CN112029718B

CN112029718B - Method for serum-free domestication culture of mesenchymal stem cells and culture medium used by same

Info

Publication number: CN112029718B
Application number: CN202010980501.6A
Authority: CN
Inventors: 肖海蓉; 魏卿; 许晓椿; 刘庆喜
Original assignee: Shenzhen Boya Perception Pharmaceutical Co ltd
Current assignee: Shenzhen Boya Perception Pharmaceutical Co ltd
Priority date: 2020-09-17
Filing date: 2020-09-17
Publication date: 2023-04-07
Anticipated expiration: 2040-09-17
Also published as: CN112029718A

Abstract

The invention relates to a method for culturing mesenchymal stem cells by serum-free domestication and a culture medium used by the method. One aspect relates to the use of a combination of 3- (2-methoxyphenoxy) -1, 2-propanediol and magnesium in the preparation of a acclimatization medium for serum-free acclimatization culture of mesenchymal stem cells, comprising: 8-12 ng/mL of transferrin, 8-12 ng/mL of insulin, 0.08-0.12 mg/mL of 3- (2-methoxyphenoxy) -1, 2-propylene glycol and the like, and a balanced DMEM/F12 culture medium. The invention also relates to a method for culturing the mesenchymal stem cells by serum-free domestication; also relates to a domestication culture medium for serum-free domestication culture of the mesenchymal stem cells; also relates to a method for preparing the domesticated culture medium for serum-free domesticated culture of the mesenchymal stem cells, which comprises the step of adding transferrin, insulin, fetal calf serum or other substances into a DMEM/F12 culture medium. The method of the invention exhibits the excellent effects described in the specification.

Description

Method for serum-free domestication culture of mesenchymal stem cells and culture medium used by same

Technical Field

The invention belongs to the fields of biotechnology and biomedicine, and relates to a culture method of human mesenchymal stem cells, in particular to a method for serum-free in-vitro domestication culture of human mesenchymal stem cells.

Background

Mesenchymal Stem Cells (MSCs), such as human mesenchymal stem cells, were first isolated from bone marrow, a class of tissue stem cells derived from the mesoderm, with multipotent differentiation potential and self-renewal capacity, and have the ability to differentiate into various adult cells, such as osteoblasts, chondrocytes, adipocytes, endothelial cells, nerve cells, muscle cells, hepatocytes, and the like, under specific conditions in vivo and in vitro (Caplan ai. Mesenchymal stem cells. J ortho res.1991, 9. Recent research shows that the mesenchymal stem cells have the functions of immunoregulation and hematopoietic support, and are easy to introduce and express exogenous genes. Therefore, the mesenchymal stem cells are not only seed cells in the construction of tissue engineering bone, cartilage and cardiac muscle and important carrier cells in gene therapy, but also have wide application prospect in hematopoietic stem cell transplantation and organ transplantation because the mesenchymal stem cells promote hematopoietic reconstruction and inhibit graft-versus-host reaction. Mesenchymal stem cells have the characteristic of adherent growth in vitro, and by utilizing the characteristic, the mesenchymal stem cells are successfully separated and cultured from various tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood and the like.

At present, the reported mesenchymal stem cells are mainly derived from bone marrow and are obtained by adopting a density gradient centrifugation method. Although the separation method is simple, the donor needs to undergo a painful operation for taking marrow, and has a high infection chance in and after the material is taken; because the content of MSC in human bone marrow is very rare, every 10 ⁵ ～10 ⁶ Only about 1 of the mononuclear cells are present, and the number, proliferation and differentiation capacity of the mesenchymal stem cells in the bone marrow are remarkably reduced with the increase of the age, so that the research and the application, particularly the clinical application of the mesenchymal stem cells are limited. The placenta, which originates from the extraembryonic mesoderm during the embryonic development, is composed of mesenchyme, blood vessels and trophoblasts, and contains a large amount of mesenchyme components. The latest research shows that the placenta contains abundant stem cells, and the separation and culture of the pluripotent stem cells from the placenta opens up a new and abundant source for experimental research and clinical application.

There are many reports in the literature on the establishment of a placental stem cell bank by isolating stem cells from placenta, for example, the invention entitled "method for isolating placental mesenchymal stem cells and culturing them in vitro" disclosed in CN101270349A (chinese patent application No. 200810061267.6, published 24/9/2008); CN101693884A (chinese patent application No. 200910117522.9, published 2010, 4 months and 14 days) entitled "a method for separating and extracting stem cells from placenta, umbilical cord or adipose tissue"; CN102146359A (chinese patent application No. 201110005964.1, published 2011, 8/10) discloses an invention entitled "method for extracting original mesenchymal stem cells from placenta and serum-free expansion". In addition, chinese patent application No. 201210044648X discloses a method of isolating mesenchymal stem cells from placenta.

Stem cells are progenitors of human cells, and all cells in our body are derived from stem cells. When cells in the body age, die or the lesion denatures, stem cells grow and transform out of cells that can replace them. As seed cells, the compound is mainly used for treating various refractory diseases of tissue cells and organ injuries which cannot be naturally repaired by an organism clinically; as immunoregulatory cells, for the treatment of immune rejection and autoimmune diseases. Human mesenchymal stem cells are important members of a stem cell family, are derived from mesoderm in early development, belong to pluripotent stem cells, and are discovered in bone marrow at first, so that the human mesenchymal stem cells are increasingly concerned by people due to the characteristics of multidirectional differentiation potential, hematopoietic support, promotion of stem cell implantation, immune regulation, self-replication and the like. Initial clinical studies were conducted by Lazarus et al in 1995, who collected autologous MSCs of patients with remission stage hematological tumors, cultured for 4 to 7 weeks for in vitro amplification, and then intravenously injected into the patients, who were divided into 3 groups, administered different doses of MSCs, respectively, and no toxic side effects were observed after injection, suggesting that MSCs are safe and reliable for transplantation therapy. Then, clinical reports of autologous MSCs are gradually increased, and the disease types comprise hematopoietic reconstruction after radiotherapy and chemotherapy, graft-versus-host disease (GVHD), heart system diseases and the like, and clinical intravenous infusion is proved to be safe and reliable in the reports.

The in vitro culture process of mesenchymal stem cells is a key step related to the use safety of stem cells as therapeutic drugs. The composition of the culture process, in particular of the culture medium, is a major influencing parameter. In the methods described in the prior documents, when the mesenchymal stem cells are cultured and passaged, serum such as fetal bovine serum is added to the culture medium, in particular, 10% fetal bovine serum is usually added, and for example, the mesenchymal stem cells are cultured and passaged by using an MSC complete medium (which is DMEM/F12 medium containing 10% fetal bovine serum).

On the one hand, however, the cost of fetal bovine serum is rather high, which is disadvantageous for the culture of mesenchymal stem cells; on the other hand, the presence of fetal bovine serum as an exogenous material of animal origin in stem cells poses a potential risk to the safety of clinical use of the cells. Therefore, it is meaningful to culture mesenchymal stem cells without serum acclimation.

CN108642002A (201810047983.2) discloses a method for culturing human mesenchymal stem cells by serum-free domestication. The method uses a fresh serum-free basal culture medium, adds and replaces serum-containing complete culture solution of in vitro human mesenchymal stem cells according to a certain proportion, and uses exosome and microcapsule components secreted by the mesenchymal stem cells in the culture process as supplements to enable the mesenchymal stem cells to finish the acclimation of serum-free culture, thereby finally realizing a serum-free culture system. The density and the activity rate of the domesticated mesenchymal stem cells and the main markers on the cell surface have no obvious difference with a serum culture medium, and the components and the abundance of cytokines secreted by a culture solution and exosomes are detected to find that the expression quantity of bFGF, PDGF and VEGF related to the growth of blood vessels is improved compared with that of the domesticated cells. However, in the CN108642002A method, cells are directly cultured in a serum-free medium, so that the nutrition of the cell environment is drastically reduced, and many adverse effects are exerted on the cells.

Therefore, it is still expected in the art to provide a new method for culturing mesenchymal stem cells, and in particular to provide a new method for serum-free in vitro acclimation culture of human mesenchymal stem cells.

Disclosure of Invention

The invention aims to provide a unique culture method aiming at the defects in the prior art, and provides a serum-free domestication culture method for in vitro culture of human mesenchymal stem cells. The purpose of the invention is realized by the following technical scheme.

In a first aspect of the present invention, there is provided a method for serum-free acclimation culture of mesenchymal stem cells, comprising the steps of:

(1) The placenta surface was washed with tissue washes to remove surface congestion, the umbilical cord was trimmed and the internal blood vessels were removed.

(2) Stripping Wharton's jelly from umbilical cord, cleaning, and cutting into 2-3mm pieces ³ Transferred to T75 flasks at 1mL per flask.

(3) Add slowly 10mL of MSC complete medium to the flask and gently shakeMoving the culture flask so that the umbilical cord tissue mass is spread in the flask, and the CO is 5% at 37 ℃ ₂ And (3) carrying out static culture in an incubator, supplementing liquid once every 2-3 days, and supplementing liquid for 2-3 times, removing tissue blocks, replacing a total amount of MSC complete culture medium, and continuing to culture until the cell grows to 70% of fusion degree, and then digesting the obtained cell.

(4) The culture medium in the culture bottle is sucked and discarded, PBS is sucked to rinse the culture bottle to remove residual fetal calf serum, 3mL of 0.25% trypsin solution is added for digestion, the culture bottle is tapped to enable cells to fall to the bottom of the culture bottle, 10mL of MSC complete culture medium is added to neutralize the trypsin so as to stop the digestion process, a liquid transfer machine is used for blowing off the cells, cell suspension is collected in a centrifuge tube, centrifugation is carried out, supernatant is discarded, and cells with P0 generation marked as cell sediment are obtained.

(5) Resuspending the P0 generation cell pellet with 10mL MSC acclimation medium containing less than 10% fetal calf serum, counting cells, and then inoculating at a density of 1 × 10 ⁴ Individual MSC cells/cm ² Inoculating into T75 culture flask, supplementing the above MSC acclimatization medium to 10mL, and 5% CO at 37 deg.C ₂ And (3) performing static culture in an incubator for more than 72 hours until the cell fusion degree reaches 80%, sucking a culture medium in the culture bottle, sucking PBS to rinse the culture bottle to remove residual fetal calf serum, adding 3mL of 0.25% trypsin solution for digestion, slightly tapping the culture bottle to enable cells to fall to the bottom of the culture bottle, adding 10mL of MSC acclimation culture medium used in the step to neutralize the trypsin so as to terminate the digestion process, blowing off the cells by using a pipettor, collecting cell suspension in a centrifuge tube, centrifuging, and discarding supernatant to obtain cell sediment which is marked as P1 generation cells.

