Mesenchymal stem cell preparation as well as preparation method and application thereof

Abstract

The invention provides a mesenchymal stem cell preparation as well as a preparation method and application thereof, and relates to the technical field of biology, the mesenchymal stem cell preparation contains 5 * 10 < 6 >-1.2 * 10 < 7 > cells / mL of mesenchymal stem cells, 0.1-0.6% w / v of human serum albumin and a solvent; the mesenchymal stem cells are obtained by culturing a serum-free culture medium, and the mesenchymal stem cells are obtained by resuscitating cryopreserved mesenchymal stem cells, inoculating the resuscitated mesenchymal stem cells into a culture container, culturing until the mesenchymal stem cells are completely adhered to the wall, and digesting. The mesenchymal stem cell preparation improves the treatment effect of mesenchymal stem cells.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a mesenchymal stem cell preparation and a preparation method and application thereof. Background Art

[0002] The following statements merely provide background information related to the present disclosure and do not necessarily constitute prior art.

[0003] Mesenchymal stem cells (MSCs) are adult stem cells derived from the mesoderm. They can be isolated and cultured from multiple tissues such as bone marrow, umbilical cord, placenta, fat, bone, dental pulp and endometrium, and can also be differentiated from embryonic stem cells or pluripotent stem cells. Because of their differentiation potential, ability to regulate immune response and low immunogenicity, they have become an ideal choice for the treatment of various diseases. When administered to the body, the inherent homing ability of MSCs can stimulate their migration to the site of injury. MSCs exhibit unique immunosuppression and immune tolerance properties. Because they do not express or low-express major histocompatibility complex (MHC) class II molecules and co-stimulatory molecules, they are less likely to cause immune rejection. In innate immunity, MSCs promote the polarization of macrophages to the M2 phenotype, inhibit the activation of natural killer cells (NK), stimulate the production of proinflammatory cytokines, and affect the ability of dendritic cells to induce T cells in vivo; in adaptive immunity, MSCs inhibit the proliferation of T cells by secreting factors such as transforming growth factor β1 (TGFβ1) and hepatocyte growth factor (HGF). MSCs can inhibit the proliferation, differentiation and antibody production of B cells both in vitro and in vivo. MSCs show strong anti-inflammatory activity in immune and inflammatory-mediated diseases by changing the function of immune cells, making stem cell therapy a reliable treatment method. Optimizing the formulation of mesenchymal stem cell preparations can help improve the therapeutic effect of mesenchymal stem cell preparations.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] The purpose of the present invention is to provide a mesenchymal stem cell preparation and a preparation method and application thereof, so as to improve the therapeutic effect of the mesenchymal stem cell preparation.

[0006] In order to solve the above technical problems, the present invention particularly adopts the following technical solutions:

[0007] In a first aspect, a mesenchymal stem cell preparation is provided, wherein the mesenchymal stem cell preparation contains 5×10 6 ~1.2×10 7 cells / mL, human albumin 0.1-0.6% w / v and solvent;

[0008] The mesenchymal stem cells are obtained by culturing in a serum-free medium, and the mesenchymal stem cells are mesenchymal stem cells obtained by reviving frozen mesenchymal stem cells, inoculating them into a culture container, culturing them until they are completely adherent, and then digesting them.

[0009] In a second aspect, a method for preparing the mesenchymal stem cell preparation of the first aspect is provided, the method comprising reviving frozen mesenchymal stem cells and inoculating them into a culture container and culturing them until they are completely adherent to the wall, digesting and counting them, and then mixing them with a formulated amount of human serum albumin and a solvent to obtain the mesenchymal stem cell preparation.

[0010] In a third aspect, the use of the mesenchymal stem cell preparation of the first aspect or the preparation method of the second aspect in any of the following is provided:

[0011] (I) Preparation of drugs for preventing and / or treating atopic dermatitis;

[0012] (II) Preparation of medicaments for preventing and / or treating psoriasis;

[0013] (III) Preparation of medicaments for preventing and / or treating osteoarthritis;

[0014] (IV) preparing a medicament for preventing and / or treating hair loss;

[0015] (V) Preparation of a medicament for preventing and / or treating immune rejection.

[0016] In a fourth aspect, a pharmaceutical composition is provided, comprising the mesenchymal stem cell preparation according to the first aspect and a pharmaceutically acceptable excipient.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The whole process of preparing mesenchymal stem cells of the present invention adopts a highly efficient human mesenchymal serum-free culture medium, which is a commercial culture medium specially developed for culturing human mesenchymal stem cells, which improves the efficiency of cell culture, has stable properties, is not easy to cause batch differences and affect cell quality, and improves the stability of stem cell preparations; serum-free components avoid the problem of exogenous virus contamination that may be introduced by animal-derived raw materials, and improves the safety of stem cell preparations. In order to ensure the timeliness, safety and effectiveness of clinical use, the present invention adopts the method of collecting and preparing mesenchymal stem cells again after re-applying, and develops the preparation into a preparation form of a fresh product. Compared with frozen preparations, the re-applying process of fresh preparations screens out most of the dead cells, has a higher viability, and is safer and more effective. The present invention optimizes the composition ratio in the mesenchymal stem cell preparation, so that the viability of the mesenchymal stem cells in the preparation is improved, and it is not easy to clump. The mesenchymal stem cell preparation provided by the present invention has a good therapeutic effect on atopic dermatitis and osteoarthritis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The activity results of each group of preparations at 0 hours in Example 2;

[0021] Figure 2 The activity results of each group of preparations in Example 2 after 18 hours;

[0022] Figure 3 The activity results of each group of preparations in Example 2 for 24 hours;

[0023] Figure 4 The activity results of each group of preparations in Example 2 after 42 hours;

[0024] Figure 5 The activity results of each group of preparations in Example 2 after 48 hours;

[0025] Figure 6 The agglomeration rate results of each group of preparations in Example 2 at 0 hours;

[0026] Figure 7 The agglomeration rate results of each group of preparations in Example 2 after 18 hours;

[0027] Figure 8 The agglomeration rate results of each group of preparations in Example 2 for 24 hours;

[0028] Fig. 9 The agglomeration rate results of each group of preparations in Example 2 after 42 hours;

[0029] Fig.10 The agglomeration rate results of each group of preparations in Example 2 after 48 hours;

[0030] Fig.11 The mycoplasma detection results of SEB001-Y22001, SEB002-Y22001, SEB003-Y22001 and each control group in Example 3;

