WO2021219003A1 - 一种脂肪来源干细胞制剂、制备方法及其应用 - Google Patents

一种脂肪来源干细胞制剂、制备方法及其应用 Download PDF

Info

Publication number
WO2021219003A1
WO2021219003A1 PCT/CN2021/090539 CN2021090539W WO2021219003A1 WO 2021219003 A1 WO2021219003 A1 WO 2021219003A1 CN 2021090539 W CN2021090539 W CN 2021090539W WO 2021219003 A1 WO2021219003 A1 WO 2021219003A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
adipose
stem cell
scleroderma
stem cells
Prior art date
Application number
PCT/CN2021/090539
Other languages
English (en)
French (fr)
Inventor
龙笑
王晓军
陈洁
李竹君
崇煜明
张文超
王晨羽
俞楠泽
夏泽楠
黄久佐
Original Assignee
中国医学科学院北京协和医院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from CN202010365622.XA external-priority patent/CN113577106A/zh
Priority claimed from CN202010385153.8A external-priority patent/CN113694084A/zh
Application filed by 中国医学科学院北京协和医院 filed Critical 中国医学科学院北京协和医院
Publication of WO2021219003A1 publication Critical patent/WO2021219003A1/zh

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • A01N1/02—Preservation of living parts
    • A—HUMAN NECESSITIES
    • 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
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • 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

