WO2019161590A1 - Suspension de cellules souches mésenchymateuses, son procédé de préparation et application correspondante - Google Patents

Suspension de cellules souches mésenchymateuses, son procédé de préparation et application correspondante Download PDF

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WO2019161590A1
WO2019161590A1 PCT/CN2018/080586 CN2018080586W WO2019161590A1 WO 2019161590 A1 WO2019161590 A1 WO 2019161590A1 CN 2018080586 W CN2018080586 W CN 2018080586W WO 2019161590 A1 WO2019161590 A1 WO 2019161590A1
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mesenchymal stem
stem cell
cells
culture
primary
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PCT/CN2018/080586
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Chinese (zh)
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杨涛
隋昳
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深圳至博生物科技有限公司
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    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0665—Blood-borne mesenchymal stem cells, e.g. from umbilical cord blood
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0668—Mesenchymal stem cells from other natural sources
    • 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
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • the present application relates to the field of biotechnology, and in particular, to a mesenchymal stem cell suspension and a preparation method and application thereof.
  • T2DM global type 2 diabetes
  • the existing diabetes treatment strategies are mainly therapeutic improvement of life-based and drug-based individualized treatment.
  • the main goal of drug therapy is to comprehensively and effectively control various metabolic abnormalities at an early stage, and to protect islet cells by drugs that regulate blood lipids, lower blood pressure, improve insulin resistance, and increase insulin sensitivity.
  • These treatment methods require strong compliance of the patient to maintain the therapeutic effect, and are prone to drug dependence and adverse reactions. Once the drug is discontinued, the condition may be repeated or even worse. Therefore, the traditional treatment takes a long time, and many patients have poor self-discipline, it is difficult to adhere to medication or regular medication, resulting in poor treatment results, eventually a series of complications, increasing the difficulty of treatment.
  • MSCs Mesenchymal stem cells
  • chemokines chemokines
  • IL-12 hematopoietic support and promotion of stem cell implantation, immune regulation and self-replication.
  • Adult MSCs are mainly found in bone marrow, fat, subperiosteum, and blood vessels in various organs and tissues, and have various mesoderms including osteoblasts, adipocytes, chondrocytes, stromal cells, fibroblasts, tendon cells, and the like.
  • the potential of cell lines In the normal process of injury repair, MSCs can be recruited to the injury site under the induction of chemokines, local proliferation, differentiation, and participate in injury repair and tissue regeneration through paracrine.
  • no further studies have been conducted on the treatment of diabetes.
  • the purpose of the present application is to provide a mesenchymal stem cell suspension and a preparation method and application thereof, and the mesenchymal stem cell suspension is particularly suitable for the treatment of diabetes.
  • a method for preparing a mesenchymal stem cell suspension comprising:
  • mesenchymal stem cells primary cells After 5-10 days of primary cell culture of mesenchymal stem cells, replace them with serum-free DMEM nutrient solution, and add 65 to 75% of the cells to the cells, remove the culture supernatant, and then add the first A digestive juice, digested for 0.5-2 min, the cells are shrunk, added to the removed culture supernatant and neutralized, centrifuged, re-suspended in complete medium, inoculated according to 1 to 1.5, and cultured for 4 to 5 days, When the cells are 65 to 75% confluent, they are subcultured at a ratio of 1:6 to 8;
  • Preparation of Mesenchymal Stem Cell Suspension After culture and expansion of primary cells of mesenchymal stem cells, P4 cells are cultured for 48-96 hours, and after cell fusion is less than 80%, the cells are digested with a third digestive juice. After washing, it was suspended with 5% human albumin, and then physiological saline was added to obtain a desired mesenchymal stem cell suspension.
  • the third digestive juice is 0.25% trypsin-EDTA.
  • the centrifugation is performed by centrifugation at 800 to 1200 rpm for 6 to 10 minutes.
  • the mesenchymal stem cell primary cell is a bone marrow mesenchymal stem cell primary cell or an autologous adipose-derived mesenchymal stem cell primary cell or a umbilical cord mesenchymal stem cell primary cell.
