛血清白蛋白残留量符合人用药标准，含量≤1ng/cm2，人血清白蛋白的残留量为10ng/cm2-15000ng/cm2，人纤维蛋白原的残留量为0.5ng/cm2-5ng/cm2。
上述制备方法获得的间充质细胞膜片能够分泌大量的HGF、VEGF和IL-8促血管生成功能相关因子，分泌大量IL-6和IL-8等抗炎相关因子；并具有抑制淋巴细胞Th1亚型，抑制淋巴细胞增殖和淋巴细胞TNFα分泌的功能。
在第三个方面，本公开涉及上述制备方法获得的间充质干细胞膜片用于在受试者中调节炎症反应或治疗自身免疫系统疾病中的用途，或在制备用于 调节炎症反应或治疗自身免疫系统疾病的药物中的用途。
在一些实施方案中，所述自身免疫性疾病为类风湿、过敏、红斑狼疮等。
在第四个方面，本公开涉及上述制备方法获得的间充质干细胞膜片用于对受试者中受损组织进行损伤修复的用途，或制备用于在受试者中进行受损组织的损伤修复的药物中的用途。
在一些实施方案中，所述受损组织为心脏、肝脏、胰脏和子宫的组织或其他组织。
附图说明
图1示出了血清作为包被基质对细胞膜片成膜的影响。FBS：胎牛血清。
图2示出了细胞膜片的示例性外观图。
图3示出了在无血清体系中人纤维蛋白原作为包被基质的存在或缺失对细胞膜片成膜的影响。
图4示出了在无血清体系中人血清白蛋白作为成膜培养基中粘合剂的存在或缺失对细胞膜片成膜的影响。
图5示出了细胞膜片IHC染色的结果。
图6示出了细胞膜片H&E染色的结果。
图7示出了不同批次细胞膜片的照片。样本1-3为独立的膜片样本。
图8示出了细胞膜片的牛血清白蛋白残留量的结果。样本1-3为独立的膜片样本。
图9示出了细胞膜片的人血清白蛋白残留量的结果。样本1-3为独立的膜片样本。
图10示出了细胞膜片的人纤维蛋白原的残留量的结果。样本1-3为独立的膜片样本。
图11示出了细胞培养液、膜片培养液、膜片复贴培养液中HGF分泌情况。结果表明，三者均能检测到HGF的分泌，并且膜片培养液和膜片复贴培养液中HGF的分泌量明显高于细胞培养液。样本1-3为独立的膜片样本。
图12示出了细胞培养液、膜片培养液、膜片复贴培养液中VEGF分泌情况。结果表明，三者均能检测到VEGF的分泌，并且膜片复贴培养液中VEGF分泌量远远高于细胞培养液中和膜片培养液中的量。样本1-3为独立的膜片样本。
图13示出了细胞培养液、膜片培养液、膜片复贴培养液中IL-6分泌情况。结果表明，膜片培养液和膜片复贴培养液中IL-6的分泌量明显高于细胞培养液。样本1-3为独立的膜片样本。
图14示出了细胞培养液、膜片培养液、膜片复贴培养液中IL-8分泌情况。结果表明，膜片培养液和膜片复贴培养液中IL-8的分泌量明显高于细胞培养液。样本1-3为独立的膜片样本。
图15示出了膜片对淋巴细胞亚群Th1的抑制作用。结果表明，样本1对淋巴细胞亚群Th1的抑制率为23.5％，样本2对淋巴细胞亚群Th1的抑制率为16.3％，样本3对淋巴细胞亚群Th1的抑制率为20.1％。样本1-3为独立的膜片样本。
图16A-16B示出了膜片对淋巴细胞增殖的抑制作用。图16A表明，样本1对淋巴细胞增殖的抑制率为56.2％，样本2对淋巴细胞增殖的抑制率为57.8％，样本3对淋巴细胞增殖的抑制率为67.6％。图16B示出了细胞膜片中的间充质干细胞对单个核细胞存在较明显的抑制作用。样本1-3为独立的膜片样本。
图17示出了膜片对淋巴细胞TNFα分泌的抑制作用。结果表明，膜片对炎性环境下淋巴细胞TNFα分泌有抑制作用，说明膜片具有免疫调节和抗炎作用。样本1-3为独立的膜片样本。
