CN114410579A

CN114410579A - Composition, mesenchymal stem cell serum-free culture medium containing composition, and preparation and application of mesenchymal stem cell serum-free culture medium

Info

Publication number: CN114410579A
Application number: CN202210276513.XA
Authority: CN
Inventors: 陈东煌; 陈海佳; 姜交华; 戚康艺; 王悦萌
Original assignee: Guangdong Guoke Cell Technology Co ltd; Bioisland Laboratory
Current assignee: Guangdong Guoke Cell Technology Co ltd; Bioisland Laboratory
Priority date: 2022-03-21
Filing date: 2022-03-21
Publication date: 2022-04-29
Anticipated expiration: 2042-03-21
Also published as: CN114410579B

Abstract

The invention relates to a composition, a mesenchymal stem cell serum-free culture medium containing the composition, and preparation and application of the composition. The compositions comprise beta-estradiol, G-CSF, M-CSF and IL-6. Compared with the prior art, the invention has the following beneficial effects: the invention provides a composition containing beta-estradiol, G-CSF, M-CSF and IL-6, which is added on the basis of a serum-free basic culture medium of mesenchymal stem cells so as to form a specific culture medium formula. The mesenchymal stem cells prepared by the culture medium formula have obviously stronger immunoregulation capability and high immunocompetence, can improve the clinical application effect of the mesenchymal stem cells, and reduce the dosage of the mesenchymal stem cells.

Description

Composition, mesenchymal stem cell serum-free culture medium containing composition, and preparation and application of mesenchymal stem cell serum-free culture medium

Technical Field

The invention belongs to the field of biomedicine, and relates to a mesenchymal stem cell serum-free medium, and preparation and application thereof.

Background

The stem cell therapy is to utilize stem cells or cells derived from the stem cells to transplant the stem cells or the cells into a body by a special technology to promote tissue and organ regeneration, body rehabilitation and the like so as to achieve the purpose of treating or relieving diseases. Stem cells are the cells of the engineer in the body, and when other cells, tissues and organs are damaged, inflammation or homeostasis is changed, the stem cells in the human body can become seed cells of blood, bone, skin, muscle and the like, and further differentiate into cells required by the body.

Currently, the types of stem cells used in clinical or clinical research mainly include hematopoietic stem cells, embryonic stem cells, induced pluripotent stem cells, Mesenchymal Stem Cells (MSCs), and the like. Among them, mesenchymal stem cells are the most popular cell type recently used as a novel drug or a clinical research object. Mesenchymal stem cells are derived from mesoderm in early development, and are a type of non-hematopoietic stem cells widely existing in bone marrow, subcutaneous fat, periosteum, muscle, synovium, synovial fluid, liver, peripheral tissues, umbilical cord blood, placenta and other tissues. The mesenchymal stem cells have high self-renewal capacity and multidirectional differentiation potential and can be cultured and amplified in vitro; under different induction conditions, the cells can be differentiated into bones, cartilages, muscles, nerves, cardiac muscles, endothelia, fat and the like in vitro, still have multidirectional differentiation potential after continuous subculture and cryopreservation, and can be used as ideal seed cells for repairing tissue and organ injuries caused by aging and pathological changes.

In recent two years, the types of diseases related to the research of mesenchymal stem cells are more and more abundant, including graft-versus-host disease, knee osteoarthritis, liver failure, ischemic stroke, acute respiratory distress syndrome, idiopathic pulmonary fibrosis, rheumatoid arthritis, inflammatory bowel disease, diabetic ulcer, psoriasis, chronic periodontitis and the like. In addition, in the aspect of mesenchymal stem cell clinical research projects, the method relates to various different indications, such as macular degeneration, spinal cord injury, graft-versus-host disease, heart failure, myocardial infarction, liver disease, severe acute kidney injury and other diseases.

The mesenchymal stem cells have wide clinical application prospect, are the first choice seed cells for cell replacement therapy and tissue engineering, and are the research hotspots in the field of transplantation and the treatment of autoimmune diseases. How to provide the mesenchymal stem cells with good immunoregulatory ability is one of the key factors influencing the clinical application effect of the mesenchymal stem cells.

Disclosure of Invention

In view of the above, it is an object of the present invention to provide a specific composition, which has good immunoregulatory ability, for mesenchymal stem cells prepared by using a serum-free culture medium for mesenchymal stem cells containing the composition.

The purpose of the invention can be realized by the following technical scheme:

it is an object of the present invention to provide a composition comprising beta-estradiol, G-CSF, M-CSF and IL-6.

In some of these embodiments, the composition comprises 1mg/L to 100mg/L β -estradiol, 1 μ g/L to 100 μ g/L G-CSF, 1 μ g/L to 100 μ g/L M-CSF, and 1 μ g/L to 100 μ g/L IL-6.