(6) And (5) treating the cells obtained in the previous step in the operation process by using MSC acclimation culture media which sequentially reduce the concentration of fetal calf serum to be finally 0% by adopting the culture and digestion method in the step (5) to obtain the final serum-free in-vitro acclimation-cultured mesenchymal stem cells. Optionally (c) is

(7) And (4) continuously culturing and passaging the mesenchymal stem cells of the last generation obtained in the last step by using an MSC (mesenchymal stem cell) acclimation culture medium containing 0% fetal calf serum (so as to continuously transfer to the P20 generation mesenchymal stem cells cultured in the serum-free acclimation culture).

The method according to the first aspect of the invention, wherein the tissue wash is a 0.9% physiological saline solution containing 1% penicillin, 1% streptomycin.

The method according to the first aspect of the present invention, wherein the gordonia gel is washed with the tissue washing solution in the step (2).

The method according to the first aspect of the present invention, wherein the MSC complete medium is DMEM/F12 medium containing 10% fetal bovine serum.

The method according to the first aspect of the present invention, wherein in step (3), 3 to 4mL of the MSC complete medium is supplemented every 2 to 3 days.

The method according to the first aspect of the present invention, wherein in step (4), after adding the trypsin solution, the culture flask is gently shaken to allow trypsin to sufficiently cover the cell surface, digestion is carried out for 1min until the cells are microscopically contracted into a round shape, and the culture flask is gently tapped to allow the cells to fall off in a stream of sand to the bottom of the culture flask.

The method according to the first aspect of the present invention, wherein in step (4), the mixture is centrifuged at 1400rpm for 5min in a centrifuge.

The method according to the first aspect of the present invention, wherein in step (5), the P0 generation cell pellet is resuspended in MSC acclimation medium containing 7-9.5% fetal bovine serum.

The method according to the first aspect of the present invention, wherein in step (5), the P0 generation cell pellet is resuspended in MSC acclimation medium containing 8-9.5% fetal bovine serum, for example, MSC acclimation medium containing 8%, 8.5%, 9%, or 9.5% fetal bovine serum.

The method according to the first aspect of the present invention, wherein in the repeating of step (5) in step (6), the concentration of fetal bovine serum in the MSC acclimation medium decreases as the number of generations increases until the concentration of fetal bovine serum in the MSC acclimation medium is 0.

According to the method of the first aspect of the invention, in the step (6), the serum-free mesenchymal stem cells cultured by in vitro acclimation are obtained from the generation P4 to the generation P10 by gradually reducing the concentration of the fetal calf serum.

According to the method of the first aspect of the invention, in the step (6), the serum-free mesenchymal stem cells cultured by in vitro acclimation are obtained by gradually reducing the concentration of the fetal calf serum until the generation of P4-P9.

According to the method of the first aspect of the invention, in the step (6), the serum-free mesenchymal stem cells cultured by in vitro acclimation are obtained by gradually reducing the concentration of the fetal calf serum until the generation of P4-P8.

According to the method of the first aspect of the invention, in the step (7), the mesenchymal stem cells cultured by the in vitro domestication of 0% fetal serum finally obtained in the step (6) are continuously cultured and passaged by the domestication culture medium containing 0% fetal bovine serum MSC, and are continuously transferred to the P20 generation mesenchymal stem cells cultured by the serum-free domestication culture.

The method according to the first aspect of the present invention, wherein the MSC acclimation medium comprises: 8-12 ng/mL of transferrin, 8-12 ng/mL of insulin, 0-9.5% of fetal calf serum and a balanced DMEM/F12 culture medium.

The method according to the first aspect of the present invention, wherein the MSC acclimation medium comprises: transferrin 10ng/mL, insulin 10ng/mL, fetal calf serum 0-9.5%, balancing DMEM/F12 culture medium.

The method according to the first aspect of the present invention, wherein the MSC acclimation medium further comprises 3- (2-methoxyphenoxy) -1, 2-propanediol and magnesium nitrate. In one embodiment, the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol is 0.08 to 0.12mg/mL. In one embodiment, the magnesium nitrate is present in a concentration of from 4 to 6 μ g/mL of magnesium ion. In one embodiment, the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol is 0.1mg/mL. In one embodiment, the magnesium nitrate is 5 μ g/mL in magnesium ion concentration. 3- (2-methoxyphenoxy) -1, 2-propanediol, also known as guaifenesin, having CAS registry number 93-14-1.

The method according to the first aspect of the present invention, wherein the MSC acclimation medium comprises: 8-12 ng/mL of transferrin, 8-12 ng/mL of insulin, 0.08-0.12 mg/mL of 3- (2-methoxyphenoxy) -1, 2-propylene glycol, 4-6 mu g/mL of magnesium nitrate in terms of magnesium ion concentration, 0-9.5% of fetal calf serum and a balanced DMEM/F12 culture medium. The method according to the first aspect of the present invention, wherein the MSC acclimation medium comprises: 10ng/mL of transferrin, 10ng/mL of insulin, 0.1mg/mL of 3- (2-methoxyphenoxy) -1, 2-propylene glycol, 5 mu g/mL of magnesium nitrate in terms of magnesium ion concentration, 0-9.5% of fetal calf serum and a balance of DMEM/F12 culture medium. In the present invention, the term "equilibrium amount" means an amount added to the total amount, which herein means adding the rest of the substances to their respective prescribed concentrations on the basis of DMEM/F12 medium, as a medium, unless otherwise specified.

Further, the second aspect of the present invention provides a acclimatization medium for serum-free acclimatization culture of mesenchymal stem cells, comprising: 8-12 ng/mL of transferrin, 8-12 ng/mL of insulin, 0-9.5% of fetal bovine serum and a balanced DMEM/F12 culture medium.

The acclimatisation medium according to the second aspect of the invention, comprising: transferrin 10ng/mL, insulin 10ng/mL, fetal calf serum 0-9.5%, balancing DMEM/F12 culture medium.

The acclimatization medium according to the second aspect of the present invention, wherein the acclimatization medium further comprises 3- (2-methoxyphenoxy) -1, 2-propanediol and magnesium nitrate. In one embodiment, the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol is 0.08 to 0.12mg/mL. In one embodiment, the magnesium nitrate is present in a concentration of 4 to 6 μ g/mL magnesium ions. In one embodiment, the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol is 0.1mg/mL. In one embodiment, the magnesium nitrate is 5 μ g/mL as a concentration of magnesium ions. 3- (2-methoxyphenoxy) -1, 2-propanediol, also known as guaifenesin, having CAS registry number 93-14-1.

The acclimatization medium according to the second aspect of the present invention, wherein the acclimatization medium comprises: 8 to 12ng/mL of transferrin, 8 to 12ng/mL of insulin, 0.08 to 0.12mg/mL of 3- (2-methoxyphenoxy) -1, 2-propylene glycol, 4 to 6 mu g/mL of magnesium nitrate by concentration of magnesium ions, 0 to 9.5 percent of fetal calf serum and a balanced DMEM/F12 culture medium.

The acclimatization medium according to the second aspect of the present invention, wherein the MSC acclimatization medium comprises: 10ng/mL of transferrin, 10ng/mL of insulin, 0.1mg/mL of 3- (2-methoxyphenoxy) -1, 2-propylene glycol, 5 mu g/mL of magnesium nitrate in terms of magnesium ion concentration, 0-9.5% of fetal calf serum and a balance of DMEM/F12 culture medium. In the present invention, the term "equilibrium amount" means an amount added to the total amount, which herein means adding the rest of the substances to their respective prescribed concentrations on the basis of DMEM/F12 medium, as a medium, unless otherwise specified.

The acclimatization medium according to the second aspect of the present invention, wherein the serum-free acclimatization culture of the mesenchymal stem cells comprises the steps of:

(3) Slowly adding 10mL of MSC complete medium to the flask, gently shaking the flask to allow the umbilical cord tissue pieces to be spread in the flask, 5% CO at 37 ℃% ₂ And (3) carrying out static culture in an incubator, supplementing liquid once every 2-3 days, and supplementing liquid for 2-3 times, removing tissue blocks, replacing a total amount of MSC complete culture medium, and continuing to culture until the cell grows to 70% of fusion degree, and then digesting the obtained cell.

(5) Resuspending the P0 generation cell pellet with 10mL of MSC acclimation culture medium containing less than 10% fetal calf serum, counting cells, and then inoculating at a density of 1 × 10 ⁴ Individual MSC cells/cm ² Inoculating into T75 culture flask, supplementing the above MSC acclimatization medium to 10mL, 5% CO at 37 deg.C ₂ And (3) performing static culture in an incubator for more than 72 hours until the cell fusion degree reaches 80%, sucking the culture medium in the culture bottle, sucking PBS (phosphate buffer solution) to rinse the culture bottle to remove residual fetal bovine serum, adding 3mL of 0.25% trypsin solution for digestion, slightly beating the culture bottle to enable the cells to fall to the bottom of the culture bottle, adding 10mL of MSC (mesenchymal stem cell) acclimation culture medium used in the step, neutralizing the trypsin to stop the digestion process, blowing off the cells by using a pipettor, collecting cell suspension in a centrifuge tube, centrifuging, and discarding supernatant to obtain cell sediment which is marked as P1 generation cells.

(6) And (5) treating the cells obtained in the previous step in the operation process by using the MSC acclimation culture medium for sequentially reducing the concentration of fetal calf serum to be 0% finally by adopting the culture and digestion method in the step (5) to obtain the final serum-free mesenchymal stem cells subjected to in vitro acclimation culture. Optionally (c) is

The acclimatization medium according to the second aspect of the invention, wherein the tissue washing solution is a 0.9% physiological saline solution containing 1% penicillin and 1% streptomycin.

The acclimatization medium according to the second aspect of the present invention, wherein the gordonia gum is washed with a tissue washing solution in step (2).

The acclimatization medium according to the second aspect of the present invention, wherein the MSC complete medium is DMEM/F12 medium containing 10% fetal bovine serum.

The acclimatization medium according to the second aspect of the present invention, wherein in step (3), 3-4mL of the MSC complete medium is supplemented every 2-3 days of fluid infusion.

The acclimatization medium according to the second aspect of the invention, wherein in the step (4), after adding the trypsin solution, the culture flask is gently shaken to make the trypsin sufficiently cover the cell surface, the cells are digested for 1min until the cells are microscopically observed to shrink into a round shape, and the culture flask is gently tapped to make the cells shed into a sandy shape to the bottom of the culture flask.