[0031] Fig.12 The results of the three-lineage differentiation of SEB001-Y22001 in the preparation in Example 3;

[0032] Fig.13 The results of the three-lineage differentiation of SEB002-Y22001 in the preparation in Example 3;

[0033] Fig.14 The results of the three-lineage differentiation of SEB003-Y22001 in the preparation in Example 3;

[0034] Fig.15 is the atopic dermatitis status of each group of mice in Example 5;

[0035] Fig.16 is the level of IgE in the serum of each group of mice in Example 5;

[0036] Fig.17 The X-ray images of the knee joints of rats in each group in Example 6;

[0037] Fig.18 The photographs of the knee cartilage status of rats in each group in Example 6;

[0038] Fig.19 The serum type 2 collagen level of each group of rats in Example 6;

[0039] Fig. 20 The TNF-α level of each group of rats in Example 6;

[0040] Fig.21 This is the level of IL-1β in the serum of rats in each group in Example 6. DETAILED DESCRIPTION

[0041] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0042] It should be noted that:

[0043] In this document, unless otherwise specified, all implementation methods and preferred implementation methods mentioned in this document can be combined with each other to form a new technical solution; all technical features and preferred features mentioned in this document can be combined with each other to form a new technical solution; the components involved or their preferred components can be combined with each other to form a new technical solution.

[0044] In this document, unless otherwise specified, the numerical range "a-b" is an abbreviation for any combination of real numbers between a and b, where a and b are both real numbers. For example, the numerical range "6-22" means that all real numbers between "6-22" are listed in this document, and "6-22" is just an abbreviation for these numerical combinations.

[0045]

[0046] Herein, a "range" disclosed in the form of a lower limit and an upper limit may be one or more lower limits, and one or more upper limits, respectively.

[0046] Herein, unless otherwise specified, each reaction or operation step may be performed sequentially or non-sequentially. Preferably, the reaction method herein is performed sequentially.

[0047] As used herein, unless otherwise stated, "optionally", "optional", "optional" or "optional" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not occur.

[0048] In this article, the concept of "prevention and / or treatment" refers to any measure applicable to the disease, or preventive treatment of the disease or symptoms manifested, or avoiding the recurrence of the disease, such as recurrence after the treatment period is completed or the symptoms of the disease that have already occurred are treated, or pre-intervention to prevent, inhibit or reduce the occurrence of such diseases or symptoms.

[0049] Unless otherwise specified, the professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any method or material similar or equivalent to the described content may also be applied to the present invention.

[0050] In a first aspect, a mesenchymal stem cell preparation is provided, comprising mesenchymal stem cells, human serum albumin and a solvent.

[0051] The content of mesenchymal stem cells in the mesenchymal stem cell preparation is 5×10 6 ~1.2×10 7 cells / mL, for example, but not limited to, 5×10 6 , 6×10 6 ,7×10 6 , 8×10 6 ,9×10 6 , 1×10 7 or 1.2×10 7 The mesenchymal stem cells are obtained by culturing in a serum-free medium, and the mesenchymal stem cells are mesenchymal stem cells obtained by reviving frozen mesenchymal stem cells, inoculating them into a culture container, culturing them until they are completely adherent, and then digesting them.

[0052] In an optional embodiment, the mesenchymal stem cells include mesenchymal stem cells isolated from at least one of bone marrow, umbilical cord, placenta, fat, bone, dental pulp and endometrium; and / or, the mesenchymal stem cells include mesenchymal stem cells differentiated from embryonic stem cells and / or pluripotent stem cells.

[0053] In an optional embodiment, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0054] The content of human serum albumin in the mesenchymal stem cell preparation is 0.1-0.6% w / v, for example, but not limited to 0.1, 0.2, 0.3, 0.4, 0.5 or 0.6% w / v.

[0055] In an optional embodiment, the solvent includes a composite electrolyte.

[0056] In an optional embodiment, the solvent is a compound electrolyte injection solution, and the compound electrolyte injection solution can be prepared according to a conventional formula known in the art, or a commercially available electrolyte injection solution can be selected.

[0057] In an optional embodiment, the mesenchymal stem cell preparation comprises 1.0×10 7 cells / mL, human serum albumin 0.6% w / v and compound electrolytes; the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

[0058] In a second aspect, a method for preparing the mesenchymal stem cell preparation of the first aspect is also provided, the preparation method comprising reviving the frozen mesenchymal stem cells and inoculating them into a culture container and culturing them until they are completely adhered to the wall, digesting and counting them, and then mixing them with a formulated amount of human serum albumin and a solvent to obtain the mesenchymal stem cell preparation.

[0059] In an optional embodiment, the mesenchymal stem cells include umbilical cord mesenchymal stem cells.

[0060] In an optional embodiment, the umbilical cord mesenchymal stem cells are prepared according to the following method: culturing freshly isolated human umbilical cord tissue blocks, and collecting and obtaining P0 generation mesenchymal stem cells when the cell fusion degree of mesenchymal stem cells crawling out of the umbilical cord tissue blocks is 80% to 90%; subculturing the P0 generation mesenchymal stem cells, and collecting the mesenchymal stem cells when the cell confluence degree is 80% to 90%, and continuing to subculture until the target generation of mesenchymal stem cells are obtained; the culture medium used in the umbilical cord mesenchymal stem cell culture process does not contain serum.

[0061] In an optional embodiment, the target generation is the fifth generation of mesenchymal stem cells.

[0062] In an optional embodiment, the umbilical cord mesenchymal stem cells are prepared according to the following method:

[0063] (1) Cultivating freshly isolated human umbilical cord tissue blocks, and collecting and obtaining P0 generation mesenchymal stem cells when the cell fusion degree of mesenchymal stem cells crawling out of the umbilical cord tissue blocks is 80% to 90%.

[0064] The specific operation of step (1) includes: taking human umbilical cord tissue from the body, washing it with physiological saline, cutting it into small pieces of 2 to 3 cm, taking out Wharton's jelly, cutting it into pieces, washing it, and spreading it on a 75 cm thick plate coated with an adhesion promoter. 2 At the bottom of the culture bottle, add 1 mL of tissue / bottle. Invert the culture bottle in a CO2 incubator at 37°C for 30 minutes. After the tissue block is firmly attached to the bottom of the bottle, flip the culture bottle and add 6 mL of high-efficiency human mesenchymal stem cell serum-free medium. Place in a CO2 incubator for culture at 37°C and a CO2 concentration of 5%. After 5 days of culture of the umbilical cord tissue block, aspirate the culture medium and add new culture medium. Change the medium every 4 days.