Definitions

  • the invention relates to the technical field of biomedicine, in particular to a fat-derived stem cell preparation, a preparation method and an application thereof.
  • Scleroderma is an autoimmune connective tissue disease characterized by fibrosis and sclerosis of the skin and tissues and organs. It is clinically divided into two categories: First, only skin involvement without obvious visceral system involvement is called localized scleroderma Localized sclerosis (LS), also known as Morphea; Second, extensive skin sclerosis with visceral system involvement is systemic sclerosis (SSc), also known as systemic sclerosis.
  • LS localized scleroderma Localized sclerosis
  • SSc systemic sclerosis
  • systemic sclerosis The etiology of systemic sclerosis is unknown, and factors such as genetics, infection, environment and drugs may be involved; its pathogenesis is complex, with immune disorders, vascular endothelial cell damage, fibroblast proliferation and abnormal collagen synthesis as the main pathological manifestations of the disease.
  • the early pathological features of systemic sclerosis skin are the infiltration of inflammatory cells around the blood vessels, telangiectasia, microvascular changes, and excessive accumulation of extracellular matrix in the late stage resulting in tissue fibrosis, proliferation of endothelial cells in small arteries and small arteries, and narrowing or even blockage of the vascular lumen , Accompanied by microvascular bleeding. Similar lesions can also be seen in other organs, causing damage to the responding organs.
  • the cause of localized scleroderma is unknown, and may be related to infection, drugs, chemicals, malignant tumors, trauma, genetic factors, autoimmunity, hormone levels, etc.
  • the lesion usually only involves the skin, but sometimes it can also involve the subcutaneous tissues, such as fat, muscle, and bone. In a small number of cases, internal organ damage may occur.
  • the skin lesions are manifested as hypopigmented spots on the trunk, limbs, and face.
  • the size of the plaques and the distribution of skin lesions vary from person to person. Some skin lesions can cause facial deformities. If scleroderma affects the joints, it can cause movement disorders.
  • the pathogenesis of LSC mainly includes: small blood vessel damage, T cell activation and increased connective tissue production.
  • scleroderma Early treatment of scleroderma is mainly for immune, vascular and collagen abnormalities, based on anti-inflammatory, immunosuppressive, immune regulation, improvement of blood circulation and reduction of fibrosis.
  • the remaining deformities such as skin hardening, subcutaneous lipoatrophy, and local depression often require plastic surgery treatment.
  • Orthopedic treatment of locally depressed deformities currently includes autologous tissue transplantation with vascular pedicle and autologous fat transplantation without vascular pedicle.
  • vascular pedicled tissue transplantation has large surgical trauma and high risk of micro-suturing.
  • Autologous fat transplantation has become the most popular treatment option for patients and plastic surgeons because of the small surgical trauma.
  • the fat transplantation of scleroderma patients is different from that of normal people.
  • the fat survival rate of patients with dermatosis is still significantly lower than that of normal people’s fat transplantation, which may be related to factors such as tissue atrophy and adhesion in the lesion area of scleroderma, poor local blood supply, and local inflammatory microenvironment.
  • patients with scleroderma are autologous Whether there are biological changes in fat also needs to be verified.
  • Adipose-derived stem cells are a type of cells derived from human adipose tissue that can proliferate in vitro and have multidirectional differentiation potential. This type of cell is similar to bone marrow mesenchymal stem cells (BMSCs), etc. It has similar expression and multi-directional differentiation potential, provides lifelong self-renewal of adipose tissue, and maintains the balance of fat metabolism. Compared with stem cells from other sources, ADSCs has unique advantages. For example, adipose-derived stem cells are derived from adipose tissue. They have the advantages of a wide range of sources, simple materials, rapid proliferation in vitro, and low cell aging rate, which have attracted wide attention from researchers. It has gradually become the ideal seed cell for tissue engineering.
  • Adipose-derived stem cells are fibroblast-like. Adipose-derived stem cells have low expression of HLA-ABC but not HLA-DR, which indicates that ADSCs have low immunogenicity and will not be immunized after allogeneic or xenograft transplantation. Rejection, under certain conditions, can differentiate into adipocytes, chondrocytes, muscle cells and osteoblasts. The basic morphology, proliferation cycle, immune cycle, immunogenicity and multidirectional differentiation potential of adipose-derived stem cells become tissue engineering and regeneration. The most widely used adult stem cell in the medical field. These characteristics of stem cells provide us with new ideas for the application of fat transplantation in the treatment of scleroderma.
  • adipose-derived stem cells are very fragile, and it is difficult to guarantee survival rate when they are formulated into a preparation, and cannot be stored and transported for a long time. If the survival rate of adipose-derived stem cells in the preparation is insufficient, they cannot exert a good efficacy.
  • the first object of the present invention is to provide an adipose-derived stem cell preparation and a preparation method thereof, which retain the biologically active factors of stem cells to a great extent, facilitate long-term transportation and storage, and are easy to use.
  • the second object of the present invention is to provide the use of adipose-derived stem cells in the preparation of cell preparations for the treatment of scleroderma.
  • the first aspect of the present invention provides a stem cell preparation comprising adipose-derived stem cells and a stem cell preservation solution, the content of adipose-derived stem cells is 1 ⁇ 10 5 -5 ⁇ 10 6 cells/ml, preferably, 1 ⁇ 10 6 -2x10 6 cells/ml.
  • the stem cells are in a single cell suspension state after being resuspended in a stem cell preservation solution.
  • the stem cell preservation solution is a preservation solution of TC199 containing 0.5-1% human albumin.
  • the stem cell preparation is oral or injection. Preferably, subcutaneous injection.
  • the stem cells are adipose-derived stem cells subcultured from the 3rd to the 6th passage.
  • the second aspect of the present invention provides the storage method of the stem cell preparation, the storage condition of the stem cell preparation is 4°C, the preservation solution of TC199 containing 0.5%-1% human albumin is stored in a vial, and the shelf life is It is set at 24 hours; the storage condition of the stem cell preparation is liquid nitrogen storage, and the shelf life is set at 1 year.
  • the third aspect of the present invention provides a method for preparing the stem cell preparation, including the following steps:
  • Adipose tissue separation and primary culture of adipose stem cells add physiological saline to the adipose tissue, pipette 10-20 times with a straw, let stand for 1-2 minutes, suck out the liquid from the lower layer of the tissue, add collagenase digestion solution, and digest 30 -60min, aspirate the lower liquid, filter, centrifuge, discard the supernatant, resuspend the cell pellet in the culture medium, centrifuge and discard the supernatant, in the culture system at a density of 2 ⁇ 10 6 -4 ⁇ 10 6 cells/mL Cultivate, put the culture bottle in the incubator;
  • Adipose tissue separation and primary culture of adipose stem cells when the cells grow to 70%-80% confluence, take them out, add trypsin and EDTA, when the cells begin to shrink and become round, add an equal volume of medium to terminate the digestion, pipette the bottle Cells on the wall, make the cells detached from the bottle wall and become a single-cell suspension. Transfer the single-cell suspension to a centrifuge tube, centrifuge, discard the supernatant, add complete medium, blow gently to loosen it, and pass 1:3 Cultivation; collection of adipose-derived stem cells from passage 3 to passage 6;
  • the cells are resuspended in a stem cell preservation solution to obtain a stem cell suspension.
  • the stem cell suspension comprises adipose-derived stem cells to a concentration of 1x10 6 -2x10 6 cells / ml.
  • pancreatin and 0.01% EDTA are added in the step (2).
  • it further includes adding pharmaceutically acceptable auxiliary components to the stem cell suspension.
  • the fourth aspect of the present invention provides the use of the stem cell preparation in the preparation of medicines for malignant tumors and cosmetic plastic surgery.
  • the fifth aspect of the present invention provides the use of the adipose-derived stem cell preparation in the preparation of products for assisting fat transplantation in the treatment of scleroderma.
  • the scleroderma includes systemic scleroderma and localized scleroderma.
  • the systemic sclerosis includes diffuse cutaneous sclerosis (dcSSc); limited cutaneous sclerosis (lcSSc); systemic sclerosis without skin manifestations; overlap synthesis Sign; undifferentiated connective tissue disease.
  • the localized scleroderma includes spotting scleroderma (Bullous morphea); generalized scleroderma (Generalized morphea); plaque morphea (Plaque morphea); linear scleroderma (Linear scleroderma) ) And Deep morphea.
  • linear scleroderma involves subtypes of the head and face: linear scleroderma-saber scleroderma (Linear scleroderma'en coup de saber' (LScs), progressive hemifacial atrophy (progressive) facial hemiatrophy, PFH).
  • linear scleroderma-saber scleroderma Linear scleroderma'en coup de saber' (LScs)
  • PFH progressive hemifacial atrophy
  • the adipose-derived stem cell-assisted fat transplantation can not only improve the survival rate of fat, but also reduce the deposition of skin collagen in patients with scleroderma.
  • the adipose-derived stem cells reduce the content of TGF- ⁇ 1 and the content of type III collagen in patients with scleroderma.
  • the product comprises per ml 1x10 6 -2x10 6 th adipose-derived stem cells.
  • the product is a stem cell preparation or medicine.
  • the stem cell preparation or medicine is a subcutaneous injection.
  • the drug also includes other drugs for the treatment of scleroderma.
  • the stem cell viability rate in the fat-derived stem cell preparation prepared by the present invention is as high as 90% after being stored for 24 hours with 1% human albumin concentration TC199 at 4°C; under liquid nitrogen storage, cell viability after 1 year Still up to 95% or more, the stem cell preparation has high viability, long storage period, convenient transportation and simple preparation method; in addition, the stem cell preparation is of controllable quality, safe and effective, and can be used for clinical treatment of malignant tumors or in the field of cosmetic surgery.
  • adipose-derived stem cells can not only improve the survival rate of fat, but also found that the collagen content of the skin of nude mice with scleroderma after fat transplantation is significantly reduced after adipose stem cells assisted fat transplantation.
  • the inventors studied that adipose stem cells + fat transplantation can reduce the content of TGF- ⁇ 1 and type III collagen in mice in the scleroderma group, and alleviate the symptoms of scleroderma.
  • Figure 1 The gross observation and histological section of normal nude mice and bleomycin scleroderma nude mice.
  • the skin of the model is thickened and hardened, and the collagen fibers are thickened, increased, and arranged disorderly, indicating the success of the model;
  • Figure 4 Masson staining shows skin changes after fat transplantation or CAL in normal nude mice and scleroderma nude mice;
  • TGF- ⁇ 1 immune index normal nude mice have no significant changes after fat transplantation or fat stem cell + fat transplantation. But for scleroderma mice, fat transplantation or CAL can reduce the content of TGF- ⁇ 1 in scleroderma mice, especially the effect of CAL is more obvious;
  • Type III collagen immune index normal nude mice have no significant changes after fat transplantation or fat stem cell + fat transplantation. But for scleroderma mice, fat transplantation or CAL can reduce the type III collagen content of scleroderma mice, especially the effect of CAL is more obvious.
  • the adipose tissue is derived from healthy volunteers. Before collection, the age and gender of the donor are recorded in detail. Routine physical examinations are required to understand their health status. Serological tests for HBV, HCV, HIV and syphilis are required to be negative. Take fasting and water in the early morning of the day, and check the biochemical items and blood routine before entering the operating room.
  • Fat preservation solution sterile TC199 containing heparin, each 100ml preservation solution contains 4000 units of heparin and 2000 units of gentamicin, 1ml preservation solution can anticoagulate 3-10ml adipose tissue;
  • DMEM/F12 medium was purchased from GIBICO;
  • MCDB (MCDB201medium complete with trace element) was purchased from Sigma, containing 2% fetal bovine serum, 1xA solution and 1xB solution;
  • 100xA solution DF12, containing 1000ng/ml EGF (purchased from CHEMICON), 10-6M dexamethasone (purchased from SIGMA), 100mM ascorbic acid (purchased from SIGMA);