  • the mesenchymal stem cell primary cell is a bone marrow mesenchymal stem cell primary cell
  • the obtained mesenchymal stem cell primary cell comprises:
  • the bone marrow solution was taken and diluted: the bone marrow solution was extracted with a syringe containing 500 U/ml heparin in PBS, mixed uniformly, and diluted with PBS containing 20 U/ml heparin to obtain a bone marrow buffer solution;
  • the bone marrow buffer solution was added to a lymphocyte separation liquid having a specific gravity of 1.073 g/ml, and the mononuclear cell population was purified by centrifugation, and then centrifuged to wash the DMEM medium to obtain a precipitate;
  • Cell expansion culture The precipitate obtained by centrifugation is suspended by serum-free culture medium, inoculated at a density of 5 ⁇ 10 6 /mL, and cultured under the conditions of 35-40 ° C, 5% CO 2 and saturated humidity; After 3 days of culture, the medium was changed for the first time, and the unattached cells were discarded. The culture medium was replaced once every 3 days, and the primary cells of the bone marrow mesenchymal stem cells were obtained after 6 days of co-culture.
  • the mesenchymal stem cell primary cell is an autologous adipose-derived mesenchymal stem cell primary cell, and the obtained mesenchymal stem cell primary cell comprises:
  • Autologous fat particles were extracted and cut: autologous fat particles were taken, visible microvessels and muscle tissue were removed, washed with PBS, and then the adipose tissue was cut into a paste to make it less than 1 mm 3 ;
  • Isolation and purification of cells adding a second digestive juice to the paste-like adipose tissue for digestion, digesting, collecting and collecting the filtrate by filtration, and then neutralizing with a low-sugar DMEM medium containing 10% FBS, and then centrifuging to obtain a precipitate;
  • Cell expansion culture serum-free medium is added to the precipitate obtained after separation and purification of the cells, inoculated at a density of 1 ⁇ 10 6 /mL, and at 35 to 40 ° C, 5% CO 2 and saturated humidity. The culture was carried out under the condition; after the culture for 3 days, the liquid was changed for the first time, the unattached cells were discarded, and the culture solution was changed once every 3 days, and the primary cells of the autologous adipose-derived mesenchymal stem cells were obtained after 6 days of co-culture.
  • the second digestive juice is serum-free DMEM containing 0.1% collagenase type I and 0.05% trypsin.
  • the mesenchymal stem cell primary cell is a umbilical cord mesenchymal stem cell primary cell
  • the obtained mesenchymal stem cell primary cell comprises:
  • Treatment of umbilical cord Take the umbilical cord and cut it.
  • the total length of the umbilical cord after cutting is more than 15cm.
  • Cell expansion culture The shredded Walton gum is applied to the culture dish, added to serum-free complete medium, cultured at 35-40 ° C, 5% CO 2 and saturated humidity, and cultured for 7 to 10 days.
  • the primary cells of umbilical cord mesenchymal stem cells can be obtained.
  • a mesenchymal stem cell suspension prepared by the preparation method of the above mesenchymal stem cell suspension prepared by the preparation method of the above mesenchymal stem cell suspension.
  • the present application provides a mesenchymal stem cell suspension and a preparation method thereof, and the prepared mesenchymal stem cell suspension is particularly suitable for the treatment of diabetes.
  • the present application also provides an application of a mesenchymal stem cell suspension, wherein the above-mentioned mesenchymal stem cell suspension is used for the treatment of diabetes, which can effectively alleviate the hyperglycemia state of diabetic patients, and provides insulin sensitivity significantly, and has Teach good security.
  • FIG. 1 is a schematic flow chart of a method for preparing an intermediate mesenchymal stem cell suspension according to an embodiment of the present application.
  • FIG. 2 is a schematic flow chart of obtaining mesenchymal stem cell primary cells in the first embodiment of the first embodiment of the present application.
  • FIG. 3 is a schematic flow chart of obtaining mesenchymal stem cell primary cells in a second embodiment of the first embodiment of the present application.
  • FIG. 4 is a schematic flow chart of obtaining mesenchymal stem cell primary cells in a third embodiment of the first embodiment of the present application.
  • a method for preparing a mesenchymal stem cell suspension comprising:
  • Step S1 obtaining primary cells of mesenchymal stem cells
  • Step S2 Culture and expansion of primary cells of mesenchymal stem cells: After primary culture of mesenchymal stem cells for 5-10 days, the cells are replaced with serum-free DMEM nutrient solution, and 65 to 75% of the cells are fused, and the culture supernatant is removed. Then, the first digestive juice is added, and the cells are digested for 0.5-2 min. After the cells are shrunk, the removed culture supernatant is added to neutralize, and then centrifuged, re-suspended in complete medium, inoculated according to 1 to 1.5, and cultured 4 to After 5 days, the cells were subcultured at a ratio of 1:6-8 when 65 to 75% of the cells were fused;
  • Step S3 Preparation of Mesenchymal Stem Cell Suspension: After culture and expansion of primary cells of mesenchymal stem cells, P4 cells are cultured and expanded for 48-96 hours, and after cell fusion does not exceed 80%, the third digestive solution is used. The cells were digested and washed, suspended with 5% human albumin, and then added with physiological saline to obtain a desired mesenchymal stem cell suspension.