[0065] When the cells reach 80% to 90% confluence, discard the culture medium, add saline to wash twice, and then add stem cell mild digestion enzyme to digest. When most of the cells begin to detach from the wall, add saline to neutralize and terminate digestion. Transfer the cell suspension to a 50mL centrifuge tube, add saline to rinse the bottom of the bottle, combine with the first cell suspension and mix well, filter with a 100μm filter, and then centrifuge the collected cell suspension and discard the supernatant. The centrifugation parameters are 200g and 6min. Add saline to resuspend the cells, wash and centrifuge once, resuspend the cells with saline again, take samples and count, and calculate the total number of primary (P0) cells obtained.

[0066] (2) The P0 generation mesenchymal stem cells are subcultured until the cell confluence reaches 80% to 90%, and then the P1 generation mesenchymal stem cells are obtained.

[0067] The specific operation of step (2) includes: discarding the supernatant of the cell suspension in step (1), using a high-efficiency human mesenchymal stem cell serum-free culture medium at a rate of 6000 to 8000 cells / cm 2 The cells were inoculated at a density of 1.50 μg / mL into pre-coated 175 cm2 culture flasks, 22 mL of serum-free culture medium was added to each flask, the cells were gently shaken to mix, and then cultured in a 5% CO2, 37°C environment.

[0068] When the cells grow to 80%-90% confluence, they are collected in the same manner as step (1).

[0069] (3) The P1 generation mesenchymal stem cells are subcultured until the cell confluence reaches 80% to 90%, and then the P2 generation mesenchymal stem cells are obtained.

[0070] The specific operation of step (3) is the same as step (2).

[0071] (4) The P2 mesenchymal stem cells are subcultured until the cell confluence reaches 80% to 90%, and then the P3 mesenchymal stem cells are obtained.

[0072] The specific operation of step (4) is the same as step (2).

[0073] (5) Cryopreserving the P3 mesenchymal stem cells and collecting the cryopreserved master generation bank cells.

[0074] The P3 cells collected in step (5) were frozen in the following number of tubes according to the total number of cells, with each tube containing 1×10 7 Cells were frozen in a volume of 1 mL. Serum-free cell freezing solution was added to the cell pellet, and the cells were gently blown with a pipette to make the cells uniform, and then evenly distributed into cryopreservation tubes. After being placed in a -80℃ refrigerator overnight, they were transferred to a -196℃ liquid nitrogen box to obtain the master generation bank cells.

[0075] (6) The frozen P3 master generation bank cells were revived and subcultured until the cell confluence reached 80% to 90%, and then P4 generation mesenchymal stem cells were obtained.

[0076] The specific operation of step (6) is the same as (2).

[0077] (7) The P4 mesenchymal stem cells are subcultured until the cell confluence reaches 80% to 90%, and then the P5 mesenchymal stem cells are obtained.

[0078] The specific operation of step (7) is the same as (2).

[0079] (8) Cryopreserving the P5 mesenchymal stem cells to obtain frozen P5 working bank cells.

[0080] The P5 cells collected in step (7) were frozen in the following number of tubes according to the total number of cells, with each tube containing 1×10 7 Cells were frozen in 1 mL suspension. Serum-free cell freezing solution was added to the cell pellet, and the cells were gently blown with a pipette to make the cells uniform, and then dispensed into cryopreservation tubes. After being placed in a -80℃ refrigerator overnight, they were transferred to a -196℃ liquid nitrogen box to obtain working bank cells.

[0081] In an optional embodiment, the cryopreservation tube containing the P5 generation working bank cells is placed in a 37°C water bath, and the cryopreservation tube is gently shaken to completely melt within 1 minute. The stem cell suspension is transferred to a centrifuge tube and 10 times the volume of the cell suspension is added with pre-cooled saline to dilute it. Centrifuge at 200g for 6 minutes, discard the supernatant, add an equal volume of pre-cooled saline, wash twice, and filter the cell suspension with a 100μm filter before the last centrifugation, and then take samples for counting. According to the counting results, the cells are inoculated on the coated 175cm 2 Culture flask, cell inoculation volume 1×10 7cells / bottle, then placed in a 37°C, 5% CO2 incubator for culture, discard the supernatant after the cells are completely attached (no more than 16 hours), wash the cells twice with saline, and add stem cell mild digestion enzymes for digestion. When most of the cells begin to detach from the wall, add saline to terminate the digestion, transfer the cell suspension to a clean 250mL centrifuge tube, wash the bottom of the culture bottle with saline, transfer the washed suspension to the above centrifuge tube for combined centrifugation, the centrifugation parameters are 500g, 8min, and discard the supernatant after completion. Repeat the washing twice, filter the cell suspension with a 100μm filter before the last centrifugation, and then take samples and count. Resuspend with solvent according to the counting results, take samples and count again, and adjust the cell concentration to the standard according to the counting results. Transfer the preparation that meets the standard to a 1.8mL cryopreservation tube with a specification of 1mL, and wrap the tube mouth with a sealing film with a width of 1cm for 2 circles to seal, obtain human umbilical cord mesenchymal stem cell preparation, and keep samples for testing at the same time.

[0082] In a third aspect, the invention further provides the use of the mesenchymal stem cell preparation described in the first aspect or the preparation method described in the second aspect in any of the following:

[0083] (I) Preparation of drugs for preventing and / or treating atopic dermatitis;

[0084] (II) Preparation of drugs for preventing and / or treating psoriasis;

[0085] (III) Preparation of medicaments for preventing and / or treating osteoarthritis;

[0086] (IV) preparing a medicament for preventing and / or treating hair loss;

[0087] (V) Preparation of a medicament for preventing and / or treating immune rejection.

[0088] In a fourth aspect, a pharmaceutical composition is provided, comprising the mesenchymal stem cell preparation according to the first aspect and a pharmaceutically acceptable excipient.

[0089] In an optional embodiment, the pharmaceutically acceptable excipients include, but are not limited to, one or more of a pH regulator, an osmotic pressure regulator, an antibacterial agent, a suspending agent, a preservative, a matrix and a drug carrier material.

[0090] In an optional embodiment, the pharmaceutically acceptable excipient includes gel as a drug carrier.

[0091] In an optional embodiment, the pharmaceutical composition is an injection.

[0092] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed description and should not be construed as limiting the present invention in any form.