  • 25xB solution DF12, containing 25mg/ml human serum albumin (purchased from Luo Shi Company), 10ug/ml insulin (purchased from GIBICO Company), 5.5ug/ml transferrin (purchased from Bethyl Laboratories Company);
  • Digestion solution 0.25% trypsin, filtered and sterilized by a 0.22 ⁇ m filter, divided into packages, and stored at -20°C;
  • Collagenase digestion solution 0.2% collagenase, filtered and sterilized through a 0.22 ⁇ m filter, divided into packages, and stored at -20°C;
  • Cell cryopreservation medium containing 20% DMSO, 20% fetal bovine serum, 60% DF12 culture medium;
  • Stem cell preservation solution TC199, normal saline containing 1% human albumin and compound amino acids.
  • Total cell number (n/4) ⁇ 2 ⁇ 10000 ⁇ total cell volume, the value obtained is the total number of cells, according to the cells used Adjust the cell concentration of the complete culture medium to about 1x10 6 cells/ml.
  • Adipose-derived stem cells adhere to the wall within 24 hours. Most of the adherent cells are fibroblast-like cells, mainly prismatic cells, which grow in parallel or whirlpool. Observed under the Swiss staining light microscope, the cell cytoplasm is sky blue and the nucleus is purple. Red, thick nuclear chromatin, with obvious nucleoli.
  • Tube 0.1ml 4Add 5ul of FITC-labeled mouse anti-human CD29, CD34, CD44, HLA-DR, CD105 and Flk-1 respectively; 5Incubate at 4°C in the dark for 30min, add the same amount of PBS to the blank control group; 6Incubate After finishing, wash 2 times with PBS; 7Finally resuspend the cells with 1mLPBS; 8Flow cytometry to detect cell surface markers; 9Result determination: Cellquest software statistically analyzes the percentage of positive cells labeled with fluorescent antibodies.
  • stem cells prepared by the present invention are qualified adipose-derived stem cells.
  • lipid droplets After 2 weeks of adipogenic induction culture, small foam-like structures with increased light transmittance and different surrounding structures can be seen in the cells, which are newly formed lipid droplets. Continued culture shows that the number of lipid droplets increases and begins to fuse into large lipid droplets. After oil red staining, the lipid droplets can be seen to be stained red.
  • adipose-derived stem cells can differentiate into chondrocytes.
  • stem cell products in order to avoid heat sources that may be introduced in the in vitro culture of stem cells that may cause heat in the infusion, the stem cell products are checked for bacteria, mycoplasma and endotoxin to ensure that the stem cells used for infusion are free of contamination.
  • Test product tube limulus reagent plus 0.1 mL bacterial endotoxin test water plus 0.1 mL test product solution
  • test product positive control tube limulus reagent plus 0.1 mL bacterial endotoxin test water plus 0.1 mL test product positive control solution
  • Positive control tube limulus reagent plus 0.1mL bacterial endotoxin test water plus 0.1 mL 2 ⁇ positive control solution
  • negative control tube limulus reagent plus 0.1 mL bacterial endotoxin test water plus 0.1 mL bacterial endotoxin test water
  • prepare 4 The limulus reagent was sealed with a sealing film, and then a set of parallel, a total of 8 limulus reagents, was placed in a 37 °C water bath in the super circulating water tank for 1 hour, and the agglutination was observed.
  • the cells were digested, filtered with a 120-mesh nylon mesh, and the cell density was counted, and the cells were diluted to 2 ⁇ 10 5 cells/ml.
  • the 1.25% agar prepared with distilled water in advance is boiled in a boiling water bath and placed in a 42°C water bath environment for later use.
  • the cell survival rate is detected to determine the best storage solution, container and storage conditions. It is finally determined that the best storage conditions for this product are: at 0 ⁇ 4°C, in the stem cell preservation solution, its validity period is 24 hours.
  • TC199 preparation (V/V) preservation solution containing 5%, 2%, 1% and 0.5% human albumin, preserved in a vial, placed at 4°C, After 8 hours of storage, the cell viability was counted by trypan blue staining.
  • the storage effect at 4°C is significantly better than the storage effect at room temperature; at 4°C, the 24-hour survival rate can be maintained above 90%; therefore, the storage condition for adipose-derived stem cells is 4 °C, the storage time can reach 24 hours.
  • non-composite membrane infusion bag non-PVC
  • vial storage condition: 4°C, 8 hours.
  • the TC199 preservation solution containing 1% human albumin has the best preservation effect on adipose-derived stem cells; the survival rate can reach more than 90% when stored for 24 hours; therefore, we will
  • the storage conditions were set as: 4°C, a storage solution of TC199 containing 1% human albumin, and the storage period was set at 24 hours.
  • Resuscitation, culture, digestion, passage, and expansion were performed according to conventional operating procedures, and fat-derived stem cells were harvested and prepared with TC199 with a concentration of 1% human albumin to prepare a cell suspension with a concentration of 5x10 5 /ml for later use.
  • TC199 with a concentration of 1% human albumin
  • a cell suspension with a concentration of 5x10 5 /ml for later use.
  • the cells are digested and centrifuged according to the aforementioned cell digestion method to collect the cells.
  • the cell pellet was washed three times with 10 ml of normal saline, 1000 rpm, and centrifuged at room temperature for 5 minutes.
  • Cells were resuspended using a cell storage solution, a vial charged with 20ml sterile, and the number of cells adjusted to 2 x 10 7 -4x10 7 cells / 20 ml. Seal the lid, label it, and store it at 4 degrees Celsius.
  • Example 5 Use of adipose-derived stem cell preparation
  • Acute leukemia chronic myeloid leukemia, multiple myeloma, malignant lymphoma, breast disease, neuroblastoma, myelodysplastic syndrome, polycythemia vera, paroxysmal nocturnal hemoglobinuria.
  • Aplastic anemia Fanconi anemia, thalassemia, sickle cell anemia, myelofibrosis, non-megakaryocytic thrombocytopenia.
  • Intravenous infusion infusion with blood transfusion device, remove the filter. Prepare emergency medicines and instruments before reinfusion, intravenous dexamethasone 5mg, oral chlorpheniramine 8mg, and phenacin 25mg before infusion.
  • Example 6 Adipose stem cells assisted fat transplantation to treat a nude mouse model of scleroderma
  • phosphate buffered saline PBS
  • the control group was injected with PBS solution into the back skin of nude mice, once a day, 0.1ml each time for 4 weeks. Observe the elasticity and appearance of the back skin. The general changes. At 4 weeks, 2 pre-experimental model mice and 2 control mice were sacrificed. The skin on the back of the injection area was fixed in tissue fixative, and HE staining and Masson staining were performed respectively. Compared with the PBS control group mice, it was confirmed Whether the model is successful or not.
  • PBS phosphate buffered saline
  • Fat transplantation and adipose stem cell assisted fat transplantation (Cell-assisted lipotransfer, CAL) operation
  • mice and scleroderma model nude mice were divided into three groups: control group, fat transplantation group, and CAL group. 6 patients in the control group were given subcutaneous injection of 0.3ml PBS, 6 patients in the fat transplantation group were given 0.3ml fat back subcutaneous injection, and 6 patients in the CAL group were given 0.3ml fat+1 ⁇ 10 6 fat stem cells subcutaneous injection back. Use a 1ml screw-propelled syringe to inject evenly in the receiving area.
  • the transplanted fat was weighed, and the skin of the injection site of the nude mice was cut, fixed in formaldehyde solution, embedded in paraffin, and made into tissue sections, and observed by hematoxylin-eosin (HE) staining and Masson staining.
  • HE hematoxylin-eosin
  • Masson staining was performed to determine the content of type III collagen and transforming growth factor TGF- ⁇ 1 in the skin.
  • the thickness of the slice is preferably 5um-10um, and the inclination angle is generally 20°-30°.
  • the wax sheet face up gently on the water surface of the film box at 40 ⁇ 45°C, and use the tension and temperature of the water to flatten the slightly wrinkled wax tape.
  • the wax slice is pulled to the middle section of the glass slide and the remaining water on the glass slide is poured.
  • the slices are almost completely dry before roasting.
  • Hematoxylin-eosin staining is one of the commonly used staining methods in paraffin section technology. Hematoxylin stain is alkaline, which mainly makes the chromatin in the nucleus and the ribosomes in the cytoplasm colored purple-blue; Eosin is an acid dye that mainly makes the components in the cytoplasm and extracellular matrix colored red.
  • A 0.5 to 1% eosin alcohol solution. Weigh 0.5 ⁇ 1g of Eosin Y, add a small amount of distilled water to dissolve, and then add glacial acetic acid dropwise until it becomes a paste. Filter with filter paper, dry the filter residue in an oven, and dissolve it with 100 ml of 95% alcohol.
  • Hematoxylin dye solution formula (prepared 3000ml, can be reduced in proportion) Hematoxylin 6g; absolute ethanol 100ml; aluminum potassium sulfate 150g; distilled water 2000ml; sodium iodate 1.2g; glacial acetic acid 120ml; glycerol 900ml.
  • Preparation method Dissolve hematoxylin in absolute ethanol, then dissolve potassium aluminum sulfate in distilled water, pour glycerin into the mixture after dissolution, and finally add glacial acetic acid and sodium iodate.
  • Masson staining makes collagen fibers green, elastic fibers brown to dark brown, muscle fibers and cellulose red. This staining method can better show the distribution of muscle, collagen and elastic fibers in the tissue than HE staining, and show more vividly. The characteristics of the organizational structure.
  • phosphomolybdic acid aqueous solution 1 g of phosphomolybdic acid, and distilled water to 100 ml.
  • Histologically (Masson staining) model shows that the dermis is thick, the collagen fibers are thickened, increased, and arranged disorderly, the blood vessels in the subdermal layer are thickened or even occluded, the fat layer is thinned and disappeared, while the normal dermis is thin and the collagen fibers are arranged neatly .
  • Type III collagen immunohistochemical indexes, scleroderma model nude mice induced by bleomycin (2519.17 ⁇ 774.19) were significantly higher than normal nude mice (623.85 ⁇ 113.84) ( Figure 1, Table 7).
  • laser speckle contrast imaging technology laser speckle contrast imaging system, Perimed
  • the results showed that the blood flow at the injection site of normal nude mice was 251.72 ⁇ 43.82, and scleroderma nude mice The blood flow at the injection site was 241.89 ⁇ 22.76, and the results were not significantly different.
  • the fat weight of the normal nude mouse fat transplantation group was 102.97 ⁇ 17.87mg, and the fat survival rate was 36.8%; the fat weight of the CAL group was 135.05 ⁇ 27.28mg, and the fat survival rate was 48.2%; the fat transplantation weight of scleroderma nude mice was 77.54 ⁇ 12.40mg, fat The survival rate was 27.7%; the fat weight of the CAL group was 102.48 ⁇ 23.16 mg, and the fat survival rate was 36.6%. Regardless of normal nude mice or scleroderma nude mice, the survival rate of the CAL group was higher than that of the simple fat transplantation group (Table 8, Figure 2).
  • HE staining results showed that adipose tissue was composed of A fat cells, vascular endothelial cells, fibroblasts and connective tissue, as well as macrophages and lymphocytes. Adipose tissue is filled with large fat cells, and there are scarce connective tissue and abundant capillary network. The degree of fibrosis in the scleroderma group was heavier than that in the normal group. The CAL group of normal nude mice had the largest number of adipocytes, with clear boundaries and clear contours.
  • Masson staining showed that there was no obvious difference between the normal nude mice control group, fat transplantation group, and CAL group, the degree of fibrosis was light, the collagen fibers were blue, and the fibrous tissue was arranged and shaped.
  • the degree of fibrosis in scleroderma modeled nude mice was heavier than that of normal nude mice.
  • the dermis of the control group of scleroderma modeled nude mice was the thickest, and the collagen fibers were thickened, increased, and arranged disorderly.
  • the dermis of the CAL group was significantly thinner than that of the control group. , The content of collagen fibers decreased, and the fat graft group was somewhere in between (Figure 4).
  • TGF- ⁇ 1 in the control group was 939.34 ⁇ 216.92
  • the fat transplantation group was 974.98 ⁇ 470.18
  • the CAL group was 967.66 ⁇ 448.74
  • the type III collagen content in the control group was 623.85 ⁇ 113.84
  • the fat transplantation group was 623.85 ⁇ 113.84 605.73 ⁇ 217.17
  • CAL group is 599.34 ⁇ 89.39
  • TGF- ⁇ 1 content in control group is 1945.95 ⁇ 330.77
  • fat transplantation group is 1418.86 ⁇ 376.82
  • CAL group is 1132.12 ⁇ 190.69
  • type III collagen content control was 2629.39 ⁇ 746.62
  • the fat transplant group was 1531.74 ⁇ 836.41
  • the CAL group was 946.92 ⁇ 448.90.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Biomedical Technology (AREA)
  • Biotechnology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Wood Science & Technology (AREA)
  • Cell Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Developmental Biology & Embryology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Immunology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Environmental Sciences (AREA)
  • Virology (AREA)
  • Dentistry (AREA)
  • Epidemiology (AREA)
  • Dermatology (AREA)
  • Hematology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