The acclimatization medium according to the second aspect of the present invention, wherein in step (4), the medium is centrifuged at 1400rpm for 5min in a centrifuge.

The acclimatization medium according to the second aspect of the invention, wherein in the step (5), the P0 generation cell pellet is resuspended in MSC acclimatization medium containing 7-9.5% fetal bovine serum.

The acclimatization medium according to the second aspect of the invention, wherein in step (5), the P0 generation cell pellet is resuspended in an MSC acclimatization medium containing 8-9.5% fetal bovine serum, for example, an MSC acclimatization medium containing 8%, 8.5%, 9%, or 9.5% fetal bovine serum.

According to the acclimatization culture medium of the second aspect of the invention, in the process of repeating the step (5) in the step (6), the concentration of the fetal calf serum in the MSC acclimatization culture medium is reduced along with the increase of the cell generation until the concentration of the fetal calf serum in the MSC acclimatization culture medium is 0.

The acclimation culture medium according to the second aspect of the invention, wherein in the step (6), the serum-free mesenchymal stem cells cultured in vitro are obtained by gradually reducing the concentration of fetal bovine serum until the generation of P4-P10.

The domestication medium according to the second aspect of the invention, wherein in the step (6), the serum-free mesenchymal stem cells cultured in vitro are obtained by gradually reducing the concentration of fetal calf serum until the generation of P4-P9.

The domestication culture medium according to the second aspect of the invention, wherein in the step (6), the serum-free mesenchymal stem cells cultured in vitro are obtained by gradually reducing the concentration of fetal calf serum until the generation of P4-P8.

The domestication culture medium according to the second aspect of the invention, wherein in the step (7), the mesenchymal stem cells obtained by the in vitro domestication culture of the 0% fetal serum finally obtained in the step (6) are continuously cultured and passaged by using the domestication culture medium containing 0% fetal bovine serum MSC, and are continuously transferred to the P20 generation mesenchymal stem cells obtained by serum-free domestication culture.

Further, the third aspect of the present invention provides a method for preparing an acclimatized medium for serum-free acclimatization of cultured mesenchymal stem cells, which comprises the step of adding transferrin, insulin, fetal bovine serum or other substances to a DMEM/F12 medium.

According to the method of the third aspect of the present invention, the acclimatization medium comprises: 8-12 ng/mL of transferrin, 8-12 ng/mL of insulin, 0-9.5% of fetal calf serum and a balanced DMEM/F12 culture medium.

According to the method of the third aspect of the present invention, the acclimatization medium comprises: transferrin 10ng/mL, insulin 10ng/mL, fetal calf serum 0-9.5%, balancing DMEM/F12 culture medium.

According to the method of the third aspect of the present invention, the acclimatization medium prepared by the method further comprises 3- (2-methoxyphenoxy) -1, 2-propanediol and magnesium nitrate. In one embodiment, the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol is 0.08 to 0.12mg/mL. In one embodiment, the magnesium nitrate is present in a concentration of 4 to 6 μ g/mL magnesium ions. In one embodiment, the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol is 0.1mg/mL. In one embodiment, the magnesium nitrate is 5 μ g/mL as a concentration of magnesium ions.

According to the method of the third aspect of the present invention, the acclimatization medium comprises: 8 to 12ng/mL of transferrin, 8 to 12ng/mL of insulin, 0.08 to 0.12mg/mL of 3- (2-methoxyphenoxy) -1, 2-propylene glycol, 4 to 6 mu g/mL of magnesium nitrate by concentration of magnesium ions, 0 to 9.5 percent of fetal calf serum and a balanced DMEM/F12 culture medium.

According to the method of the third aspect of the present invention, the acclimatization medium comprises: 10ng/mL of transferrin, 10ng/mL of insulin, 0.1mg/mL of 3- (2-methoxyphenoxy) -1, 2-propylene glycol, 5 mu g/mL of magnesium nitrate in terms of magnesium ion concentration, 0-9.5% of fetal calf serum and a balance of DMEM/F12 culture medium.

The method according to the third aspect of the present invention, wherein the serum-free acclimated culture of the mesenchymal stem cells comprises the steps of:

(2) Stripping Wharton's jelly from umbilical cord, cleaning, and cutting into 2-3mm pieces ³ The small pieces of the rubber,transfer to T75 flasks, 1mL per flask.

(3) Slowly adding 10mL of MSC complete medium to the flask, gently shaking the flask to allow the umbilical cord tissue mass to lay flat in the flask, 5% CO at 37 ℃% ₂ And (3) performing static culture in an incubator, supplementing liquid once every 2-3 days, and supplementing liquid for 2-3 times, removing tissue blocks, replacing a total amount of MSC complete culture medium, and continuously culturing until the cells grow to 70% of fusion degree, and then digesting the obtained cells.

(5) Resuspending the P0 generation cell pellet with 10mL of MSC acclimation culture medium containing less than 10% fetal calf serum, counting cells, and then inoculating at a density of 1 × 10 ⁴ Individual MSC cells/cm ² Inoculating into T75 culture flask, supplementing the above MSC acclimatization medium to 10mL, 5% CO at 37 deg.C ₂ And (3) performing static culture in an incubator for more than 72 hours until the cell fusion degree reaches 80%, sucking a culture medium in the culture bottle, sucking PBS to rinse the culture bottle to remove residual fetal calf serum, adding 3mL of 0.25% trypsin solution for digestion, slightly tapping the culture bottle to enable cells to fall to the bottom of the culture bottle, adding 10mL of MSC acclimation culture medium used in the step to neutralize the trypsin so as to terminate the digestion process, blowing off the cells by using a pipettor, collecting cell suspension in a centrifuge tube, centrifuging, and discarding supernatant to obtain cell sediment which is marked as P1 generation cells.

(7) And (4) continuously culturing and subculturing the last generation of the mesenchymal stem cells obtained in the last step by using an MSC (mesenchymal stem cell) acclimation culture medium containing 0% fetal calf serum, and carrying out passage (so as to continuously transfer the mesenchymal stem cells to the P20 generation of the serum-free acclimation culture).

The method according to the third aspect of the present invention, wherein said tissue washing solution is a 0.9% physiological saline solution containing 1% penicillin and 1% streptomycin.

The method according to the third aspect of the present invention, wherein the gordonia gel is washed with the tissue washing solution in the step (2).

The method according to the third aspect of the present invention, wherein the MSC complete medium is DMEM/F12 medium containing 10% fetal bovine serum.

The method according to the third aspect of the present invention, wherein in step (3), 3 to 4mL of the MSC complete medium is supplemented each time every 2 to 3 days of fluid infusion.

According to the third aspect of the present invention, in the step (4), after adding the trypsin solution, the culture flask is gently shaken to allow the trypsin to sufficiently cover the cell surface, the cells are digested for 1min until the cells are microscopically observed to shrink into a circular shape, and the culture flask is gently tapped to allow the cells to fall off in a stream of sand to the bottom of the culture flask.

The method according to the third aspect of the present invention, wherein in the step (4), the mixture is centrifuged at 1400rpm for 5min in a centrifuge.

The method according to the third aspect of the present invention, wherein in step (5), the P0 generation cell pellet is resuspended in MSC acclimation medium containing 7-9.5% fetal bovine serum.

The method according to the third aspect of the present invention, wherein in step (5), the P0 generation cell pellet is resuspended in MSC acclimation medium containing 8-9.5% fetal bovine serum, for example, 8%, 8.5%, 9%, or 9.5% fetal bovine serum.

According to the method of the third aspect of the present invention, in the process of repeating step (5) in step (6), as the number of generations of cells increases, the concentration of fetal bovine serum in the MSC acclimation medium decreases until the concentration of fetal bovine serum in the MSC acclimation medium becomes 0.

According to the method of the third aspect of the invention, in the step (6), the serum-free mesenchymal stem cells cultured in vitro are obtained by gradually reducing the concentration of the fetal bovine serum until the generation of P4-P10.

According to the method of the third aspect of the invention, in the step (6), the serum-free in vitro domestication cultured mesenchymal stem cells are obtained by gradually reducing the concentration of the fetal calf serum until the generation of P4-P9.

According to the method of the third aspect of the invention, in the step (6), the serum-free in vitro domestication cultured mesenchymal stem cells are obtained by gradually reducing the concentration of the fetal calf serum until the generation of P4-P8.

According to the method of the third aspect of the invention, in the step (7), the mesenchymal stem cells cultured by in vitro domestication of 0% fetal serum finally obtained in the step (6) are continuously cultured and passaged by using a domestication culture medium containing 0% fetal bovine serum MSC, and are continuously transferred to the mesenchymal stem cells of generation P20 which are cultured by serum-free domestication.

Further, the fourth aspect of the present invention provides the use of a combination of 3- (2-methoxyphenoxy) -1, 2-propanediol and magnesium nitrate in the preparation of a acclimation medium for serum-free acclimation culture of mesenchymal stem cells.

Use according to the fourth aspect of the invention, wherein the acclimatizing medium comprises: 8-12 ng/mL of transferrin, 8-12 ng/mL of insulin, 0-9.5% of fetal bovine serum and a balanced DMEM/F12 culture medium.

Use according to the fourth aspect of the invention, wherein the acclimatizing medium comprises: 10ng/mL transferrin, 10ng/mL insulin, 0-9.5 percent fetal bovine serum and balance DMEM/F12 culture medium.

The use according to the fourth aspect of the invention, wherein the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol in the acclimatizing medium is 0.08-0.12 mg/mL. In one embodiment, the magnesium nitrate is present in a concentration of 4 to 6 μ g/mL magnesium ions. In one embodiment, the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol is 0.1mg/mL. In one embodiment, the magnesium nitrate is 5 μ g/mL as a concentration of magnesium ions.

Use according to the fourth aspect of the invention, wherein the acclimatizing medium comprises: 8 to 12ng/mL of transferrin, 8 to 12ng/mL of insulin, 0.08 to 0.12mg/mL of 3- (2-methoxyphenoxy) -1, 2-propylene glycol, 4 to 6 mu g/mL of magnesium nitrate by concentration of magnesium ions, 0 to 9.5 percent of fetal calf serum and a balanced DMEM/F12 culture medium.

Use according to the fourth aspect of the invention, wherein the acclimatizing medium comprises: 10ng/mL of transferrin, 10ng/mL of insulin, 0.1mg/mL of 3- (2-methoxyphenoxy) -1, 2-propylene glycol, 5 mu g/mL of magnesium nitrate in terms of magnesium ion concentration, 0-9.5% of fetal calf serum and a balance of DMEM/F12 culture medium.