In some of these embodiments, the composition comprises 5mg/L to 50mg/L β -estradiol, 5 μ g/L to 50 μ g/L G-CSF, 5 μ g/L to 50 μ g/L M-CSF, and 5 μ g/L to 50 μ g/L IL-6.

In some of these embodiments, the composition comprises 5mg/L to 35mg/L β -estradiol, 5 μ g/L to 35 μ g/L G-CSF, 5 μ g/L to 35 μ g/L M-CSF, and 5 μ g/L to 35 μ g/L IL-6.

In some of these embodiments, the composition comprises 6mg/L to 20mg/L β -estradiol, 8 μ g/L to 15 μ g/L G-CSF, 8 μ g/L to 18 μ g/L M-CSF, and 5 μ g/L to 20 μ g/L IL-6.

The invention also provides a mesenchymal stem cell serum-free culture medium, which comprises the composition and the mesenchymal stem cell serum-free basal culture medium.

The invention also provides a preparation method of the serum-free culture medium for the mesenchymal stem cells, which comprises the step of mixing the composition and the serum-free basal culture medium for the mesenchymal stem cells.

The fourth purpose of the invention is to provide a preparation method of the mesenchymal stem cells, which comprises the step of culturing the mesenchymal stem cells by using the mesenchymal stem cell serum-free culture medium.

The fifth purpose of the invention is to provide the mesenchymal stem cell prepared by the preparation method.

The invention also aims to provide an application of the mesenchymal stem cells in preparing an immunity regulating medicament.

In some of these embodiments, the immune modulating drug is a drug that inhibits the immune activity or/and proliferation of lymphocytes.

The seventh object of the present invention is to provide a method for inhibiting the immunological activity or/and proliferation of lymphocytes, which comprises co-culturing the mesenchymal stem cells and the lymphocytes.

In some embodiments, the culture medium used for co-culture is the serum-free culture medium of the mesenchymal stem cells.

Compared with the prior art, the invention has the following beneficial effects:

the invention provides a composition comprising beta-estradiol, G-CSF, M-CSF and IL-6, which is added on the basis of a serum-free basal medium of mesenchymal stem cells, thereby forming a specific culture medium formula. The mesenchymal stem cells prepared by the culture medium formula have obviously stronger immunoregulation capability and high immunocompetence, can improve the clinical application effect of the mesenchymal stem cells, and reduce the dosage of the mesenchymal stem cells.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a summary of the proportion (%) of dividing cells in each medium group obtained in example 3 of the present invention;

FIG. 2 is a summary of the proportion (%) of CD4+ cells in each medium group obtained in example 4 of the present invention;

FIG. 3 is a summary of the proportion (%) of CD8+ cells in each medium group obtained in example 4 of the present invention;

FIG. 4 is a graph summarizing the TNF- α contents (pg/mL) of each of the culture medium groups obtained in example 5 of the present invention;

FIG. 5 is a graph showing the total IL-1. beta. content (pg/mL) in each of the medium groups obtained in example 5 of the present invention;

FIG. 6 is a graph showing the total IL-4 content (pg/mL) in each of the medium groups obtained in example 6 of the present invention;

FIG. 7 is a graph showing the total IL-10 content (pg/mL) in each medium group obtained in example 6 of the present invention.

Detailed Description

In order that the invention may be more fully understood, reference will now be made to the following description. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

Unless otherwise specified, the reagents used in the present invention are commercially available or can be prepared by known methods; unless defined otherwise or clearly indicated by the background, all technical and scientific terms used in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

Term(s) for

Unless otherwise stated or contradicted, terms or phrases used herein have the following meanings:

the term "and/or", "and/or" as used herein is intended to be inclusive of any one of the two or more items listed in association, and also to include any and all combinations of the items listed in association, including any two or more of the items listed in association, any more of the items listed in association, or all combinations of the items listed in association. It should be noted that when at least three items are connected by at least two conjunctive combinations selected from "and/or", "or/and", "and/or", it should be understood that, in the present application, the technical solutions definitely include the technical solutions all connected by "logic and", and also the technical solutions all connected by "logic or". For example, "A and/or B" includes A, B and A + B. For example, the embodiments of "a, and/or, B, and/or, C, and/or, D" include any of A, B, C, D (i.e., all embodiments using "logical or" connection "), any and all combinations of A, B, C, D (i.e., any two or any three of A, B, C, D), and four combinations of A, B, C, D (i.e., all embodiments using" logical and "connection).

The present invention relates to "plural", etc., and indicates that it is 2 or more in number, unless otherwise specified. For example, "one or more" means one or two or more.

As used herein, "a combination thereof," "any combination thereof," and the like, includes all suitable combinations of any two or more of the listed items.