一种干细胞制剂，包含脂肪来源干细胞和干细胞保存液，其中脂肪来源干细胞含量为1×10 5-5×10 6个细胞/毫升，干细胞保存液为含0.5-1%人血白蛋白的TC199的保存液，剂型为口服剂或注射剂。脂肪干细胞的制备方法包括脂肪组织分离、脂肪干细胞原代培养等步骤，收集第3-6代脂肪来源干细胞。干细胞制剂可用于制备用于恶性肿瘤、美容整形、辅助脂肪移植治疗硬皮病的药物或产品，能够提高脂肪存活率，减轻硬皮病患者的皮肤胶原沉积，降低硬皮病患者的TGF-β1含量及III型胶原含量。

Description

一种脂肪来源干细胞制剂、制备方法及其应用 技术领域
本发明涉及生物医药技术领域，具体涉及一种脂肪来源干细胞制剂、制备方法及其应用。
背景技术
硬皮病是以皮肤和组织器官纤维化、硬化为特征的自身免疫性结缔组织病，临床上主要分为两类：其一，单纯只有皮肤受累而无明显内脏系统受累称为局限性硬皮病(Localized sclerosis,LS)，又称硬斑病(Morphea)；其二，广泛皮肤硬化伴内脏系统受累为系统性硬皮病(Systemic sclerosis,SSc)，也称为系统性硬化症。
系统性硬化症病因不明，遗传、感染、环境及药物等因素均可能参与其中；其发病机制复杂，免疫紊乱、血管内皮细胞损伤、成纤维细胞增殖及胶原合成异常为疾病的主要病理表现。系统性硬化症皮肤早期的病理特点是血管周围炎症细胞的浸润、毛细血管扩张、微血管改变，晚期细胞外基质过度积聚造成组织纤维化，微小动脉和小动脉内皮细胞增生，血管腔变狭窄甚至堵塞，伴以微血管性出血。类似病变亦可见于其他脏器，导致响应器官损害。系统性硬化症患者临床表现异质性较大。80％患者以雷诺现象为早期首发症状，其本质为间歇性周围血管痉挛，到晚期快速进展的弥漫性皮肤硬化伴多器官功能衰竭。临床统计显示，几乎所有系统性硬化症患者皮肤硬化都是从手开始，进而进展到面颈部及骨、关节、消化系统、肺、肾脏、心脏等多个内脏器官。
局限性硬皮病病因不明，可能与感染、药物、化学物品、恶性肿瘤、创伤、遗传因素、自身免疫、激素水平等有关。病变通常仅累及皮肤，但有时也可累及皮下组织，例如，脂肪、肌肉、骨骼，少部分病例可出现内脏的损害。皮损表现为躯干，四肢，面部等处色素减退斑。斑块的大小及皮损的分布因人而异，有些皮损可引起面部畸形，若硬皮病累及关节，可引起活动障碍。根据以往文献报道，LSC的发病机制主要包括：小血管损伤、T细胞活化以及结缔组织生成增加。
硬皮病的早期治疗主要针对免疫、血管及胶原的异常，以抗炎、免疫抑制、免疫调节、 改善血循环和减少纤维化为基础。后期遗留皮肤硬化、皮下脂肪萎缩、局部凹陷等畸形，常常需要整形科治疗。局部凹陷畸形的整形外科治疗目前包括带血管蒂的自体组织移植及不带血管蒂的自体脂肪移植。带血管蒂的组织移植手术创伤大，显微缝合风险高等，自体脂肪移植因手术创伤小，成为最受患者及整形科医师欢迎的治疗方案。
影响脂肪注射移植存活率的相关因素众多，包括供区的选择、血供、受区活动度及黏连情况，以及脂肪颗粒的获取方式、抽吸技术、脂肪颗粒的纯化和患者的全身因素等。技术层面上，在抽取脂肪、清洗和移植中，凡有可能对脂肪颗粒造成破坏的物理、化学等因素均应避免。同时，为促进脂肪颗粒的存活，在移植中，我们应尽量使脂肪颗粒与组织有更大的接触面。因此，多层次、多隧道、多点移植已成为脂肪移植技术上的共识。但以往研究针对的大部分都是正常的脂肪标本，硬皮病患者研究比较少，硬皮病患者的脂肪移植同正常人群的脂肪移植有所不同，即使遵循上述脂肪移植技术上的原则，硬皮病患者的脂肪存活率仍明显低于正常人群的脂肪移植，可能与硬皮病病变区域组织萎缩粘连，局部血液供应差，局部炎性微环境等因素有关，同时硬皮病患者自体供区脂肪有无生物学改变也需要验证。
脂肪来源干细胞(Adipose-derived stem cells)，简称ADSCs，是来源于人脂肪组织中一类可以在体外增殖并具有多向分化潜能的细胞，这种细胞与骨髓间充质干细胞(BMSCs)等都有相似的表达和多向分化潜能，提供脂肪组织终生的自我更新，维持脂肪代谢的平衡。与其他来源的干细胞相比，ADSCs因其具有独特的优势如脂肪来源干细胞取自于脂肪组织，具有来源广泛、取材简单、体外增殖迅速、细胞老化率低等优点，引起研究者的广泛关注，逐渐成为组织工程学的理想种子细胞。
脂肪来源干细胞为成纤维细胞样，脂肪来源干细胞低表达HLA-ABC，而不表达HLA-DR，从而说明ADSCs具有低免疫原性，用于同种异体、异种异体移植后，均不会发生免疫排斥反应，在一定条件下，可以向脂肪细胞、软骨细胞、肌肉细胞和成骨细胞分化，脂肪来源干细胞的基本形态、增殖周期、免疫周期、免疫原性及多向分化潜能成为组织工程和再生医学领域应用最广泛的成体干细胞。干细胞的这些特点，为我们应用脂肪移植治疗硬皮病提供了新的思路。
然而，脂肪来源干细胞十分脆弱，将其制成制剂很难保证存活率，且不能长时间保存及运输而如果制剂中脂肪来源干细胞的存活率不足则不能发挥很好的药效。
发明内容
为了解决上述问题，本发明第一个目的在于提供了一种脂肪来源干细胞制剂及其制备方法，极大程度的保留了干细胞生物活性因子，便于长时间运输和储存，便于使用。
本发明的第二个目的在于提供脂肪来源的干细胞在制备治疗硬皮病的细胞制剂中的用途。
为实现上述目的，本发明第一方面提供了一种干细胞制剂，所述制剂包含脂肪来源干细胞和干细胞保存液，所述脂肪来源干细胞含量为1x10 5-5x10 6个细胞/毫升，优选的，1x10 6-2x10 6个细胞/毫升。
在本发明的一些实施方式中，所述干细胞为经干细胞保存液重悬后的单细胞悬液状态。
在本发明的一些实施方式中，所述干细胞保存液为含0.5-1％人血白蛋白的TC199的保存液。
在本发明的一些实施方式中，所述干细胞制剂为口服剂或注射剂。优选的，皮下注射剂。
在本发明的一些实施方式中，所述干细胞为传代培养第3代至第6代的脂肪来源干细胞。
本发明第二方面提供了所述的干细胞制剂的储存方法，所述干细胞制剂储存条件为4℃，含0.5％-1％人血白蛋白的TC199的保存液，储存于西林瓶中，保存期定为24小时；所述干细胞制剂储存条件为液氮保存，保存期定为1年。
本发明第三方面提供了一种所述的干细胞制剂的制备方法，包括以下步骤：
(1)脂肪组织分离及脂肪干细胞原代培养：将脂肪组织中加入生理盐水，用吸管吹打10-20次，静置1-2min，将组织下层的液体吸出，加入胶原酶消化液，消化30-60min，吸出下层液体，过滤、离心，弃上清，将细胞沉淀以培养基重悬后离心弃上清液，以2×10 6-4×10 6个细胞/mL的密度在培养体系内培养，将培养瓶放入培养箱中；
(2)脂肪组织分离及脂肪干细胞原代培养：细胞生长至70％-80％汇合时取出，加入胰酶和EDTA，当细胞开始回缩变圆后，加入等体积培养基终止消化，吹打瓶壁上的细胞， 使细胞脱离瓶壁后呈单细胞悬液，将单细胞悬液移至离心管中，离心，弃上清，加入完全培养基，轻吹使之松散，按1:3传代培养；收集第3代至第6代脂肪来源干细胞；
(3)最后，细胞用干细胞保存液重悬脂肪来源干细胞得到干细胞悬液。
在本发明的一些实施方式中，所述干细胞悬液中包括脂肪来源干细胞浓度为1x10 6-2x10 6个细胞/毫升。
在本发明的一些实施方式中，所述步骤(2)中加入0.125％的胰酶和0.01％的EDTA。
在本发明的一些实施方式中，还包括在所述干细胞悬液中加入药学上可接受的辅助成分。
本发明第四方面提供了所述的干细胞制剂在制备用于恶性肿瘤、美容整形的药物中的用途。
本发明第五方面提供了上述脂肪来源干细胞制剂在制备辅助脂肪移植治疗硬皮病的产品中的用途。
在本发明的一些实施方式中，所述硬皮病包括系统硬皮病、局限性硬皮病。优选的，所述系统性硬皮病包括弥漫性皮肤系统硬化症(Diffuse cutaneous sclerosis,dcSSc)；局限性皮肤系统硬化症(Limited cutaneous sclerosis,lcSSc)；无皮肤表现的系统性硬化症；重叠综合征；未分化结缔组织病。
优选的，所述局限性硬皮病包括点滴型硬皮病(Bullous morphea)；泛发型硬皮病(Generalized morphea)；斑块型硬斑病(Plaque morphea)；线状硬皮病(Linear scleroderma)和深部硬皮病(Deep morphea)。
所述线状硬皮病涉及头部和面部的亚型：线性硬皮病-军刀型硬皮病(Linear scleroderma‘en coup de saber’)(LScs)、进行性面肌偏侧萎缩症(progressive facial hemiatrophy,PFH)。
在本发明的一些实施方式中，所述脂肪来源干细胞辅助脂肪移植不仅可提高脂肪存活率，同时还可减轻硬皮病患者的皮肤胶原沉积。
在本发明的一些实施方式中，所述脂肪来源干细胞降低硬皮病患者的TGF-β1含量及 Ⅲ型胶原含量。
在本发明的一些实施方式中，每毫升所述产品包含1x10 6-2x10 6个脂肪来源干细胞。
在本发明的一些实施方式中，所述产品为干细胞制剂或药物。
在本发明的一些实施方式中，所述的干细胞制剂或药物为皮下注射剂。
在本发明的一些实施方式中，所述药物还包括其他治疗硬皮病的药物。
有益效果
1、本发明制备的脂肪来源干细胞制剂中的干细胞活率在4℃下，用1％人血白蛋白浓度的TC199保存24h后仍高达90％以上；在液氮保存下，1年后细胞活性仍高达95％以上，该干细胞制剂活率高、保存周期长、方便运输且制备方法简单；此外，该干细胞制剂质量可控、安全有效，可用于临床治疗恶性肿瘤或在美容整形领域应用。
2、本发明发现脂肪来源干细胞除了可提高脂肪存活率，还发现了脂肪干细胞辅助脂肪移植后硬皮病裸鼠的皮肤胶原含量明显降低。发明人通过构建博来霉素硬皮病裸鼠模型，研究脂肪干细胞+脂肪移植可降低硬皮病组小鼠的TGF-β1含量及Ⅲ型胶原含量，减轻硬皮病的症状。
附图说明
图1正常裸鼠与博来霉素硬皮博裸鼠大体观察及组织学切片，造模组皮肤皮肤增厚，变硬，胶原纤维增粗、增多、排列紊乱，显示造模成功；
图2无论正常裸鼠还是硬皮病裸鼠，CAL组存活率均较单纯的脂肪移植存活率高；
图3移植脂肪HE染色；
图4 Masson染色显示正常裸鼠及硬皮病裸鼠脂肪移植或CAL后皮肤改变；
图5 TGF-β1免疫指数：正常裸鼠经脂肪移植或脂肪干细胞+脂肪移植后无明显改变。但对于硬皮病小鼠，脂肪移植或者CAL可降低硬皮病组小鼠的TGF-β1含量，尤其是CAL效果更加明显；
图6 III型胶原免疫指数：正常裸鼠经脂肪移植或脂肪干细胞+脂肪移植后无明显改变。但对于硬皮病小鼠，脂肪移植或者CAL可降低硬皮病组小鼠的III型胶原含量，尤其是CAL效果更加明显。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1脂肪来源干细胞的制备
一、实验前准备
1、脂肪组织供体
脂肪组织来源于健康志愿者，在采集前详细记录供体的年龄、性别，常规体检了解其健康状况，要求其HBV，HCV，HIV及梅毒血清学检测应均为阴性。采集当日清晨禁食、水，进手术室前检查生化全项及血常规。
2、材料与药品
脂肪保存液：为含有肝素的无菌TC199，每100ml保存液含肝素4000单位、庆大霉素2000单位，1ml保存液可抗凝3至10ml脂肪组织；
细胞完全培养液：DMEM/F12培养基购于GIBICO公司；
MCDB(MCDB201medium complete with trace element)购于Sigma公司，含2％胎牛血清、1xA液及1xB液；
100xA液：DF12，含1000ng/mlEGF(购于CHEMICON公司)、10-6M地塞米松(购于SIGMA公司)、100mM抗坏血酸(购于SIGMA公司)；
25xB液：DF12，含25mg/ml人血清白蛋白(购于罗式公司)、10ug/ml胰岛素(购于GIBICO公司)、5.5ug/ml转铁蛋白(购于Bethyl Laboratories公司)；
消化液：0.25％胰蛋白酶，经0.22μm滤器过滤除菌后分装，置-20℃保存；
胶原酶消化液：0.2％胶原酶，经0.22μm滤器过滤除菌后分装，置-20℃保存；
细胞冻存液：含20％DMSO、20％胎牛血清、60％DF12培养液；
干细胞保存液：TC199，为含1％人血白蛋白及复方氨基酸的生理盐水。
二、脂肪来源干细胞的制备
1、脂肪的采集
选择脐周、髂前上棘做切口；将吸脂管从手术的切口插入腹中，进行抽脂；当抽出的脂肪的量基本达到预期的量以后，停止吸脂，将吸脂管从腹中抽出，放入脂肪保存液中，最后对伤口进行缝合。
2、体细胞的分离
(1)加等体积的生理盐水洗脂肪三次：上层脂肪转移到50ml无菌离心管内，每管约20ml，用盐水补充到40ml每管，水平离心机室温下离心，500转/分钟，5分钟。
(2)每管加入胶原酶(II型胶原酶)消化液10ml，用盐水补充到35ml每管，37℃振荡消化15～35分钟，直到脂肪消化为均匀浑浊液，不可见明显块状物体存在。
(3)用100μm的滤网过滤至50ml无菌离心管内，生理盐水冲洗原离心管及滤网。用DPBS或生理盐水补足过滤后液体至40ml每管，水平离心机室温下离心，1500转/分钟，10分钟。