  • the method for preparing a mesenchymal stem cell suspension provided by the present application is suitable for the treatment by determining the culture expansion of the mesenchymal stem cell primary cells and the specific operation in the preparation of the mesenchymal stem cell suspension.
  • the mesenchymal stem cell primary cell may be a bone marrow mesenchymal stem cell primary cell or an autologous adipose-derived mesenchymal stem cell primary cell or a umbilical cord mesenchymal stem cell primary cell. Therefore, there are three embodiments for obtaining mesenchymal stem cell primary cells. All three implementation methods need to evaluate and screen the donor's health, strictly exclude HBV, syphilis-positive, high-risk groups susceptible to HIV (such as drug abusers, homosexuals, multiple sexual partners), various Patients with tuberculosis (such as tuberculosis, kidney tuberculosis, lymphatic tuberculosis, and bone tuberculosis).
  • the obtaining the mesenchymal stem cell primary cell comprises:
  • Step S11 extracting the bone marrow solution and diluting: the bone marrow solution is extracted with a syringe containing 500 U/ml heparin in PBS, mixed uniformly, and diluted with PBS containing 20 U/ml heparin to obtain a bone marrow buffer solution;
  • Step S12 isolation and purification of cells: the bone marrow buffer solution is added to a lymphocyte separation liquid having a specific gravity of 1.073 g/ml, and the mononuclear cell population is purified by centrifugation, and then washed by DMEM culture solution to obtain a precipitate;
  • Step S13 Cell expansion culture: The precipitate obtained by centrifugation is suspended by a serum-free culture solution, and inoculated at a density of 5 ⁇ 10 6 /mL and at 35 to 40 ° C, 5% CO 2 and saturated humidity. The culture was carried out for the first time after the culture for 3 days, and the unadhered cells were discarded, and the culture medium was replaced once every 3 days, and the primary cells of the bone marrow mesenchymal stem cells were obtained after 6 days of co-culture.
  • the bone marrow fluid is extracted specifically: the prone position is taken, the upper spine is taken as the puncture point, the iodophor is routinely disinfected, and the 2% lidocaine is locally anesthetized. Puncture the upper spine with a bone through the needle, and pull out the needle core after a breakthrough.
  • the bone marrow solution was extracted with a 20 ml sterile syringe containing 2 ml of PBS containing 500 U/ml heparin, shaken, and poured into a 50 ml sterile centrifuge tube to extract about 50 ml. Immediately after the bone marrow fluid is taken, it can be sent to the laboratory for dilution.
  • the dilution of the bone marrow fluid is preferably: diluting the bone marrow solution with an equal amount of PBS containing 20 U/ml heparin to obtain a bone marrow buffer.
  • step S12 it is preferred that the bone marrow buffer is slowly added to the lymphocyte separation solution along the wall of the tube. More preferably, the bone marrow buffer and the lymphocyte separation solution are in equal volume.
  • Purification of the mononuclear cell population by centrifugation is preferably carried out by centrifugation at 2000 rpm for 30 minutes to purify the mononuclear cell population.
  • the precipitate was preferably washed by centrifugation with DMEM medium, and washed twice with DMEM (L) medium for 1000 rpm, centrifuged for 5 minutes, and the supernatant was discarded to obtain a precipitate.
  • step S13 it is preferred to cultivate at 37 ° C, 5% CO 2 and saturated humidity after inoculation.
  • the specific embodiment may be: taking a prone position, taking the upper spine as a puncture point, iodophor conventional disinfection, and 2% lidocaine local anesthesia. Puncture the upper spine with a bone through the needle, and pull out the needle core after a breakthrough.
  • the bone marrow solution was extracted with a 20 ml sterile syringe containing 2 ml of PBS containing 500 U/ml heparin, shaken, and poured into a 50 ml sterile centrifuge tube to extract about 50 ml.
  • the collected bone marrow fluid is stored in a refrigerator at 4 ° C for a short period of time (no more than 24 hours) or immediately sent to the laboratory.