In the present specification, the term "suitable" in "a suitable combination, a suitable manner," any suitable manner "and the like shall be construed to mean that the technical solution of the present invention can be implemented, the technical problem of the present invention can be solved, and the technical effect of the present invention can be achieved.

The terms "preferably", "better" and "suitable" are used herein only to describe preferred embodiments or examples, and it should be understood that the scope of the present invention is not limited by these terms.

In the present invention, "further", "still further", "specifically" and the like are used for descriptive purposes to indicate differences in content, but should not be construed as limiting the scope of the present invention.

In the present invention, "optionally", "optional" and "optional" refer to the presence or absence, i.e., to any one of two juxtapositions selected from "present" and "absent". If multiple optional parts appear in one technical scheme, if no special description exists, and no contradiction or mutual constraint relation exists, each optional part is independent.

In the present invention, the terms "first", "second", "third", "fourth", etc. in the terms of "first aspect", "second aspect", "third aspect", "fourth aspect", etc. are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or quantity, nor as implying that importance or quantity indicating the technical feature being indicated. Also, "first," "second," "third," "fourth," etc. are used for non-exhaustive enumeration of description purposes only and should not be construed as a closed limitation to the number.

In the present invention, the technical features described in the open type include a closed technical solution composed of the listed features, and also include an open technical solution including the listed features.

In the present invention, where a range of values (i.e., a numerical range) is recited, unless otherwise specified, alternative distributions of values within the range are considered to be continuous, and include both the numerical endpoints of the range (i.e., the minimum and maximum values), and each numerical value between the numerical endpoints. Unless otherwise specified, when a numerical range refers to integers only within the numerical range, both endpoints of the numerical range, and each integer between the two endpoints are included, and herein, it is equivalent to reciting each integer directly, such as t being an integer selected from 1 to 10, meaning t being any integer selected from the group of integers consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10. Further, when multiple range-describing features or characteristics are provided, the ranges may be combined. In other words, unless otherwise indicated, the ranges disclosed herein are to be understood to include any and all subranges subsumed therein.

The temperature parameter in the present invention is not particularly limited, and may be a constant temperature treatment or a variation within a certain temperature range. It will be appreciated that the described thermostatic process allows the temperature to fluctuate within the accuracy of the instrument control. Allowing fluctuations in the range of, for example,. + -. 5 deg.C,. + -. 4 deg.C,. + -. 3 deg.C,. + -. 2 deg.C, + -. 1 deg.C.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. The citation referred to herein is incorporated by reference in its entirety for all purposes unless otherwise in conflict with the present disclosure's objectives and/or technical solutions. Where a citation is referred to herein, the definition of a reference in the document, including features, terms, nouns, phrases, etc., that is relevant, is also incorporated by reference. In the present invention, when the citation is referred to, the cited examples and preferred embodiments of the related art features are also incorporated by reference into the present application, but the present invention is not limited to the embodiments. It should be understood that where the citation conflicts with the description herein, the application will control or be adapted in accordance with the description herein.

The mesenchymal stem cells have wide clinical application prospect, are the first choice seed cells for cell replacement therapy and tissue engineering, and are the research hotspots in the field of transplantation and the treatment of autoimmune diseases. However, conventional mesenchymal stem cell culture media generally maintain only the normal proliferation and essential properties of cells.

To this end, the present invention provides a composition comprising β -estradiol, G-CSF, M-CSF and IL-6, which is added on the basis of a serum-free basal medium of mesenchymal stem cells to form a specific medium formulation. The mesenchymal stem cells prepared by the culture medium formula have obviously stronger immunoregulation capability and high immunocompetence, can improve the clinical application effect of the mesenchymal stem cells, and reduce the dosage of the mesenchymal stem cells.

"M-CSF": macrophage colony stimulating factor, macrophage stimulating factor.

"G-CSF": granulocyte colony factor, granulocyte-colony stimulating factor, granulocyte colony stimulating factor, colony stimulating factor.

As a first aspect of the present invention,the present invention provides a composition comprising beta-estradiol, G-CSF, M-CSF and IL-6.

Preferably, the composition comprises 1mg/L to 100mg/L beta-estradiol, 1 μ g/L to 100 μ g/LG-CSF, 1 μ g/L to 100 μ g/L M-CSF and 1 μ g/L to 100 μ g/L IL-6.

Preferably, the composition comprises 5mg/L to 50mg/L β -estradiol, 5 μ g/L to 50 μ g/L G-CSF, 5 μ g/L to 50 μ g/L M-CSF, and 5 μ g/L to 50 μ g/L IL-6;

preferably, the composition comprises 5mg/L to 35mg/L β -estradiol, 5 μ g/L to 35 μ g/L G-CSF, 5 μ g/L to 35 μ g/L M-CSF, and 5 μ g/L to 35 μ g/L IL-6;

more preferably, the composition comprises 6mg/L to 20mg/L of beta-estradiol, 8 μ g/L to 15 μ g/LG-CSF, 8 μ g/L to 18 μ g/L M-CSF and 5 μ g/L to 20 μ g/L of IL-6.