图18A-18C示出了样本1、样本2、样本3细胞膜片表面标志物的流式细胞术的结果。
图19A-19C示出了样本1、样本2、样本3细胞膜片中间充质干细胞三向诱导分化的结果。图19A示出了细胞膜片中间充质干细胞成脂分化的结果，图19B示出了细胞膜片中间充质干细胞成骨分化的结果，图19C示出了细胞膜片中间充质干细胞成软骨分化的结果。
图20A-20D示出了无血清成膜体系中采用不同包被基质以及含有HSA的成膜培养基制备的细胞膜片的照片。图20A示出了采用明胶作为包被基质制备的细胞膜片的照片，图20B示出了采用玻连蛋白XF作为包被基质制备的细胞膜片的照片，图20C示出了采用PDL作为包被基质制备的细胞膜片的照片，图20D示出了采用重组纤连蛋白作为包被基质制备的细胞膜片的照片。
具体实施方式
本发明通过下述实施例进一步阐明，但任何实施例或其组合不应当理解为对本发明的范围或实施方式的限制。本发明的范围由所附权利要求书限定，结合本说明书和本领域一般常识，本领域普通技术人员可以清楚地明白权利要求书所限定的范围。在不偏离本发明的精神和范围的前提下，本领域技术人员可以对本发明的技术方案进行任何修改或改变，这种修改和改变也包含在本发明的范围内。
实施例1.脐带间充质干细胞的分离和培养
将采集的新生儿脐带用生理溶液洗净后去除动脉、静脉、外膜，分离出华通氏胶，剪碎成0.1～2mm小组织块，将组织块均匀铺在包被有基质的培养容器中，组织块之间的间距为2～30mm。然后将培养容器置于细胞培养箱中，2～7天后加入适量完全培养基(补充有20IU/mL bFGF的和10％胎牛血清的α-MEM)覆盖组织块，8～21天后可观察到脐带间充质干细胞爬出。
待细胞扩增至70～100％汇合时，移除组织块，细胞进行传代培养。通过胰酶消化和细胞刮等方式将细胞与培养容器分离。然后通过搅拌、涡旋等方法将细胞分散于培养基中，并按照500～100000个细胞/cm2的密度将细胞接种于培养容器中。加入适量补充有20IU/mL bFGF和10％胎牛血清的α-MEM，根据细胞生长状态每1～5天更换适量新鲜的上述培养基，待细胞扩增至70～100％汇合时，重复传代操作。培养的脐带间充质干细胞呈成纤维状贴壁生长，形态均一。
实施例2.脐带间充质干细胞膜片制备
在制备脐带间充质干细胞膜片前，首先使用0.1-5mg/mL的人纤维蛋白原包被温敏培养皿，其有利于间充质干细胞贴服在培养皿内表面。将所述温敏培养皿在37℃包被2小时，而后弃去包被液。
从实施例1获得的脐带间充质干细胞培养液中除去旧的培养基，之后用PBS清洗1-3次，加入消化酶TryPLE(购自Life technologies，货号12604021)消化至细胞呈单细胞状态，加入PBS终止或者降低酶的消化作用，离心后，弃除上清，获得细胞沉淀。
用PBS缓冲液洗涤消化的细胞，以小于等于5×10^5细胞/mL的清洗密 度清洗3遍。
细胞经过清洗去除脐带间充质干细胞培养基的残留，之后加入成膜培养基(α-MEM，并含有50μM至200μM的非必需氨基酸、0.5mM至4mM的L-谷氨酰胺、0.1％-10％人血清白蛋白)重悬细胞，预热至37℃后接种在事先包被好的利于细胞贴壁基质的温度敏感培养皿中，细胞在培养皿中贴壁生长。
将培养液降至室温，细胞呈单层片状自动脱离培养皿，即可收获细胞膜片。从培养箱中取出温敏培养皿，除去培养基，并加入于4℃预冷的成膜培养基，0.25-1小时后则可观察到细胞膜片从温皿边缘开始剥离。图2示出了收获的细胞膜片的照片，可见膜片呈灰白色，结构致密，表面光滑平整。
