CN113832099A - Mesenchymal stem cell preparation for preparing medicine for treating rheumatoid arthritis - Google Patents
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Abstract
本发明公开了一种间充质干细胞的培养方法，包括以下步骤：将贴壁培养的间充质干细胞加入含有细胞因子的无血清完全培养基中培养，其中，细胞因子包括IL‑1β，IL‑4，TNF‑α,bFGF,EGF,PDGF或IGF中的至少一种。本发明还公开了一种间充质干细胞制剂。本发明还公开了一种间充质干细胞制剂在制备类风湿性关节炎的药物中的应用。本发明中采用无血清加细胞因子的培养体系，间充质干细胞可以快速的增殖，并且批次间更稳定。无血清加细胞因子的体系，不仅解决了细胞增殖和稳定性的问题，同时还提高了间充质干细胞的促血管再生能力和免疫调节能力，提高细胞稳定性的同时提高了细胞的活性。本发明提供的高活性间充质干细胞制剂可应用于制备类风湿性关节炎的药物。The invention discloses a method for culturing mesenchymal stem cells, comprising the following steps: adding the adherent cultured mesenchymal stem cells into a serum-free complete medium containing cytokines for culture, wherein the cytokines include IL-1β, IL -4, at least one of TNF-α, bFGF, EGF, PDGF or IGF. The invention also discloses a mesenchymal stem cell preparation. The invention also discloses the application of the mesenchymal stem cell preparation in the preparation of a drug for rheumatoid arthritis. In the present invention, the culture system of serum-free and cytokines is adopted, and the mesenchymal stem cells can proliferate rapidly and are more stable between batches. The serum-free and cytokine-free system not only solves the problems of cell proliferation and stability, but also improves the angiogenesis-promoting and immunoregulatory abilities of mesenchymal stem cells, improving cell stability and increasing cell activity. The highly active mesenchymal stem cell preparation provided by the present invention can be applied to the preparation of drugs for rheumatoid arthritis.
Description
Technical Field
The invention relates to the technical field of biomedicine, in particular to a mesenchymal stem cell preparation for preparing a medicament for treating rheumatoid arthritis.
Background
Rheumatoid Arthritis (RA) is an autoimmune disease in which the patient's immune system falsely recognizes tissues around the joint as "non-self" to attack, causing symptoms such as joint inflammation, pain, swelling, and stiffness, seriously affecting the patient's physical health and quality of life, and reducing the patient's life expectancy. The pathological features of RA are mainly synovial lining cell proliferation, interstitial massive inflammatory cell infiltration, neovascularization of microvessels and destruction of cartilage and bone tissues, and the pathogenesis is complex, genetic, infectious, hormonal and the like.
The existing treatment means mainly comprises joint damage prevention and joint function protection, and mainly comprises drug treatment such as non-steroidal anti-inflammatory drugs, slow-acting antirheumatic drugs, immunosuppressant and the like, and other operation treatment means such as joint replacement and the like, however, the drug treatment cannot be well controlled, the radical treatment cannot be realized, the operation treatment has risks, the use time is limited, and no better treatment means exists at present.
Mesenchymal Stem Cells (MSCs) have a wide source, can be obtained from bone marrow, umbilical cord, placenta, fat and other tissues, not only have self-renewal ability and can be cultured in vitro in large quantities, but also have the three-line differentiation ability of differentiating bone, cartilage and fat, and have very low immunogenicity so as to be used by foreign bodies, and in recent years, the Mesenchymal stem cells are widely used for clinical research and new drug research and development.
At present, more than ten mesenchymal stem cell medicines are approved by clinical experiments of the State food and drug administration in China, and mainly relate to transplantation anti-host disease, diabetes, myocardial infarction, cerebral apoplexy, lower limb ischemia, pulmonary fibrosis, novel coronavirus pneumonia and the like.
Mesenchymal stem cells have two characteristics, so that the mesenchymal stem cells have theoretical basis and unique advantages in RA treatment. The mesenchymal stem cells are used as pharmacodynamic signal cells and secrete certain cytokines so as to promote the regeneration of blood vessels and improve microcirculation, such as VEGF factors; meanwhile, the mesenchymal stem cells have good immunoregulation capability, and inflammation is improved by inhibiting T cell proliferation, up-regulating Treg cell proportion, inhibiting B cell function, reducing B cell number and the like; in addition, it has paracrine function and trilineage differentiation ability, and can promote regeneration of cartilage and synovial tissue and inhibit inflammatory reaction. Therefore, the mesenchymal stem cells can well inhibit inflammation and regulate the immune system, thereby treating RA.
At present, many mesenchymal stem cells adopt a serum culture system, so that many risks are brought to clinical experiments, and some mesenchymal stem cells in the serum-free culture system are aged quickly and have low proliferation speed.
In view of the above, there is a need for a new culture system that can ensure rapid proliferation of mesenchymal stem cells and is more stable from batch to batch.
Disclosure of Invention
An object of the present invention is to solve at least the above problems and to provide at least the advantages described later.