[0093] Example 1 Preparation of human umbilical cord mesenchymal stem cells

[0094] 1. Umbilical cord collection standards

[0095] The umbilical cords of full-term newborns without congenital diseases were obtained. The maternal age of the newborns was 20 to 35 years old, the gestational period was 38 to 42 weeks, and they had no infectious diseases such as syphilis, AIDS, hepatitis, Epstein-Barr virus, etc. The delivery method was caesarean section. The maternal and family members were informed and agreed to the use of the umbilical cords for experimental research.

[0096] 2. Isolation of human umbilical cord mesenchymal stem cells

[0097] After fully soaking and cleaning the fresh umbilical cord with saline in a biosafety cabinet, use scissors to cut it into small pieces of 2 to 3 cm in length. Take a small piece of umbilical cord, cut it along the outer epidermal texture of the umbilical cord and spread the umbilical cord, remove the veins and arteries in the umbilical cord, and separate the Walton's jelly. Cut the Walton's jelly into pieces, clean it, and evenly spread it on the bottom of the culture bottle, and invert the culture bottle for half an hour. After the tissue block is firmly attached to the bottom of the bottle, turn the culture bottle over, add mesenchymal stem cell serum-free culture medium, and place it in a 37°C, 5% CO2 incubator for culture. Change the medium every four days. When the cell growth reaches 90% confluence, discard the culture medium, add saline to wash twice, and add stem cell mild digestion enzyme for 2 minutes. When the cells shrink and become round under the microscope and most of the cells begin to detach from the wall, add an appropriate amount of physiological saline to terminate digestion, transfer the cell suspension to a clean centrifuge tube, add physiological saline to the original culture bottle to rinse the bottom of the bottle, combine and mix with the first cell suspension, filter with a 100μm filter, and sample into a 1.5mL EP tube for counting cell number and viability. Then centrifuge and wash the collected cell suspension, with centrifugation parameters of 200g, 6min, and discard the supernatant. Repeat the washing with physiological saline once, discard the supernatant to obtain the cell pellet, i.e. the primary (P0) cells.

[0098] 3. Building the main database

[0099] (1) Preparation of P1 cells

[0100] According to the P0 cell counting results, the obtained P0 cells were resuspended in mesenchymal stem cell serum-free medium at 8000 cells / cm 2The cells were inoculated into culture flasks at a density of 100 μg / mL, and each flask was supplemented with mesenchymal stem cell serum-free medium to a standard volume, and then placed in a 37°C, 5% CO2 incubator for continued culture. After 3 to 4 days of culture, when the cell confluence reached 80% to 90%, the cell supernatant was discarded, and saline was added to the culture flask to gently rinse, and stem cell mild digestion enzyme was added to each flask for 2 minutes. Under the microscope, the cells retracted into a round shape and most of the cells began to detach from the wall. An appropriate amount of saline was added to each flask to terminate the digestion, and the cell suspension was transferred to a centrifuge tube. The original culture flask was then added with saline to rinse the bottom of the flask, and then mixed with the first cell suspension, centrifuged at 200g for 6 minutes, and the supernatant was discarded. The cells were resuspended with saline, washed once, and the supernatant was discarded. The cells were suspended with mesenchymal stem cell serum-free medium, suspended and mixed, filtered with a 100μm filter, and sampled into a 1.5mL EP tube for counting cell number and viability. The collected cell suspension was centrifuged at 200 g for 6 min, the supernatant was discarded, and the obtained cells were the P1 generation cells.

[0101] (2) Preparation of P2 cells

[0102] According to the P1 cell counting results, the obtained P1 cells were resuspended in mesenchymal stem cell serum-free medium at 8000 / cm 2 The cells were inoculated into culture flasks at a density of 100 μm, and each flask was supplemented with serum-free medium for mesenchymal stem cells to a standard volume, and then placed in a 37°C, 5% CO2 incubator for further culture. After culturing for 4 days, when the cell confluence reached 90%, digestion and washing were performed, and before the last centrifugation, the cell suspension was filtered through a 100 μm filter, and then the cell number and viability were sampled and counted, and the supernatant was discarded to obtain P2 cells.

[0103] (3) Building the master database

[0104] According to the P2 cell counting results, the obtained P2 cells were resuspended in mesenchymal stem cell serum-free medium at 8000 / cm 2 The cells were inoculated into culture flasks at a density of 100 μm, and each flask was supplemented with serum-free medium for mesenchymal stem cells to a standard volume, and then placed in a 37°C, 5% CO2 incubator for further culture. After culturing for 4 days, when the cell confluence reached 90%, digestion and washing were performed, and before the last centrifugation, the cell suspension was filtered through a 100 μm filter, and then the cell number and viability were sampled and counted, and the supernatant was discarded to obtain P3 cells.

[0105] Add freezing solution to the P3 cell pellet, gently blow to make the cells uniform, and then evenly distribute them into clean freezing tubes. Place them in a -80℃ refrigerator overnight and then transfer them to a liquid nitrogen box to obtain the master generation bank cells.

[0106] 4. Establishment of work library

[0107] (1) Preparation of P4 cells

[0108] Place the cryopreservation tube containing the primary cell bank in a 37°C water bath, gently shake the cryopreservation tube to melt it completely within 1 minute, wipe the outside of the cryopreservation tube with 75% alcohol, and move it into a sterile operating table. Transfer the stem cell suspension to a centrifuge tube and add 10 times the volume of pre-cooled saline to dilute it, centrifuge at 200g for 6 minutes, discard the supernatant, add an equal volume of pre-cooled saline, wash twice, and filter the cell suspension with a 100μm filter before the last centrifugation, then take a sample to count the cell number and viability. 2 Inoculate into culture flasks and culture in a 37°C, 5% CO2 incubator for 3-4 days. After the cells reach 90% confluence, digest and wash. Before the last centrifugation, filter the cell suspension with a 100 μm filter, take samples to count the cell number and viability, and discard the supernatant to obtain P4 cells.

[0109] (2) Work database construction

[0110] According to the P4 cell counting results, the obtained P4 cells were resuspended in mesenchymal stem cell serum-free medium at 8000 / cm 2 The cells were inoculated into culture flasks at a density of 100 μg / mL, and each flask was supplemented with serum-free medium for mesenchymal stem cells to a standard volume, and then placed in a 37°C, 5% CO2 incubator for further culture. After culturing for 4 days, when the cell confluence reached 90%, the cells were digested, washed, and resuspended, and the supernatant was discarded to obtain P5 cells.