结果表明，提前在温敏培养皿上包被人纤维蛋白原，可以促使细胞贴壁成膜，如果不使用包被基质进行包被，细胞膜片会提前起膜，膜片状态较差(图3)。通过在成膜培养基中加入人血清白蛋白可以促使细胞间相互连接。在高细胞接种量的基础上不添加人血清白蛋白到成膜培养基的情况下，尽管可以保持膜片形态，但细胞间连接比较松散，膜片边缘破碎不堪(图4)。
将上述得到的间充质干细胞膜片用4％多聚甲醛进行固定，随后通过冰冻切片法将细胞膜片制成组织切片进行免疫荧光染色(图5)。切片及染色步骤如下：取出固定完成后的间充质干细胞膜片放入OCT包埋剂进行包埋，而后使用冰冻切片机切成10μm厚度切片。使用纤连蛋白或整合素-β1的单克隆抗体对切片进行染色，过夜孵育，而后使用FITC标记的荧光二抗对切片进行染色。染色完成后滴加含有Hoechst33258的封片剂覆盖组织切片。最后使用盖玻片覆盖组织切片并在荧光显微镜下观察。
通过石蜡包埋、切片，将细胞膜片制成组织切片进行H&E染色(图6)。切片及染色步骤如下：将固定好的细胞膜片经过程序脱水后使用石蜡包埋。而后使用切片机切成4-7μm厚度切片，制成石蜡切片。依次将切片放入二甲苯Ⅰ10min-二甲苯Ⅱ10min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min-95％酒精5min-90％酒精5min-80％酒精5min-70％酒精5min-蒸馏水洗。切片放入苏木素染3-8min,自来水洗，1％的盐酸酒精分化数秒，自来水冲洗，0.6％氨水返蓝，流水冲洗。切片放入伊红染液中染色1-3min。将切片依次放入95％酒精Ⅰ5min-95％酒精Ⅱ5min-无水乙醇Ⅰ5min-无水乙醇Ⅱ5min,二甲苯Ⅰ5min-二甲苯Ⅱ5min中脱水透明，将切片从二甲苯拿出来稍晾干，中性树胶封片。在显微镜下进行观察并拍照。
结果可以看出，细胞膜片为单纯细胞及细胞所分泌的外基质成分组成，厚度约为10个细胞堆叠厚度。其中细胞外基质成分可以被纤连蛋白和整合素-β1染色。
实施例3.细胞膜片的特性
针对所制备的三个批次(样本1、样本2、样本3)的膜片(图7)，对其细胞活率、总细胞量以及细胞凋亡情况进行检测。
凋亡检测步骤如下：将脐带间充质干细胞膜片消化至单细胞，重悬至细胞浓度2～5×105/ml。而后使用Annexin V/PI凋亡染色试剂盒，参照说明进行染色，步骤如下：1、使用Binding Buffer清洗细胞2次；2、使用195μL Binding Buffer重悬细胞，并向其中加入5μL Annexin V染液。避光孵育30分钟。3、使用Binding Buffer清洗细胞2次，重悬细胞至190μL Binding Buffer。向其中加入10μL PI染液。4、使用流式细胞仪检测染色完成后的细胞，其中Annexin V/PI染色均为阴性的为未凋亡的正常细胞。
此外，针对细胞膜片在制备过程中所引入的物质的残留进行检测。
1.牛血清白蛋白残留量检测
将间充质干细胞膜片从保护液中取出后，用1mL TryPLE 37℃消化3min，消化过程中加入50μL 20％人血清白蛋白注射液以保证牛血清白蛋白在消化过程中不被破坏，然后用1mL成膜培养基终止消化，将膜片用1mL移液枪吹打成单细胞，300g离心5分钟后，取上清，使用抗牛血清白蛋白抗体用ELISA方法检测牛血清白蛋白的含量。结果参见图8。图8表明，膜片中残留的牛血清白蛋白的含量低于0.5ng/cm2。
2.人血清白蛋白残留量检测