The use according to the fourth aspect of the present invention, wherein the serum-free acclimated cultured mesenchymal stem cells comprises the steps of:

(5) Resuspending the P0 generation cell pellet with 10mL MSC acclimation medium containing less than 10% fetal calf serum, counting cells, and then inoculating at a density of 1 × 10 ⁴ Individual MSC cells/cm ² Inoculating into T75 culture flask, supplementing the above MSC acclimatization medium to 10mL, 5% CO at 37 deg.C ₂ Static culture in incubatorAnd (3) more than 72 hours until the cell fusion degree reaches 80%, absorbing the culture medium in the culture bottle, sucking PBS to rinse the culture bottle to remove residual fetal calf serum, adding 3mL of 0.25% trypsin solution for digestion, slightly beating the culture bottle to enable the cells to fall to the bottom of the culture bottle, adding 10mL of MSC acclimation culture medium used in the step to neutralize the trypsin so as to stop the digestion process, blowing off the cells by using a pipettor, collecting cell suspension in a centrifuge tube, centrifuging, and discarding supernatant to obtain cell sediment which is marked as P1 generation cells.

Use according to the fourth aspect of the invention, wherein the tissue washing solution is a 0.9% physiological saline solution containing 1% penicillin and 1% streptomycin.

Use according to the fourth aspect of the present invention, wherein the gordonia gum is washed with the tissue washing solution in the step (2).

The use according to the fourth aspect of the present invention, wherein the MSC complete medium is DMEM/F12 medium containing 10% fetal bovine serum.

The use according to the fourth aspect of the present invention, wherein in step (3), 3 to 4mL of the MSC complete medium is supplemented each time every 2 to 3 days of fluid infusion.

The use according to the fourth aspect of the invention, wherein in the step (4), after adding the trypsin solution, the culture flask is gently shaken to make the trypsin sufficiently cover the cell surface, the cell surface is digested for 1min until the cells are microscopically observed to shrink into a round shape, and the culture flask is gently tapped to make the cells shed into a sandy shape to the bottom of the culture flask.

Use according to the fourth aspect of the invention, wherein in step (4), the mixture is centrifuged in a centrifuge at 1400rpm for 5min.

The use according to the fourth aspect of the present invention, wherein in step (5), the P0 generation cell pellet is resuspended in MSC acclimation medium containing 7-9.5% fetal bovine serum.

The use according to the fourth aspect of the present invention, wherein in step (5), the P0 generation cell pellet is resuspended in MSC acclimation medium containing 8 to 9.5% fetal bovine serum, for example, 8%, 8.5%, 9%, or 9.5% fetal bovine serum.

The use according to the fourth aspect of the present invention, wherein in the repeating of step (5) in step (6), the concentration of fetal bovine serum in the MSC acclimation medium decreases as the number of generations increases until the concentration of fetal bovine serum in the MSC acclimation medium becomes 0.

The use of the fourth aspect of the present invention, wherein in the step (6), the serum-free mesenchymal stem cells cultured by in vitro acclimation are obtained by gradually reducing the concentration of the fetal bovine serum until the generation of P4-P10.

The use of the fourth aspect of the invention, wherein in the step (6), the serum-free mesenchymal stem cells cultured in vitro are obtained by gradually reducing the concentration of the fetal bovine serum until the generation P4-P9.

The use of the fourth aspect of the present invention, wherein in the step (6), the serum-free mesenchymal stem cells cultured by in vitro acclimation are obtained by gradually reducing the concentration of the fetal bovine serum until the generation of P4-P8.

According to the fourth aspect of the invention, in the step (7), the mesenchymal stem cells cultured by the in vitro domestication of the 0% fetal serum finally obtained in the step (6) are continuously cultured and passaged by the domestication culture medium containing 0% fetal bovine serum MSC, and are continuously transferred to the P20 generation mesenchymal stem cells cultured by the serum-free domestication culture.

Of the various process steps described in this invention, those skilled in the art can, however, readily generalize the above-described process steps from the detailed disclosure throughout the present invention, even though the specific steps described therein may differ in some detail or in language specific to the steps described in the preparation examples of the detailed description section below.

Any embodiment of any aspect of the present invention may be combined with any other embodiment, as long as they do not contradict each other. Furthermore, in any embodiment of any aspect of the invention, any feature may be applicable to that feature in other embodiments, so long as they do not contradict. The invention is further described below.

All documents cited herein are incorporated by reference in their entirety and to the extent such documents do not conform to the meaning of the present invention, the present invention shall control. Further, the various terms and phrases used herein have the ordinary meaning as is known to those skilled in the art, and even though such terms and phrases are intended to be described or explained in greater detail herein, reference is made to the term and phrase as being inconsistent with the known meaning and meaning as is accorded to such meaning throughout this disclosure.

It is well known that MSC are abundant in origin, mainly organized in perinatal: placenta, umbilical cord, amnion, amniotic fluid, etc. Especially, the MSC from the umbilical cord is more convenient to prepare and has high quality. The umbilical cord comprises a vein and two arteries, wherein Wharton's jelly is arranged around the veins, the outer layer of the umbilical cord is wrapped by the epithelium from the source of amnion, and from the development perspective, the umbilical cord is a passage for forming and passing stem cells, and the research shows that the connective tissue of the human umbilical cord is a tissue source rich in mesenchymal stem cells. The treatment method comprises the steps of firstly stripping umbilical cord arteriovenous and outer epithelium to leave gelatinous tissue (Wharton's jelly), and cutting off the part with clamping marks and extravasated blood on both sides.

The method for serum-free in-vitro domestication culture of the human mesenchymal stem cells, provided by the invention, has the advantages that the prepared mesenchymal stem cells have excellent technical effects in one or more aspects as described herein.

Drawings

FIG. 1: as a result of osteogenic induced differentiation, a is a serum-free acclimation experimental group (the dark part in the original photograph is orange red), and b is a control group.

FIG. 2: and (b) a serum-free domestication experimental group (the dark part in the original photo picture is red), and b a control group.

FIG. 3: chondrogenic induced differentiation results, a serum-free acclimation experimental group (dark blue part in the middle of original photograph, b control group).

FIG. 4: flow cytophenotype results (from top to bottom, CD73, CD90, CD105, HLA-DR, left panel for serum-free acclimatized group, right panel for conventional culture group).

Detailed Description

The present invention will be further described by the following examples, however, the scope of the present invention is not limited to the following examples. It will be understood by those skilled in the art that various changes and modifications may be made to the invention without departing from the spirit and scope of the invention. The present invention generally and/or specifically describes the materials used in the tests, as well as the test methods. The following provides specific materials and sources thereof used in embodiments of the present invention. However, it should be understood that these are exemplary only and not intended to limit the invention, and that materials of the same or similar type, quality, nature or function as the following reagents and instruments may be used in the practice of the invention. The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

All percentages used in the present invention are by weight unless otherwise indicated. In this context, reference to PBS buffer or PBS solution or PBS, etc., unless otherwise specified, refers to phosphate buffer at pH6.8, which is prepared by: 250mL of 0.2mol/L potassium dihydrogen phosphate solution is taken, 118mL of 0.2mol/L sodium hydroxide solution is added, the solution is diluted to 1000mL by water, and the mixture is shaken up to obtain the potassium dihydrogen phosphate.

Preparation of MSC complete culture medium: adding fetal calf serum into DMEM/F12 culture medium to reach concentration of 10%.

Preparation of MSC acclimatization medium (also called reduced serum medium): to DMEM/F12 medium, transferrin (to a concentration of 10ng/mL, from Transferrin, proSpec-Tany), insulin (to a concentration of 10ng/mL, from Sigma-Aldrich), fetal bovine serum (to a concentration of 0 to 9.5%, for example, to a concentration of 9.5%, 9%, 8%, 7.5%, 7%, 6%, 5.5%, 5%, 4%, 3%, 2%, 1%, 0%, etc., from Gibco) was added to prepare MSC acclimation medium containing Transferrin, insulin, and fetal bovine serum at various concentrations.

1. Preparation of mesenchymal Stem cells

Example 1: serum-free domesticated culture MSC

(1) The surface of the placenta was washed with a tissue cleanser (0.9% normal saline containing 1% penicillin, 1% streptomycin) to remove surface congestion, the umbilical cord was cut and the internal blood vessels were removed.

The present embodiment specifically operates as follows: in the biological safety cabinet, articles such as a stainless steel tray, a filter screen, a stainless steel cup, a lunch box, a tissue cleaning fluid (0.9% physiological saline containing 1% penicillin and 1% streptomycin), a pipettor and the like are placed at proper positions. The placenta tissue is taken out by using a surgical forceps and is placed in a stainless steel tray, and the surface of the placenta is cleaned by using a tissue cleaning solution to remove the extravasated blood on the surface of the placenta. The umbilical cord at the connection with the placenta is cut off by using surgical scissors and forceps, and the other end of the umbilical cord is cut off from the back of the umbilical cord sterile clamp. The umbilical cord was removed and washed 3 times with tissue washes to remove surface clots and clots. One end of the cord is grasped with forceps and the cord is grasped with another forceps and slid along the cord tissue to remove residual cord blood from the blood vessels. The umbilical cord is cut into several sections with the length of 2-3cm, then the umbilical cord is cut open along the longitudinal section of the umbilical cord by scissors, and the umbilical cord is flattened to remove 3 blood vessels (2 umbilical arteries and 1 umbilical vein) inside the umbilical cord. Transfer to a new plate.

(2) Stripping Wharton's jelly from umbilical cord, cleaning with tissue cleaning solution, and cutting into 2-3mm pieces ³ Transferred to T75 flasks at 1mL per flask.

The present embodiment specifically operates as follows: stripping the Wharton's jelly with forceps, placing into a culture dish filled with a tissue washing solution, washing with the tissue washing solution, and tearing the Wharton's jelly into strips. Transferring the Wharton's jelly into a 50mL centrifuge tube by using forceps after the treatment is finished, and shearing the Wharton's jelly to 2-3mm by using scissors ³ Small pieces of (2), transferred with a 3mL sterile dropperIn T75 culture bottles, each bottle is 1mL, and the tip of a dropper needs to be cut off by using sterile scissors before use.

