CN112029717B - Serum-free in vitro domestication culture of human mesenchymal stem cells - Google Patents

Serum-free in vitro domestication culture of human mesenchymal stem cells Download PDF

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CN112029717B
CN112029717B CN202010980495.4A CN202010980495A CN112029717B CN 112029717 B CN112029717 B CN 112029717B CN 202010980495 A CN202010980495 A CN 202010980495A CN 112029717 B CN112029717 B CN 112029717B
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许晓椿
肖海蓉
魏卿
刘庆喜
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Shenzhen Boya Perception Pharmaceutical Co ltd
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Abstract

The invention relates to serum-free in vitro domestication culture of human mesenchymal stem cells. In one aspect, the acclimation medium for serum-free in vitro acclimation of human mesenchymal stem cells of the present invention comprises: transferrin 8-12 ng/mL, insulin 8-12 ng/mL, fetal bovine serum 0-9.5%, and balance DMEM/F12 culture medium. In another aspect, a method for serum-free acclimation culture of mesenchymal stem cells comprises the steps of: collecting Wheatstone gum, culturing the Wheatstone gum in MSC complete culture medium, and then digesting the Wheatstone gum with trypsin solution to obtain cell sediment which is marked as P0 generation cell; culturing by using MSC acclimation culture medium containing less than 10% of fetal calf serum, digesting by using trypsin solution to obtain P1 generation cells, treating the cells obtained in the last step in the operation process by using MSC acclimation culture medium which sequentially reduces the fetal calf serum concentration to 0% finally, and obtaining the final serum-free in vitro acclimation cultured mesenchymal stem cells. The method of the invention presents the excellent effects described in the specification.

Description

Serum-free in vitro domestication culture of human mesenchymal stem cells
Technical Field
The invention belongs to the field of biotechnology and biological medicine, relates to a culture method of human mesenchymal stem cells, and in particular relates to a serum-free in vitro domestication culture method of human mesenchymal stem cells.
Background
Mesenchymal stem cells (mesenchymal stem cell, MSC), such as human mesenchymal stem cells, were originally isolated from bone marrow, were derived from a class of tissue stem cells of mesoderm that have multipotent differentiation potential and self-renewal capacity, and were capable of differentiating into various adult cells such as osteoblasts, chondrocytes, adipocytes, endothelial cells, neural cells, myocytes, hepatocytes, etc., under specific conditions in vivo and in vitro (Caplan AI. Mesenchyal stem cells J ortho Res.1991,9:641-650.Pittenger MF,Mackay AM,Beck SC,et al.Multilineage potential of adult human mesenchymal stem cells.Science.1999;284:143-147). Recent studies have shown that mesenchymal stem cells have immunoregulatory and hematopoietic support effects and are easy for exogenous gene expression. Therefore, the mesenchymal stem cells not only tissue engineer seed cells in bone, cartilage and myocardial construction and carrier cells important in gene therapy, but also have wide application prospects in hematopoietic stem cell transplantation and organ transplantation because the mesenchymal stem cells promote hematopoietic reconstruction and inhibit graft versus host reaction functions. Mesenchymal stem cells have the characteristic of in vitro adherent growth, and by utilizing the characteristic, people have successfully isolated and cultured the mesenchymal stem cells from various tissues such as liver, kidney, pancreas, muscle, cartilage, skin, peripheral blood and the like.
The mesenchymal stem cells reported at present are mainly derived from bone marrow and are obtained by adopting a density gradient centrifugation method. Although the separation method is simple, the marrow taking of the donor needs to be subjected to a painful operation, and has a high infection chance in the process of taking materials and after taking materials; since the content of MSC in human bone marrow is extremely rare, every 10 5 ～10 6 Only about 1 single nuclear cell, and the number, proliferation and differentiation capacity of bone marrow mesenchymal stem cells are significantly reduced with the increase of age, so that they are limited in research and application, especially clinical application. Placenta, which originates from the ectomesoderm of embryo at embryonic development stage, is composed of the interstitium, blood vessels and trophoblasts, and contains a large amount of mesenchymal components. Recent researches show that the placenta contains abundant stem cells, and the isolation and culture of the pluripotent stem cells from the placenta opens up a brand-new and abundant source for experimental research and clinical application.
The isolation of stem cells from placenta to establish a placental stem cell bank has been reported in many documents, for example, CN101270349a (chinese patent application No. 200810061267.6, publication No. 2008, 9, 24) published invention titled "isolation and in vitro expansion culture method of placental mesenchymal stem cells"; CN101693884a (chinese patent application No. 200910117522.9, publication No. 2010, 4, 14) discloses an invention titled "a method for separating and extracting stem cells from placenta, umbilical cord or adipose tissue"; CN102146359a (chinese patent application No. 201110005964.1, publication date 2011, month 8, 10) discloses the invention titled "method for extracting primitive 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 ancestors of human cells, and all cells in our body are derived from stem cells. When cells in the body die or the injury degenerates, stem cells grow and transform into cells that can replace them. As seed cells, the seed cells are mainly used for treating various refractory diseases of tissue cell and organ injury which cannot be naturally repaired by organisms clinically; as immunoregulatory cells, for the treatment of immune rejection and autoimmune diseases. Human mesenchymal stem cells are important members of stem cell families, originate from early-stage mesoderm, belong to multipotent stem cells, and MSCs are originally found in bone marrow, and are increasingly attracting attention due to their multipotent differentiation potential, hematopoietic support, promotion of stem cell implantation, immune regulation, self replication and the like. Initial clinical studies were carried out by Lazarus et al in 1995 who collected autologous MSCs from patients with hematological neoplasms in remission, amplified in vitro for 4-7 weeks, and then injected intravenously into patients, and patients were divided into 3 groups, each given different doses of MSCs, with no adverse side effects observed after injection, suggesting that MSCs are safe and reliable for transplantation therapy. Clinical reports of autologous MSCs are gradually increased, and disease types relate to hematopoietic reconstitution after radiotherapy and chemotherapy, graft Versus Host Disease (GVHD), heart system diseases and the like, and all the reports prove that the clinical intravenous infusion is safe and reliable.
The in vitro culture process of mesenchymal stem cells is a key step in relation to the safety of stem cells used 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 art, when the mesenchymal stem cells are cultured and passaged, the culture medium used is supplemented with serum, for example, fetal bovine serum, and in particular, 10% fetal bovine serum is usually added, for example, MSC complete medium (DMEM/F12 medium containing 10% fetal bovine serum) is usually used for culturing and passaging the mesenchymal stem cells.
However, on the one hand, the cost of fetal bovine serum is quite high, which is disadvantageous for the culture of mesenchymal stem cells; on the other hand, fetal bovine serum, an exogenous substance of animal origin, is present in stem cells and presents a potential risk to the safety of the clinical use of the cells. Therefore, it is interesting to culture mesenchymal stem cells without serum acclimation.
CN 108642002A (201810047983.2) discloses a method for serum-free acclimation culture of human mesenchymal stem cells. The method uses fresh serum-free basic culture medium, adds and replaces serum-containing complete culture solution of in vitro human mesenchymal stem cells according to a certain proportion, and utilizes exosomes and microcapsule components secreted by the mesenchymal stem cells in the culture process as supplements, so that the mesenchymal stem cells finish domestication of serum-free culture, and finally a serum-free culture system is realized. It is believed that the density, the activity rate and the cell surface of the mesenchymal stem cells after acclimation of the invention have no obvious difference from that of a serum culture medium, and the components and the abundance of cytokines secreted by a detection culture solution and an exosome find that the bFGF, PDGF, VEGF expression quantity related to the vascular growth is increased compared with that of cells which are not acclimated. However, since the CN 108642002a method is that cells are directly cultured in a serum-free medium, the nutrition of the cell environment is drastically reduced, and thus the cells are adversely affected.
Accordingly, there remains a need in the art to provide a novel method for culturing mesenchymal stem cells, and in particular to provide a novel method for serum-free in vitro acclimation of human mesenchymal stem cells.
Disclosure of Invention
The invention aims at overcoming the defects in the prior art, provides a unique culture method and provides a serum-free domestication culture method for in-vitro culture of human mesenchymal stem cells. The aim of the invention is achieved by the following technical scheme.
In a first aspect of the present invention, there is provided a method for serum-free acclimating and culturing mesenchymal stem cells, the method comprising the steps of:
(1) The placenta surface is washed with a tissue washing solution to remove surface congestion, the umbilical cord is sheared and internal blood vessels are removed.
(2) Stripping Wharton's jelly in umbilical cord, cleaning and shearing to 2-3 mm 3 Transferred to T75 flasks with 1mL of each flask.