As a second aspect of the present invention,the invention provides a serum-free culture medium for mesenchymal stem cells, which comprises the composition of the first aspect and a serum-free basal culture medium for mesenchymal stem cells.

In one example, the mesenchymal stem cell serum-free medium comprises 1mg/L-100mg/L beta-estradiol, 1 μ g/L-100 μ g/L G-CSF, 1 μ g/L-100 μ g/L M-CSF, 1 μ g/L-100 μ g/L IL-6, and a mesenchymal stem cell serum-free basal medium.

Preferably, the mesenchymal stem cell serum-free culture medium comprises 5mg/L-50mg/L beta-estradiol, 5 mug/L-50 mug/L G-CSF, 5 mug/L-50 mug/L M-CSF, 5 mug/L-50 mug/L IL-6 and a mesenchymal stem cell serum-free basal culture medium.

Preferably, the serum-free culture medium of the mesenchymal stem cells comprises 5mg/L-35mg/L beta-estradiol, 5 mug/L-35 mug/L G-CSF, 5 mug/L-35 mug/L M-CSF, 5 mug/L-35 mug/L IL-6 and a serum-free basal culture medium of the mesenchymal stem cells.

Preferably, the serum-free culture medium of the mesenchymal stem cells comprises 6mg/L-20mg/L beta-estradiol, 8 mug/L-15 mug/L G-CSF, 8 mug/L-18 mug/L M-CSF, 5 mug/L-20 mug/L IL-6 and a serum-free basal culture medium of the mesenchymal stem cells.

The serum-free basic culture medium for the mesenchymal stem cells is not particularly limited, can be any serum-free culture medium suitable for culturing the mesenchymal stem cells, can well maintain the proliferation activity and dryness of the mesenchymal stem cells, and can be selected from, but not limited to: PremCyls chemical refined Serum-free Media, a product of Guangdong national cell technology Co., Ltd, or Serum-free culture medium of any one of the same types of MSCs.

The concentrations of the components in the first and second aspects of the present invention are used as reference concentrations.

As a third aspect of the present invention,the invention provides a preparation method of the serum-free culture medium for the mesenchymal stem cells, which comprises the step of mixing the composition of the first aspect and the serum-free basal culture medium for the mesenchymal stem cells.

It will be appreciated that the preparation method may further include the step of adding antibiotics to the mixture obtained by mixing, as necessary. The antibiotic of the present invention, such as penicillin and streptomycin, may be added in one kind or in plural kinds.

It will be appreciated that the method of preparation may also include the step of sterilisation, for example by membrane filtration, as required.

AsIn a fourth aspect of the present invention,the invention provides a preparation method of mesenchymal stem cells, which comprises the step of culturing the mesenchymal stem cells by using the mesenchymal stem cell serum-free culture medium of the second aspect.

Optionally, the mesenchymal stem cell of the present invention is an umbilical cord-derived mesenchymal stem cell.

As a fifth aspect of the present invention,the invention provides the mesenchymal stem cell prepared by the preparation method of the fourth aspect.

As a sixth aspect of the present invention,the invention provides an application of the mesenchymal stem cell in the fifth aspect in preparing an immunity regulating medicament.

In one example, the immune modulating drug is a drug that inhibits the immune activity or/and proliferation of lymphocytes.

As a seventh aspect of the present invention,the present invention provides a method of inhibiting the immunological activity or/and proliferation of lymphocytes, the method comprising co-culturing the mesenchymal stem cells and lymphocytes of the fifth aspect.

In one example, the medium employed for co-cultivation may be selected from, but is not limited to: the mesenchymal stem cell serum-free culture medium.

In the invention, the components and reagents are conventional commercial products. Such as beta-estradiol (cat # E2758) from Sigma; G-CSF (cat # 300-23), M-CSF (cat # 300-25), IL-6 (cat # 200-06) were purchased from Peprotech.