(4)用培养基将细胞悬浮，用台盼蓝染色方法计数活细胞，具体步骤如下：①吸取20微升细胞悬液，再吸取20微升的白细胞稀释液与之混合；②将细胞计数板与盖片放到显微镜下，用移液器吸取上面的细胞悬液从盖片与细胞计数板之间加入细胞计数板。计数四个大格中的细胞数量n；③按下面的公式计算细胞总数：Total cell number＝(n/4)×2×10000×细胞总体积，所得数值即为细胞总数，根据其用细胞完全培养液调整细胞浓度至约1x10 6个/毫升。
3、脂肪来源干细胞的分离培养
(1)将细胞以约1x10 6个/ml的密度接种于一次性细胞培养瓶中，置于37℃，5％CO 2孵箱中培养。
(2)培养24小时后，有少量细胞贴壁，原代脂肪来源干细胞呈克隆样生长，细胞呈梭形。弃掉悬浮细胞，加入新鲜配制的细胞完全培养液，继续培养贴壁细胞。
(3)每天观察细胞生长状态，每隔3-4天全量换液。
(4)细胞消化传代：
①当细胞长至70％－80％融合时，吸去培养基，加入10ml生理盐水洗涤细胞一次；②吸去洗液，加入5-10毫升0.125％的胰酶和0.01％的EDTA，在显微镜下观察，待细胞边缘回缩，细胞变圆时，加入0.5毫升的血清终止消化反应；③用移液管轻轻吹打细胞，将细胞吹打下来，移至新的15毫升离心管中；④将离心管在天平上配平，放入水 平离心机中，1200转/分钟，离心10分钟；⑤离心结束后，取出离心管，用移液管小心地将上清吸出弃掉，拍打离心管，使细胞沉淀分散；⑥加入培养液重新悬浮细胞，按照1:3比例进行传代。本发明均在第3-6代进行。
实施例2脂肪来源干细胞的鉴定
1、细胞计数及成活率检测
细胞计数和台盼蓝染色法进行细胞数量和成活率的检测。
台盼蓝染色法，具体操作如下：
将血球计数板及盖片擦拭干净，并将盖片盖在计数板上。将0.5ml细胞悬液加入试管中，并加入0.5ml 0.2％台盼蓝染液，染色2～3分钟。将染色的细胞悬液混匀后吸出10μl，充池(滴加在盖片边缘)，使悬液充满盖片和计数板之间，满而不溢。静置1-2分钟。镜下(10×)任意取几个视野，计细胞总数300，分别计数着色(死)细胞和(非着色)活细胞数，按下式计算细胞存活率：
存活率＝[细胞总数300－着色(死)细胞]/细胞总数300×100％
结果显示，细胞数量应不低于2～5×10 6/毫升，细胞成活率为95％不低于90％，合格。
2、细胞形态检查、细胞周期及表面标志检测
(1)细胞形态检查
①接种1×10 5细胞于放有无菌盖玻片的6孔板中，培养基同前。②待细胞达到60％融合，PBS冲洗2-3遍，洗净培养基。③加入瑞氏染液，能覆盖住玻片为止。④30秒之后加入等体积PBS(PH值6.4)，混匀。⑤30分钟后流水冲洗，取出玻片，甘油明胶封片。⑥光镜下观察，判定结果：
脂肪来源干细胞在24小时内贴壁，贴壁细胞大多呈成纤维细样，以棱形细胞为主，平行排列或漩涡样生长，瑞士染色光镜下观察细胞胞浆呈天蓝色，核呈紫红色，核染色质较粗，有明显核仁。
(2)流式细胞分析仪检测细胞周期
①取接近融合的第3代细胞，70％冷乙醇40℃固定过夜；②PBS液洗涤两次；③加入的RNA酶(10ug/mL)37℃孵育30min；④再加入50ug/mL碘化丙啶(PI)4℃避光孵育15min；⑤用流式细胞仪检测细胞周期。
结果显示，大部分细胞处于静止期，GO/G1期细胞为60％以上，表明所得细胞具有干细胞特征。
(3)流式细胞仪检测细胞表面标志
①将生长状态良好且密度达到80％-90％的细胞制成单细胞悬液，800r/min离心5min；②PBS洗2次；③调整细胞密度至10 6/ml，分装入试管中，每管0.1ml；④分别加入5ul结合有FITC标记的小鼠抗人CD29、CD34、CD44、HLA-DR、CD105和Flk-1；⑤4℃避光孵育30min，空白对照组加入等量PBS；⑥孵育完毕后，PBS洗涤2次；⑦最后用lmLPBS重悬细胞；⑧流式细胞仪检测细胞表面标志；⑨结果判定:Cellquest软件统计分析各荧光抗体标记阳性细胞的百分率。
结果显示，CD34(0.3％)、HLA-DR(0.2％)，呈阴性表达，CD29(99.8％)、CD44(99.9％)及CD105(99.9％)呈阳性,呈阳性表达。说明本发明所制备的干细胞为合格的脂肪来源干细胞。
3、油红O染色鉴定成脂肪分化
取融合接近60％～70％的第3代脂肪来源干细胞,培养7d后去除原培养液；更换成脂诱导培养液，加入含10％FBS及成脂诱导剂(1umoL/L地塞米松、10mg/L胰岛素)的DMEM培养液继续培养，进行成脂诱导；诱导2周后进行油红染色；甲醛固定2min；50％乙醇漂洗；加入油红O染料染色10min；50％乙醇漂洗；自来水冲洗；苏木精复染lmin；显微镜下观察结果并拍照。
成脂诱导培养2周后可见细胞内产生透光度增高与周围其他结构不同的小泡沫样结构，即为新生成的脂滴；继续培养可见脂滴数量增加并开始融合成大的脂滴，油红染色后可见脂滴染为红色。
4、茜素红染色鉴定成骨分化
取第3代生长状态良好的细胞，消化收集培养；每个实验样本两复孔，一个空白对照孔；待细胞达到80％-100％融合；实验孔用适量的hMSC成骨诱导培养基每3-4天换液，对照组用适量细胞完全培养液换液；保持诱导条件，持续培养2-3周；培养结束后，PBS清洗细胞；95％乙醇固定10分钟；纯水清洗2次；茜素红染色30min；水清洗2次，显微镜下观察拍照。
结果显示，经诱导后，脂肪来源干细胞可分化为成骨细胞。
5、翻红O染色鉴定成软骨分化
取第3代生长状态良好的细胞，消化收集培养；每个实验样本两复孔，一个空白对照孔；待细胞达到80％-100％融合；应用成软骨诱导液定向分化诱导培养3周，每48～72h换液。对照组用适量细胞完全培养液换液基。倒置显微镜观察细胞形态变化，诱导3周后行番红O染色观察。
结果显示，脂肪来源干细胞可分化为成软骨细胞。
6、MSC微生物及内毒素检测
根据对干细胞制品的质量要求，为了避免干细胞体外培养中可能引入的导致输液发热的热源，对干细胞制品进行细菌、支原体和内毒素的检查，保证用于输注的干细胞无污染。
(1)培养法无菌检测
在生物安全柜内，打开培养瓶盖，用酒精棉球擦拭瓶口，用无菌注射器取16mL待测样本分别等量打入需氧培养瓶和厌氧培养瓶各8mL；每批次培养瓶取需氧培养瓶和厌氧培养瓶各1瓶做为阴性对照进行培养；做好标记后将注有样本的培养瓶及对照培养瓶放入全自动细菌培养系统中培养5天，观察是否染菌。
(2)鲎试剂检测内毒素残留
供试品管：鲎试剂加0.1mL细菌内毒素检查水加0.1mL供试品溶液；供试品阳性对照管：鲎试剂加0.1mL细菌内毒素检查用水加0.1mL供试品阳性对照液；阳性对照管：鲎试剂加0.1mL细菌内毒素检查用水加0.1mL 2λ阳性对照液；阴性对照管：鲎试剂加0.1mL细菌内毒素检查用水加0.1mL细菌内毒素检查用水；将4支制备好的鲎试剂用封口膜封口，再做一组平行，共8支鲎试剂，放到超级循环水箱37℃水浴1h，观察凝集情况。
(3)一步法检测支原污染检测
取培养两天以上的细胞培养上清2mL，200×g离心5分钟；离心后取上清100μL转移到白色96孔板中，做上标记；在细胞培养上清空中先加入100μL MycoAlert PLUS Reagent，作用5分钟，用分光光度计读数A；再加入100μL MycoAlert PLUS Substrate，作用10分钟，用分光光度计读数B。计算B比A的比率为Ratio；Ratio＜1，支原体检测为阴性；Ratio＞1.2，支原体检测为阳性；由此来判断支原体感染情况。
取第3代的脂肪来源干细胞进行细菌、内毒素、支原体等项目检测。检测结果全部符合规定，可以用于后续干细胞制剂的制备。
7、致瘤性检测
消化细胞，用120目尼龙网过滤后计数细胞密度，将细胞稀释成2×10 5个/ml。将预先用蒸馏水配制的1.25％的琼脂置沸水浴煮溶，放在42℃水浴环境备用。将30ml胎牛血清加入90ml二倍浓度的RPMI1640培养液中，混匀，置42℃水浴中。将80ml1.25％琼脂加入步骤3.2.4.3配好的含血清的培养液中。置42℃水浴中。吸取7ml在步骤3.2.4.4配好的琼脂营养液放入直径为5cm的培养皿中。注意避免气泡。在室温静置约10分钟，即凝固为底层琼脂。将1ml配好的琼脂营养液放入一离心管底部，再将0.5ml细胞悬液(含10 5个脂肪来源干细胞或基底细胞癌BCC细胞)加入离心管中混匀。用吸管吸出均匀地滴在铺有底层琼脂的平皿。静置片刻。在显微镜下观察细胞分布是否均匀。将平皿置饱和湿度的CO 2培养箱中培养。每隔1周取出，用显微镜观察有否克隆形成。检查后加入0.3ml培养液以避免琼脂干燥。如需进行亚克隆分离，可用吸管将克隆小心吸出，在少量培养液吹打后继续培养。
实验结果：培养2周后应无克隆形成，而阳性对照组则可见明显的克隆形成。
实施例3脂肪来源干细胞稳定性研究
通过不同保存液，不同保存容器，不同保存温度保存相同的时间检测的细胞存活率高低，确定最佳保存液、容器及保存条件。最终确定本品最佳保存条件为：在0～4℃条件下，在干细胞保存液中，其有效期为24小时。
1、脂肪来源干细胞的最适保存条件：
取复苏后新鲜制备的脂肪来源干细胞成品，分组：含5％、2％、1％及0.5％人血白蛋白的TC199的配制(V/V)保存液，西林瓶保存，置于4℃，8小时保存后，台盼蓝染色计数细胞存活率。
表1细胞存活率
Figure PCTCN2021090539-appb-000001
在1％及以上白蛋白浓度的保存液条件下，细胞存活率均较高，各组之间没有显著性 差异(表1)；随着人血白蛋白浓度增高，细胞成团现象加重，可能与保存液粘度增加有关；因此，选择1％人血白蛋白浓度的TC199，为脂肪来源干细胞的保存液。
2、保存条件及时间对脂肪来源干细胞的影响
取复苏后新鲜制备的脂肪来源脂肪来源干细胞成品，在室温、4℃的条件下保存。分别在保存的2小时、4小时、6小时、8小时、10小时、12小时、24小时时，取样测定细胞存活率。取样测定细胞存活率，表2。
表2细胞存活率
Figure PCTCN2021090539-appb-000002
在同样的保存液条件下(TC199)，4℃保存效果明显优于室温的保存效果；在4℃条件下，保存24小时存活率可维持在90％以上；因此脂肪来源干细胞的保存条件为4℃，保存时间可达24小时。
3、保存容器对脂肪来源干细胞的影响
分组：非复合膜输液袋(非PVC)；西林瓶；保存条件：4℃，8小时。
表3细胞存活率
Figure PCTCN2021090539-appb-000003
结果显示(表3)，4℃保存条件下，两种保存容器对细胞活率的影响无明显差别， 但非PVC复合膜材料的输液袋对细胞数量损失较大。总体来说，西林瓶对脂肪来源干细胞的保存效果，优于非PVC复合膜材料的输液袋。
综上，在4℃条件下，含1％人血白蛋白的TC199保存液对脂肪来源干细胞的保存效果最好；保存24小时时存活率可达90％以上；所以，我们将脂肪来源干细胞的保存条件定为：4℃，含1％人血白蛋白的TC199的保存液，保存期定为24小时。
4、脂肪来源干细胞的稳定性实验
(1)保存期内脂肪来源干细胞的存活率的稳定性
按照常规的操作程序复苏、培养、消化、传代、扩增，收获脂肪来源干细胞并用1％人血白蛋白浓度的TC199配制成5x10 5/ml浓度的细胞悬液备用。取混匀后的细胞平均分成三份，装于西林瓶中，置于4℃冰箱，在保存的24小时时，取样测定细胞存活率，取均数。
表4细胞存活率
Figure PCTCN2021090539-appb-000004
结果显示(表4)，4℃下，在1％人血白蛋白浓度的TC199保存液中，24小时的保存期内5x10 5/ml浓度的细胞的存活率保持在90％以上，因此干细胞制剂在保存期内的存活率稳定。
(2)保存期内液氮深低温保存对脂肪来源干细胞存活率的影响实验
将细胞分成六份，分别装于六只冻存管中，经液氮深低温冻存后，分别于六个月、一年分两次各取三只测定细胞存活率，并分别取均数。
表5冻存六个月的细胞存活率
Figure PCTCN2021090539-appb-000005
表6冻存十二个月的细胞存活率
Figure PCTCN2021090539-appb-000006
结果显示(表5、6)，保存期内脂肪来源干细胞以5x10 6/ml浓度在经过液氮深低温保存后，一年之内复苏，细胞的存活率可以保持在95％以上，存活率稳定。完全可以适应临床回输的要求。
实施例4脂肪来源干细胞制剂的制备
待细胞扩增总数达所需细胞数量后，按前述细胞消化的方法消化离心，收集细胞。细胞沉淀用10ml生理盐水重复洗涤3次，1000转/分，室温离心5分钟。细胞用细胞储存液重悬，装入20ml无菌西林瓶中，并调整细胞数量为2 x 10 7-4x10 7个细胞/20毫升。封好盖子，贴上标签，置于4摄氏度中保存。
实施例5脂肪来源干细胞制剂的用途
能够在体内、体外重建造血组织功能，且具有较低免疫原性和诱导免疫耐受等特点。获得足够临床应用量的脂肪来源干细胞，经HLA配型后静脉输注于患者，用于多种血液系统恶性肿瘤的临床治疗以及其它非恶性的严重造血障碍性疾病。具有降低异体移植免疫排斥反应，提高骨髓移植存活率的作用。