(3) Add slowly 10mL of MSC complete medium (DMEM/F12 medium containing 10% fetal bovine serum) to the flask, gently shake the flask to allow the umbilical cord tissue pieces to lay flat in the flask, 5% CO at 37 ℃% ₂ And (3) carrying out static culture in an incubator, supplementing liquid once every 2-3 days (supplementing 3-4mL of MSC complete culture medium every time), removing tissue blocks and replacing the MSC complete culture medium in full quantity to continue culturing until the cell grows to reach 70% fusion degree after 2-3 times of total liquid supplementation, and then digesting the obtained cell.

The present embodiment specifically operates as follows: 10mL of MSC complete medium (DMEM/F12 medium containing 10% fetal bovine serum) was slowly added to the flask, and the flask was gently shaken to flatten the umbilical cord tissue pieces into the flask. Placing the T75 flask at 37 ℃ 5% ₂ And (5) standing and culturing in an incubator. The flasks were removed from the incubator by the third day of culture, supplemented with 3.5mL of MSC complete medium, and replenished once every 3 days thereafter. After 3 times of fluid replacement, cells at the bottom of the culture flask climbed out, the tissue blocks were removed and the complete culture medium of MSC was replaced in full for further culture. When the cells have grown to 70% confluence, the resulting cells are then digested.

(4) The medium in the flask was discarded by aspiration, the flask was rinsed with PBS (20 mL) to remove residual fetal bovine serum, 3mL of 0.25% trypsin solution was added for digestion, the flask was tapped to detach the cells to the bottom of the flask, 10mL of MSC complete medium was added to neutralize trypsin to stop the digestion process, the cells were blown off by a pipette, the cell suspension was collected in a centrifuge tube, centrifuged, and the supernatant was discarded to obtain the cell pellet marked as P0 passage.

The present embodiment specifically operates as follows: prior to cell subculture, 0.25% trypsin, MSC complete medium (DMEM/F12 medium containing 10% fetal bovine serum), PBS were placed at room temperature at equilibrium temperature. The culture flask was taken out of the incubator, the medium in the flask was removed by pipetting, and the flask was rinsed with 20mL of PBS to remove residual fetal bovine serum. Adding 3mL of 0.25% trypsin solution into a culture bottle for digestion, slightly shaking the culture bottle to enable the trypsin to fully cover the cell surface, digesting for about 1min, observing the cells to shrink into a circle under a microscope, slightly beating the culture bottle to enable the cells to shed to the bottom of the culture bottle in a flowing sand shape, adding 10mL of MSC complete culture medium to neutralize the trypsin to stop the digestion process, blowing off the cells by using a pipettor, and collecting cell suspension in a 15mL centrifuge tube. The tube was placed in a centrifuge and centrifuged at 1400rpm for 5min to remove the supernatant and the pellet was scored as P0 passage.

(5) The P0 cell pellet was resuspended in 10mL of MSC acclimatization medium containing 9% fetal bovine serum, cell counting was performed, and then the cell pellet was suspended at an inoculation density of 1X 10 ⁴ Individual MSC cells/cm ² Inoculating into T75 culture flask, supplementing the above MSC acclimatization medium to 10mL, and 5% CO at 37 deg.C ₂ And (3) performing static culture in an incubator for more than 72 hours until the cell fusion degree reaches 80%, sucking the culture medium in the culture bottle, sucking (20 mL) PBS to rinse the culture bottle to remove residual fetal calf serum, adding 3mL of 0.25% trypsin solution for digestion, slightly beating the culture bottle to enable the cells to fall to the bottom of the culture bottle, adding 10mL of MSC domestication culture medium used in the step to neutralize the trypsin to terminate the digestion process, blowing off the cells by using a pipettor, collecting cell suspension in a centrifuge tube, centrifuging, and discarding supernatant to obtain cell precipitates as P1 generation cells.

(6) And (2) treating the cells obtained in the previous step in the operation process by using the culture and digestion method of the step (5) by using MSC domestication culture media which sequentially reduce the concentration of fetal calf serum to 0% finally to obtain the final serum-free in-vitro domesticated cultured mesenchymal stem cells (in the embodiment, the culture and digestion are sequentially performed on P1 generation cells by using the domestication culture medium containing 7.5% of fetal calf serum MSC (to obtain P2 generation cells), the culture and digestion are performed on P2 generation cells by using the domestication culture medium containing 5.5% of fetal calf serum MSC (to obtain P3 generation cells), the culture and digestion are performed on P3 generation cells by using the domestication culture medium containing 3% of fetal calf serum MSC (to obtain P4 generation cells), the culture and digestion are performed on P4 generation cells by using the domestication culture medium containing 0% of fetal calf serum MSC (to obtain P5 generation cells), and the obtained P5 generation cells can be subjected to subsequent detection or used for subsequent serum-free culture and passage. Optionally (c) is

In the present embodiment, the first and second electrodes are,

culturing with MSC complete culture medium (containing 10% fetal calf serum) to obtain P0 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 9% fetal calf serum) to obtain P1 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 7.5% fetal calf serum) to obtain P2 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 5.5% fetal calf serum) to obtain P3 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, and 3% fetal calf serum) to obtain P4 generation cells,

culturing in serum-free culture medium (containing transferrin, insulin, and 0% fetal calf serum) to obtain P5 generation cells; in various assays subsequent to the present invention, the generation of cells (which are also referred to as test cells) is performed, if not otherwise stated.

The reduced serum medium or MSC acclimation medium described in this example, e.g., reduced serum medium for obtaining P1 generation cells, was formulated as follows: adding transferrin (to a concentration of 10 ng/mL), insulin (to a concentration of 10 ng/mL) and fetal calf serum (to a concentration of 9%) into a DMEM/F12 culture medium to prepare MSC acclimation culture media containing transferrin, insulin and fetal calf serum with different concentrations; in other words, the reduced serum medium used to obtain the P1 generation cells of this example contained: 10ng/mL of transferrin, 10ng/mL of insulin, 9% of fetal bovine serum and a balanced DMEM/F12 culture medium. In the present invention, reduced serum medium or MSC acclimatization medium has similar meaning unless otherwise stated.

Example 2: serum-free domesticated culture MSC

The same procedures as in example 1 were carried out for steps (1) to (4), to obtain cells in which the cell pellet was counted as P0 generation.

Steps (5) to (6) were conducted in accordance with the procedures in example 1 except that the gradient change of the decrease in fetal bovine serum concentration in the MSC acclimation medium was used. Optionally (c) is

In the present embodiment, the first and second electrodes are,

culturing with reduced serum culture medium (containing transferrin, insulin, 7% fetal calf serum) to obtain P2 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 5% fetal calf serum) to obtain P3 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin and 3% fetal calf serum) to obtain P4 generation cells,

culturing in serum-free culture medium (containing transferrin, insulin, and 0% fetal calf serum) to obtain P5 generation cells; in various assays that follow the present invention, the generation of cells (which are also referred to as test cells) is used unless otherwise indicated.

Example 3: serum-free domesticated culture MSC

(7) And (4) continuously culturing and passaging the mesenchymal stem cells of the last generation obtained in the last step by using an MSC (mesenchymal stem cell) acclimation culture medium containing 0% fetal calf serum (so as to continuously transfer to the P18 generation mesenchymal stem cells cultured in the serum-free acclimation culture).

In the present embodiment of the present invention,

culturing with reduced serum culture medium (containing transferrin, insulin, and 8% fetal calf serum) to obtain P2 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 6% fetal calf serum) to obtain P3 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin and 4% fetal calf serum) to obtain P4 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 2% fetal calf serum) to obtain P5 generation cells,

culturing in serum-free culture medium (containing transferrin, insulin and 0% fetal calf serum) to obtain P6 generation cells; in various assays subsequent to the present invention, the generation of cells (which are also referred to as test cells) is performed, if not otherwise stated.

Example 4: serum-free domesticated culture MSC

In the present embodiment, the first and second electrodes are,

culturing with reduced serum culture medium (containing transferrin, insulin, 9.5% fetal calf serum) to obtain P1 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, and 8.5% fetal calf serum) to obtain P2 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 7.5% fetal calf serum) to obtain P3 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 6% fetal calf serum) to obtain P4 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 4% fetal calf serum) to obtain P5 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 2% fetal calf serum) to obtain P6 generation cells,

culturing in serum-free culture medium (containing transferrin, insulin and 0% fetal calf serum) to obtain P7 generation cells; in various assays subsequent to the present invention, the generation of cells (which are also referred to as test cells) is performed, if not otherwise stated.

Example 5: serum-free domesticated culture MSC

In the same manner as in example 1, the cell pellets in steps (1) to (4) were counted as P0 generation cells.

(7) And (4) continuously culturing and passaging the mesenchymal stem cells of the last generation obtained in the last step by using an MSC (mesenchymal stem cell) acclimation culture medium containing 0% fetal calf serum (so as to continuously transfer to the mesenchymal stem cells of the P15 generation cultured in the serum-free acclimation culture).

In the present embodiment of the present invention,

culturing with reduced serum culture medium (containing transferrin, insulin, and 8% fetal calf serum) to obtain P1 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 6% fetal calf serum) to obtain P2 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, and 3% fetal calf serum) to obtain P3 generation cells,

culturing in serum-free culture medium (containing transferrin, insulin, and 0% fetal calf serum) to obtain P4 generation cells; in various assays that follow the present invention, the generation of cells (which are also referred to as test cells) is used unless otherwise indicated.

Example 6: serum-free domesticated culture MSC

In the present embodiment, the first and second electrodes are,

culturing with reduced serum culture medium (containing transferrin, insulin, 9% fetal calf serum) to obtain P2 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, and 8.5% fetal calf serum) to obtain P3 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 7.5% fetal calf serum) to obtain P4 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 6% fetal calf serum) to obtain P5 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 4.5% fetal calf serum) to obtain P6 generation cells,

culturing with reduced serum culture medium (containing transferrin, insulin, 2.5% fetal calf serum) to obtain P7 generation cells,

culturing in serum-free culture medium (containing transferrin, insulin and 0% fetal calf serum) to obtain P8 generation cells; in various assays subsequent to the present invention, the generation of cells (which are also referred to as test cells) is performed, if not otherwise stated.

Example 7: serum-free domesticated culture MSC

Referring to the procedures of examples 1 to 6, respectively, except that 3- (2-methoxyphenoxy) -1, 2-propanediol (to a concentration of 0.1mg/mL in each MSC acclimation medium) and magnesium nitrate (to a concentration of 5. Mu.g/mL in each MSC acclimation medium) were additionally added to MSC acclimation media of various fetal bovine serum concentrations, to finally obtain 6 types of serum-free acclimated cultured mesenchymal stem cells, which were referred to as the respective generation cells of examples 71 to 76, respectively; in the various detection tests which follow the present invention, the corresponding passage of cells (which are also referred to as detection cells) for detection in the working examples referred to is carried out, if not stated otherwise.