[0111] Add freezing solution to the P5 cell pellet, gently blow to make the cells uniform, and then evenly distribute them into clean freezing tubes, place them in a -80℃ refrigerator overnight, and then transfer them to a liquid nitrogen box to obtain working bank cells.

[0112] Example 2 Human umbilical cord mesenchymal stem cell preparation formulation design

[0113] The P5 cells obtained in Example 1 were taken and reattached overnight. The cells were collected and resuspended with a solvent to count, and cell preparations containing different cell concentrations and different human albumin concentrations were prepared. The specific grouping is shown in Table 1. Finally, the prepared cell preparations were stored in a 4°C medical refrigerator, and samples were taken at 0h, 18h, 24h, 42h and 48h to detect the aggregation and viability, and the effects of different human albumin and cell concentrations on the cell viability and aggregation rate of human umbilical cord mesenchymal stem cell preparations were compared.

[0114] Table 1 Grouping of human umbilical cord mesenchymal stem cell preparation formulation design

[0115] Group Human serum albumin concentration Cell concentration Group 1 0.1％ <![CDATA[1.2×10 7 cells / mL]]> Group 2 0.6％ <![CDATA[1.2×10 7 cells / mL]]> Group 3 1.2％ <![CDATA[1.2×10 7 cells / mL]]> Group 4 0.1％ <![CDATA[1.0×10 7 cells / mL]]> Group 5 0.6％ <![CDATA[1.0×10 7 cells / mL]]> Group 6 1.2％ <![CDATA[1.0×10 7 cells / mL <!-- 7 -->]]> Group 7 0.1％ <![CDATA[5.0×10 6 cells / mL]]> Group 8 0.6％ <![CDATA[5.0×10 6 cells / mL]]> Group 9 1.2％ <![CDATA[5.0×10 6 cells / mL]]>

[0116] The results are as follows Figures 1 to 10The results showed that: comprehensive evaluation of cell viability and aggregation rate showed that within 48 hours, group 5, i.e., human albumin concentration was 0.6% and cell concentration was 1.0×10 7 The results of cell agglomeration rate and viability in the 100 / mL group were better than those in other groups. When the concentration of human serum albumin was 1.2%, the human umbilical cord mesenchymal stem cell preparation could not significantly reduce the cell agglomeration rate and failed to significantly increase the cell viability. Therefore, the prescription parameters of the human umbilical cord mesenchymal stem cell preparation were: 0.6% human serum albumin, cell concentration of 1.0×10 7 Pieces / mL, specification is 1mL.

[0117] Example 3 Preparation of fresh human umbilical cord mesenchymal stem cell preparation and performance testing

[0118] 1. Preparation of human umbilical cord mesenchymal stem cell preparations

[0119] According to the data results of Example 2, the preparation was prepared: the cryopreservation tube containing the P5 generation working bank cells was placed in a 37°C water bath, and the cryopreservation tube was gently shaken to melt it completely within 1 minute. The stem cell suspension was transferred to a centrifuge tube and 10 times the volume of the cell suspension was added with pre-cooled saline to dilute it. Centrifuge at 200g for 6 minutes, discard the supernatant, add an equal volume of pre-cooled saline, wash twice, and filter the cell suspension with a 100μm filter before the last centrifugation, and then sample and count the cell number and viability. According to the P5 generation cell counting results, 8000 / cm 2 Inoculate into a culture bottle and culture in a 37°C, 5% CO2 incubator. After the cells are completely attached to the wall (no more than 16 hours), discard the supernatant in the culture bottle, gently rinse the cells twice with saline, add stem cell mild digestion enzyme, observe under the microscope that the cells shrink back to a round shape and most of the cells begin to detach from the wall, add saline to stop digestion, transfer the cell suspension to a clean centrifuge tube, add saline to the original culture bottle to rinse the bottom of the bottle, combine and mix with the first cell suspension, centrifuge at 200g for 6 minutes, and discard the supernatant. Repeat the washing twice, filter the cell suspension with a 100μm filter before the last centrifugation, and then take a sample to count the number and viability of cells. According to the counting results, use a solvent (compound electrolyte solution (Bopuli A) containing 0.6% w / v human albumin) to resuspend to 1×10 7 cells / mL, made into stem cell injection.

[0120] 2. Safety testing of subcutaneous injection of human umbilical cord-derived mesenchymal stem cells

[0121] (1) Sterility test of human umbilical cord-derived mesenchymal stem cell subcutaneous injection

[0122] Three batches of umbilical cord mesenchymal stem cell working bank cells from different sources were resuscitated, with batch numbers: SEB001-Y22001, SEB002-Y22001, and SEB003-Y22001, respectively, and three batches of preparations were prepared respectively. 20 mL of the supernatant of the preparations was collected by centrifugation and sterility test was performed by membrane filtration. Fungi and bacteria were cultured in tryptic soy liquid medium and thioethanol liquid medium, respectively. The results are shown in Table 2. The results showed that no bacterial and fungal contamination was detected in the three batches of stem cell preparations.

[0123] Table 2 Sterility test results of human umbilical cord mesenchymal stem cell preparations

[0124]

[0125] (2) Endotoxin test for subcutaneous injection of human umbilical cord-derived mesenchymal stem cells

[0126] Three batches of umbilical cord mesenchymal stem cell working bank cells from different sources were resuscitated, with batch numbers: SEB001-Y22001, SEB002-Y22001, and SEB003-Y22001, respectively, and three batches of preparations were prepared, and 1.5 mL of the suspension was taken for endotoxin detection. Gel method detection was used. The horseshoe crab reagent was dissolved in 650 μL of inspection water and mixed by oscillation. The bacterial endotoxin working standard was dissolved in 1000 μL of inspection water to prepare an endotoxin standard of 10 EU / mL. 100 μL of the mixed endotoxin standard was taken and then added to 900 μL of inspection water (or 900 μL of test sample) to dilute and prepare an endotoxin standard with a concentration of 1 EU / mL. Take 100 μL of 1EU / mL endotoxin standard and mix it with 100 μL of inspection water and 100 μL of test sample solution. After gently mixing the solution in the reaction tube, seal the tube mouth with sealing film, vertically place it in a 37℃±1℃ test tube thermostat, and keep it warm for 60±2 minutes. Gently take out the test tube and slowly turn it 180°. Gel is formed in the tubes of the positive group and the interference confirmation group, and the gel does not deform or slip from the tube wall. No gel is formed in the negative control group and the preparation group.