(3) Adding MSC complete culture medium 10mL slowly into the culture flask, gently shaking the culture flask to spread umbilical cord tissue blocks in the culture flask, and adding 5% CO at 37deg.C 2 And (3) carrying out stationary culture in an incubator, supplementing liquid once every 2-3 days, supplementing liquid 2-3 times, removing tissue blocks, and continuously culturing by completely replacing MSC complete culture medium until the cells grow to 70% of fusion degree, wherein the obtained cells are digested next.
(4) The culture medium in the culture flask is sucked and removed, PBS is sucked and used for rinsing the culture flask to remove residual fetal calf serum, 3mL of 0.25% trypsin solution is added for digestion, the culture flask is tapped to enable cells to fall off to the bottom of the culture flask, 10mL of MSC complete medium is added for neutralizing trypsin to terminate the digestion process, a pipettor is used for blowing off the cells, cell suspension is collected in a centrifuge tube, and the supernatant is centrifuged and discarded to obtain cells with cell sediment recorded as P0 times.
(5) The P0 cell pellet was resuspended in 10mL of MSC-conditioned medium containing less than 10% fetal bovine serum, counted and then plated at a plating density of 1X 10 4 Individual MSC cells/cm 2 Inoculating into T75 culture flask, and supplementing MSC acclimation culture medium to 10mL, 37deg.C 5% CO 2 And (3) standing and culturing in an incubator for more than 72 hours until the cell fusion degree reaches 80%, sucking and discarding the culture medium in the culture flask, sucking PBS, rinsing the culture flask to remove residual fetal calf serum, adding 3mL of 0.25% trypsin solution for digestion, tapping the culture flask to enable cells to fall off to the bottom of the culture flask, adding 10mL of MSC domestication culture medium used in the step to neutralize trypsin so as to terminate the digestion process, blowing off the cells by a pipette, collecting cell suspension in a centrifuge tube, centrifuging, discarding supernatant, and obtaining cell sediment which is marked as P1 generation cells.
(6) And (3) adopting the culture and digestion method in the step (5), and treating the cells obtained in the last step in the operation process with MSC domestication culture medium which sequentially reduces the serum concentration of the fetal calf to 0% at last, so as to obtain the final serum-free in-vitro domestication cultured mesenchymal stem cells. Optionally (I)
(7) The last generation of mesenchymal stem cells obtained in the last step of serum-free domestication culture are continuously cultured and passaged by a domestication culture medium containing 0% of fetal bovine serum MSC (the last generation of mesenchymal stem cells can be continuously transferred to the P20 generation of serum-free domestication culture).
The method according to the first aspect of the invention, wherein the tissue cleanser 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 step (2) is washing the Wharton's jelly with a tissue washing liquid.
The method according to the first aspect of the invention, wherein the MSC complete medium is DMEM/F12 medium with 10% fetal bovine serum.
The method according to the first aspect of the present invention, wherein in step (3), 3-4mL of complete culture medium of MSC is fed once every 2-3 days.
According to the method of the first aspect of the invention, in the step (4), after the trypsin solution is added, the culture flask is gently shaken to enable trypsin to fully cover the cell surface, the trypsin is digested for 1min, the cells are observed to shrink into a round shape under a microscope, and the culture flask is gently tapped to enable the cells to fall off to the bottom of the culture flask in a quicksand shape.
The method according to the first aspect of the present invention, wherein in step (4), centrifugation is performed at 1400rpm for 5min in a centrifuge.
The method according to the first aspect of the invention, wherein in step (5), the P0 generation cell pellet is resuspended in MSC habituation medium containing 7-9.5% fetal bovine serum.
The method according to the first aspect of the invention, wherein in step (5) the P0 generation cell pellet is resuspended in MSC-acclimating medium containing 8-9.5% fetal bovine serum, e.g. 8%, 8.5%, 9%, or 9.5% fetal bovine serum.
The method according to the first aspect of the invention, wherein during the repetition of step (5) of step (6), the concentration of fetal bovine serum in the MSC-acclimating medium decreases with increasing cell passages until the concentration of fetal bovine serum in the MSC-acclimating medium is 0.
According to the method of the first aspect of the invention, in the step (6), the mesenchymal stem cells which are domesticated and cultured in vitro without serum are obtained from the generation P4 to the generation P10 by gradually reducing the serum concentration of the fetal bovine.
According to the method of the first aspect of the invention, in the step (6), the mesenchymal stem cells which are domesticated and cultured in vitro without serum are obtained from the generation P4 to the generation P9 by gradually reducing the serum concentration of the fetal bovine.
According to the method of the first aspect of the invention, in the step (6), the mesenchymal stem cells which are domesticated and cultured in vitro without serum are obtained from the generation P4 to the generation P8 by gradually reducing the serum concentration of the fetal bovine.
According to the method of the first aspect of the invention, in the step (7), the mesenchymal stem cells which are finally obtained in the step (6) and are subjected to in vitro domestication culture by 0% fetal serum are continuously cultured and passaged by a domestication culture medium containing 0% fetal bovine serum MSC, and are continuously transferred to the P20 generation mesenchymal stem cells subjected to serum-free domestication culture.
The method according to the first aspect of the invention, wherein the MSC acclimation medium comprises: transferrin 8-12 ng/mL, insulin 8-12 ng/mL, fetal bovine serum 0-9.5%, and balance DMEM/F12 culture medium.
The method according to the first aspect of the invention, wherein the MSC acclimation medium comprises: transferrin 10ng/mL, insulin 10ng/mL, fetal bovine serum 0-9.5%, and balance DMEM/F12 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 chloride. In one embodiment, the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol is 0.08 to 0.12mg/mL. In one embodiment, magnesium chloride is present at a magnesium ion concentration of 4 to 6 μg/mL. In one embodiment, the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol is 0.1mg/mL. In one embodiment, magnesium chloride is 5 μg/mL as magnesium ion concentration. 3- (2-methoxyphenoxy) -1, 2-propanediol, also known as guaifenesin, has a CAS number of 93-14-1.
The method according to the first aspect of the invention, wherein the MSC acclimation medium comprises: transferrin 8-12 ng/mL, insulin 8-12 ng/mL, 3- (2-methoxyphenoxy) -1, 2-propanediol 0.08-0.12 mg/mL, magnesium chloride 4-6 mug/mL based on magnesium ion concentration, 0-9.5% fetal bovine serum, and balance DMEM/F12 culture medium. The method according to the first aspect of the invention, wherein the MSC acclimation medium comprises: transferrin 10ng/mL, insulin 10ng/mL, 3- (2-methoxyphenoxy) -1, 2-propanediol 0.1mg/mL, magnesium chloride 5 mug/mL based on magnesium ion concentration, 0-9.5% foetal calf serum, balance DMEM/F12 medium. In the present invention, the term "balance" means an amount added to the total amount, which herein means adding the remaining substance basis to their respective prescribed concentrations in DMEM/F12 medium, unless otherwise specified.
Further, the second aspect of the present invention relates to a acclimation medium for serum-free in vitro acclimation culture of human mesenchymal stem cells, comprising: transferrin 8-12 ng/mL, insulin 8-12 ng/mL, fetal bovine serum 0-9.5%, and balance DMEM/F12 culture medium.
The acclimatization medium according to the second aspect of the present invention, comprising: transferrin 10ng/mL, insulin 10ng/mL, fetal bovine serum 0-9.5%, and balance DMEM/F12 medium.
The acclimatized medium according to the second aspect of the present invention, further comprising 3- (2-methoxyphenoxy) -1, 2-propanediol and magnesium chloride. In one embodiment, the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol is 0.08 to 0.12mg/mL. In one embodiment, magnesium chloride is present at a magnesium ion concentration of 4 to 6 μg/mL. In one embodiment, the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol is 0.1mg/mL. In one embodiment, magnesium chloride is 5 μg/mL as magnesium ion concentration. 3- (2-methoxyphenoxy) -1, 2-propanediol, also known as guaifenesin, has a CAS number of 93-14-1.
The acclimatization medium according to the second aspect of the present invention, comprising: transferrin 8-12 ng/mL, insulin 8-12 ng/mL, 3- (2-methoxyphenoxy) -1, 2-propanediol 0.08-0.12 mg/mL, magnesium chloride 4-6 mug/mL based on magnesium ion concentration, 0-9.5% fetal bovine serum, and balance DMEM/F12 culture medium.
The acclimatization medium according to the second aspect of the present invention, comprising: transferrin 10ng/mL, insulin 10ng/mL, 3- (2-methoxyphenoxy) -1, 2-propanediol 0.1mg/mL, magnesium chloride 5 mug/mL based on magnesium ion concentration, 0-9.5% foetal calf serum, balance DMEM/F12 medium. In the present invention, the term "balance" means an amount added to the total amount, which herein means adding the remaining substance basis to their respective prescribed concentrations in DMEM/F12 medium, unless otherwise specified.