Example 1 UC-MSCs (umbilical cord-derived mesenchymal Stem cells) culture

1. Rinsing the umbilical cord with PBS, and removing meconium and the like; rinsing with 75% ethanol for 1-2 min, and rinsing with PBS;

2. removing arteriovenous and epidermis from umbilical cord, and cutting into 1mm³Tissue mass;

3. the tissue blocks were inoculated in 10cm petri dishes (about 2cm long umbilical cord per dish) and evenly distributed with plastic pasteur pipettes;

4. standing at room temperature for 15-30 min to make the tissue block stick on the bottom of the culture dish;

5. add 5mL of complete medium slowly (note add liquid strength, do not wash away tissue mass);

6. standing at 37 deg.C and 5% CO₂Culturing in a cell culture box;

7. supplementing 5mL of complete culture medium after culturing for 48 h;

8. cell climbing out is observed, and half amount of liquid change is carried out;

9. standing at 37 deg.C and 5% CO₂Continuously culturing in a cell culture box, removing tissue blocks when the observed cell clone number is more than 10, and replacing 10mL of fresh culture medium;

10. continuously culturing for 2-3 days until the cell confluency reaches more than 80%, and performing subculture.

The cells were used for subsequent experiments after 3 serial passages.

Example 2 preparation of serum-free Medium for mesenchymal Stem cells and design of Experimental group

Control group: the PremCUlt MSCs chemical ly Defined Serum-free Media (brand: BI; product name: MSC) was cultured using a conventional Serum-free medium

XF Medium)。

Experimental group 1 to experimental group 5: the mesenchymal stem cell Serum-free basal medium used was PremCUlt MSCs chemical refined Serum-free Media, to which mixtures of different concentration combinations were added, see Table 1.

TABLE 1

Experimental groups	Beta-estradiol	G-CSF	M-CSF	IL-6
					Control group	——	——	——	——
Experimental group 1	1mg/L	1μg/L	1μg/L	1μg/L
					Experimental group 2	5mg/L	5μg/L	5μg/L	5μg/L
Experimental group 3	10mg/L	10μg/L	10μg/L	10μg/L
					Experimental group
4	50mg/L	50μg/L	50μg/L	50μg/L
					Experimental group 5	100mg/L	100μg/L	100μg/L	100μg/L

Experimental group 6: the change of the serum-free medium of mesenchymal stem cells with respect to experimental group 5 was that the concentration of G-CSF was 150. mu.g/L.

Experimental group 7: the change of the serum-free medium of mesenchymal stem cells with respect to experimental group 5 was that the concentration of M-CSF was 150. mu.g/L.

Experimental group 8: the change of the serum-free medium of mesenchymal stem cells with respect to experimental group 5 was that the concentration of IL-6 was 150. mu.g/L.

Comparative group 1: the change of the serum-free culture medium of the mesenchymal stem cells relative to the experimental group 5 is that GM-CSF is used to replace G-CSF.

Comparative group 2: the change of the serum-free culture medium of the mesenchymal stem cells relative to the experimental group 5 is that GM-CSF is used to replace M-CSF.

Comparative group 3: the mesenchymal stem cell serum-free medium was changed relative to experimental group 5 by replacing IL-6 with IL-17.

The mesenchymal stem cell serum-free culture medium of each experimental group and each comparative group can be prepared by the following method:

dissolving the above components according to their respective dissolution characteristics to obtain 1000 × solution, filtering with 0.22 μm filter membrane for sterilization, and storing at-20 deg.C or below.

When in use, the mixture is redissolved at room temperature, and added into the mesenchymal stem cell serum-free basal medium according to the ratio of 1:1000 to be mixed uniformly.

Example 3 Effect of MSCs on lymphocyte proliferative Activity

1. MSCs treatment

The P3 generation MSCs are expressed by 1 × 10⁴/cm²Inoculating at 75cm²Culturing in a culture bottle, and adding beta-estradiol, G-CSF, M-CSF and IL-6 into each experimental group according to the experimental group of the example 2 when the cell confluency reaches above 60% and the control group is not treated; continuing the cultureAfter 48 hours, adding 800 mu L of mitomycin C with the concentration of 1 mu g/mu L into the MSCs of the control group and each experimental group for pretreatment for 1 to 2 hours; the cells of each group were digested and collected and prepared into cell suspensions at 1X 10⁵And inoculating the seeds in a 6-well plate for 24 h.

2. PBMCs (peripheral blood mononuclear cells) treatment

Taking 50mL of fresh peripheral blood, and separating and extracting PBMCs by using Ficoll (lymph separation medium); resuspending PBMCs to 1X 10 with culture Medium⁷Adding 1mmol of CFSE (carboxyfluorescein diacetate succinimidyl ester) fluorescent dye into the suspension per mL, uniformly mixing, incubating at room temperature in a dark place, and slightly shaking the suspension once at intervals of 3-5 min; after incubation for 10 minutes, putting the mixture into a refrigerator at 4 ℃ for refrigeration for 5 minutes to stop marking, then centrifuging the mixture at 1000rpm/5min, and removing supernatant; the same centrifugation parameters were washed 3 times with medium.