[0127] The endotoxin test results are shown in Table 3. No bacterial endotoxin was detected in the three batches of stem cell preparations.

[0128] Table 3 Endotoxin test results of human umbilical cord mesenchymal stem cell preparations

[0129]

[0130]

[0131] (3) Mycoplasma testing of human umbilical cord-derived mesenchymal stem cell subcutaneous injection

[0132] Three batches of umbilical cord mesenchymal stem cell working bank cells from different sources were resuscitated, with batch numbers: SEB001-Y22001, SEB002-Y22001, and SEB003-Y22001. Three batches of preparations were prepared respectively, and the supernatant was removed by centrifugation. The cells were resuspended in human mesenchymal stem cell serum-free medium and cultured at 8000 cells / cm 2 Inoculate to 175cm 2 The cells were cultured in a culture flask for 4 days, and the culture supernatant was taken for mycoplasma detection. The negative control group was inoculated with mycoplasma dilution into a culture flask pre-plated with indicator cells; the test sample was inoculated into a culture flask pre-plated with indicator cells; the positive control was inoculated with swine nasal mycoplasma and oral mycoplasma into a culture flask pre-plated with just cells. All cell cultures were cultured for 3 days, and after 3 days of culture, the cells were subcultured into a six-well plate containing a coverslip or other container. After subculture, the cells were cultured for 3 days. After the subculture, DNA binding dye was used for staining, and the results were observed under a microscope. The results are as follows. Fig.11 As shown, mycoplasma contamination was not detected in the three batches of stem cell preparations.

[0133] (4) Examination of cell number and viability of subcutaneous injection of human umbilical cord-derived mesenchymal stem cells

[0134] Three batches of umbilical cord mesenchymal stem cell working bank cells from different sources were resuscitated, with batch numbers: SEB001-Y22001, SEB002-Y22001, and SEB003-Y22001. Three batches of preparations were prepared, 100 μL was mixed with 100 μL of trypan blue, and 20 μL of the mixture was tested on the machine. The results are shown in Table 4. The number of preparations in the three batches was 1×10 7 cells / mL±10%, the viability was greater than 95%.

[0135] Table 4 Viability and quantity test results of human umbilical cord mesenchymal stem cell preparations

[0136] Group Live rate quantity SEB001-Y22001 98.27％ <![CDATA[1.02×10 7 cells]]> SEB002-Y22001 99.03％ <![CDATA[9.88×10 6 cells]]> SEB003-Y22001 98.52％ <![CDATA[1.05×10 7 cells]]>

[0137] (5) Tri-lineage differentiation assay of subcutaneous injection of human umbilical cord-derived mesenchymal stem cells

[0138] Three batches of umbilical cord mesenchymal stem cell working bank cells from different sources were resuscitated, with batch numbers: SEB001-Y22001, SEB002-Y22001, SEB003-Y22001, and three batches of preparations were prepared respectively. The differentiation ability of stem cells was tested using adipogenic, osteogenic, and chondrogenic kits.

[0139] The results are as follows Fig.12 , 13As shown in Figures 14, adipogenic induction showed lipid droplets by Oil Red O staining, osteogenic induction showed red by Alizarin Red staining with calcium nodules formed, and chondrogenic induction showed blue by Alecin Blue staining, indicating that the three batches of cell preparations all had the ability to induce differentiation into adipogenesis, osteoblastogenesis and chondrogenesis.

[0140] (6) Detection of surface markers and non-target cells of subcutaneous injection of human umbilical cord-derived mesenchymal stem cells

[0141] Three batches of umbilical cord mesenchymal stem cell working bank cells from different sources were resuscitated. The batch numbers were SEB001-Y22001, SEB002-Y22001, and SEB003-Y22001. Three batches of preparations were prepared respectively. 3×10 6 Add 10 μL of antibody to each tube, add 100 μL of the cell suspension to be tested, shake and mix, and incubate at room temperature and away from light for 20 min. Add 2 mL of 1× PBS to each tube for washing and centrifugation, centrifugation parameters: 300g, 5 min. Discard the supernatant, add 100 μL of 1× PBS to each tube for resuspending, mix and use BD FACSCalibur flow cytometer for detection.

[0142] The results are shown in Tables 5, 6 and 7. The results show that the preparation prepared by the method of the present invention expresses on the surface of stem cells.

[0143] Table 5 Detection results of surface markers and non-target cells of SEB001-Y22001 preparation

[0144]

[0145] Table 6 Detection results of surface markers and non-target cells of SEB002-Y22001 preparation

[0146]

[0147] Table 7 Detection results of surface markers and non-target cells of SEB003-Y22001 preparation

[0148]

[0149] As shown in Tables 5 to 7, CD73, CD90, CD29 and CD105 are all greater than 95%, showing positive; CD14, CD29, CD34, CD45, and HLADR are all less than 2%, showing negative.+ The proportion is less than 2%.

[0150] (7) Detection of factor secretion ability of subcutaneous injection of human umbilical cord-derived mesenchymal stem cells

[0151] Three batches of umbilical cord mesenchymal stem cell working bank cells from different sources were resuscitated, with batch numbers of SEB001-Y22001, SEB002-Y22001, and SEB003-Y22001, respectively, and three batches of preparations were prepared, centrifuged and the supernatant was discarded, and the cells were resuspended in serum-free medium and heated to 4 × 10 5 cells / well were inoculated in a 6-well plate. After overnight adherence, the medium was discarded, the cells were washed twice with saline, and 1 mL of serum-free basal medium was added to each well for 48 hours. The culture supernatant was then collected and tested with HGF and PGE2 kits. As shown in Table 8, the three batches of stem cell preparations all had the ability to secrete HGF and PGE2.

[0152] Table 8 Results of secretion of HGF and PGE2 by human umbilical cord mesenchymal stem cell preparation

[0153] batch number HGF (pg / mL) PGE2 (pg / mL) SEB001-Y22001 625.37±2.85 1028.06±25.33 SEB002-Y22001 543.28±1.67 1125.37±18.67 SEB003-Y22001 557.26±2.47 958.05±32.43

[0154] Example 4 Preparation of frozen human umbilical cord mesenchymal stem cell preparation and partial performance testing

[0155] 1. Preparation of frozen human umbilical cord mesenchymal stem cell preparations

[0156] The preparation steps of working bank cells are the same as those in Example 2. Preparation preparation: Place the cryopreserved tube containing the P5 generation working bank cells in a 37°C water bath, gently shake the cryopreserved tube to completely melt it within 1 minute, transfer the stem cell suspension to a centrifuge tube and add 10 times the volume of the cell suspension of pre-cooled saline to dilute, centrifuge at 200g for 6 minutes, discard the supernatant, add an equal volume of pre-cooled saline, wash twice, and filter the cell suspension with a 100μm filter before the last centrifugation, then take a sample to count the number of cells and the viability. According to the P5 generation cell counting results, resuspend with a solvent to prepare a frozen stem cell injection.