The acclimation medium according to the second aspect of the present invention, wherein the serum-free in vitro acclimation culture of human mesenchymal stem cells is performed according to the method of:
(1) The placenta surface is washed with a tissue washing solution to remove surface congestion, the umbilical cord is sheared and internal blood vessels are removed.
(2) Stripping Wharton's jelly in umbilical cord, cleaning and shearing to 2-3 mm 3 Transferred to T75 flasks with 1mL of each flask.
(3) Adding MSC complete culture medium 10mL slowly into the culture flask, gently shaking the culture flask to spread umbilical cord tissue blocks in the culture flask, and adding 5% CO at 37deg.C 2 And (3) carrying out stationary culture in an incubator, supplementing liquid once every 2-3 days, supplementing liquid 2-3 times, removing tissue blocks, and continuously culturing by completely replacing MSC complete culture medium until the cells grow to 70% of fusion degree, wherein the obtained cells are digested next.
(4) The culture medium in the culture flask is sucked and removed, PBS is sucked and used for rinsing the culture flask to remove residual fetal calf serum, 3mL of 0.25% trypsin solution is added for digestion, the culture flask is tapped to enable cells to fall off to the bottom of the culture flask, 10mL of MSC complete medium is added for neutralizing trypsin to terminate the digestion process, a pipettor is used for blowing off the cells, cell suspension is collected in a centrifuge tube, and the supernatant is centrifuged and discarded to obtain cells with cell sediment recorded as P0 times.
(5) The P0 cell pellet was resuspended in 10mL of MSC-conditioned medium containing less than 10% fetal bovine serum, counted and then plated at a plating density of 1X 10 4 Individual MSC cells/cm 2 Inoculating into T75 culture flask, and supplementing MSC acclimation culture medium to 10mL, 37deg.C 5% CO 2 And (3) standing and culturing in an incubator for more than 72 hours until the cell fusion degree reaches 80%, sucking and discarding the culture medium in the culture flask, sucking PBS, rinsing the culture flask to remove residual fetal calf serum, adding 3mL of 0.25% trypsin solution for digestion, tapping the culture flask to enable cells to fall off to the bottom of the culture flask, adding 10mL of MSC domestication culture medium used in the step to neutralize trypsin so as to terminate the digestion process, blowing off the cells by a pipette, collecting cell suspension in a centrifuge tube, centrifuging, discarding supernatant, and obtaining cell sediment which is marked as P1 generation cells.
(6) And (3) adopting the culture and digestion method in the step (5), and treating the cells obtained in the last step in the operation process with MSC domestication culture medium which sequentially reduces the serum concentration of the fetal calf to 0% at last, so as to obtain the final serum-free in-vitro domestication cultured mesenchymal stem cells. Optionally (I)
(7) The last generation of mesenchymal stem cells obtained in the last step of serum-free domestication culture are continuously cultured and passaged by a domestication culture medium containing 0% of fetal bovine serum MSC (the last generation of mesenchymal stem cells can be continuously transferred to the P20 generation of serum-free domestication culture).
The acclimation medium according to the second aspect of the present invention, wherein the tissue washing fluid is a 0.9% physiological saline solution containing 1% penicillin, 1% streptomycin.
The acclimation medium according to the second aspect of the present invention, wherein the step (2) is washing the Whatman gel with a tissue washing liquid.
The acclimatized medium according to the second aspect of the present invention, wherein the MSC complete medium is DMEM/F12 medium with 10% fetal bovine serum.
The acclimation medium according to the second aspect of the present invention, wherein in step (3), 3-4mL of complete medium of MSC is supplemented once every 2-3 days.
According to the acclimatization culture medium of the second aspect of the present invention, in the step (4), after adding a trypsin solution, the culture flask is gently shaken to allow trypsin to fully cover the cell surface, the trypsin is digested for 1min, the cells are observed under a microscope to shrink into a round shape, and the culture flask is gently patted to allow the cells to fall off to the bottom of the culture flask in a quicksand shape.
The acclimatized medium according to the second aspect of the present invention, wherein in step (4), is centrifuged at 1400rpm for 5min in a centrifuge.
According to the second aspect of the invention, wherein in step (5), the P0 generation cell pellet is resuspended in MSC acclimation medium containing 7-9.5% fetal bovine serum.
According to the second aspect of the invention, wherein in step (5), the P0 generation cell pellet is resuspended in MSC acclimation medium containing 8-9.5% fetal bovine serum, such as 8%, 8.5%, 9%, or 9.5% fetal bovine serum.
According to the acclimation medium of the second aspect of the present invention, wherein the concentration of fetal bovine serum in the MSC acclimation medium decreases with increasing cell passages during the repetition of step (5) of step (6) until the concentration of fetal bovine serum in the MSC acclimation medium is 0.
According to the acclimation medium of the second aspect of the present invention, in step (6), the serum-free in vitro acclimation-cultured mesenchymal stem cells are obtained from generation P4 to generation P10 by gradually decreasing the serum concentration of the fetal bovine.
According to the acclimation medium of the second aspect of the present invention, in step (6), the serum-free in vitro acclimation-cultured mesenchymal stem cells are obtained from generation P4 to generation P9 by gradually decreasing the serum concentration of the fetal bovine.
According to the acclimation medium of the second aspect of the present invention, in step (6), the serum-free in vitro acclimation-cultured mesenchymal stem cells are obtained from generation P4 to generation P8 by gradually reducing the serum concentration of the fetal bovine.
According to the acclimation culture medium of the second aspect of the present invention, in step (7), the mesenchymal stem cells obtained in step (6) and subjected to in vitro acclimation culture with 0% fetal serum are continuously cultured and passaged by using an MSC acclimation culture medium containing 0% fetal bovine serum, and the mesenchymal stem cells are continuously transferred to P20 generation mesenchymal stem cells subjected to serum-free acclimation culture.
Of the various operational steps described herein, although specific steps are described in some detail or language specific to those described in the preparation examples of the detailed description section below, those skilled in the art may well summarise the above described method steps in light of the detailed disclosure of the present invention as a whole.
Any of the embodiments of any of the aspects of the invention may be combined with any of the other embodiments, provided that they do not contradict. Furthermore, in any of the embodiments of any of the aspects of the present invention, any technical feature may be applied to the technical feature in other embodiments as long as they do not contradict. The present invention is further described below.
All documents cited herein are incorporated by reference in their entirety and are incorporated by reference herein to the extent they are not inconsistent with this invention. Furthermore, various terms and phrases used herein have a common meaning known to those skilled in the art, and even though they are still intended to be described and explained in greater detail herein, the terms and phrases used herein should not be construed to be inconsistent with the ordinary meaning in the sense of the present invention.
It is well known that MSCs are abundant in source, mainly organized in perinatal groups: placenta, umbilical cord, amniotic membrane, amniotic fluid, etc. And the MSC from umbilical cord is more convenient and high-quality to prepare. The umbilical cord comprises a vein and two arteries, the periphery is made of Wharton's jelly, the outer layer is wrapped by an amniotic membrane-derived epithelium, and from the development point of view, the umbilical cord is a stem cell formation and a passage, and the prior studies show that the connective tissue of the human umbilical cord is a tissue source rich in mesenchymal stem cells. The treatment method comprises firstly stripping umbilicus to drive vein and outer epithelium, leaving gelatinous tissue (Whatman's jelly), and cutting off the parts with clamp marks and blood stasis on the two sides.
The serum-free in vitro domestication method for culturing human mesenchymal stem cells, which is provided by the invention, has excellent technical effects in one or more aspects as described herein.
Drawings
Fig. 1: osteoinductive differentiation results, a is serum-free acclimation experimental group (the dark part of the original photo picture is orange), and b is control group.
Fig. 2: adipogenic induced differentiation results, a serum-free acclimated experimental group (dark part of original photo picture is red), b control group.
Fig. 3: results of chondrogenic induced differentiation, a serum-free acclimatization experimental group (dark blue in middle dark part of original photo graph, b control group.
Fig. 4: flow cell phenotype results (CD 73, CD90, CD105, HLA-DR, panel left, serum-free acclimated group, panel right, conventional culture group in order from top to bottom).
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. Those skilled in the art will appreciate that various changes and modifications can be made to the invention without departing from the spirit and scope thereof. The present invention generally and/or specifically describes the materials used in the test as well as the test methods. Specific materials and sources thereof used in embodiments of the present invention are provided below. However, it should be understood that these are merely exemplary and are not intended to limit the present invention, as materials that are the same as or similar to the type, model, quality, nature, or function of the reagents and instruments described below may be used in the practice of the present invention. The experimental methods used in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below were obtained commercially unless otherwise specified.