将间充质干细胞膜片从保护液中取出后，用3mL TryPLE 37℃消化3min，然后用7mL PBS终止消化，将膜片用1mL移液枪吹打成单细胞，300g离心5分钟后，取上清，用抗人血清白蛋白HSA的抗体使用ELISA方法检测人血清白蛋白的含量。结果参见图9。图9表明，膜片中残留的人血清白蛋白的含量为53-62ng/cm2。
3.人纤维蛋白原残留量检测
将间充质干细胞膜片从保护液中取出后，用5mL裂解液(生理盐水+0.5％Triton+2.5％蛋白酶抑制剂)，4℃裂解20分钟，12000g 4℃离心20分 钟后，取上清，用抗人纤维蛋白原的抗体使用ELISA方法检测人纤维蛋白原的含量。结果参见图10。图10表明，膜片中残留的人纤维蛋白原的含量为1.2-3.4ng/cm2。
实施例4.细胞膜片的功能检测
1.细胞膜片分泌细胞因子的检测
细胞培养液为将传代培养之后膜片制作之前培养中的细胞培养液更换新鲜培养基(补充有20IU/mL bFGF和10％FBS的α-MEM)，37℃5％二氧化碳培养24h后获得的培养液。
膜片培养液为成膜后获得的不包含细胞的培养液。
膜片复贴培养液为将膜片成品经过清洗后(如PBS清洗3次)，使其贴附在培养容器，加入脐带间充质干细胞培养液(补充有10％FBS和20IU/mL bFGF的α-MEM)，37℃5％二氧化碳培养24小时后获得的培养液。
按照酶联免疫试剂盒的使用说明书，检测细胞培养液、膜片培养液和膜片复贴培养液中的细胞因子的含量。这些细胞因子为肝细胞生长因子(HGF)(参见图11)、血管内皮生长因子(VEGF)(参见图12)、白细胞介素-6(IL-6)(参见图13)和白细胞介素-8(IL-8)(参见图14)。
上述结果表明，膜片经过保存之后，分泌细胞因子HGF的能力未下降，并且细胞膜片分泌细胞因子HGF的能力比细胞强；膜片经过保存之后，分泌细胞因子VEGF的能力未下降，并且细胞膜片分泌细胞因子VEGF的能力比细胞强。
2.膜片对Th1淋巴细胞亚群的抑制作用
膜片成品经过PBS清洗3次后，使其贴附在培养容器，然后和淋巴细胞共培养。分别设置实验组(膜片+淋巴细胞+淋巴细胞激活剂)、阴性对照组(膜片+淋巴细胞)和阳性对照组(淋巴细胞+淋巴细胞激活剂)，通过流式细胞术检测膜片对Th1淋巴细胞亚群的抑制作用。结果参见图15。图15表明，膜片对炎性环境下淋巴细胞亚型Th1细胞具有抑制作用，说明膜片具有免疫调节和抗炎作用。
3.膜片对淋巴细胞增殖的抑制作用
膜片成品经过PBS清洗3次后，使其贴附在培养容器，然后和淋巴细胞共培养。分别设置实验组(膜片+淋巴细胞+淋巴细胞刺激剂)、阴性对照组 (膜片+淋巴细胞)和阳性对照组(淋巴细胞+淋巴细胞刺激剂)，淋巴细胞用BrdU标记，通过流式细胞术检测膜片淋巴细胞BrdU阳性细胞群的抑制作用。结果参见图16A-16B。图16A-16B表明，膜片对炎性环境下淋巴细胞增殖有抑制作用，说明膜片具有免疫调节和抗炎作用。
4.膜片对淋巴细胞TNFα分泌的抑制作用
膜片成品经过PBS清洗3次后，使其贴附在培养容器，然后和淋巴细胞共培养。分别设置实验组(膜片+淋巴细胞+淋巴细胞刺激剂)、阴性对照组(膜片+淋巴细胞)和阳性对照组(淋巴细胞+淋巴细胞刺激剂)，收集培养上清，使用抗TNFα的抗体用ELISA方法检测每组TNFα的分泌量，从而计算膜片对淋巴细胞TNFα量分泌的抑制作用。结果参见图17。图17表明，膜片对炎性环境下淋巴细胞TNFα分泌具有抑制作用，说明膜片具有免疫调节和抗炎作用。
5.表面标志物的流式检测