[0157] 2. Performance testing of frozen human umbilical cord-derived mesenchymal stem cell injection

[0158] (1) Examination of cell number and viability of subcutaneous injection of human umbilical cord-derived mesenchymal stem cells

[0159] Three batches of umbilical cord mesenchymal stem cell working bank cells from different sources were resuscitated, with batch numbers: SEB001-Y22002, SEB002-Y22002, and SEB003-Y22002, respectively, and three batches of frozen preparations were prepared. 100 μL was mixed with 100 μL of trypan blue, and 20 μL of the mixed solution was tested on the machine. The results are shown in Table 9. The number of preparations in the three batches was 1×10 7 cells / mL±10%, the viability was less than 90%.

[0160] Table 9 Viability and quantity test results of frozen human umbilical cord mesenchymal stem cell preparations

[0161] Group Live rate quantity SEB001-Y22002 81.28％ <![CDATA[9.52×10 6 cells]]> SEB002-Y22002 83.22％ <![CDATA[9.88×10 6 cells]]> SEB003-Y22002 84.51％ <![CDATA[9.47×10 6 cells]]>

[0162] (2) Detection of surface markers and non-target cells of subcutaneous injection of human umbilical cord-derived mesenchymal stem cells

[0163] Three batches of umbilical cord mesenchymal stem cell working bank cells from different sources were resuscitated, with batch numbers of SEB001-Y22002, SEB002-Y22002, and SEB003-Y22002, and three batches of frozen preparations were prepared respectively. 3×10 6 Cells were added to ep tubes, and IgG1, κ-PE, LgG2α-FITC, CD19-PE, CD34-PE, CD29-PE, CD45-PE, CD73-PE, CD90-PE, CD105-PE, CD31-FITC, CD14-FITC, HLA-DR-FITC, CD326-PE were added in sequence. The amount of antibody added was 10 μL, and 100 μL of the cell suspension to be tested was added respectively, oscillated and mixed, and incubated at room temperature and away from light for 20 minutes. 2 mL 1×PBS was added to each tube for washing and centrifugation, and the centrifugation parameters were: 300g, 5min. The supernatant was discarded, 100 μL 1×PBS was added to each tube for resuspending, and after mixing, the BD FACSCalibur flow cytometer was used for detection. The results are shown in Tables 10, 11 and 12. The results show that the preparation prepared by the method of the present invention expresses on the surface of stem cells.

[0164] Table 10 Detection results of surface markers and non-target cells of SEB001-Y22002 preparation

[0165]

[0166]

[0167] Table 11 Detection results of surface markers and non-target cells of SEB002-Y22002 preparation

[0168]

[0169] Table 12 Detection results of surface markers and non-target cells of SEB003-Y22002 preparation

[0170]

[0171] The results showed that CD73, CD90 and CD29 were all greater than 95%, positive, CD105 was less than 95%, not meeting the quality standards; CD14, CD29, CD34 and HLADR were all less than 2%, negative, CD45 was greater than 2%, not meeting the quality standards. + The proportion is less than 2%.

[0172] Example 5 Application of fresh human umbilical cord mesenchymal stem cell subcutaneous injection in the treatment of atopic dermatitis (AD)

[0173] 1. Experimental conditions and materials

[0174] SPF grade 4-6 week old BALB / c female mice, weighing 18-22 g, were purchased from Shanghai Slake Company by Hangzhou Lead Pharmaceutical Technology Co., Ltd. and raised in the animal center of Hangzhou Lead Pharmaceutical Technology Co., Ltd.

[0175] Ovalbumin OVA (grade V; Sigma, St Louis, MO, USA, catalog number: A5003), alum adjuvant (Thermo Scientific Pierce, Rockford, IL, USA, catalog number: 77161), sodium pentobarbital, sterile gauze, medical bandage, sterile syringe (1 mL), mouse serum immunoglobulin E (IgE) ELISA kit (Shanghai ELISA Biology Co., Ltd.).

[0176] 2. Experimental methods

[0177] The atopic dermatitis animal model was prepared by combined intraperitoneal and skin sensitization: the sensitization mixture for each mouse was as follows: 10 μg OVA was dissolved in 100 μL PBS, then mixed with 100 μL alum adjuvant (40 mg / mL), and after thorough mixing for 30 minutes, the mixture was intraperitoneally injected once a week for three weeks (days 0, 7, and 14). Each mouse was injected with 200 μL.

[0178] Skin irritation: On day 21, mice were anesthetized (1% sodium pentobarbital, 200 μL intraperitoneal injection), the back and neck were shaved, disinfected, and 100 μL of OVA solution (10 μg OVA in 100 μL PBS) was added to sterile gauze and fixed on the shaved skin surface (1 cm × 1 cm). The dressing was changed every day for one week.

[0179] Experimental groups: blank group, model group and treatment group of mice subcutaneously injected with stem cells. Each group had 5 mice, randomly assigned. The treatment groups of mice subcutaneously injected with stem cells were divided into 0.6% human white preparation group (Group 5 in Example 2), 1.2% human white preparation group (Group 6 in Example 2) and frozen preparation group (frozen stem cell injection prepared in Example 4).

[0180] Cell therapy: 2-3 days after AD-like symptoms were induced, stem cell therapy was performed (days 31, 33 and 35). After treatment, the back lesions were stimulated again in the same way as above, also for 7 days, and follow-up testing was performed 3 days after the stimulation was completed.

[0181] 3. Experimental results: Fig.15 The skin condition of experimental mice. Fig.16 is the level of IgE in the serum of experimental mice. The above results show that fresh human umbilical cord mesenchymal stem cell subcutaneous injection containing 0.6% human serum albumin (0.6% human albumin preparation group) can effectively relieve the symptoms of atopic dermatitis. Fresh human umbilical cord mesenchymal stem cell subcutaneous injection containing 1.2% human serum albumin (1.2% human albumin preparation group) has no inhibitory effect on the inflammatory factors of atopic dermatitis, and the skin damage is also improved; it fails to reduce the levels of IL-1β and TNF-α, the inflammatory factors of osteoarthritis, and fails to increase the level of type 2 collagen. Frozen human umbilical cord mesenchymal stem cell subcutaneous injection has no inhibitory effect on the inflammatory factors of atopic dermatitis, and the skin damage is also improved, indicating that frozen human umbilical cord mesenchymal stem cell subcutaneous injection has no effect on relieving the symptoms of atopic dermatitis.