The percentages used in the present invention are weight percentages unless otherwise indicated. Herein, the reference to a PBS buffer or PBS solution or PBS or the like, unless otherwise specified, refers to a phosphate buffer having a pH of 6.8, and is prepared by the following method: taking 250mL of 0.2mol/L potassium dihydrogen phosphate solution, adding 118mL of 0.2mol/L sodium hydroxide solution, diluting to 1000mL with water, and shaking uniformly to obtain the product.
Preparation of MSC complete medium: adding fetal bovine serum to DMEM/F12 medium to a concentration of 10%.
Preparation of MSC acclimation Medium (also referred to as reduced serum Medium): to DMEM/F12 medium, transferrin (to a concentration of 10ng/mL, product of Transferrin, prospec-Tany Co.), insulin (to a concentration of 10ng/mL, product of Sigma-Aldrich Co.), 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., product of Gibco) are added to prepare MSC domestication medium containing Transferrin, insulin, and fetal bovine serum of different concentrations.
1. Preparation of mesenchymal Stem cells
Example 1: serum-free domestication culture MSC
(1) The placenta surface was washed with tissue washing solution (0.9% normal saline containing 1% penicillin, 1% streptomycin) to remove surface congestion, and umbilical cord was cut and internal blood vessels were removed.
The present embodiment specifically operates as follows: in the biosafety cabinet, articles such as a stainless steel tray, a filter screen, a stainless steel cup, a lunch box, tissue cleaning liquid (0.9% physiological saline containing 1% penicillin and 1% streptomycin), a pipettor and the like are put at proper positions. The placenta tissue is taken out and put into a stainless steel tray by using surgical forceps, and the placenta surface is cleaned by tissue cleaning liquid to remove the blood stasis on the placenta surface. The umbilical cord at the connecting placenta is cut off by using surgical scissors and forceps, and the other end is cut off from the rear of the umbilical cord sterile clamp. The umbilical cord was removed, and washed 3 times with a tissue washing solution to remove blood stasis and coagulation lumps on the surface. One end of the umbilical cord is clamped with forceps, and the umbilical cord is clamped with the other forceps and slid along the umbilical cord tissue to remove residual cord blood in the blood vessel. The umbilical cord is firstly cut into sections with the length of 2-3cm, then the sections are cut along the longitudinal section of the umbilical cord by scissors, the umbilical cord is flattened, and 3 blood vessels (2 umbilical arteries and 1 umbilical vein) in the umbilical cord are removed. Transfer to a new plate.
(2) Stripping Wharton's jelly in umbilical cord, cleaning with tissue cleaning liquid, cutting into 2-3mm 3 Transferred to T75 flasks with 1mL of each flask.
The present embodiment specifically operates as follows: the Whatman's jelly is peeled off by forceps, placed in a petri dish containing a tissue washing liquid, washed with the tissue washing liquid and torn into a long strip. Transferring Whatman's jelly into 50mL centrifuge tube with forceps after treatment, and shearing with scissors to 2-3mm 3 Transfer to T75 flasks with 3mL sterile droppers, 1mL each, with sterile scissors to cut off the tips before use.
(3) Adding MSC complete medium (DMEM/F12 medium containing 10% fetal bovine serum) 10mL slowly into the flask, gently shaking the flask, spreading umbilical cord tissue blocks in the flask, and adding 5% CO at 37deg.C 2 And (3) carrying out stationary culture in an incubator, supplementing liquid once every 2-3 days (supplementing 3-4mL of MSC complete medium each time), removing tissue blocks after supplementing liquid 2-3 times, and continuously culturing by replacing the MSC complete medium in full quantity until the cells grow to 70% of fusion degree, wherein the obtained cells are digested next.
The present embodiment specifically operates as follows: to the flask, 10mL of MSC complete medium (DMEM/F12 medium containing 10% fetal bovine serum) was slowly added, and the flask was gently shaken to spread umbilical cord tissue blocks in the flask. T75 flask was placed in 5% CO at 37 ℃C 2 And (5) standing and culturing in an incubator. The flask was removed from the incubator at day three, 3.5mL of MSC complete medium was supplemented, after which 3 days of fluid replacement were spaced. After 3 times of fluid infusion, cells can climb out of the bottom of the culture flask, remove tissue blocks and replace the complete culture medium of MSC in full quantity for continuous culture. When the cells grew to a confluence of 70%, the cells obtained were then digested.
(4) The culture medium in the culture flask is sucked and removed, PBS (20 mL) is sucked and washed to remove residual fetal calf serum, 3mL of 0.25% trypsin solution is added for digestion, the culture flask is tapped to enable cells to fall off to the bottom of the culture flask, 10mL of MSC complete medium is added for neutralizing trypsin to terminate the digestion process, a pipettor is used for blowing off the cells, cell suspension is collected in a centrifuge tube, and the supernatant is centrifuged and removed to obtain cells with the cell sediment recorded as P0 generation.
The present embodiment specifically operates as follows: before cell subculture, 0.25% trypsin, MSC complete medium (DMEM/F12 medium containing 10% foetal calf serum), PBS were placed at room temperature equilibrium temperature. The flask was removed from the incubator, the medium in the flask was pipetted off, and 20mL of PBS was pipetted off to rinse the flask to remove residual fetal bovine serum. 3mL of 0.25% trypsin solution is added into a culture bottle for digestion, the culture bottle is gently shaken to enable trypsin to fully cover the cell surface, the digestion is carried out for about 1min, the cells are observed under a microscope to shrink into a round shape, the culture bottle is gently tapped to enable the cells to fall off to the bottom of the culture bottle in a quicksand shape, 10mL of MSC complete medium is added to neutralize trypsin to terminate the digestion process, a pipettor is used for blowing off the cells, and cell suspension is collected in a 15mL centrifuge tube. The tube was placed in a centrifuge and centrifuged at 1400rpm for 5min, the supernatant removed and the precipitated cells were designated as P0 generation.
(5) The P0 cell pellet was resuspended in 10mL of MSC-conditioned medium containing 9% fetal bovine serum, and the cells were counted and then plated at a plating density of 1X 10 4 Individual MSC cells/cm 2 Inoculating into T75 culture flask, and supplementing MSC acclimation culture medium to 10mL, 37deg.C 5% CO 2 And (3) standing and culturing in an incubator for more than 72 hours until the cell fusion degree reaches 80%, sucking and discarding the culture medium in the culture flask, sucking (20 mL) PBS, rinsing the culture flask to remove residual fetal calf serum, adding 3mL of 0.25% trypsin solution for digestion, tapping the culture flask to enable the cells to fall off to the bottom of the culture flask, adding 10mL of MSC domestication culture medium used in the step to neutralize trypsin so as to terminate the digestion process, blowing off the cells by a liquid transfer device, collecting cell suspension in a centrifuge tube, centrifuging, discarding supernatant, and obtaining cell sediment which is marked as P1 generation cells.
(6) The method of culturing and digesting in the step (5) is adopted, the cells obtained in the last step in the operation process are treated by MSC domestication culture medium which sequentially reduces the concentration of fetal bovine serum to 0% finally, and finally, the mesenchymal stem cells which are subjected to final serum-free in vitro domestication culture are obtained (in the embodiment, the P1 generation cells are sequentially cultured and digested by MSC domestication culture medium containing 7.5% fetal bovine serum (obtaining P2 generation cells), the P2 generation cells are cultured and digested by MSC domestication culture medium containing 5.5% fetal bovine serum (obtaining P3 generation cells), the P3 generation cells are cultured and digested by MSC domestication culture medium containing 3% fetal bovine serum (obtaining P4 generation cells), the P4 generation cells are cultured and digested by MSC domestication culture medium containing 0% fetal bovine serum (obtaining 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 (I)
(7) The last generation of mesenchymal stem cells obtained in the last step of serum-free domestication culture are continuously cultured and passaged by a domestication culture medium containing 0% of fetal bovine serum MSC (the last generation of mesenchymal stem cells can be continuously transferred to the P20 generation of serum-free domestication culture).
In the present embodiment of the present invention,
culturing with MSC complete culture medium (containing 10% fetal bovine serum) to obtain P0 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 9% foetal calf serum) to obtain P1 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 7.5% foetal calf serum) to obtain P2 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 5.5% foetal calf serum) to obtain P3 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 3% foetal calf serum) to obtain P4 generation cell,
culturing with serum-free medium (containing transferrin, insulin, 0% bovine serum) to obtain P5 generation cells; in various subsequent detection assays of the present invention, cells of this generation (which are also referred to as detection cells) are used, unless otherwise indicated.