  • the bone marrow was diluted with an equal volume of 20 U/ml heparin in PBS, and a lymphocyte separation solution having an equal volume specific gravity of 1.073 g/ml was slowly added dropwise along the wall of the tube.
  • the mononuclear cell population was purified by centrifugation for 30 minutes at 2000 rpm.
  • the cells were washed twice with DMEM (L), centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded to take a precipitate.
  • the serum-free medium was suspended, inoculated into a culture flask at 5 ⁇ 10 6 /mL, and cultured at 37 ° C under 5% CO 2 saturated humidity. After 3 days, the medium was changed for the first time, and the unattached cells were discarded, and the culture solution was changed once every 3 days. A total of 6d, for transplantation, passaging or cryopreservation.
  • the obtaining mesenchymal stem cell primary cell includes:
  • Step S11' extracting autologous fat particles and cutting: extracting autologous fat particles, removing visible microvessels and muscle tissue, washing with PBS, and then cutting the adipose tissue into a paste to make it less than 1 mm 3 ;
  • Step S12' separation and purification of cells: adding a second digestive juice to the paste-like adipose tissue for digestion, digesting, collecting and collecting the filtrate by filtration, and then neutralizing with a low-sugar DMEM medium containing 10% FBS, and then centrifuging to obtain a precipitate. ;and
  • Step S13' Cell expansion culture: a serum-free medium is added to the precipitate obtained after separation and purification of the cells, and inoculated at a density of 1 ⁇ 10 6 /mL, and at 35 to 40 ° C, 5% CO 2 The culture was carried out under the condition of saturated humidity; after the culture for 3 days, the cells were first changed, the unattached cells were discarded, and the culture solution was replaced once every 3 days, and the primary cells of autologous adipose-derived mesenchymal stem cells were obtained after 6 days of co-culture.
  • step S11' it is preferred to wash three times with PBS.
  • the second digestive juice is preferably serum-free DMEM containing 0.1% collagenase type I and 0.05% trypsin. More preferably, the digestion is carried out in a water bath at 37 ° C and stirred for a period of 40 to 45 minutes. The post-digestion filtration is preferably carried out by filtration using a 200 mesh filter. Preferably, the low sugar DMEM medium is used in an amount equal to the filtrate; the centrifugation after neutralization using the low sugar DMEM medium is preferably carried out by centrifugation at 1500 rpm for 10 minutes.
  • step S13' it is preferred to cultivate at 37 ° C, 5% CO 2 and saturated humidity after inoculation.
  • a specific embodiment may be: 15 to 20 mL of autologous fat particles are taken from the lower abdomen of the patient, visible microvessels and muscle tissue are removed, PBS is washed 3 times, and then the adipose tissue is cut into a paste with scissors, and the requirement is ⁇ 1mm 3 . Transfer the paste adipose tissue to the Erlenmeyer flask, add the digestive solution (serum-free DMEM containing 0.1% collagenase type I and 0.05% trypsin), stir at 40 ° C for 40 to 45 minutes in a 37 ° C water bath until the tissue block is digested. .
  • the filtrate was collected by filtration through a 200-mesh sieve, and then neutralized in an equal amount using a low-sugar DMEM medium containing 10% FBS, and centrifuged at 1500 rpm for 10 min, and the supernatant was discarded.
  • Fresh serum-free medium was added to the precipitate, and the cells were cultured at a cell density of 1 ⁇ 10 6 /mL at 37 ° C under 5% CO 2 . After 3 days, the medium was changed for the first time, the unattached cells were discarded, and the culture solution was changed once every 3 days. A total of 6d, for transplantation, passaging or cryopreservation.
  • the obtaining mesenchymal stem cell primary cell includes:
  • Step S11′′ Processing the umbilical cord: taking the umbilical cord and cutting it.
  • the total length of the umbilical cord after cutting is more than 15 cm.
  • the blood stain is removed by using 2 times double-anti-normal saline, then the blood vessel and the outer membrane are removed, and the Walton is removed. Glue and cut into 0.5 ⁇ 1mm 3 ;
  • Step S12′′ cell expansion culture: the shredded Walton gum is applied to the culture dish, added to the serum-free complete medium, cultured at 35-40 ° C, 5% CO 2 and saturated humidity, and cultured.
  • Primary cells of umbilical cord mesenchymal stem cells can be obtained in 7 to 10 days.