The reduced serum medium or MSC acclimation medium described in this example, for example, in reference example 1, the reduced serum medium for obtaining P1 generation cells was prepared as follows: adding transferrin (to a concentration of 10 ng/mL), insulin (to a concentration of 10 ng/mL), fetal calf serum (to a concentration of 9%), 3- (2-methoxyphenoxy) -1, 2-propanediol (to a concentration of 0.1 mg/mL) and magnesium nitrate (to a concentration of 5 mug/mL) into a DMEM/F12 medium to prepare MSC acclimation media containing transferrin, insulin, 3- (2-methoxyphenoxy) -1, 2-propanediol, magnesium nitrate and fetal calf serum with different concentrations; in other words, the reduced serum medium used to obtain P1 generation cells in this example with reference to example 1 contained: 10ng/mL of transferrin, 10ng/mL of insulin, 9% of fetal bovine serum, 0.1mg/mL of 3- (2-methoxyphenoxy) -1, 2-propanediol, 5 mu g/mL of magnesium nitrate in terms of magnesium ions and a balance of DMEM/F12 medium. In the present invention, reduced serum medium or MSC acclimatization medium has similar meaning unless otherwise stated.

Example 8: serum-free acclimatization cultureMSC

With reference to the procedures of examples 1 to 6, respectively, except that 3- (2-methoxyphenoxy) -1, 2-propanediol was additionally added to MSC acclimation media of various fetal bovine serum concentrations (to a concentration of 0.1mg/mL in each MSC acclimation media), to finally obtain 6 types of serum-free acclimation-cultured mesenchymal stem cells, which were referred to as the generation cells of examples 81 to 86, respectively; in the various detection tests which follow the present invention, the corresponding passage of cells (which are also referred to as detection cells) for detection in the working examples referred to is carried out, if not stated otherwise.

Example 9: serum-free domesticated culture MSC

With reference to the procedures of examples 1 to 6, except that magnesium nitrate was additionally added to MSC acclimation media of various fetal bovine serum concentrations (to a magnesium ion concentration of 5 μ g/mL in each MSC acclimation media), 6 types of serum-free acclimation-cultured mesenchymal stem cells were finally obtained, which were referred to as the generation cells of examples 91 to 96, respectively; in the various detection tests which follow the present invention, the corresponding passage of cells (which are also referred to as detection cells) for detection in the working examples referred to is carried out, if not stated otherwise.

Example 10: serum cultured MSC

In steps (5) and (6) of example 1, the cells were all cultured and digested in the MSC complete medium instead of the respective MSC acclimation media, and the cells of the P5 generation were mesenchymal stem cells cultured with serum and used as control cells for detection.

2. Testing of mesenchymal stem cells

MSCs are pluripotent stem cells derived from early-developing mesoderm and having high self-renewal ability and multipotentiality, and can be differentiated into hepatocyte-like cells, fibroblasts, osteoblasts, chondrocytes, adipocytes, myoblasts, extraneural blastocysts, and the like. Therefore, differentiation experiments of osteogenic, adipogenic and chondrogenic of the MSC are mostly used in laboratories to verify the multidirectional differentiation potential of the cells, thereby reflecting the quality of the cells.

Test example 1: serum-free domesticated MSC cell osteogenic induced differentiation and staining experiment

1. Inoculation and osteogenic Induction culture

1. Preparing a high-sugar complete culture medium: adding 56mL of fetal calf serum into 500mL of high-glucose-DMEM culture medium to prepare a high-glucose DMEM culture medium containing 10% of fetal calf serum;

2. the serum-free acclimated MSC cells in the logarithmic growth phase of the P5 generation obtained in example 1 were digested with 0.25% trypsin solution, and the flask was gently shaken to lay trypsin on the bottom surface of the flask. Digesting for about 1min, observing cell shrinkage and clustering under a microscope, slightly beating the culture flask to make the cells shed to the bottom of the culture flask in a flowing sand shape, adding a complete culture medium with the same volume as that of trypsin to stop digestion, blowing off the cells by using a pipette, and collecting cell suspension in a 15mL centrifuge tube.

3. After centrifugation at 1400rpm for 5min, the supernatant was removed. Cells were resuspended in 10mL of complete medium and counted.

4. According to 6X 10 ⁴ Inoculum density of/mL was inoculated into 24-well plates, 1mL per well, for a total of 4 wells. 1 well is a control well and 3 wells are experimental wells. Placing the plates at 30 ℃ 5% CO ₂ Culturing in an incubator.

5. After the cell fusion rate reached 80%, the medium was washed away, and 0.5mL of high-glucose DMEM complete medium containing 10% fetal bovine serum was added to the control well, and 0.5mL of MSC osteogenic induction medium was added to the other 3 well experimental wells. Repositioning the plates at 30 ℃ 5% ₂ And continuing culturing in the incubator.

6. The liquid is changed every 2 to 3 days. When the culture medium is changed, the original culture medium in the hole is firstly sucked and discarded, then 0.5mL of corresponding culture medium is added, and the induction time is 15-20 days.

2. Dyeing identification

1. The staining identification of the osteogenesis induced differentiation culture adopts a alizarin S staining method.

2. And (3) sample fixation treatment: the culture medium was carefully aspirated from the 24-well plate, and 400. Mu.L of PBS buffer was added to each well to wash the cell surface. PBS wash was carefully aspirated off and 300. Mu.L of BI's Fixation Solution was added to each well, covering the entire growth surface. Standing at room temperature for 30min, carefully absorbing and discarding the BI Fixation Solution, adding 300 mu L of BI wash I lotion into each hole, and cleaning the cell surface for 2-3 times.

3. And (3) sample dyeing treatment: wash I wash was discarded and 300. Mu.L of BI stabilizing Solution was added and the bottom was soaked uniformly. Staining for 30min at room temperature, carefully discarding the stabilizing Solution of BI, adding 300 μ L of wash II of BI, and rinsing the cells for 2-3 times. Wash II was aspirated away, 300. Mu.L of an infection Solution was added, and the cells were observed under a microscope and photographed (the differentiated cells were stained orange-red).

The results are shown in FIG. 1, in which a serum-free acclimation experimental group showed that the cells were stained in a typical orange color, and in FIG. 1 b control group cells were not stained in a typical orange color.

The results of examining 5 test cells of examples 2 to 6, 6 test cells of example 7, test cell of example 81, and test cell of example 91 by referring to the method of test example 1 showed that all of these cells were stained in a typical orange color.

Test example 2: serum-free domesticated MSC cell adipogenic induction differentiation and staining experiment

1. Preparing a solution reagent:

1. before the experiment, trypsin, MSCgo additive basic Medium, MSCgo additive SF, XF Supplement Mix I, MSCgo additive SF and XF Supplement Mix II are placed in an incubator at 37 ℃ for preheating.

2. Preparing a high-sugar complete culture medium: adding 56mL of fetal calf serum into 500mL of high-glucose-DMEM culture medium to prepare a high-glucose DMEM culture medium containing 10% of fetal calf serum;

3. preparation of fat induction medium: the Supplement Mix I and Supplement Mix II were added to the mscgaadiplogic Differentiation base Medium by pipette.

4. The serum-free acclimated MSC cells in the logarithmic growth phase of the P5 generation obtained in example 1 were digested with trypsin, and the flask was gently shaken to allow trypsin to spread on the bottom surface of the flask. Digesting for about 1min, observing cell shrinkage and clustering under a microscope, slightly beating the culture flask to make the cells shed to the bottom of the culture flask in a flowing sand shape, adding a complete culture medium with the same volume as that of trypsin to stop digestion, blowing off the cells by using a pipette, and collecting cell suspension in a 15mL centrifuge tube.

5. After centrifugation at 1400rpm for 5min, the supernatant was removed. Cells were resuspended in 10mL of complete medium and counted.

6. According to 6X 10 ⁴ Inoculum density of/mL was inoculated into 24-well plates, 1mL per well, for a total of 4 wells. 1 well is a control well and 3 wells are experimental wells. Placing the plate at 30 ℃ 5% ₂ Culturing in an incubator.

7. After the cell fusion rate reached 80%, the medium was washed away, 0.5mL of high-glucose DMEM complete medium containing 10% fetal bovine serum was added to the control wells, and 0.5mL of MSC adipogenic induction medium was added to the other 3 well experimental wells. Repositioning the plates at 30 ℃ 5% ₂ And continuing culturing in the incubator.

8. The liquid is changed every 2 to 3 days. When the culture medium is changed, the original culture medium in the hole is firstly sucked and discarded, then 0.5mL of corresponding culture medium is added, and the induction time is 15-20 days.

2. Dyeing identification

1. The dyeing identification of adipogenic induction differentiation culture adopts a neutral lipid oil red O dyeing method.

2. And (3) sample fixation treatment: the culture medium was carefully aspirated from the 24-well plate, and 300. Mu.L of PBS buffer was added to each well to wash the cell surface. PBS wash was carefully aspirated off and 300. Mu.L of BI's Adipo-Fixation was added to each well, covering the entire growth surface. And (3) after standing for 30min at room temperature, carefully absorbing BI Adipo-Fixation, adding 300 mu L of BI Adipo-wash I washing liquor into each hole, cleaning the surface of the cells, and standing for 2-3 min. The BI's Adipo-Wash I was carefully withdrawn.

3. Sample staining treatment: adding an Adipo-Staining A and an Adipo-Staining B into a mixture of 3:2, and obtaining the dyeing working solution. Carefully add 300. Mu.L of BI's staining solution to cover the entire growth surface and let stand at room temperature for 30min. Carefully remove the BI staining solution, carefully add Wash II at 300. Mu. LBI, rinse the surface, aspirate Wash II, repeat the previous steps, and rinse the cells again (if the supernatant is still very red, the rinse time can be extended or rinsed once more, taking care not to rinse the cells off). Add 300. Mu.L of Adipo-Insection to each well. Upon observation with an optical microscope, the lipid material exhibited a typical red color.

The results are shown in fig. 2, wherein a in fig. 2 shows the lipid substances with typical red color in the serum-free domesticated experimental group, and b in fig. 2 shows no lipid substances with typical red color in the control group.

The results of examining 5 test cells of examples 2 to 6, 6 test cells of example 7, test cell of example 81, and test cell of example 91 by referring to the method of this test example 2 showed that these cells all exhibited lipid substances of a typical red color.