适应症
1、恶性肿瘤
急性白血病、慢性髓系白血病、多发性骨髓瘤、恶性淋巴瘤、乳腺病、神经母细胞瘤、骨髓增生异常综合症、真性红细胞增多症、阵发性睡眠性血红蛋白尿。
2、血液系统非恶性肿瘤
再生障碍性贫血、Fanconi贫血、地中海贫血、镰状红细胞性贫血、骨髓纤维化、无巨核细胞性血小板减少症。
3、其他
急性放射病、重症联合免疫缺陷症、严重自身免疫性疾病、器官移植、基因治疗。
用法与剂量
输注非冻存的细胞
1、静脉应用地塞米松5mg，口服扑尔敏8mg、非那根25mg。
2、细胞不加处理直接从静脉快速输注，用生理盐水冲管，同时心电、血压监护，观察临床表现。
输注冻存的细胞
1、从静脉输注，应用输血器输注，去掉滤网。回输前备齐急救药品及器械，输前静脉应用地塞米松5mg，口服扑尔敏8mg、非那根25mg。
2、从液氮中取出深低温保存的细胞立即置于39-41℃水浴中解冻，在1min内融化，将细胞置于无菌离心管中，400g离心5分钟，弃上清，加入10ml生理盐水洗涤细胞二次，弃上清，在细胞沉淀中加入20ml干细胞保存液TC199重悬细胞。然后不加处理从静脉快速输注，用生理盐水冲管，同时心电、血压监护，观察临床表现。
实施例6脂肪干细胞辅助脂肪移植治疗硬皮病裸鼠模型
1、博莱霉素硬皮病裸鼠造模
用磷酸盐缓冲液(PBS)将博莱霉素粉剂稀释成200ug/ml，经0.22um的无菌滤膜过滤除菌后，保存备用。造模组将博莱霉素稀释液注射到裸鼠背部皮下，对照组将PBS液注射到裸鼠背部皮下，每日1次，每次0.1ml，共4周，观察背部皮肤弹性、外观等大体变化，4周时处死预实验造模组2只及对照组2只，取注射区背部皮肤置于组织固定液中固定，分别行HE染色和Masson染色，与PBS对照组小鼠比较，证实造模成功与否。
2、脂肪移植及脂肪干细胞辅助脂肪移植(Cell-assisted lipotransfer,CAL)操作
4周后，以2％戊巴比妥钠50mg/kg腹腔注射麻醉，1ml注射器行脂肪及干细胞皮下注射。正常裸鼠及硬皮病造模裸鼠均分为三组：对照组，脂肪移植组，CAL组。对照组6例，给予0.3ml PBS皮下注射，脂肪移植组6例，给予0.3ml脂肪背部皮下注射，CAL组6例，给予0.3ml脂肪+1×10 6脂肪干细胞背部皮下注射。应用1ml螺旋推进注射器在受区均匀推注，采用退针时推注，防止推注中阻力的突然变化导致移植脂肪量的不准确，切勿粗暴高压注射。单点注射，使注射脂肪呈原团状，避免散在注射后与裸鼠自身脂肪组织混淆，便于术后测量。
术后1月取移植脂肪称重，切取裸鼠的注射部位皮肤，固定于甲醛液，石蜡包埋，制成组织切片，苏木素-伊红(HE)染色和Masson染色观察。同时作免疫组织化学染色以测定皮肤中Ⅲ型胶原及转化生长因子TGF-β1的含量。
3、标本切片的取材
3.1标本固定
在固定液的选取上，通常使用中性10％福尔马林作为常规固定液。
3.2流水冲洗
经过福尔马林固定后，应充分用流动自来水冲洗，时间约为6～8小时。
3.3脱水
逐级从70％酒精—80％酒精—95％酒精—95％酒精—100％酒精，共历时10小时。
3.4透明
将材料放于二甲苯与无水乙醇比例为1：1的透明剂中，然后再放于二甲苯中。时间共计是2h左右即可。
3.5浸蜡
将透明后的材料放人溶解状态的普通石蜡中，使石蜡浸入组织中。
3.6包埋
先将熔化的石蜡注入包埋盒内，然后用镊子将组织块从60℃石蜡中取出立即放入包埋模具里，使切面朝下进行直立包埋，用镊子将组织固定在石蜡中央稍停片刻，使组织在蜡凝时立住，达到立埋的要求，待其慢慢冷却凝固。
3.7切片
切片时为保持蜡块硬度，可把蜡块放于冰柜中。切时取出，组织蜡块四周在切片前应修齐，大小适当。切片时左手执毛笔，右手旋转切片机转轮。切片的厚度以5um-10um为宜，倾斜角，一般以20°—30°为宜。
3.8贴片、烘片
将腊片正面朝上轻轻平铺在展片箱40～45℃的水面上，借水的张力和温度将略皱的蜡带自然展平。待切片在恒温水面上充分展平后，将蜡片捞到载玻片的中段处倾去载玻片上的余水。切片捞出后可待切片几乎完全干后再进行烤片。一般在烤片仪上烤片一小时或 置入60～65℃恒温箱内，脱去切片中部分石蜡。
4、HE染色
苏木精—伊红染色法(Hematoxylin-eosin staining)，简称HE染色法，石蜡切片技术里常用的染色法之一。苏木精染液为碱性，主要使细胞核内的染色质与胞质内的核糖体着紫蓝色；伊红为酸性染料，主要使细胞质和细胞外基质中的成分着红色。
试剂配置
A：0.5～1％的伊红酒精溶液。称取伊红Y 0.5～1g，加少量蒸馏水溶解后，再滴加冰醋酸直至浆糊状。以滤纸过滤，将滤渣在烘箱中烤干后，以95％酒精100毫升溶解。
B：苏木素染液配方：(配制3000ml，可按比例减少)苏木精6g；无水乙醇100ml；硫酸铝钾150g；蒸馏水2000ml；碘酸钠1.2g；冰醋酸120ml；甘油900ml。
配制方法：将苏木素溶于无水乙醇，再将硫酸铝钾溶于蒸馏水，溶解后将甘油倾入一起混合，最后加入冰醋酸和碘酸钠。
C：1％盐酸酒精分化液：将1毫升浓盐酸加入99毫升70％酒精中即可。
染色步骤：
(1)二甲苯(Ⅰ)15min；(2)二甲苯(Ⅱ)15min；(3)100％乙醇(Ⅰ)5min；(4)100％乙醇(Ⅱ)5min；(5)80％乙醇5min；(6)蒸馏水5min；(7)苏木精液染色5min；(8)流水稍洗去苏木精液1-3s；(9)1％盐酸乙醇1-3s；(10)稍水洗10-30s；(11)促蓝液返蓝10-30s；(12)流水冲洗10-15min；(13)蒸馏水过洗1-2s；(14)0.5％伊红液染色1-3min；(15)蒸馏水稍洗1-2s；(16)80％乙醇稍洗1-2s；(17)95％乙醇(Ⅰ)2-3s；(18)95％乙醇(Ⅱ)3-5s；(19)无水乙醇5-10min；(20)石炭酸二甲苯5-10min；(21)二甲苯(Ⅰ)2min；(22)二甲苯(Ⅱ)2min；(23)二甲苯(Ⅲ)2min；(24)中性树胶封固。
5、Masson染色
Masson染色将胶原纤维呈绿色，弹性纤维呈棕色至深棕色，肌纤维、纤维素红色，这种染色方法比HE染色法能更好的显示组织内肌肉、胶原及弹性纤维的分布，更形象的展现了组织结构特点。
试剂配制：
(1)苏木素染液(见HE染色法)；
(2)Masson复合染色液：丽春红(ponceau 2R)0.7g，酸性品红(acid fuchsin)0.3g， 蒸馏水99ml，冰醋酸(glacial acetic acid)1ml；
(3)亮绿染色液：亮绿(light green)1g，冰醋酸(glacial acetic acid)1ml，蒸馏水99ml；
(4)1％磷钼酸水溶液：磷钼酸(phosphomolybdic acid)1g，蒸馏水加至100ml。
染色步骤：
(1)二甲苯(Ⅰ)15min；(2)二甲苯(Ⅱ)15min；(3)100％乙醇(Ⅰ)5min；(4)100％乙醇(Ⅱ)5min；(5)80％乙醇5min；(6)蒸馏水5min；(7)苏木素染液5～10min；(8)流水稍洗，1％盐酸分化；(9)流水冲洗数分钟；(10)Masson复合染色液5～10min；(11)蒸馏水稍冲洗；(12)1％磷钨酸液处理约5min；(13)不用水洗，直接用亮绿染色液(或苯胺蓝液)复染5min；(14)1％冰醋酸水处理1min；(15)80％乙醇稍洗1-2s；(16)95％乙醇(Ⅰ)2-3s；(17)95％乙醇(Ⅱ)3-5s；(18)无水乙醇5-10min；(19)石炭酸二甲苯5-10min；(20)二甲苯(Ⅰ)2min；(21)二甲苯(Ⅱ)2min；(22)二甲苯(Ⅲ)2min；(23)中性树胶封固。
6、TGF-β1及III型胶原免疫组化染色
(1)烤片：
选取组织完整的切片，70℃烤片机加热30min；
(2)脱蜡、水化：
二甲苯15min→二甲苯15min→无水乙醇3min→95％乙醇3min→85％乙醇3min→75％乙醇3min→纯水3min→3％H2O2，10min(用于阻断内源性过氧化物酶)→纯水2min；
(3)抗原修复：
加入足量抗原修复液(1×CB)加热，95℃，15min；
冷却至室温(20℃左右)；
(4)封闭：
滴加山羊血清覆盖组织→37℃温箱孵育30min；
(5)加I抗(按I抗说明书比例稀释抗体)
4℃过夜(12-16h)→恢复至室温；
(5)清洗：
1×PBS清洗三次，每次3min；
(6)加II抗：
滴加二抗试剂1→37℃孵育20min→1×PBS清洗三次，每次3min；
滴加二抗试剂2→37℃孵育20min→1×PBS清洗三次，每次3min；
(7)DAB(3-3二氨基苯联胺)显色：
稀释至1×DAB液滴加组织10s，显微镜下观察显色程度；
(8)蒸馏水冲洗：
冲洗三次，每次1min；
(9)复染：
苏木素复染5min→自来水冲洗；
(10)1％盐酸酒精溶液分化：
1％盐酸酒精溶液分化组织，10s；
(11)自来水浸泡反蓝，15min
(12)脱水+透明处理：
蒸馏水3min→75％乙醇3min→85％乙醇3min→95％乙醇3min→无水乙醇3min→二甲苯，8min→二甲苯，8min；
(13)中性树胶封片处理；
(14)镜检。
7、结果
7.1博莱霉素硬皮博裸鼠造模
太体上观察，每日博莱霉素皮下注射可诱导皮肤硬化，注射3天到1周出现急性皮肤反应及结痂，1月后注射部位皮肤出现皮肤增厚，变硬、弹性差，而PBS对照组小鼠背部皮肤未见明显以上变化。
组织学上(Masson染色)造模组显示真皮厚，胶原纤维增粗、增多、排列紊乱，真皮下层血管增粗或甚至血管闭塞，脂肪层变薄、消失，而正常真皮薄，胶原纤维排列整齐。III型胶原免疫组化指标，博莱霉素诱导的硬皮病模型裸鼠(2519.17±774.19)与正常裸鼠 (623.85±113.84)相比显著升高(图1，表7)。
表7造模组与对照组III型胶原免疫指数
Figure PCTCN2021090539-appb-000007
应用激光散斑对比成像技术(激光散斑对比成像系统，Perimed)测定正常裸鼠及硬皮博裸鼠血流，结果显示：正常裸鼠注射部位血流为251.72±43.82，硬皮病裸鼠注射部位血流为241.89±22.76，结果无显著性差异。
7.2脂肪移植与CAL的脂肪存活率比较
正常裸鼠脂肪移植组脂肪重量102.97±17.87mg，脂肪存活率为36.8％；CAL组脂肪重量135.05±27.28mg，脂肪存活率为48.2％；硬皮病裸鼠移植脂肪重量77.54±12.40mg，脂肪存活率为27.7％；CAL组脂肪重量102.48±23.16mg，脂肪存活率为36.6％。无论正常裸鼠还是硬皮病裸鼠，CAL组存活率均较单纯的脂肪移植组存活率高(表8，图2)。
表8脂肪移植及CAL脂肪存活率比较
Figure PCTCN2021090539-appb-000008
HE染色结果显示，脂肪组织由A脂肪细胞，血管内皮细胞，成纤维细胞和结缔组织，以及巨噬细胞和淋巴细胞等构成。脂肪组织内充满体积大的脂肪细胞，并有稀少的结缔组织和丰富的毛细血管网。硬皮病组较正常组纤维化程度重，正常裸鼠CAL组脂肪细胞数量最多，境界清楚、轮廓清晰，脂肪细胞周围可见细小的纤维组织包绕，纤维化程度轻；而硬皮病脂肪移植组的脂肪细胞数量最少，多个脂肪细胞相互融合成空洞的大疱，周围纤维组织粗大，纤维化程度最重。纤维组织之间可见小的脂肪细胞，可能为新生的脂肪干细胞转化而来(图3)。
7.3脂肪移植或CAL后皮肤改变
Masson染色显示：正常裸鼠对照组、脂肪移植组、CAL组三者之间差异不明显，纤维化程度轻，胶原纤维呈蓝色，纤维组织排列整形。硬皮病造模裸鼠较正常裸鼠纤维化程度重，其中硬皮病造模裸鼠对照组真皮最厚，胶原纤维增粗、增多、排列紊乱，CAL组真皮明显较对照组明显变薄，胶原纤维含量减少，脂肪移植组介于两者之间(图4)。
对于正常裸鼠，免疫组化显示，TGF-β1含量对照组为939.34±216.92，脂肪移植组为974.98±470.18，CAL组为967.66±448.74，Ⅲ型胶原含量对照组为623.85±113.84，脂肪移植组为605.73±217.17，CAL组为599.34±89.39；对于硬皮病裸鼠，TGF-β1含量对照组为1945.95±330.77，脂肪移植组为1418.86±376.82，CAL组为1132.12±190.69，Ⅲ型胶原含量对照组2629.39±746.62，脂肪移植组为1531.74±836.41，CAL组为946.92±448.90。正常裸鼠经脂肪移植或脂肪干细胞+脂肪移植后TGF-β1含量及Ⅲ型胶原含量无明显改变(表9，图5，图6)。但对于硬皮病小鼠，脂肪移植或者CAL可降低硬皮病组小鼠的TGF-β1含量及Ⅲ型胶原含量，尤其是CAL效果更加明显。
表9正常裸鼠及硬皮病裸鼠TGF-β1含量及Ⅲ型胶原含量
Figure PCTCN2021090539-appb-000009
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。