The serum reduction medium or MSC acclimation medium described in this example, for example, was used to obtain P1 generation cells, which was prepared as follows: adding transferrin (to a concentration of 10 ng/mL), insulin (to a concentration of 10 ng/mL) and fetal bovine serum (to a concentration of 9%) to a DMEM/F12 medium to prepare MSC domestication mediums containing transferrin, insulin and fetal bovine serum with different concentrations; in other words, the reduced serum medium used to obtain the P1 generation cells in this example contains: transferrin 10ng/mL, insulin 10ng/mL, fetal bovine serum 9%, balanced amount of DMEM/F12 medium. In the present invention, the reduced serum medium or the MSC-acclimated medium has similar meanings unless otherwise indicated.
Example 2: serum-free domestication culture MSC
The steps (1) to (4) were the same as in example 1, and cells were obtained in which the cell pellet was counted as P0 passages.
Steps (5) - (6) refer to the corresponding steps of example 1, except that the gradient of decrease in fetal bovine serum concentration in the MSC acclimation medium was changed. Optionally (I)
(7) The last generation of mesenchymal stem cells obtained in the last step of serum-free domestication culture are continuously cultured and passaged by a domestication culture medium containing 0% of fetal bovine serum MSC (the last generation of mesenchymal stem cells can be continuously transferred to the P20 generation of serum-free domestication culture).
In the present embodiment of the present invention,
culturing with MSC complete culture medium (containing 10% fetal bovine serum) to obtain P0 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 9% foetal calf serum) to obtain P1 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 7% foetal calf serum) to obtain P2 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 5% foetal calf serum) to obtain P3 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 3% foetal calf serum) to obtain P4 generation cell,
culturing with serum-free medium (containing transferrin, insulin, 0% bovine serum) to obtain P5 generation cells; in various subsequent detection assays of the present invention, cells of this generation (which are also referred to as detection cells) are used, unless otherwise indicated.
Example 3: serum-freeDomesticated culture of MSC
The steps (1) to (4) were the same as in example 1, and cells were obtained in which the cell pellet was counted as P0 passages.
Steps (5) - (6) refer to the corresponding steps of example 1, except that the gradient of decrease in fetal bovine serum concentration in the MSC acclimation medium was changed. Optionally (I)
(7) The last generation of mesenchymal stem cells obtained in the last step of serum-free domestication culture are continuously cultured and passaged by a domestication culture medium containing 0% of fetal bovine serum MSC (the last generation of mesenchymal stem cells can be continuously transferred to the P18 generation of mesenchymal stem cells obtained in the serum-free domestication culture).
In the present embodiment of the present invention,
culturing with MSC complete culture medium (containing 10% fetal bovine serum) to obtain P0 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 9% foetal calf serum) to obtain P1 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 8% foetal calf serum) to obtain P2 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 6% foetal calf serum) to obtain P3 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 4% foetal calf serum) to obtain P4 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 2% foetal calf serum) to obtain P5 generation cell,
Culturing with serum-free medium (containing transferrin, insulin, 0% bovine serum) to obtain P6 generation cells; in various subsequent detection assays of the present invention, cells of this generation (which are also referred to as detection cells) are used, unless otherwise indicated.
Example 4: serum-free domestication culture MSC
The steps (1) to (4) were the same as in example 1, and cells were obtained in which the cell pellet was counted as P0 passages.
Steps (5) - (6) refer to the corresponding steps of example 1, except that the gradient of decrease in fetal bovine serum concentration in the MSC acclimation medium was changed. Optionally (I)
(7) The last generation of mesenchymal stem cells obtained in the last step of serum-free domestication culture are continuously cultured and passaged by a domestication culture medium containing 0% of fetal bovine serum MSC (the last generation of mesenchymal stem cells can be continuously transferred to the P20 generation of serum-free domestication culture).
In the present embodiment of the present invention,
culturing with MSC complete culture medium (containing 10% fetal bovine serum) to obtain P0 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 9.5% foetal calf serum) to obtain P1 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 8.5% foetal calf serum) to obtain P2 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 7.5% foetal calf serum) to obtain P3 generation cell,
Culturing with reduced serum culture medium (containing transferrin, insulin, 6% foetal calf serum) to obtain P4 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 4% foetal calf serum) to obtain P5 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 2% foetal calf serum) to obtain P6 generation cell,
culturing with serum-free medium (containing transferrin, insulin, 0% bovine serum) to obtain P7 generation cells; in various subsequent detection assays of the present invention, cells of this generation (which are also referred to as detection cells) are used, unless otherwise indicated.
Example 5: serum-free domestication culture MSC
The steps (1) to (4) were the same as in example 1, and cells were obtained in which the cell pellet was counted as P0 passages.
Steps (5) - (6) refer to the corresponding steps of example 1, except that the gradient of decrease in fetal bovine serum concentration in the MSC acclimation medium was changed. Optionally (I)
(7) The last generation of mesenchymal stem cells obtained in the last step of serum-free domestication culture are continuously cultured and passaged by a domestication culture medium containing 0% fetal bovine serum MSC (the last generation of mesenchymal stem cells can be continuously transferred to the P15 generation of mesenchymal stem cells obtained in the serum-free domestication culture).
In the present embodiment of the present invention,
culturing with MSC complete culture medium (containing 10% fetal bovine serum) to obtain P0 generation cell,
Culturing with reduced serum culture medium (containing transferrin, insulin, 8% foetal calf serum) to obtain P1 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 6% foetal calf serum) to obtain P2 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 3% foetal calf serum) to obtain P3 generation cell,
culturing with serum-free medium (containing transferrin, insulin, 0% bovine serum) to obtain P4 generation cells; in various subsequent detection assays of the present invention, cells of this generation (which are also referred to as detection cells) are used, unless otherwise indicated.
Example 6: serum-free domestication culture MSC
The steps (1) to (4) were the same as in example 1, and cells were obtained in which the cell pellet was counted as P0 passages.
Steps (5) - (6) refer to the corresponding steps of example 1, except that the gradient of decrease in fetal bovine serum concentration in the MSC acclimation medium was changed. Optionally (I)
(7) The last generation of mesenchymal stem cells obtained in the last step of serum-free domestication culture are continuously cultured and passaged by a domestication culture medium containing 0% of fetal bovine serum MSC (the last generation of mesenchymal stem cells can be continuously transferred to the P20 generation of serum-free domestication culture).
In the present embodiment of the present invention,
culturing with MSC complete culture medium (containing 10% fetal bovine serum) to obtain P0 generation cell,
Culturing with reduced serum culture medium (containing transferrin, insulin, 9.5% foetal calf serum) to obtain P1 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 9% foetal calf serum) to obtain P2 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 8.5% foetal calf serum) to obtain P3 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 7.5% foetal calf serum) to obtain P4 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 6% foetal calf serum) to obtain P5 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 4.5% foetal calf serum) to obtain P6 generation cell,
culturing with reduced serum culture medium (containing transferrin, insulin, 2.5% foetal calf serum) to obtain P7 generation cell,
culturing with serum-free medium (containing transferrin, insulin, 0% bovine serum) to obtain P8 generation cells; in various subsequent detection assays of the present invention, cells of this generation (which are also referred to as detection cells) are used, unless otherwise indicated.
Example 7: serum-free domestication culture MSC
Referring to the operations of examples 1 to 6, except that 3- (2-methoxyphenoxy) -1, 2-propanediol (to a concentration of 0.1mg/mL in each MSC-acclimating medium) and magnesium chloride (to a concentration of 5. Mu.g/mL of magnesium ions in each MSC-acclimating medium) were additionally added to each MSC-acclimating medium used, 6 types of serum-free acclimating-cultured mesenchymal stem cells, which were called each generation of cells of examples 71 to 76, were finally obtained, respectively; in the various subsequent detection assays of the present invention, the corresponding generation of secondary cells (which are also referred to as detection cells) for detection are performed with the referenced working examples, unless otherwise specified.
The serum-reduced medium or the MSC-acclimated medium described in this example, for example, in reference example 1, was used to obtain P1-generation cells, which were prepared as follows: adding 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), magnesium chloride (to a magnesium ion concentration of 5 μg/mL) to a DMEM/F12 medium to prepare MSC domestication media containing transferrin, insulin, 3- (2-methoxyphenoxy) -1, 2-propanediol, magnesium chloride, and fetal bovine serum of different concentrations; in other words, the present example refers to example 1 in which the reduced serum medium for obtaining P1 generation cells contains: transferrin 10ng/mL, insulin 10ng/mL, fetal bovine serum 9%, 3- (2-methoxyphenoxy) -1, 2-propanediol 0.1mg/mL, magnesium chloride 5. Mu.g/mL as magnesium ion, balance DMEM/F12 medium. In the present invention, the reduced serum medium or the MSC-acclimated medium has similar meanings unless otherwise indicated.