[0182] Example 6 Application of fresh human umbilical cord mesenchymal stem cell subcutaneous injection in the treatment of osteoarthritis

[0183] 1. Experimental conditions and materials: 20 SPF male SD rats, weighing 180-220 g, were purchased from Shanghai Slake and raised in the Animal Experiment Center of Zhejiang University.

[0184] Iodoacetic acid (Shanghai Bioengineering), sodium pentobarbital, normal saline, sterile cotton balls, 75% medical alcohol, 1 mL sterile syringe, PBS, paraformaldehyde, type 2 collagen Elisa kit.

[0185] 2. Experimental groups: blank group, model group and treatment group. Each group had 5 mice, randomly assigned. The treatment groups for local subcutaneous injection of stem cells in mice were divided into 0.6% human white preparation group (Group 5 in Example 2), 1.2% human white preparation group (Group 6 in Example 2) and frozen preparation group (frozen stem cell injection prepared in Example 4).

[0186] 3. Experimental methods:

[0187] (1) Rats were fasted for 12 h before anesthesia, but not water, and anesthetized by intraperitoneal injection of 0.2 mL / 100 g of 3% sodium pentobarbital.

[0188] (2) After anesthesia, the rat was placed in a lateral lying position with its body stretched out for routine skin preparation and ethanol disinfection of the right knee of the rat. The knee joint was flexed 45°, and the concave part of the knee joint was found. A 1 mL syringe was inserted into the concave part of the knee joint, and a hollow feeling was felt. 50 μL of 25 mg / mL iodoacetic acid was injected into the rat knee joint cavity with a syringe to induce early rat knee osteoarthritis.

[0189] (3) One week after the model was established, stem cell therapy was performed. The stem cells were combined with hydrogels and implanted subcutaneously near the joints for treatment.

[0190] 4. Experimental results

[0191] like Fig.17 As shown, the articular cartilage was repaired after stem cell treatment. Fig.18 As shown, the content of type 2 collagen, a cartilage marker, in the serum of rats treated with stem cells was increased, and the levels of inflammatory factors IL-1β and TNF-α were reduced. This indicates that subcutaneous injection of fresh human umbilical cord mesenchymal stem cells can effectively relieve the symptoms of osteoarthritis.

[0192] The articular cartilage treated with frozen stem cell preparations did not show significant repair. Fig.19 , 20 As shown in Figure 21, the levels of cartilage markers type 2 collagen, IL-1β and TNF-α in the serum of rats treated with frozen stem cells were not significantly different from those in the model group, and the level of type 2 collagen protein was not increased. This indicates that subcutaneous injection of frozen human umbilical cord mesenchymal stem cells cannot effectively relieve the symptoms of osteoarthritis.

[0193] Table 13 Information on materials required for the preparation of the above human umbilical cord mesenchymal stem cells or human umbilical cord mesenchymal stem cell preparations

[0194]

[0195]

[0196] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mesenchymal stem cell preparation, characterized in that: Contains 5×10 mesenchymal stem cells 6 ~1.2×10 7 cells / mL, human albumin 0.1-0.6% w / v and solvent; The mesenchymal stem cells are obtained by culturing in a serum-free medium, and the mesenchymal stem cells are mesenchymal stem cells obtained by reviving frozen mesenchymal stem cells, inoculating them into a culture container, culturing them until they are completely adherent, and then digesting them.

2. The mesenchymal stem cell preparation according to claim 1, characterized in that The solvent includes a composite electrolyte.

3. The mesenchymal stem cell preparation according to claim 1, characterized in that The mesenchymal stem cells include mesenchymal stem cells separated from at least one of bone marrow, umbilical cord, placenta, fat, bone, dental pulp and endometrium; and / or, the mesenchymal stem cells include mesenchymal stem cells differentiated from embryonic stem cells and / or pluripotent stem cells; Optionally, the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

4. The mesenchymal stem cell preparation according to any one of claims 1 to 3, characterized in that Contains 1.0×10 mesenchymal stem cells 7 cells / mL, human serum albumin 0.6% w / v and compound electrolytes; the mesenchymal stem cells are umbilical cord mesenchymal stem cells.

5. The method for preparing the mesenchymal stem cell preparation according to any one of claims 1 to 4, characterized in that: The method comprises the steps of resuscitating frozen mesenchymal stem cells and inoculating them into a culture container until they are completely attached to the wall, digesting and counting them, and then mixing them with a formula amount of human serum albumin and a solvent to obtain the mesenchymal stem cell preparation.

6. The method for preparing a mesenchymal stem cell preparation according to claim 5, characterized in that: The mesenchymal stem cells include umbilical cord mesenchymal stem cells, and the umbilical cord mesenchymal stem cells are prepared according to the following method: Cultivating freshly isolated human umbilical cord tissue blocks, and when the cell confluence of mesenchymal stem cells crawling out of the umbilical cord tissue blocks is 80% to 90%, collecting and obtaining P0 generation mesenchymal stem cells; subculturing the P0 generation mesenchymal stem cells, and when the cell confluence is 80% to 90%, collecting the mesenchymal stem cells, and continuing to subculture until obtaining the target generation of mesenchymal stem cells; Optionally, the target generation is the fifth generation of mesenchymal stem cells.

7. Use of the mesenchymal stem cell preparation according to any one of claims 1 to 4, or the preparation method according to claim 5 or 6 in any of the following: (I) Preparation of drugs for preventing and / or treating atopic dermatitis; (II) Preparation of medicaments for preventing and / or treating psoriasis; (III) Preparation of medicaments for preventing and / or treating osteoarthritis; (IV) preparing a medicament for preventing and / or treating hair loss; (V) Preparation of a medicament for preventing and / or treating immune rejection.

8. A pharmaceutical composition, characterized in that The invention comprises the mesenchymal stem cell preparation according to any one of claims 1 to 4 and a pharmaceutically acceptable excipient.

9. The pharmaceutical composition according to claim 8, characterized in that The pharmaceutical composition is an injection.

10. The pharmaceutical composition according to claim 9, characterized in that The auxiliary material includes gel.

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