将脐带间充质干细胞膜片消化至单细胞，重悬至细胞浓度1×106/ml。取200μL至流式管中，每管中根据说明书加入对应的CD73、CD90、CD105、CD11b、CD19、CD34、CD45、HLA-DR荧光染色抗体。避光孵育30分钟后，使用PBS缓冲溶液清洗细胞2次，而后使用流式细胞仪检测染色完成后的细胞。
细胞膜片表面标志物的流式细胞术结果如图18A-18C所示，细胞膜片内细胞表面标志物均符合要求(CD73、CD90、CD105阳性率>95％，CD11b、CD19、CD34、CD45、HLA-DR阴性率<2％)。说明本公开制备的细胞膜片中所包含的细胞具有干细胞特定的表面标志物。
6.膜片中的细胞具备三向诱导分化能力
将脐带间充质干细胞膜片消化至单细胞，并按照三向诱导分化试剂说明书的比例接种于合适的培养器皿中，待成骨诱导检测的细胞生长至70％左右汇合，成脂肪诱导检测的细胞生长至90％以上汇合时，分别加入成骨和成脂肪诱导培养基。成软骨诱导时将一定数量的细胞离心至离心管底部，而后加入成软骨诱导培养基，待细胞团成小球后，使细胞小球离开管底，保证与诱导培养基完全接触。全部诱导培养21天以上时对细胞进行检测。成骨诱导可用茜素红等染色，成脂肪诱导可用油红O等染色，成软骨诱导可以用阿尔新蓝等染色。
三向诱导分化的结果如图19A-19C所示，其中可以看出，细胞膜片中所包含的细胞在保存前后均可实现成脂、成骨、成软骨分化，染色呈阳性。说明本公开制备的细胞膜片具有良好的分化潜能。
实施例5.其他包被基质和成膜培养基
采用不同的包被基质与成膜培养基的组合，根据如上所述的制备过程制备细胞膜片，结果如下表1。
表1采用不同的包被基质与成膜培养基的组合制备的细胞膜片
图20A-20D分别示出了明胶(浓度0.1％w/w)包被+含有HSA的成膜培养基(图20A)、玻连蛋白XF(浓度10μg/mL)包被+含有HSA的成膜培养基(图20B)、PDL(浓度0.1mg/mL)包被+含有HSA的成膜培养基(图20C)和重组纤连蛋白(浓度10μg/mL)包被+含有HSA的成膜培养基(图20D)制备的膜片的照片。明胶、玻连蛋白XF和PDL成本较低，有利于大规模生产和应用。

Claims (24)

  1. 一种制备细胞膜片的方法，所述方法包括以下步骤：
    a.培养和传代细胞；
    b.将所述细胞转移到用基质包被的温敏培养皿中，在成膜培养基中培养从而在培养皿中形成膜片，其中所述成膜培养基包含基础培养基和人血清白蛋白且不包含动物源性成分和外源生长因子；和
    c.通过降低温度使细胞从所述温敏培养皿脱离。
  2. 根据权利要求1所述的方法，其中所述细胞膜片是干细胞膜片，例如间充质干细胞膜片。
  3. 根据权利要求1或2所述的方法，其中所述基质选自纤连蛋白、层粘连蛋白、明胶、胶原、玻连蛋白和人纤维蛋白原。
  4. 根据权利要求1或2所述的方法，其中所述基质是多聚D赖氨酸(PDL)。
  5. 根据权利要求1或2所述的方法，其中所述基质是人纤维蛋白原。
  6. 根据权利要求5所述的方法，所述人纤维蛋白原的浓度为0.1-10mg/mL，例如0.2-5mg/mL，例如1-2.5mg/mL。
  7. 根据权利要求1-6中任一项所述的方法，其中在步骤b中，成膜培养基中的基础培养基选自DMEM(高糖)、DMEM(低糖)、RPMI1640、α-MEM、DMEM/F12和F12，优选为α-MEM。
  8. 根据权利要求1-7中任一项所述的方法，其中在步骤b中，成膜培养基中的人血清白蛋白的浓度为0.1-10％，例如0.1-5％，例如0.5-2％。
  9. 根据权利要求1-8中任一项所述的方法，其中在步骤b中，成膜培养基还包含：