Claims (24)

  1. 一种干细胞制剂，其特征在于，所述制剂包含脂肪来源干细胞和干细胞保存液，所述脂肪来源干细胞含量为1x10 5-5x10 6个细胞/毫升。
  2. 如权利要求1所述的干细胞制剂，其特征在于，所述脂肪来源干细胞含量为1x10 6-2x10 6。
  3. 如权利要求2所述的干细胞制剂，其特征在于，所述干细胞为经干细胞保存液重悬后的单细胞悬液状态。
  4. 如权利要求3所述的干细胞制剂，其特征在于，所述干细胞保存液为含0.5-1％人血白蛋白的TC199的保存液。
  5. 如权利要求4所述的干细胞制剂，其特征在于，所述干细胞制剂为口服剂或注射剂。
  6. 如权利要求5所述的干细胞制剂，其特征在于，所述干细胞为传代培养第3代至第6代的脂肪来源干细胞。
  7. 权利要求1～6中任一项所述的干细胞制剂的储存方法，其特征在于，所述干细胞制剂储存条件为4℃，含0.5％-1％人血白蛋白的TC199的保存液，储存于西林瓶中，保存期定为24小时；所述干细胞制剂储存条件为液氮保存，保存期定为1年。
  8. 一种如权利要求1～6中任一项所述的干细胞制剂的制备方法，包括以下步骤：
    (1)脂肪组织分离及脂肪干细胞原代培养：将脂肪组织中加入生理盐水，用吸管吹打10-20次，静置1-2min，将组织下层的液体吸出，加入胶原酶消化液，消化30-60min，吸出下层液体，过滤、离心，弃上清，将细胞沉淀以培养基重悬后离心弃上清液，以2-4×10 6个细胞/mL的密度在培养体系内培养，将培养瓶放入培养箱中；
    (2)脂肪组织分离及脂肪干细胞原代培养：细胞生长至70％-80％汇合时取出，加入胰酶和EDTA，当细胞开始回缩变圆后，加入等体积培养基终止消化，吹打瓶壁上的细胞，使细胞脱离瓶壁后呈单细胞悬液，将单细胞悬液移至离心管中，离心，弃上清，加入完全培养基，轻吹使之松散，按1:3传代培养；收集第3代至第6代脂肪来源干细胞；
    (3)最后，细胞用干细胞保存液重悬脂肪来源干细胞得到干细胞悬液。
  9. 如权利要求8所述的方法，其特征在于，所述干细胞悬液中包括脂肪来源干细胞浓度为1-2x10 6个细胞/毫升。
  10. 如权利要求9所述的方法，其特征在于，所述步骤(2)中加入0.125％的胰酶 和0.01％的EDTA。
  11. 如权利要求10所述的方法，其特征在于，还包括在所述干细胞悬液中加入药学上可接受的辅助成分。
  12. 权利要求1～6中任一项所述的干细胞制剂在制备用于恶性肿瘤、美容整形的药物中的用途。
  13. 权利要求1～6中任一项所述的干细胞制剂在制备辅助脂肪移植治疗硬皮病的产品中的用途。
  14. 如权利要求13所述的应用，其特征在于，所述硬皮病包括系统硬皮病和局限性硬皮病。
  15. 如权利要求14所述的应用，其特征在于，所述系统性硬皮病包括弥漫性皮肤系统硬化症、局限性皮肤系统硬化症、无皮肤表现的系统性硬化症、重叠综合征和未分化结缔组织病。
  16. 如权利要求15所述的应用，其特征在于，所述系统性硬皮病为局限性皮肤系统硬化症。
  17. 如权利要求14所述的应用，其特征在于，所述局限性硬皮病包括点滴型硬皮病、泛发型硬皮病、线状硬皮病、斑块型硬斑病和深部硬皮病。
  18. 如权利要求17所述的应用，其特征在于，所述局限性硬皮病为线状硬皮病。
  19. 如权利要求14-18中任一项所述的用途，其特征在于，所述脂肪来源干细胞辅助脂肪移植不仅可提高脂肪存活率，同时还可减轻硬皮病患者的皮肤胶原沉积。
  20. 如权利要求19所述的用途，其特征在于，所述脂肪来源干细胞降低硬皮病患者的TGF-β1含量及Ⅲ型胶原含量。
  21. 如权利要求20所述的用途，其特征在于，每毫升所述产品包含1×10 6-2×10 6个脂肪来源干细胞。
  22. 如权利要求21所述的用途，其特征在于，所述产品为干细胞制剂或药物。
  23. 如权利要求22所述的干细胞制剂，其特征在于，所述的干细胞制剂或药物为皮下注射剂。
  24. 如权利要求23所述的应用，其特征在于，所述药物还包括其他治疗硬皮病的药物。
PCT/CN2021/090539 2020-04-30 2021-04-28 一种脂肪来源干细胞制剂、制备方法及其应用 WO2021219003A1 (zh)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN202010365622.XA CN113577106A (zh) 2020-04-30 2020-04-30 一种脂肪来源干细胞制剂及其制备方法
CN202010365622.X 2020-04-30
CN202010385153.8 2020-05-09
CN202010385153.8A CN113694084A (zh) 2020-05-09 2020-05-09 脂肪来源干细胞在局限性硬皮病中的用途