3. Cell seeding

Negative group: CFSE treated PBMCs at 1X 10⁶Inoculate/well into new 6-well plate;

positive group: CFSE treated PBMCs at 1X 10⁶Perwell into a new 6-well plate, while adding 20. mu.L of activator PHA (phytohemagglutinin) at a concentration of 1. mu.g/. mu.L;

and (3) co-culture group: CFSE-treated PBMCs were inoculated into 6-well plates previously seeded with each group of MSCs and co-cultured while 20. mu.L of activator PHA (phytohemagglutinin) was added at a concentration of 1. mu.g/. mu.L.

After 5 days of culture, each set of PBMCs was collected and CFSE fluorescence intensity was measured using flow cytometry.

The results of the inhibition efficiency of the compositions to be protected according to the present invention on the proliferative activity of lymphocytes are shown in table 2 and fig. 1. The results showed that experimental group 3 was very significantly different from each of the other groups (. about.p < 0.01); the experimental group 1, the experimental group 2, the experimental group 4 and the experimental group 5 have significant differences (# #, p <0.01) with the control group, the comparison group 1, the comparison group 2 and the comparison group 3; the experimental group 8 has significant difference (delta, p is less than 0.05) with the control group, the control group 1, the control group 2 and the control group 3; the experimental group 6 and the experimental group 7 have no significant difference from the control group.

The results show that the compositions to be protected according to the invention are effective in increasing the inhibition of lymphocyte division activity by MSCs.

TABLE 2

# # denotes p <0.01, Δ denotes p <0.05

Example 4 modulation of different immune cell subsets by MSCs

1. MSCs treatment

The P3 generation MSCs are expressed by 1 × 10⁴/cm²Inoculating at 75cm²Culturing in a culture bottle, and adding beta-estradiol, G-CSF, M-CSF and IL-6 into each experimental group according to the experimental group of the example 2 when the cell confluency reaches above 60% and the control group is not treated; after continuing culturing for 48h, adding 800 μ L mitomycin C with concentration of 1 μ g/μ L into the MSCs of the control group and each experimental group for pretreatment for 1h-2 h; the cells of each group were digested and collected and prepared into cell suspensions at 1X 10⁵And inoculating the seeds in a 6-well plate for 24 h.

2. PBMCs treatment

Taking 50mL of fresh peripheral blood, and separating and extracting PBMCs by using Ficoll (lymph separation medium); resuspending PBMCs to 1X 10 with culture Medium⁷/mL。

3. Cell seeding

Negative group: PBMCs at 1 × 10⁶Inoculate/well into new 6-well plate;

positive group: PBMCs at 1 × 10⁶Perwell into a new 6-well plate, while adding 20. mu.L of activator PHA (phytohemagglutinin) at a concentration of 1. mu.g/. mu.L;

and (3) co-culture group: PBMCs were inoculated into the above 6-well plates already seeded with each group of MSCs for co-culture, while 20. mu.L of activator PHA (phytohemagglutinin) at a concentration of 1. mu.g/. mu.L was added.

After 3 days of culture, groups of PBMCs were collected and the proportion of CD4+ cells and CD8+ cells was determined for each group using flow cytometry.

The results of the modulation of different subpopulations of immune cells by the compositions to be protected according to the invention are shown in table 3 and fig. 2 and 3.

From table 3 and fig. 2, it can be seen that the MSCs have a modulating effect on the CD4+ cell subset, and the experimental group 3 is very different from each of the other groups (x, p < 0.01); the experimental group 1, the experimental group 2, the experimental group 4 and the experimental group 5 have significant differences (###, p <0.01) with the control group, the comparison group 1 and the comparison group 2; the experimental group 8 has significant difference (delta, p is less than 0.05) with the control group, the comparison group 1 and the comparison group 2; the experimental group 6, the experimental group 7 and the comparative group 3 have no significant difference from the control group.

The results show that the composition to be protected can effectively improve the upregulation effect of the MSCs on the CD4+ cell subset.

From table 3 and fig. 3, it can be seen that MSCs have a modulating effect on CD8+ cell subsets, and the experimental group 3 has significant differences (Δ, p <0.05) from the experimental group 1, the experimental group 2, the experimental group 4, and the experimental group 5, and has significant differences (x, p <0.01) from the control group, the experimental group 6, the experimental group 7, the experimental group 8, the control group 1, the control group 2, and the control group 3; the experimental group 1, the experimental group 2, the experimental group 4 and the experimental group 5 have significant differences (###, p <0.01) with the control group, the experimental group 6, the experimental group 7, the experimental group 8, the comparison group 1, the comparison group 2 and the comparison group 3; the control group 1, the control group 2 and the control group 3 have very significant differences (Δ Δ, p < 0.01).

The results show that the composition to be protected can effectively improve the inhibition effect of the MSCs on the CD8+ cell subset.