Test example 3: serum-free domesticated MSC cell chondrogenic induced differentiation and staining experiment

1. Reagent configuration and cell culture treatment

1. The experiments were started by placing trypsin, MSCgo Chondrogenic Differentiation basic Medium, MSCgo Chondrogenic Differentiation Supplement Mix in a 37 ℃ incubator for preheating.

3. cartilage induction culture and configuration: 10mL of Supplement Mix was pipetted into 100mL of Chondrogenic Differentiation basic Medium.

2. Cartilage induction

The first scheme is as follows:

1. according to 1X 10 ⁷ cells/mL were seeded at a density of 10. Mu.L per well in 3 culture wells of a 24-well cell culture plate. Adding 1mL of sterile PBS into the peripheral blank holes, and sealing the edges for keeping humidity;

2. putting the 24-well cell culture plate into 5% of CO ₂ Culturing in incubator for 2hr;

3. after the cells adhere to the wall, taking 1 hole of the cells as a control group, and adding 1mL of high-glucose DMEM culture medium containing 10% FBS; in addition, 3 holes are used as experimental groups, and 0.5mL of MSC is added to form a cartilage induction culture medium;

4. charging 5% CO ₂ Continuously culturing in an incubator;

5. the culture medium is replaced every 3 days, and the induction time is 14-21 days.

Scheme II:

1. according to 6X 10 ⁵ cell/mL, resuspending the MSC cell pellet, and preparing 4mL of MSC cell suspension;

2. inoculating the cell suspension into 24-well plates, 1mL per well, 5% CO at 37 ℃ ₂ Continuously culturing in an incubator;

3. 24hr later, the culture medium was washed off, and 0.5mL of high sugar medium containing 10% FBS was added to 1 well of the control well, and 0.5mL of MSC cartilage induction medium was added to the other 3 wells of the test group, and then 5% CO was added ₂ Culturing at 37 deg.C in incubator.

4. Changing the culture medium every 3-4 days, wherein the culture medium in the holes is firstly sucked and discarded when the culture medium is changed, then 0.5mL of the corresponding culture medium is added, and the induction time is 14-21 days.

3. Dyeing identification

1. The dyeing identification of the chondrogenesis induced differentiation culture adopts an alcian dyeing method;

2. and (3) sample fixation treatment: the culture medium was carefully aspirated from the 24-well plate, and 400. Mu.L of PBS buffer was added to each well to wash the cell surface. PBS wash was carefully aspirated off and 400. Mu.L of BI's Fixation Solution was added to each well, covering the entire growth surface. Standing for 30min at room temperature, carefully absorbing and discarding the BI Fixation Solution, adding 200 mu L of BI wash I lotion into each hole, and standing for 2-3 min;

3. and (3) sample dyeing treatment: absorbing and removing Wash I, adding 300 mu L of stabilizing solution of BI, uniformly soaking the bottom surface, and dyeing overnight at room temperature in a dark place;

4. discarding the stabilizing solution, adding 300 mu L of BI Wash II, and rinsing the cells;

5. discarding the Wash II, adding 300. Mu.L of BI of Wash II, rinsing again (if the supernatant is still very blue, the rinsing time can be prolonged and rinsing again);

6. adding 300 mu L of BI observation Solution; the cells successfully differentiated by chondrogenesis induction appear dark blue when observed by an optical microscope.

The results of the first protocol are shown in fig. 3, in which a serum-free acclimated experimental group showed cells exhibiting a typical deep blue color, and b control group cells showed no cells exhibiting a typical deep blue color in fig. 3. The results of protocol two are essentially the same as those of protocol one.

The results of examining 5 test cells of examples 2 to 6, 6 test cells of example 7, test cell of example 81, and test cell of example 91 were examined by the method of this test example 3 (protocol one), and all of these cells exhibited typical dark blue cells.

The results of the above test examples 1 to 3 show that the human mesenchymal stem cells obtained by the serum-free in vitro acclimation culture method according to examples 1 to 9 of the present invention exhibited the typical differentiation performance of mesenchymal stem cells into osteoblasts, chondroblasts and adipocytes.

Test example 4: using Annexin V/PI reagent for detecting early/late apoptosis of P5 generation domesticated cells

Apoptosis is one of the basic characteristics of cells, and plays an important role in embryonic development, tissue repair, internal environment stabilization and the like of organisms. Annexin V is Ca2 with molecular weight of 35.8KD ⁺ Dependent phospholipid binding proteins, phosphatidylserines (Phosphositidyls) that are capable of turning outside the membrane during early apoptosis of cellsein, PS) binds specifically with high affinity. After FITC-Annexin V is combined with the apoptotic cells, under the excitation of blue light, green fluorescence is emitted, and the apoptotic cells and normal cells are distinguished. Propidium Iodide (PI) is a nucleic acid dye that does not penetrate the intact cell membrane, but penetrates damaged membranes of late apoptotic and dead cells and red stains the nucleus. By matching FITC-Annexin V to PI, cells in the early stage of apoptosis can be distinguished from cells in the late stage.

Test materials in this test example: mesenchymal stem cells obtained by serum-free acclimation culture according to the method of the present invention (the test cells of examples 1 to 9, for example, the P5 mesenchymal stem cells of example 1) and P5 mesenchymal stem cells obtained by serum-free acclimation culture according to the method of example 1 with reference to CN108642002A (201810047983.2).

1. Taking the serum-free domesticated P5-generation mesenchymal stem cells to be tested, sucking and discarding the complete culture medium by using a pipette, rinsing the culture bottle for 30s by using 20mL PBS, and then sucking and discarding;

2. 2mL of trypsin solution (0.25%) pre-warmed to 37 ℃ was added to the flask, and the flask was gently shaken to allow trypsin to spread on the bottom surface of the flask. Digesting for about 1min, observing cell shrinkage and clustering under a microscope, slightly beating the culture bottle to enable the cells to fall off to the bottom of the culture bottle in a flowing sand shape, adding a complete culture medium with the same volume as that of trypsin to stop digestion, blowing off the cells by using a pipette, and collecting cell suspension in a 15mL centrifuge tube;

3. centrifuging the cell suspension in a centrifuge at 1000rpm for 5min, removing supernatant, re-suspending cell precipitate with PBS pre-cooled to 4 deg.C, and centrifuging at 1000rpm for 5min to rinse the cells;

4. diluting 3mL of Binding Buffer (10X) to 30mL with 27mL of deionized water;

5.1 × Binding Buffer suspension cells are used for 1mL, centrifugation is carried out for 10min at 1000rpm, and the supernatant is discarded;

6. resuspend cells with 1mL of 1 × Binding Buffer to a cell density of 1 × 10 ⁷ cell/mL;

7. a1.5 mL EP tube was filled with 100. Mu.L of cell suspension, i.e., 1X 10 cells ⁶ cell/mL;

8. adding 5 μ L Annexin V-FITC into the tube, mixing, incubating at room temperature in dark for 10min;

9. adding 5 mu L of PI, and incubating for 5min at room temperature in a dark place;

10. adding PBS to 500 mu L, gently mixing uniformly, detecting the cell expression quantity at 488nm by using a flow machine, simultaneously collecting 75 ten thousand cells in each test group, and calculating the proportion of early apoptosis and late apoptosis/death of the cells (the mean value of n = 5);

11. the results of determination and calculation show that the mesenchymal stem cells obtained by the serum-free domestication culture method have excellent results in two parameters of early apoptosis cell ratio and late apoptosis/death cell ratio.

As a result: the 6 test cells of examples 1 to 6 had an early apoptotic cell ratio in the range of 23.5% to 25.2% and an late apoptotic/dead cell ratio in the range of 13.6% to 15.1%, for example, the P5 stem cell of example 1 had an early apoptotic cell ratio of 24.3% and an late apoptotic/dead cell ratio of 14.4%; the 6 test cells of example 7 had an early apoptotic cell ratio in the range of 12.8% to 13.7% and an late apoptotic/dead cell ratio in the range of 6.5% to 7.4%, for example, the P5 stem cells of example 71 had an early apoptotic cell ratio of 13.4% and an late apoptotic/dead cell ratio of 7.0%; the 6 test cells of example 8 had an early apoptotic cell ratio in the range of 23.7% to 25.7% and an late apoptotic/dead cell ratio in the range of 13.7% to 15.3%, for example, the P5 stem cells of example 81 had an early apoptotic cell ratio of 23.9% and an late apoptotic/dead cell ratio of 14.8%; the 6 test cells of example 9 had an early apoptotic cell ratio in the range of 23.5% to 24.3% and an late apoptotic/dead cell ratio in the range of 14.0% to 15.4%, for example, the P5 stem cells of example 91 had an early apoptotic cell ratio of 23.9% and an late apoptotic/dead cell ratio of 14.3%; CN108642002A has an early apoptotic cell ratio of 33.4% and a late apoptotic/dead cell ratio of 21.7%.

According to the results of the test example 4, the mesenchymal stem cells obtained by the serum-free acclimation culture method of the invention have better performance than the mesenchymal stem cells obtained by the prior art. In particular, it was unexpectedly found that the simultaneous addition of a diol compound and a magnesium salt to an MSC acclimation medium supplemented with transferrin and insulin with a gradual depletion of fetal bovine serum can significantly improve the performance of the cells.

Test example 5: detection of phenotype of serum-free acclimated MSC cells

Serum-free domesticated cultured cells of the P5 generation obtained in example 1 and serum-cultured mesenchymal stem cells of the P5 generation obtained in example 10 are taken respectively, a cell surface marker is detected by flow cytometry, and whether the change of the cell surface marker in the domestication culture process is different from that in conventional culture is observed. The specific implementation mode is as follows: digesting and collecting P5 generation serum-free domesticated MSC cells and conventionally cultured MSC cells, counting, and taking 8 × 10 ⁶ Each cell is divided into 16 tubes; washing with PBS once, and centrifuging at 1500rpm for 10min; discarding the supernatant, and blowing and beating the uniformly mixed cells, wherein 100-200 mu L of the supernatant is remained; adding 10 μ L of CD73 antibody marked by FITC, 10 μ L of CD90 antibody marked by PerCP-Cy5.5, 10 μ L of CD105 antibody marked by PerCP-Cy5.5 and 10 μ L of HLA-DR antibody marked by PE, and setting a tube as a blank control; reacting at 4 ℃ in the dark for 30min; washing with PBS once, and centrifuging at 1500rpm for 10min; discarding supernatant of directly labeled cells, adding 200 μ L PBS, blowing and mixing the cells, fixing 200 μ L1% paraformaldehyde, standing at 4 ℃ for detection, and detecting by an up-flow cytometer within 3 days.