  • step S11 after washing with physiological saline, it can be immediately sent to the laboratory for dilution, and if it needs to be temporarily stored, it can be temporarily stored in a refrigerator at 4 ° C.
  • step S12 serum-free complete medium is added. Thereafter, it is preferably cultured under the conditions of 37 ° C, 5% CO 2 and saturated humidity.
  • the specific embodiment may be: within 1 minute after the placenta is delivered, the umbilical cord is cut after ligation near the placenta, and the total length of the collected umbilical cord is not less than 15 cm.
  • the umbilical cord was transferred to a sterile Petri dish by sterile forceps, and the blood stain was substantially removed with 2 times of double-resistant physiological saline. Remove the blood vessels and the outer membrane, peel off the Walton gum, and cut into 0.5 to 1 mm 3 .
  • the above-mentioned shredded Walton gum was evenly spread on a 10 cm culture dish, attached at room temperature for 5 to 10 minutes, and then added with 5 ml of complete medium (without serum).
  • the CO 2 saturated humidity incubator at 37 ° C and a volume fraction of 5% was cultured for 7 to 10 days.
  • the first digestive juice is preferably 0.25% trypsin-EDTA.
  • the centrifugation is performed by centrifugation at 800 to 1200 rpm for 6 to 10 minutes.
  • the primary cells are cultured for 7 days, then changed to medium (serum-free DMEM (L)), 70% of the cells are fused, and the culture supernatant is removed to a 50 ml centrifuge tube, and 0.25% trypsin is added to the culture flask.
  • the third digestive juice is preferably 0.25% trypsin-EDTA.
  • the P4 generation cell culture is expanded for 72 hours. After digesting the cells with the three digestive juices, it is preferred to repeat the washing three times with physiological saline. Generally, to the second washing, a cell sample is required for counting. After the desired mesenchymal stem cell suspension is obtained, it can be placed in a refrigerator at 4 ° C for a short period of time.
  • the cell fusion is not more than 80%, the cells are digested with 0.25% trypsin-EDTA, and washed repeatedly 3 times with physiological saline.
  • cell samples were taken for counting.
  • the cells after centrifugation washing were suspended in 10 ml of 5% human albumin (diluted physiological saline), and the cell suspension was injected into 100 ml of physiological saline with a syringe and mixed. Mark the number, preparation time (accurate to the minute), and the total number of cells, and keep the transport in the refrigerator at 4 °C for a short time.
  • the mesenchymal stem cell suspension provided in the second embodiment is used for the treatment of diabetes. After infusion, it can effectively alleviate the hyperglycemia state of diabetic patients, significantly improve insulin sensitivity, and effectively improve insulin resistance in peripheral tissues such as muscle, fat and liver. In addition, there is no serious adverse reaction, and it has better safety.

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Abstract

L'invention concerne un procédé de préparation d'une suspension de cellules souches mésenchymateuses, comprenant les étapes suivantes : l'obtention d'une culture primaire de cellules souches mésenchymateuses ; la culture et le développement de la culture primaire de cellules souches mésenchymateuses ; et la préparation de la suspension de cellules souches mésenchymateuses. L'invention concerne en outre une suspension de cellules souches mésenchymateuses préparée selon le procédé de préparation de la suspension de cellules souches mésenchymateuses, la suspension de cellules souches mésenchymateuses pouvant être utilisée pour une injection thérapeutique pour le traitement du diabète sucré.
PCT/CN2018/080586 2018-02-23 2018-03-27 Suspension de cellules souches mésenchymateuses, son procédé de préparation et application correspondante WO2019161590A1 (fr)

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CN109453199A (zh) * 2018-11-05 2019-03-12 北京世纪劲得生物技术有限公司 间充质干细胞、组合物及注射液在制备治疗糖尿病药物的应用
CN111346051A (zh) * 2020-03-19 2020-06-30 瑞太干细胞中心(沈阳)有限公司 一种用于治疗脑梗死的脐带间充质干细胞注射液制备方法
CN112111448B (zh) * 2020-08-21 2022-06-10 深圳市弘际生物科技有限责任公司 改良的间充质干细胞培养基、骨髓间充质干细胞及其培养方法和应用
CN115554478A (zh) * 2021-07-01 2023-01-03 高山艳 一种生物组织修复用自体活细胞制剂及其制备方法和应用
CN113755433A (zh) * 2021-08-09 2021-12-07 合肥滴碧云生物科技有限公司 一种滑膜间充质干细胞的悬浮液及其制备方法

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