TABLE 3

#, Δ Δ denotes p <0.01, Δ denotes p <0.05

Example 5 modulation of lymphocyte proinflammatory factor secretion by MSCs

1. MSCs treatment

The P3 generation MSCs are expressed by 1 × 10⁴/cm²Inoculating at 75cm²Culturing in a culture bottle, and adding beta-estradiol, G-CSF, M-CSF and IL-6 into each experimental group according to the experimental group of the example 2 when the cell confluency reaches above 60% and the control group is not treated; after continuing culturing for 48h, adding 800 μ L mitomycin C with concentration of 1 μ g/μ L into the MSCs of the control group and each experimental group for pretreatment for 1-2 h; the cells of each group were digested and collected and prepared into cell suspensions at 1X 10⁵And inoculating the seeds in a 6-well plate for 24 h.

2. PBMCs treatment

Taking 50mL of fresh peripheral blood, and separating and extracting PBMCs by using Ficoll (lymph separation medium); resuspending PBMC to 1X 10 with culture Medium⁷/mL。

3. Cell seeding

Negative group: PBMCs at 1 × 10⁶Inoculate/well into new 6-well plate;

After 3 days of culture, culture supernatants of the cells of each group were collected, and the secretion amounts of proinflammatory factors TNF-a and IL-1. beta. were measured by ELISA kit.

The results of the compositions of the present invention on MSCs to regulate the secretion of pro-inflammatory factors from lymphocytes are shown in table 5 and fig. 4 and 5. According to table 5 and fig. 4, the regulation effect of MSCs on the secretion of the proinflammatory factor TNF- α of lymphocytes is known, and the experimental group 3 is significantly different from other experimental groups (x, p < 0.01); the experimental group 1, the experimental group 2, the experimental group 4, the experimental group 5, the experimental group 6, the experimental group 7 and the experimental group 8 have significant differences (### # and p <0.01) with the control group, the comparison group 1, the comparison group 2 and the comparison group 3; experimental group 1, experimental group 2, experimental group 4, experimental group 5 were significantly different from experimental group 6, experimental group 7, and experimental group 8 (, p < 0.05).

The result shows that the composition to be protected can effectively improve the inhibition effect of the MSCs on the secretion of the proinflammatory factor TNF-alpha of lymphocytes.

According to table 5 and fig. 5, the regulation effect of MSCs on the secretion of proinflammatory factor IL-1 β of lymphocytes is known, and the experimental group 3 is significantly different from other experimental groups (x, p < 0.01); the experimental group 1, the experimental group 2, the experimental group 4 and the experimental group 5 have significant differences (###, p <0.01) with the control group, the experimental group 6, the experimental group 7, the experimental group 8, the comparison group 1, the comparison group 2 and the comparison group 3; the experimental group 6, the experimental group 7 and the experimental group 8 have significant difference (delta, p is less than 0.05) with the control group; the comparative groups 1, 2 and 3 have very significant differences (Δ Δ, p <0.01) from the control group and each experimental group.

The result shows that the composition to be protected can effectively improve the inhibition effect of the MSCs on the secretion of the proinflammatory factor IL-1 beta of lymphocytes.

TABLE 5

#, Δ Δ denotes p <0.01, Δ denotes p <0.05

Example 6 modulation of lymphocyte anti-inflammatory factor secretion by MSCs

1. MSCs treatment

2. PBMC treatment

Taking 50mL of fresh peripherySeparating blood, and extracting PBMC with Ficoll (lymph separating medium); resuspending PBMC to 1X 10 with culture Medium⁷/mL。

3. Cell seeding

Negative group: PBMC at 1X 10⁶Inoculate/well into new 6-well plate;

positive group: PBMC at 1X 10⁶Perwell into a new 6-well plate, while adding 20. mu.L of activator PHA (phytohemagglutinin) at a concentration of 1. mu.g/. mu.L;

and (3) co-culture group: PBMC were inoculated into the above 6-well plate with each group of MSCs for co-culture, while 20. mu.L of activator PHA (phytohemagglutinin) was added at a concentration of 1. mu.g/. mu.L.

After 3 days of culture, culture supernatants of the respective groups of cells were collected, and the amounts of the secretion of the anti-inflammatory factors IL-4 and IL-10 were measured by using an ELISA kit.

The results of the compositions of the present invention on MSCs to regulate the secretion of lymphokine are shown in table 6 and fig. 7. From table 6 and fig. 6, it can be seen that MSCs have a significant difference (x, p <0.01) in the effect of modulating the secretion of the lymphocyte anti-inflammatory factor IL-4 from the experimental group 3; the experimental group 1, the experimental group 2, the experimental group 4 and the experimental group 5 have significant differences (##, p <0.01) with the control group, the experimental group 6, the experimental group 7, the experimental group 8, the comparison group 1, the comparison group 2 and the comparison group 3; the experimental group 6, the experimental group 7 and the experimental group 8 have no significant difference (p is more than 0.05) from the control group; the comparative groups 1, 2 and 3 have very significant differences (Δ Δ, p <0.01) from the control group and each experimental group.