Publications (1)

Publication Number Publication Date
WO2021219003A1 true WO2021219003A1 (zh) 2021-11-04

Family

ID=78331798

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2021/090539 WO2021219003A1 (zh) 2020-04-30 2021-04-28 一种脂肪来源干细胞制剂、制备方法及其应用

Country Status (1)

Country Link
WO (1) WO2021219003A1 (zh)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115125196A (zh) * 2022-08-24 2022-09-30 蚌埠医学院第一附属医院（蚌埠医学院附属肿瘤医院） 一种脂肪干细胞成脂诱导分化方法
CN115624571A (zh) * 2022-12-21 2023-01-20 北京瑷格干细胞科技有限公司 人脂肪间充质干细胞在制备治疗硬皮病药物中的应用
CN115624570A (zh) * 2022-12-21 2023-01-20 北京瑷格干细胞科技有限公司 人脂肪间充质干细胞在制备治疗结缔组织疾病药物中的应用

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101210232A (zh) * 2006-12-28 2008-07-02 天津昂赛细胞基因工程有限公司 一种间充质干细胞保存液及其用途
CN104498433A (zh) * 2014-11-28 2015-04-08 广州赛莱拉干细胞科技股份有限公司 一种脂肪干细胞的提取方法以及制剂和应用
CN104771414A (zh) * 2015-01-30 2015-07-15 广州赛莱拉干细胞科技股份有限公司 一种脂肪干细胞制剂及其制备方法
CN105132370A (zh) * 2015-09-28 2015-12-09 丛秀丽 一种临床级别脂肪干细胞的制备和储存方法
CN105168251A (zh) * 2015-09-09 2015-12-23 广州赛莱拉干细胞科技股份有限公司 一种干细胞制剂及其制备方法与应用
CN106047805A (zh) * 2016-08-22 2016-10-26 湖州赛托森生物科技发展有限公司 一种脂肪干细胞的提取方法
CN110846273A (zh) * 2019-11-19 2020-02-28 山东省齐鲁细胞治疗工程技术有限公司 一种脂肪组织来源的间充质干细胞培养及三系分化诱导方法

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101210232A (zh) * 2006-12-28 2008-07-02 天津昂赛细胞基因工程有限公司 一种间充质干细胞保存液及其用途
CN104498433A (zh) * 2014-11-28 2015-04-08 广州赛莱拉干细胞科技股份有限公司 一种脂肪干细胞的提取方法以及制剂和应用
CN104771414A (zh) * 2015-01-30 2015-07-15 广州赛莱拉干细胞科技股份有限公司 一种脂肪干细胞制剂及其制备方法
CN105168251A (zh) * 2015-09-09 2015-12-23 广州赛莱拉干细胞科技股份有限公司 一种干细胞制剂及其制备方法与应用
CN105132370A (zh) * 2015-09-28 2015-12-09 丛秀丽 一种临床级别脂肪干细胞的制备和储存方法
CN106047805A (zh) * 2016-08-22 2016-10-26 湖州赛托森生物科技发展有限公司 一种脂肪干细胞的提取方法
CN110846273A (zh) * 2019-11-19 2020-02-28 山东省齐鲁细胞治疗工程技术有限公司 一种脂肪组织来源的间充质干细胞培养及三系分化诱导方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
CHEN, BO: "Cell-Assisted Lipotransfer for Localized Scleroderma:Supportive Use of Adipose Derived Stem Cells", CHINESE DOCTORAL DISSERTATIONS FULL-TEXT DATABASE, MEDICINE & PUBLIC HEALTH, 1 May 2017 (2017-05-01), pages 1 - 88, XP055862072 *
LI YICHANG: "Research progress on the relationship between adipose-derived stem cells and tumors", RESEARCH PROGRESS ON THE RELATIONSHIP BETWEEN ADIPOSE-DERIVED STEM CELLS AND TUMORS, vol. 30, no. 4, 15 April 2019 (2019-04-15), pages 224 - 226+258, XP055862061, ISSN: 1673-7040, DOI: 10.3969/j.issn.1673-7040.2019.04.009 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN115125196A (zh) * 2022-08-24 2022-09-30 蚌埠医学院第一附属医院（蚌埠医学院附属肿瘤医院） 一种脂肪干细胞成脂诱导分化方法
CN115624571A (zh) * 2022-12-21 2023-01-20 北京瑷格干细胞科技有限公司 人脂肪间充质干细胞在制备治疗硬皮病药物中的应用
CN115624570A (zh) * 2022-12-21 2023-01-20 北京瑷格干细胞科技有限公司 人脂肪间充质干细胞在制备治疗结缔组织疾病药物中的应用

Similar Documents

Publication Publication Date Title
WO2021219003A1 (zh) 一种脂肪来源干细胞制剂、制备方法及其应用
Kern et al. Isolation and culture of microvascular endothelium from human adipose tissue.
Du et al. Immuno-inflammatory regulation effect of mesenchymal stem cell transplantation in a rat model of myocardial infarction
CN106434557B (zh) 由脐带间充质干细胞制备cd34阳性细胞的方法
TWI626939B (zh) 細胞包裝產品及其之製備及使用方法
Chen et al. Effect of suction pressures on cell yield and functionality of the adipose-derived stromal vascular fraction
CN109481466A (zh) 使用胎盘间充质干细胞治疗卵巢早衰的方法和细胞制剂
CN106265740B (zh) 脐带间充质干细胞联合黄芪多糖在制备治疗高血糖和糖尿病肾病药物中的应用
CN109893541B (zh) 经血干细胞来源的外泌体在制备治疗宫腔粘连的药物中的应用
CN109652366A (zh) 用于治疗卵巢早衰的胎盘间充质干细胞制剂
CN108642002A (zh) 一种无血清驯化培养人间充质干细胞的方法
Taha et al. Adipose-derived stem/progenitor cells from lipoaspirates: A comparison between the Lipivage200-5 liposuction system and the Body-Jet liposuction system
WO2022063027A1 (zh) 一种药物组合物及其治疗剂和应用
Luo et al. Manufacturing and banking canine adipose-derived mesenchymal stem cells for veterinary clinical application
CN112029717B (zh) 无血清体外驯化培养人间充质干细胞
TR201911506A2 (tr) Trombosi̇tten zengi̇n fi̇bri̇n kaynakli mezenki̇mal kök hücreler
CN108066750A (zh) 干细胞及其分泌物用于治疗皮肤烧烫伤的新用途
CN113694084A (zh) 脂肪来源干细胞在局限性硬皮病中的用途
CN112972495A (zh) 一种面部萎缩干细胞治疗剂及其应用
CN115786247B (zh) 一种无血清培养基及其在维持毛囊活性、毛发养护及移植方面的应用
CN112342191A (zh) hMSC生产试剂盒开发和质量管理标准优化和体系建立
BR112020004517A2 (pt) células tronco derivadas de porco neonato e processo para sua preparação
Chandel et al. Isolation and culture of human umbilical vein endothelial cells (HUVECs)
Hsu et al. Co-culture of human mesenchymal stromal cells and primary mouse hepatocytes
CN113577106A (zh) 一种脂肪来源干细胞制剂及其制备方法

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 21797056

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 21797056

Country of ref document: EP

Kind code of ref document: A1