It is still another object of the present invention to provide a method for culturing mesenchymal stem cells, wherein the mesenchymal stem cells can be rapidly proliferated and are more stable in batches using a serum-free and cytokine-free culture system.
It is still another object of the present invention to provide a mesenchymal stem cell culture medium.
It is still another object of the present invention to provide a highly active mesenchymal stem cell preparation which can be applied to the preparation of a medicament for rheumatoid arthritis.
To achieve these objects and other advantages in accordance with the present invention, there is provided a method for culturing mesenchymal stem cells, comprising the steps of:
adding the mesenchymal stem cells after 16-24 hours of adherent culture into a serum-free complete culture medium containing cytokines for culturing for 36-71 hours, wherein the cytokines comprise at least one of IL-1 beta, IL-4, TNF-alpha, bFGF, EGF, PDGF or IGF.
Preferably, the cytokine is IL-1 β, IL-4L, TNF- α, bFGF, EGF, PDGF and IGF.
Preferably, the cytokines are added in amounts of IL-1 β: 0ng/mL-20ng/mL, IL-4: 0ng/mL-20ng/mL, TNF- α: 0ng/mL-20ng/mL, 0ng/mL-50ng/mL bFGF, 0ng/mL-100ng/mL EGF, 0ng/mL-100ng/mL PDGF and 0ng/mL-50ng/mL IGF.
A mesenchymal stem cell culture medium comprising: a serum-free complete medium supplemented with a cytokine, wherein the cytokine comprises at least one of IL-1 β, IL-4, TNF- α, bFGF, EGF, PDGF or IGF.
Preferably, the cytokine is IL-1 β, IL-4L, TNF- α, bFGF, EGF, PDGF and IGF.
Preferably, the cytokines are added in amounts of IL-1 β: 0ng/mL-20ng/mL, IL-4: 0ng/mL-20ng/mL, TNF- α: 0ng/mL-20ng/mL, 0ng/mL-50ng/mL bFGF, 0ng/mL-100ng/mL EGF, 0ng/mL-100ng/mL PDGF and 0ng/mL-50ng/mL IGF.
A preparation method of a mesenchymal stem cell preparation comprises the following steps:
a) collecting mesenchymal stem cells obtained by the culture method of mesenchymal stem cells according to any one of claims 1 to 3, washing twice with DPBS, and counting;
b) the suspension cells are re-suspended by 10 to 20 percent DMSO and 5 to 10 percent human albumin plus compound electrolyte injection to prepare 5x107In one unit, 20ml-25ml is packed in a transfusion bag and frozen by a programmed cooling instrument.
A mesenchymal stem cell preparation.
An application of mesenchymal stem cell preparation in preparing medicine for treating rheumatoid arthritis is disclosed.
The invention at least comprises the following beneficial effects:
in the invention, a culture system without serum and cell factors is adopted, the mesenchymal stem cells can be rapidly proliferated, and the batch is more stable.
The invention adopts a system without serum and cell factors, not only solves the problems of cell proliferation and stability, but also improves the angiogenesis promoting capability and the immunoregulation capability of the mesenchymal stem cells, improves the cell stability and simultaneously improves the activity of the cells.
The high-activity mesenchymal stem cell preparation provided by the invention can be applied to preparation of medicines for treating rheumatoid arthritis.
Additional advantages, objects, and features of the invention will be set forth in part in the description which follows and in part will become apparent to those having ordinary skill in the art upon examination of the following or may be learned from practice of the invention.
Detailed Description
The present invention is further described in detail below with reference to examples so that those skilled in the art can practice the invention with reference to the description.
It will be understood that terms such as "having," "including," and "comprising," as used herein, do not preclude the presence or addition of one or more other elements or groups thereof.
Example 1
1. Preparation of umbilical cord mesenchymal stem cells
The specific separation method of the human umbilical cord mesenchymal stem cells used in the experiment is as follows:
a) after obtaining the authorization agreement of the guardian, taking out the umbilical cord of the newborn under the aseptic condition, soaking the umbilical cord in the DPBS buffer solution, and temporarily storing at the temperature of 4 ℃.
b) The cord was transferred to a sterile container and rinsed thoroughly 2 times with DPBS containing 1% cyan/streptomycin for 2 minutes each.
c) The umbilical cord is cut into small segments of about 2-3cm in length, the umbilical cord is dissected along the inside of the umbilical vein, and the umbilical artery, umbilical vein and umbilical cord envelope are removed using toothed forceps.
d) The umbilical cord tissue was minced and digested continuously for 30 minutes in a 37 ℃ water bath with DMEM/F12 medium containing 0.1% collagenase type II.
e) The tissue suspension was transferred to a new centrifuge tube, centrifuged at 2000rpm for 30 minutes at room temperature and the supernatant discarded, and the tissue mass was washed repeatedly with PBS buffer several times, and then after digestion was continued for 30 minutes using 0.125% pancreatin in a 37 ℃ water bath, digestion was terminated with 10% serum replacement (PALL) DMEM/F12.
f) The digested tissue suspension was filtered using a 200 mesh cell filter to remove incompletely digested tissue impurities, and then the filtrate was collected again into a new centrifuge tube and centrifuged at 2000rpm for 20 minutes at room temperature.
h) The supernatant was discarded and Nuwacell was usedTMAdding 1% streptomycin into ncMission hMSC Medium (RP02010), suspending cell precipitate by full culture Medium, blowing uniformly, counting cells, inoculating into T75 cell culture bottle, and placing in saturated humidity and 5% CO237 ℃ cell incubatorAnd (5) culturing.
i) The culture solution is replaced every 72 hours, the cell morphology and the cell density are observed under an inverted microscope, and the cell is subjected to passage amplification after the cell reaches more than 80 percent of fusion degree.