The results are shown in FIG. 4. As can be seen from the results in the figure, the phenotypic results of the serum-free acclimated cultured cells are consistent with those of the flow cytometry cultured cells with serum.

The results of 5 test cells of examples 2 to 6, 6 test cells of example 7, 81 and 91 were examined by referring to the method of test example 5, and the results of fig. 4 were almost the same, showing that all of these serum-free acclimatized cultured cells were consistent with the phenotypic results of flow cells of serum-containing cultured cells, indicating that the mesenchymal stem cells obtained by the serum-free acclimatized culture methods of examples 1 to 9 of the present invention exhibited excellent cell performance.

In addition, in supplementary example a, with reference to the method of example 1, except that transferrin concentration thereof was changed to 8ng/mL and insulin concentration thereof was changed to 12ng/mL, or transferrin concentration thereof was changed to 12ng/mL and insulin concentration thereof was changed to 8ng/mL, two kinds of P5-generation serum-free acclimatized cultured mesenchymal stem cells respectively referred to as a complement 1 and a complement 2 were obtained; in addition, in supplementary example b, with reference to the method of example 71, except that the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol was changed to 0.08mg/mL and the concentration of magnesium nitrate was changed to 6. Mu.g/mL in terms of magnesium ion, or the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol was changed to 0.12mg/mL and the concentration of magnesium nitrate was changed to 4. Mu.g/mL in terms of magnesium ion, two types of P5 generation serum-free acclimatized cultured mesenchymal stem cells, which were referred to as complementary b1 and complementary b2, respectively, were obtained. The results of the measurements of the parameters of the four cells in the test examples 1-5 are substantially the same as those of the P5 cell in example 1 for the two cells in complement a1 and a2, and the results of the measurements of the parameters of the two cells in complement b1 and b2 are substantially the same as those of the P5 cell in example 71, for example, the proportion of early apoptotic cells in complement a1 cell is 23.7% and the proportion of late apoptotic/dead cells in complement a1 cell is 14.6%.

3. Preparation of acclimatization Medium

Acclimatization medium preparation example 1: transferrin (to a concentration of 10 ng/mL), insulin (to a concentration of 10 ng/mL), fetal bovine serum (to a concentration of 9%), 3- (2-methoxyphenoxy) -1, 2-propanediol (to a concentration of 0.1 mg/mL), and magnesium nitrate (to a concentration of 5. Mu.g/mL) were added to DMEM/F12 medium to prepare MSC acclimation medium.

Acclimatization medium preparation example 2: transferrin (to a concentration of 10 ng/mL), insulin (to a concentration of 10 ng/mL), fetal bovine serum (to a concentration of 7%), 3- (2-methoxyphenoxy) -1, 2-propanediol (to a concentration of 0.1 mg/mL), and magnesium nitrate (to a concentration of 5. Mu.g/mL) were added to DMEM/F12 medium to prepare MSC acclimation medium.

Acclimatization medium preparation example 3: transferrin (to a concentration of 10 ng/mL), insulin (to a concentration of 10 ng/mL), fetal bovine serum (to a concentration of 5%), 3- (2-methoxyphenoxy) -1, 2-propanediol (to a concentration of 0.1 mg/mL), and magnesium nitrate (to a concentration of 5. Mu.g/mL) were added to DMEM/F12 medium to prepare MSC acclimation medium.

Acclimatization medium preparation example 4: transferrin (to a concentration of 10 ng/mL), insulin (to a concentration of 10 ng/mL), fetal bovine serum (to a concentration of 2.5%), 3- (2-methoxyphenoxy) -1, 2-propanediol (to a concentration of 0.1 mg/mL), and magnesium nitrate (to a concentration of 5. Mu.g/mL) were added to a DMEM/F12 medium to prepare an MSC acclimation medium.

Acclimatization medium preparation example 5: transferrin (to a concentration of 10 ng/mL), insulin (to a concentration of 10 ng/mL), fetal bovine serum (to a concentration of 0%), 3- (2-methoxyphenoxy) -1, 2-propanediol (to a concentration of 0.1 mg/mL), and magnesium nitrate (to a concentration of 5. Mu.g/mL) were added to DMEM/F12 medium to prepare MSC acclimation medium.

Acclimatization medium preparation example 6: transferrin (to a concentration of 8 ng/mL), insulin (to a concentration of 12 ng/mL), fetal bovine serum (to a concentration of 6%), 3- (2-methoxyphenoxy) -1, 2-propanediol (to a concentration of 0.08 mg/mL), and magnesium nitrate (to a concentration of 6. Mu.g/mL) were added to DMEM/F12 medium to prepare an MSC acclimation medium.

Acclimatization medium preparation example 7: transferrin (to a concentration of 12 ng/mL), insulin (to a concentration of 8 ng/mL), fetal bovine serum (to a concentration of 5%), 3- (2-methoxyphenoxy) -1, 2-propanediol (to a concentration of 0.12 mg/mL), and magnesium nitrate (to a concentration of 4. Mu.g/mL) were added to DMEM/F12 medium to prepare MSC acclimation medium.

The examples of the present invention should be construed as merely illustrative and not a limitation of the scope of the present invention. After reading the description of the invention, one skilled in the art can make various changes and modifications to the invention, and such equivalent changes and modifications also fall into the scope of the invention defined by the claims.

Claims

Use of a combination of 3- (2-methoxyphenoxy) -1, 2-propanediol and magnesium nitrate in the preparation of a acclimation medium for serum-free acclimation culture of mesenchymal stem cells; the domestication culture medium comprises: 10ng/mL of transferrin, 10ng/mL of insulin, 0.1mg/mL of 3- (2-methoxyphenoxy) -1, 2-propylene glycol, 5 mug/mL of magnesium nitrate in terms of magnesium ion concentration, 0 to 9.5% of fetal calf serum and a balanced DMEM/F12 culture medium; the serum-free domestication culture of the mesenchymal stem cells comprises the following steps:

(1) Cleaning placenta surface with tissue cleaning solution to remove surface blood stasis, cutting umbilical cord and removing internal blood vessel,

(2) Stripping Wharton's jelly from umbilical cord, cleaning, and cutting into 2-3mm pieces ³ Transferred to T75 flasks, 1mL per flask,

(3) Add slowly 10mL of MSC complete medium to the flask, gently shake the flask to allow the umbilical cord tissue pieces to lay flat in the flask, 5% CO at 37 deg.C ₂ Performing static culture in an incubator, supplementing liquid once every 2-3 days, supplementing liquid for 2-3 times, removing tissue blocks, replacing MSC complete culture medium completely, culturing until the cell grows to 70%, digesting the obtained cell,

(4) Removing the culture medium in the culture bottle by suction, sucking PBS to rinse the culture bottle to remove residual fetal calf serum, adding 3mL of 0.25% trypsin solution for digestion, tapping the culture bottle to make the cells fall to the bottom of the culture bottle, adding 10mL of MSC complete culture medium to neutralize the trypsin to stop the digestion process, blowing off the cells by a pipette, collecting cell suspension in a centrifuge tube, centrifuging, removing the supernatant to obtain the cells with the cell precipitation marked as P0 generation,

(5) Resuspending the P0 generation cell pellet with 10mL acclimation medium containing less than 10% fetal calf serum, counting cells, and then inoculating at a density of 1 × 10 ⁴ Individual MSC cells/cm ² Inoculating into T75 culture flask, supplementing the above MSC acclimatization medium to 10mL, culturing at 37 deg.C with 5% CO ₂ Standing and culturing for more than 72h in an incubator until the cell fusion degree reaches 80%, sucking and removing the culture medium in the culture bottle, sucking PBS to rinse the culture bottle to remove residual fetal calf serum, adding 3mL of 0.25% trypsin solution for digestion, tapping the culture bottle to make the cells fall to the bottom of the culture bottle, adding 10mL of the domesticated culture medium used in the step to neutralize the trypsin to stop the digestion process, blowing off the cells by using a pipettor, collecting cell suspension in a centrifuge tube, centrifuging, removing the supernatant to obtain cell sediment which is marked as P1 generation cells,

(6) Adopting the culture and digestion method in the step (5), treating the cells obtained in the last step in the operation process by using MSC (mesenchymal stem cell) acclimation culture medium for sequentially reducing the concentration of fetal calf serum to be finally 0 percent to obtain the final serum-free in-vitro acclimation culture mesenchymal stem cells, and optionally carrying out serum-free in-vitro acclimation culture

(7) And (4) continuously culturing the mesenchymal stem cells of the last generation obtained in the last step and subjected to serum-free domestication culture by using a domestication culture medium containing 0% fetal calf serum MSC, and carrying out passage.
2. The use according to claim 1, wherein the MSC complete medium is DMEM/F12 medium containing 10% fetal bovine serum.
3. The use according to claim 1, wherein in step (3), 3-4mL of MSC complete medium is supplemented each time with fluid additions every 2-3 days.
4. The use according to claim 1, wherein in step (4), after adding the trypsin solution, the culture flask is gently shaken to allow the trypsin to sufficiently cover the cell surface, the cells are digested for 1min until the cells are microscopically observed to shrink into a circular shape, and the culture flask is gently tapped to allow the cells to shed into a stream of sands at the bottom of the culture flask.
5. Use according to claim 1, step (4), in a centrifuge at 1400rpm for 5min.
6. The use according to claim 1, in step (5), the P0 generation cell pellet is resuspended in MSC acclimation medium containing 7 to 9.5% fetal bovine serum.
7. The use according to claim 1, in step (5), the P0 generation cell pellet is resuspended in MSC acclimation medium containing 8 to 9.5% fetal bovine serum.
8. The use according to claim 1, in step (5), the P0 cell pellet is resuspended in MSC acclimation medium containing 8%, 8.5%, 9%, or 9.5% fetal bovine serum.
9. The use according to claim 1, wherein in the repeating of step (5) in step (6), the concentration of fetal bovine serum in the MSC acclimation medium is decreased with the increase of the passage of cells until the concentration of fetal bovine serum in the MSC acclimation medium is 0.
10. The use according to claim 1, wherein in the step (6), the mesenchymal stem cells cultured by serum-free in vitro acclimation are obtained by gradually reducing the concentration of fetal bovine serum until generation P4-P10.
11. The use according to claim 1, wherein in the step (7), the mesenchymal stem cells cultured by the in vitro domestication of the 0% fetal serum finally obtained in the step (6) are continuously cultured and passaged by using the domestication culture medium containing 0% fetal bovine serum MSC, and are continuously transferred to the P20 generation mesenchymal stem cells cultured by the serum-free domestication.