The result shows that the composition to be protected can effectively improve the secretion promotion effect of the MSCs on the lymphocyte anti-inflammatory factor IL-4.

From table 6 and fig. 7, it can be seen that MSCs have a significant difference (x, p <0.01) in the effect of modulating the secretion of the lymphocyte anti-inflammatory factor IL-10 from each of the other experimental groups in experimental group 3; the experimental group 1, the experimental group 2, the experimental group 4 and the experimental group 5 have significant differences (##, p <0.01) with the control group, the experimental group 6, the experimental group 7, the experimental group 8, the comparison group 1, the comparison group 2 and the comparison group 3; the experimental group 6, the experimental group 7, the experimental group 8 and the comparative group 3 have no significant difference (p is more than 0.05) from the control group; the comparison groups 1 and 2 have very significant differences (delta, p <0.01) with the comparison group, each experiment group and the comparison group 3.

The result shows that the composition to be protected can effectively improve the secretion promotion effect of the MSCs on the lymphocyte anti-inflammatory factor IL-10.

TABLE 6

Experimental groups	IL-4(pg/mL)	IL-10(pg/mL)
			Negative group	23.27±1.03	112.27±3.32
Positive group	45.03±1.11	211.36±4.31
			Control group	64.71±1.97	240.39±3.16
Experimental group 1	75.03±3.17##	258.95±3.98##
			Experimental group 2	77.26±1.89##	271.78±3.63##
Experimental group 3	115.30±2.86**	337.42±4.11**
			Experimental group 4	98.30±1.03##	299.52±4.63##
Experimental group 5	89.30±1.98##	288.95±3.82##
			Experimental group 6	63.92±0.85	242.64±2.74
Experimental group 7	64.17±1.67	240.49±1.74
			Experimental group 8	63.75±2.20	243.16±3.72
Comparative group 1	43.04±0.65ΔΔ	211.36±4.85ΔΔ
			Comparative group 2	43.55±2.47ΔΔ	215.89±4.45ΔΔ
Comparative group 3	44.69±3.68ΔΔ	242.70±4.03

Δ Δ denotes p <0.01

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A composition comprising β -estradiol, G-CSF, M-CSF and IL-6.

2. The composition of claim 1, wherein the composition comprises 1mg/L to 100mg/L β -estradiol, 1 μ g/L to 100 μ g/L G-CSF, 1 μ g/L to 100 μ g/L M-CSF, and 1 μ g/L to 100 μ g/LIL-6;

preferably, the composition comprises 5mg/L to 50mg/L β -estradiol, 5 μ g/L to 50 μ g/L G-CSF, 5 μ g/L to 50 μ g/L M-CSF, and 5 μ g/L to 50 μ g/LIL-6;

preferably, the composition comprises 5mg/L to 35mg/L β -estradiol, 5 μ g/L to 35 μ g/L G-CSF, 5 μ g/L to 35 μ g/L M-CSF, and 5 μ g/L to 35 μ g/LIL-6;

more preferably, the composition comprises 6mg/L to 20mg/L β -estradiol, 8 μ g/L to 15 μ g/LG-CSF, 8 μ g/L to 18 μ g/L M-CSF, and 5 μ g/L to 20 μ g/LIL-6.

3. A serum-free culture medium for mesenchymal stem cells, comprising the composition of claim 1 or 2 and a serum-free basal medium for mesenchymal stem cells.

4. The method for preparing the serum-free culture medium of the mesenchymal stem cells, which is characterized by comprising the step of mixing the composition with the serum-free basic culture medium of the mesenchymal stem cells.

5. A method for preparing mesenchymal stem cells, comprising the step of culturing mesenchymal stem cells in the mesenchymal stem cell serum-free medium according to claim 3.

6. Mesenchymal stem cells produced by the production method according to claim 5.

7. Use of the mesenchymal stem cell of claim 6 in the preparation of an immune modulating medicament.

8. The use according to claim 7, wherein the immune modulating medicament is a medicament for inhibiting the immune activity or/and proliferation of lymphocytes.

9. A method of inhibiting the immune activity or/and proliferation of lymphocytes, comprising co-culturing the mesenchymal stem cells of claim 6 with lymphocytes.

10. The method for inhibiting the immunological activity or/and proliferation of lymphocytes according to claim 9, wherein the culture medium used for co-culture is the serum-free culture medium of mesenchymal stem cells according to claim 3.