2. Passage amplification of human umbilical cord mesenchymal stem cells
a) After the primary cultured cells grew to 80% confluence, the original culture medium supernatant was aspirated and the adherent cells were rinsed gently 2 times with 10ml pbs.
b) 3mL of TrpLE digest was added, and the flask was then placed in a 37 ℃ incubator for digestion for 3-5 minutes.
c) Cells were observed under a microscope and when cells started to round and brighten and the cell gap gradually increased, DPBS complete medium was added rapidly to stop digestion.
d) The cells were repeatedly blown up and the cell suspension was transferred to a new 50mL centrifuge tube and centrifuged at room temperature.
e) After discarding the supernatant, add the appropriate amount of NuwacellTMCarrying out passage according to the proportion of 1:3-1:4 on the cell resuspended by the ncMission hMSC Medium, transferring 10ng/mL-15mL of cell suspension into a new T75 culture bottle, and placing the bottle into a constant temperature incubator for continuous culture.
f) When the cell fusion degree reaches about 80% again, the steps are repeated for subculture.
3. Cryopreservation of human umbilical cord mesenchymal stem cells
a) The cell freezing box is put into a refrigerator at 4 ℃ in advance for precooling for more than 30 minutes.
b) The procedure for digesting cells was as above, after centrifugation and discarding of supernatant, the cells were resuspended using cell freezing medium from Youkang and transferred to a 2mL freezing tube. c) The tube was placed in a cell cryopreservation box and stored overnight at-80 ℃ and then transferred to a liquid nitrogen tank for long term cryopreservation.
4. Resuscitation of human umbilical cord mesenchymal stem cells
a) The frozen cells are taken out from the liquid nitrogen tank quickly and continuously shaken in a water bath at 37-42 ℃ so as to be melted as soon as possible in a short time.
b) Cell cryopreservation suspension is transferred to a cell prepared beforehand with NuwacellTMncMission Centrifugation in fresh tubes of hMSC Medium.
c) After discarding the supernatant, Nuwacell was used againTMAnd (3) resuspending the cells by the ncMission hMSC Medium, uniformly blowing and then transferring the cells to a T75 culture flask for continuous culture.
d) After 24 hours, the non-adherent dead cells and old medium were discarded and the complete medium was replaced with new one.
Example 2
Treatment of human umbilical cord mesenchymal stem cells
a) At 1 × 106Density of individual cells/well UC-MSC prepared in example 1 was seeded into T75 and cultured adherently for 16-24 hours.
b) Preparing a Youkang serum-free culture medium containing different cytokines in advance, and adding one or more of the following cytokines, wherein the final concentration of each cytokine is: IL-1. beta.: 0ng/mL-20ng/mL, IL-4: 0ng/mL-20ng/mL, TNF- α: 0ng/mL-20ng/mL, 0ng/mL-50ng/mL bFGF, 0ng/mL-100ng/mL EGF, 0ng/mL-100ng/mL PDGF and 0ng/mL-50ng/mL IGF.
c) Adding cytokine-containing Youkang serum-free complete medium containing cytokine after 16-24 hr, and culturing for 36-71 hr
d) After 48 hours of culture, the cells were collected to prepare preparations and examined.
Example 3
1. Preparation of cell preparations
a) Collecting cells, washing the cells twice by using DPBS, and counting the cells;
b) the suspension cells are re-suspended by 10 to 20 percent DMSO and 5 to 10 percent human albumin plus compound electrolyte injection to prepare 5x10720-25 ml of the single unit is frozen in a transfusion using a programmed cooling instrument.
2. Cell detection
a) Flow detection of positive and negative surface markers specific to mesenchymal stem systems such as CD73+/CD90+/CD105+, CD14-/CD34-/CD45-/CD79 alpha-/HLA-DR-, and the like
b) Detection of sterility, endotoxin, mycoplasma, etc.;
c) detecting a Treg regulation experiment;
d) the detection of cell factors is mainly related to the promotion of angiogenesis and immunoregulation, VEGF, TGF-beta and IL-6 are detected by adopting an ELISA kit.
While embodiments of the invention have been described above, it is not limited to the applications set forth in the description and the embodiments, which are fully applicable in various fields of endeavor to which the invention pertains, and further modifications may readily be made by those skilled in the art, it being understood that the invention is not limited to the details shown and described herein without departing from the general concept defined by the appended claims and their equivalents.
Claims (9)
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