Example 8: serum-free domestication culture MSC
Referring to the operations of examples 1 to 6, except that 3- (2-methoxyphenoxy) -1, 2-propanediol was additionally added to each of the MSC-acclimating mediums of fetal bovine serum concentrations (to a concentration of 0.1mg/mL in each of the MSC-acclimating mediums), 6 types of serum-free acclimating-cultured mesenchymal stem cells, respectively referred to as each of the generation cells of examples 81 to 86, were finally obtained; in the various subsequent detection assays of the present invention, the corresponding generation of secondary cells (which are also referred to as detection cells) for detection are performed with the referenced working examples, unless otherwise specified.
Example 9: serum-free domestication culture MSC
Referring to the operations of examples 1 to 6, except that magnesium chloride was additionally added to each of the MSC-acclimating mediums of fetal bovine serum concentrations (to a concentration of 5 μg/mL of magnesium ions in each of the MSC-acclimating mediums), 6 kinds of serum-free acclimating-cultured mesenchymal stem cells were finally obtained, which were called each of the cell generations of examples 91 to 96, respectively; in the various subsequent detection assays of the present invention, the corresponding generation of secondary cells (which are also referred to as detection cells) for detection are performed with the referenced working examples, unless otherwise specified.
Example 10: MSC cultured with serum
In the steps (5) and (6) of example 1, the culture and digestion of cells were carried out by changing all of the various MSC-acclimating mediums to MSC complete mediums, and P5-generation cells were obtained as mesenchymal stem cells cultured with serum as control cells for detection.
2. Testing of mesenchymal stem cells
MSCs are multipotent stem cells derived from early stage mesoderm of development and having high self-renewal capacity and multipotent differentiation potential, and can differentiate into hepatocyte-like cells, fibroblasts, osteoblasts, chondrocytes, adipocytes, myogenic cells, neuroblastocysts, and the like. Therefore, the multi-directional differentiation potential of the cells is verified by the differentiation experiments of the osteogenesis, the adipogenesis and the chondrogenesis of the MSC in a laboratory, so that the cell quality is reflected.
Test example 1: serum-free domesticated MSC cell osteogenic induced differentiation and staining experiment
1. Inoculation and osteogenesis induction culture
1. Preparing a high sugar complete medium: 56mL of fetal bovine serum is added into 500mL of high-sugar DMEM culture medium to prepare a high-sugar DMEM culture medium containing 10% of fetal bovine serum;
2. MSC cells in the logarithmic growth phase after serum-free acclimation of the P5 generation obtained in example 1 were digested with 0.25% trypsin solution, and the flask was gently shaken to spread trypsin on the bottom surface of the flask. Digesting for about 1min, observing cell shrinkage and agglomeration under a microscope, gently beating a culture bottle to enable the cells to fall off to the bottom of the culture bottle in a quicksand shape, adding a complete culture medium with the same volume as 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 complete medium and counted.
4. According to 6X 10 4 The inoculation density per mL was inoculated into 24-well plates with 1mL per well and 4 wells total inoculated. 1 well is control well and 3 wells are experimental wells. The plates were placed in 5% CO at 30 ℃C 2 Culturing in an incubator.
5. After the cell fusion rate reached 80%, the medium was washed away, 0.5mL of high-sugar DMEM complete medium containing 10% fetal bovine serum was added to the control well, and 0.5mL of MSC osteogenesis induction medium was added to the other 3 experimental wells. The plates were re-placed at 30℃with 5% CO 2 Culturing is continued in the incubator.
6. The liquid is changed every 2-3 days. When the liquid is changed, the Kong Nayuan culture medium is firstly sucked away, then 0.5mL of the corresponding culture medium is added, and the induction time is 15-20 days.
2. Dyeing identification
1. The staining identification of osteogenic induced differentiation culture adopts alizarin S staining method.
2. Sample fixation treatment: the culture medium in the 24-well plate was carefully aspirated, and 400. Mu.L of PBS buffer was added to each well to wash the cell surface. The PBS wash was carefully pipetted off, 300. Mu.L BI Fixation Solution was added to each well, covering the entire growth surface. After 30min of standing at room temperature, fixation Solution of BI was carefully pipetted, 300. Mu.L of BI wash I was added to each well, and the cell surface was washed 2-3 times.
3. Sample dyeing treatment: wash I was pipetted off and 300 μl BI Staining Solution was added and the bottom surface soaked evenly. Staining at room temperature for 30min, carefully pipetting out Staining Solution of BI, adding 300. Mu.L of wash II of BI, rinsing the cells 2-3 times. Wash II was pipetted, 300 μl of Inspection Solution was added, the cells were observed under a microscope and photographed (the differentiated cells were stained orange).
The results are shown in FIG. 1. A serum-free acclimation test group in FIG. 1 shows cells were stained with a typical orange color, and b control group in FIG. 1 did not stain with a typical orange color.
Referring to the method of this test example 1, the 5 test cells of examples 2 to 6, the 6 test cells of example 7, the test cell of example 81, and the test cell of example 91 were each examined, and the results showed that these cells exhibited the result that the cells were stained with a typical orange color.
Test example 2: serum-free domesticated MSC cell adipogenic induction differentiation and staining experiment
1. Solution reagent preparation:
1. trypsin, MSCgo Adipogenic Differentiation Basal Medium, MSCgo advertisement SF, XF supply Mix I, MSCgo advertisement SF, XF Supplement Mix II were preheated in a 37 ℃ incubator prior to the start of the experiment.
2. Preparing a high sugar complete medium: 56mL of fetal bovine serum is added into 500mL of high-sugar DMEM culture medium to prepare a high-sugar DMEM culture medium containing 10% of fetal bovine serum;
3. configuration of fat induction medium: the support Mix I, supplement Mix II was added to MSCgo Adipogenic Differentiation Basal Medium with a pipette.
4. MSC cells in the logarithmic growth phase after serum-free acclimation of the P5 generation obtained in example 1 were digested with trypsin, and the flask was gently shaken to spread the trypsin on the bottom surface of the flask. Digesting for about 1min, observing cell shrinkage and agglomeration under a microscope, gently beating a culture bottle to enable the cells to fall off to the bottom of the culture bottle in a quicksand shape, adding a complete culture medium with the same volume as 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 complete medium and counted.
6. According to 6X 10 4 The inoculation density per mL was inoculated into 24-well plates with 1mL per well and 4 wells total inoculated. 1 well is control well and 3 wells are experimental wells. The plates were placed in 5% CO at 30 ℃C 2 Culturing in an incubator.
7. After the cell fusion rate reached 80%, the culture medium was washed off, 0.5mL of high-sugar DMEM complete medium containing 10% fetal bovine serum was added to the control well, and 0.5mL of MSC fat induction medium was added to the other 3 experimental wells. The plates were re-placed at 30℃with 5% CO 2 Culturing is continued in the incubator.
8. The liquid is changed every 2-3 days. When the liquid is changed, the Kong Nayuan culture medium is firstly sucked away, then 0.5mL of the corresponding culture medium is added, and the induction time is 15-20 days.
2. Dyeing identification
1. The dyeing identification of the adipogenic induced differentiation culture adopts a neutral lipid oil red O dyeing method.
2. Sample fixation treatment: the culture medium in the 24-well plate was carefully aspirated, and 300. Mu.L of PBS buffer was added to each well to wash the cell surface. The PBS wash was carefully pipetted off, and 300. Mu.L of BI's Adipo-Fixation was added to each well to cover the entire growth surface. After 30min of standing at room temperature, 300. Mu.L of BI Adipo-wash I wash was added to each well to carefully blot the BI Adipo-wash, and the cell surface was washed and left standing for 2-3 min. The Adipo-Wash I of the BI was carefully withdrawn.
3. Sample dyeing treatment: adipo-starting A and Adipo-starting B were mixed according to 3:2, and mixing the volumes to obtain the dyeing working solution. 300. Mu.L of BI staining solution was carefully added to cover the entire growth surface and allowed to stand at room temperature for 30min. The BI staining solution was carefully withdrawn, 300 μLBI of Wash II was carefully added, the surface was rinsed, wash II was blotted, the above procedure repeated, and the cells were rinsed again (as clear as red, the rinsing time could be prolonged or the rinsing time was repeated, taking care not to Wash the cells down). mu.L of Adipo-injection was added to each well. Immediately upon observation with an optical microscope, the lipid material would appear typically red.
The results are shown in FIG. 2, in which the serum-free acclimated experimental group a in FIG. 2 shows lipid substances exhibiting typical red color, and in which the control group b in FIG. 2 cells do not show lipid substances exhibiting typical red color.
Referring to the method of this test example 2, the results of the detection of 5 kinds of detection cells of examples 2 to 6, 6 kinds of detection cells of example 7, detection cell of example 81, and detection cell of example 91 showed that these cells all exhibited typical red lipid substances.