    (1)甘氨酸、L-丙氨酸、L-天冬氨酸、L-天门冬酰胺、L-谷氨酸、L-脯氨酸和L-丝氨酸，和/或
    (2)L-谷氨酰胺。
  10. 根据权利要求9所述的方法，其中L-谷氨酰胺的浓度为0.5mM至4mM，优选为2mM。
  11. 根据权利要求9所述的方法，其中甘氨酸、L-丙氨酸、L-天冬氨酸、L-天门冬酰胺、L-谷氨酸、L-脯氨酸和L-丝氨酸各自的浓度为50μM至 200μM，优选为100μM。
  12. 根据权利要求1-11中任一项所述的方法，其中在步骤a中用于培养和传代的培养基为有血清培养基或补充有一种或多种外源生长因子的无血清培养基。
  13. 根据权利要求12所述的方法，所述无血清培养基选自：RPMI1640、DMEM、α-MEM、DMEM/F12和F12无血清培养基，并且所述外源生长因子选自以下中的一种或多种：维生素C、硒酸钠、氢化可的松、胰岛素、转铁蛋白、人血清白蛋白、孕酮、腐胺、生物素、丙酮酸钠、乙醇胺、肉毒碱、氨基酸、维生素、谷胱甘肽、亚油酸和亚麻酸。
  14. 根据权利要求12所述的方法，其中所述无血清培养基为选自以下的商业化培养基：CTSTMStem ProTMMSC SFM、MesenCultTM-ACF培养基、MesenCultTM-ACF Plus培养基和MesenCultTM-XF培养基。
  15. 根据权利要求1-14中任一项所述的方法，所述方法在步骤a之后步骤b之前还包括对细胞进行清洗的步骤。
  16. 根据权利要求2-15中任一项所述的方法，其中所述间充质干细胞可以来源于选自以下的组织：羊水、羊膜、绒毛膜、绒毛膜绒毛、蜕膜、胎盘、脐带血、华通氏胶、脐带、成人骨髓、成人外周血和成人脂肪组织。
  17. 根据权利要求2-15中任一项所述的方法，其中所述间充质干细胞可以选自脐带间充质干细胞、胎盘间充质干细胞、脂肪间充质干细胞和骨髓间充质干细胞。
  18. 根据权利要求17所述的方法，其中所述间充质干细胞为脐带间充质干细胞，且其中所述方法在步骤a)之前还包括从脐带获得间充质干细胞的步骤。
  19. 根据权利要求18所述的方法，其中从脐带获得脐带间充质干细胞包括以下步骤：
    i.从脐带分离华通氏胶；
    ii.将华通氏胶剪碎成小组织块，并在无血清培养体系中培养所述组织块足够的时间，使得间充质干细胞从组织块爬出；和
    iii.待所述间充质干细胞生长至50％-100％汇合，例如70％-100％汇合或80％-100％汇合时，移除组织块，从而获得脐带间充质干细胞。
  20. 根据权利要求1-19中任一项所述的方法，在步骤b中，成膜培养温 度为约37℃，通过将温度降低至4-32℃从而使细胞从所述温敏培养皿脱离。
  21. 一种细胞膜片，其是通过权利要求1-20中任一项所述的方法制备的。
  22. 根据权利要求21所述的细胞膜片，其中所述细胞膜片中的牛血清白蛋白的残留量≤1ng/cm2，人血清白蛋白的残留量为10ng/cm2-15000ng/cm2，人纤维蛋白原的残留量为0.5ng/cm2-5ng/cm2。
  23. 通过权利要求1-20中任一项所述的方法制备的细胞膜片在制备用于在受试者中调节炎症反应或治疗自身免疫系统疾病的药物中的用途。
  24. 通过权利要求1-20中任一项所述的方法制备的细胞膜片在制备用于在受试者中进行受损组织的损伤修复的药物中的用途。
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