Test example 3: serum-free domesticated MSC cell chondrogenic induced differentiation and staining experiment
1. Reagent preparation and cell culture treatment
1. Trypsin, MSCgo Chondrogenic Differentiation Basal Medium, MSCgo Chondrogenic Differentiation Supplement Mix was preheated in an incubator at 37 ℃ before the experiment started.
2. Preparing a high sugar complete medium: 56mL of fetal bovine serum is added into 500mL of high-sugar DMEM culture medium to prepare a high-sugar DMEM culture medium containing 10% of fetal bovine serum;
3. configuration of cartilage induction culture: 10mL Supplement Mix was added to 100mL of Chondrogenic Differentiation Basal Medium using a pipette.
4. MSC cells in the logarithmic growth phase after serum-free acclimation of the P5 generation obtained in example 1 were taken and digested with trypsin, and the flask was gently shaken to spread the trypsin on the bottom surface of the flask. Digesting for about 1min, observing cell shrinkage and agglomeration under a microscope, gently beating a culture bottle to enable the cells to fall off to the bottom of the culture bottle in a quicksand shape, adding a complete culture medium with the same volume as 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 complete medium and counted.
2. Cartilage induction
Scheme one:
1. according to 1X 10 7 cells/mL were seeded at a density of 10. Mu.L per well into 3 wells in a 24-well cell culture plate. 1mL of sterile PBS was added to the peripheral blank wells to seal the edges for humidity;
2. placing the 24-well cell culture plate into 5% CO 2 Culturing in incubator for 2hr;
3. after the cells are attached, 1 hole is taken as a control group, and 1mL of high-sugar DMEM culture medium containing 10% FBS is added; in addition, 3 holes are experimental groups, and 0.5mL of MSC is added into the experimental groups to form cartilage induction culture medium;
4. put in 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 density, re-suspending MSC cell pellet, and preparing 4mL MSC cell suspension;
2. the cell suspension was seeded in 24-well plates, each well was seeded with 1mL of 5% CO at 37 ℃ 2 Continuously culturing in an incubator;
3. after 24hr, the culture solution was washed off, 1 well was used as a control well, 0.5mL of 10% FBS-containing high sugar medium was added, and the other 3 wells were used as experimental groups, and 0.5mL of MSC cartilage induction medium was added, and 5% CO was added 2 Culturing at 37deg.C in incubator.
4. The liquid is changed once every 3 to 4 days, the culture medium in the hole is firstly sucked and removed when the liquid is changed, then 0.5mL of the corresponding culture medium is added, and the induction time is 14 to 21 days.
3. Dyeing identification
1. The staining identification of the chondrogenic induced differentiation culture adopts an Abl Xin Lan staining method;
2. sample fixation treatment: the culture medium in the 24-well plate was carefully aspirated, and 400. Mu.L of PBS buffer was added to each well to wash the cell surface. The PBS wash was carefully pipetted off, and 400. Mu.L BI Fixation Solution was added to each well to cover the entire growth surface. After standing for 30min at room temperature, carefully absorbing Fixation Solution of BI, adding 200 mu L of BI wash I lotion into each hole, and standing for 2-3 min;
3. sample dyeing treatment: absorbing Wash I, adding 300 mu L of BI Staining solution, soaking the bottom surface uniformly, and dyeing overnight at room temperature in a dark place;
4. staining solution was pipetted off, 300 μl of Wash II was added to the BI, and the cells were rinsed;
5. wash II is pipetted and rinsed once again by adding 300. Mu.L of BI (if the supernatant is still blue, the rinsing time can be prolonged again);
6. inspection Solution with 300 μl BI added; cells successfully differentiated into cartilage were observed by optical microscopy to appear dark blue.
The results of protocol one are shown in FIG. 3, where the serum-free acclimated experimental group a in FIG. 3 shows cells exhibiting a typical deep blue color, and the control group b in FIG. 3 does not show cells exhibiting a typical deep blue color. The results of scheme two are substantially the same as those of scheme one.
Referring to the method of this test example 3, the results of the detection of 5 kinds of detection cells of examples 2 to 6, 6 kinds of detection cells of example 7, detection cell of example 81, and detection cell of example 91 (scheme one) showed that these cells all exhibited typical deep 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 of examples 1 to 9 of the present invention exhibit typical properties of differentiating the mesenchymal stem cells into osteoblasts, chondroblasts, and adipocytes.
Test example 4: by means of Annexin Detection of early/late apoptosis of P5 generation domesticated cells by V/PI reagent
Apoptosis is one of the basic characteristics of cells, and plays an important role in the aspects of embryo development, tissue repair, internal environment stabilization and the like of organisms. Annexin V is Ca2 with a molecular weight of 35.8KD + The dependent phospholipid binding protein can be specifically combined with Phosphatidylserine (PS) which is turned outside a membrane in the early apoptosis process. After FITC-Annexin V is combined to apoptotic cells, green fluorescence is emitted under the excitation of blue light, and apoptotic cells and normal cells are distinguished. Propidium Iodide (PI) is a nucleic acid dye that does not penetrate intact cell membranes but is capable of penetrating damaged cell membranes of cells in late apoptosis and dead cells and redstains the nucleus. FITC-Annexin V was used in combination with PI to distinguish early and late apoptotic cells.
Test article of this test example: the mesenchymal stem cells obtained by serum-free acclimation culture in the method of the present invention (the test cells of examples 1 to 9, e.g., the P5 mesenchymal stem cells of example 1) and the P5 mesenchymal stem cells obtained by serum-free acclimation culture in the method of example 1 were used in reference to CN108642002A (201810047983.2).
1. Taking P5 generation mesenchymal stem cells to be tested which are subjected to serum-free domestication, sucking and discarding the complete culture medium by a pipette, and washing a culture bottle for 30s by 20mL of PBS and then sucking and discarding;
2. 2mL of trypsin solution (0.25%) preheated to 37℃was added to the flask, and the flask was gently shaken to spread trypsin on the bottom surface of the flask. Digesting for about 1min, observing cell shrinkage and agglomeration under a microscope, lightly beating a culture bottle to enable the cells to fall off to the bottom of the culture bottle in a quicksand shape, adding a complete culture medium with the same volume as trypsin to terminate digestion, blowing off the cells by using a liquid transfer device, 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 4deg.C, and rinsing cells again at 1000rpm for 5 min;
4. 3mL of Binding Buffer (10X) was diluted to 30mL with 27mL of deionized water;
5. The cells were suspended with 1mL of 1 Xbinding Buffer, centrifuged at 1000rpm for 10min, and the supernatant was discarded;
6. the cells were resuspended with 1mL of 1 Xbinding Buffer to a cell density of 1X 10 7 cells/mL;
7. an EP tube of 1.5mL was taken, and 100. Mu.L of the cell suspension, i.e., 1X 10 in cell number, was added to the tube 6 cells/mL;
8. then 5 mu L of Annexin V-FITC is added into the tube, the mixture is gently mixed, and the mixture is incubated for 10min at room temperature in a dark place;
9. adding 5 mu L of PI, and incubating at room temperature in a dark place for 5min;
10. adding PBS to 500 mu L, lightly mixing, loading in a flow mode, detecting cell expression quantity at 488nm, collecting 75 ten thousand cells simultaneously in each test group, and calculating the ratio of early apoptosis and late apoptosis/death of the cells (average value of n=5);
11. the measurement and calculation result shows that the mesenchymal stem cells obtained by the serum-free domestication culture method have excellent results for the two parameters of early apoptosis cell proportion and late apoptosis/death cell proportion.
Results: the 6 test cells of examples 1 to 6 have an early apoptotic cell ratio in the range of 23.5% to 25.2%, a late apoptotic/dead cell ratio in the range of 13.6% to 15.1%, e.g., the P5 stem cell of example 1 has an early apoptotic cell ratio of 24.3% and a late apoptotic/dead cell ratio of 14.4%; the 6 test cells of example 7 have an early apoptotic cell ratio in the range of 12.4% to 13.3%, a late apoptotic/dead cell ratio in the range of 6.8% to 7.7%, e.g., the P5 stem cell of example 71 has an early apoptotic cell ratio of 12.7% and a late apoptotic/dead cell ratio of 7.4%; the 6 test cells of example 8 have an early apoptotic cell ratio in the range of 23.7% to 25.7%, a late apoptotic/dead cell ratio in the range of 13.7% to 15.3%, e.g., the P5 stem cell of example 81 has an early apoptotic cell ratio of 23.9% and a late apoptotic/dead cell ratio of 14.8%; the 6 test cells of example 9 have an early apoptotic cell ratio in the range of 23.4% to 24.7%, a late apoptotic/dead cell ratio in the range of 13.8% to 15.0%, e.g., the P5 stem cell of example 91 has an early apoptotic cell ratio of 23.9% and a late apoptotic/dead cell ratio of 14.4%; the proportion of early apoptotic cells in CN108642002a was 33.4% and the proportion of late apoptotic/dead cells was 21.7%.
From the results of this test example 4, it was found that the mesenchymal stem cells obtained by the serum-free acclimation culture method of the present invention exhibited superior performance to the prior art. In particular, it has been unexpectedly found that the simultaneous addition of a glycol compound and a magnesium salt to an MSC acclimation medium supplemented with transferrin and insulin and with a progressive decrease in fetal bovine serum can significantly improve the performance of the cells.
Test example 5: detection of serum-free acclimated MSC cell phenotype
Taking the P5 generation obtained in the example 1 and the serum-cultured mesenchymal stem cells of the P5 generation obtained in the example 10 respectively, detecting cell surface markers by flow cytometry, and observing whether the change of the cell surface markers in the domestication culture process is different from that in the conventional culture. The specific implementation mode is as follows: digesting and collecting P5 generation serum-free domesticated MSC cells and conventional cultured MSC cells, counting, and collecting 8×10 6 Individual cells, 16 tubes; washing once with PBS, and centrifuging at 1500rpm for 10min; discarding the supernatant, leaving 100-200 mu L of residue, and blowing and beating uniformly mixed cells; FITC-labeled CD73 antibody 10 mu L, perCP-Cy5.5-labeled CD90 antibody 10 mu L, perCP-Cy5.5-labeled CD105 antibody 10 mu L, PE-labeled HLA-DR antibody 10 mu L was added, and a tube was set as a blank; reacting in dark at 4 ℃ for 30min; washing once with PBS, and centrifuging at 1500rpm for 10min; directly labeling cells, discarding supernatant, adding 200 mu L of PBS, blowing and mixing the cells, fixing 200 mu L of 1% paraformaldehyde, placing at 4 ℃ for detection, and detecting by an up-flow cytometer within 3 days.
The results are shown in FIG. 4. From the results, it can be seen that serum-free acclimated cells are consistent with flow cell phenotype results with serum-cultured cells.
Referring to the method of test example 5, the results of the test of 5 test cells of examples 2 to 6, 6 test cells of example 7, 81 and 91 were substantially the same as those of FIG. 4, and it was revealed that the serum-free cultured cells were identical to the flow cell phenotype results of the serum-free cultured cells, indicating that the mesenchymal stem cells obtained by the serum-free cultured methods of examples 1 to 9 of the present invention exhibited excellent cell properties.
In addition, in supplementary example a, referring to the method of example 1, except that the mesenchymal stem cells cultured by serum-free acclimation of two generations of P5, respectively referred to as complement a1 and complement a2, were obtained in which the transferrin concentration was changed to 8ng/mL and the insulin concentration was changed to 12ng/mL, or in which the transferrin concentration was changed to 12ng/mL and the insulin concentration was changed to 8 ng/mL; in addition, in supplementary example b, the method of reference example 71 was different only in that the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol was changed to 0.08mg/mL, the concentration of magnesium chloride was changed to 6. Mu.g/mL, or the concentration of 3- (2-methoxyphenoxy) -1, 2-propanediol was changed to 0.12mg/mL, and the concentration of magnesium chloride was changed to 4. Mu.g/mL, to obtain two P5-generation serum-free acclimated cultured mesenchymal stem cells called as supplementary b1 and supplementary b2, respectively. The test results of the parameters of the two cells of the supplement a1 and the supplement a2, which were measured by the methods of test examples 1 to 5, are substantially identical to those of the P5 cell of example 1, the experimental results of parameters of both the cells of complement b1 and complement b2 are substantially identical to those of the P5 cell of example 71, for example, the early apoptotic cell ratio of the cells of complement a1 is 23.7% and the late apoptotic/dead cell ratio is 14.6%.
The examples of the present invention should be understood as merely illustrative of the present invention and not as limiting the scope of the present invention. Various changes and modifications to the present invention may be made by one skilled in the art after reading the description herein, and such equivalent changes and modifications are intended to fall within the scope of the present invention as defined in the appended claims.

Claims (7)

1. A method for serum-free acclimation of mesenchymal stem cells, the method comprising the steps of:
(1) Cleaning the placenta surface with a tissue cleaning solution to remove surface congestion, shearing umbilical cord and removing internal blood vessels; the tissue washing liquid is 0.9% physiological saline solution containing 1% penicillin and 1% streptomycin;
(2) Stripping Wharton's jelly in umbilical cord, cleaning and shearing to 2-3 mm 3 Transferring to T75 flasks, 1mL per flask;
(3) Adding MSC complete culture medium 10mL slowly into the culture flask, gently shaking the culture flask to spread umbilical cord tissue blocks in the culture flask, and spreading 5% CO at 37deg.C 2 Performing stationary culture in an incubator, supplementing 3-4mL of MSC complete culture medium each time after 2-3 days of liquid supplementing, removing tissue blocks and completely replacing the MSC complete culture medium to continue culturing until the cells grow to 70% of fusion degree, and then digesting the obtained cells; the MSC complete culture medium is DMEM/F12 culture medium containing 10% fetal calf serum;
(4) Sucking and discarding the culture medium in the culture flask, sucking PBS to rinse the culture flask to remove residual fetal calf serum, adding 3mL of 0.25% trypsin solution for digestion, tapping the culture flask to enable cells to fall off to the bottom of the culture flask, adding 10mL of MSC to complete the culture medium and trypsin to terminate the digestion process, blowing off the cells by a pipette, collecting cell suspension in a centrifuge tube, centrifuging, discarding supernatant, and obtaining cells with cell sediment recorded as P0 times;
(5) The P0 generation cell sediment is resuspended by 10mL of domesticated culture medium containing 7-9.5% of fetal bovine serum, and the cell count is carried out, and then the inoculation density is 1 multiplied by 10 4 Individual MSC cells/cm 2 Inoculating into T75 culture flask, and supplementing the acclimatized culture medium to 10mL, 37deg.C 5% CO 2 Standing in incubator for more than 72 hr until cell fusion degree reaches 80%, sucking culture medium in culture bottle, sucking PBS, washing culture bottle to remove residual fetal bovine serum, adding 3mL of 0.25% trypsin solution for digestion, tapping culture bottle to make cells fall to bottom of culture bottle, adding 10mL of acclimation culture medium for use in the step to neutralize trypsin to terminate digestion process, blowing off cells with a pipette, collecting cell suspension in centrifuge tube, centrifuging, discarding supernatant to obtain cell precipitate, and recording as P1 Replacing cells;
(6) Adopting the method of culturing and digesting in the step (5), treating the cells obtained in the last step in the operation process with a domestication culture medium which sequentially reduces the serum concentration of the fetal bovine to 0% at last, and obtaining the final serum-free in-vitro domestication-cultured mesenchymal stem cells; optionally (I)
(7) Continuously culturing and passaging the last generation mesenchymal stem cells obtained in the last step and subjected to serum-free domestication culture by using a domestication culture medium containing 0% fetal bovine serum;
wherein, the composition of the domestication culture medium is as follows: 10ng/mL of transferrin, 10ng/mL of insulin, 0-9.5% of fetal bovine serum, 0.1mg/mL of 3- (2-methoxyphenoxy) -1, 2-propanediol and 5 mug/mL of magnesium chloride by magnesium ion concentration, and the balance of DMEM/F12 culture medium.
2. The method according to claim 1, wherein in the step (4), after adding the trypsin solution, the flask is gently shaken to allow trypsin to fully cover the cell surface, and the trypsin is digested for 1min until the cells are observed under a microscope to shrink into a round shape, and the flask is gently tapped to allow the cells to fall off to the bottom of the flask in a quicksand shape.
3. The method according to claim 1, wherein in step (5), the P0 cell pellet is resuspended in a acclimatizing medium containing 8 to 9.5% fetal bovine serum.
4. The method according to claim 1, wherein in step (5), the P0 generation cell pellet is resuspended in acclimated medium containing 8%, 8.5%, 9%, or 9.5% fetal bovine serum.
5. The method according to claim 1, wherein the concentration of fetal bovine serum in the MSC-acclimating medium decreases with increasing cell passage during the repetition of step (5) of step (6) until the concentration of fetal bovine serum in the MSC-acclimating medium is 0.
6. The method according to claim 1, wherein in the step (6), the serum-free in vitro domesticated and cultured mesenchymal stem cells are obtained from the generation of P4 to P10 by gradually reducing the concentration of fetal bovine serum.
7. The method according to claim 1, wherein in the step (7), the mesenchymal stem cells which are finally obtained in the step (6) and are subjected to in vitro domestication culture by 0% fetal serum are continuously cultured and passaged by using MSC domestication culture medium containing 0% fetal bovine serum, and the mesenchymal stem cells are continuously transferred to P20 generation mesenchymal stem cells subjected to serum-free domestication culture.
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