CN107043745B - Method for maintaining multipotency of mesenchymal stem cells in vitro - Google Patents
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Abstract
The invention discloses a method for maintaining the pluripotency of mesenchymal stem cells in vitro, which comprises the following steps: 1) extracting, primary culturing and subculturing the mesenchymal stem cells; 2) preparing a composite membrane culture substrate with an oriented microstructure on the surface by a method combining rubbing orientation and epiphytic crystallization; 3) culturing the mesenchymal stem cells on the composite membrane culture substrate prepared in the step 2). The method disclosed by the invention is simple to operate, low in cost and easy to implement in a large area, can be used for obtaining the mesenchymal stem cells with multidirectional differentiation potential in a large scale, and has very high potential clinical application value in the field of tissue engineering.
Description
Technical Field
The present invention relates to the field of tissue engineering. More particularly, relates to a method for maintaining the pluripotency of mesenchymal stem cells in vitro.
Background
In response to the problems of poor transplantable organs and large allograft rejection faced by clinical organ transplantation, researchers have focused on the study of artificial tissues and organs. Bone Marrow Mesenchymal Stem Cells (BMMSCs) have attracted much attention from researchers because of their ability to differentiate into various Cells such as osteoblasts, chondrocytes, adipocytes, and the like. However, one of the problems that needs to be solved first for the use of BMMSCs for clinical treatment is how to obtain sufficient numbers of BMMSCs. The quantity of BMMSCs in human bone marrow is only 0.001% -0.01%, which is far less than the quantity of stem cells required by clinical treatment, so that the BMMSCs need to be proliferated in vitro for clinical treatment. The initial clinical research is to collect autologous stem cells of a patient, expand and culture the autologous stem cells in vitro for several weeks to a certain amount, and then use the autologous stem cells, however, the autologous stem cells are gradually exposed to various inconveniences in the application process, such as large individual difference of expansion capacity, potential tumor cell pollution risk, incapability of timely adapting to the needs of diseases and the like, so that the use of the autologous stem cells is severely restricted, and due to the change of the growth environment, the stem cells are easy to age in the in vitro culture process, namely the conditions of cell morphology change, homeostasis damage and pluripotency reduction occur, so that the pluripotency of the stem cells is finally lost, the value of clinical application is lost, and therefore, the maintenance of good growth state, especially the pluripotency potential of the BMMSCs in the in vitro proliferation culture process becomes very important.
In vitro culture of BMMSCs is mainly directly acted by adjacent cells and a contacted culture substrate, and the culture substrate is widely concerned and deeply researched by researchers due to the complexity of factors such as chemical composition, topological structure, mechanical property, surface bioactive molecules and the like. Morphologically analyzing the adherent growth of the primary extracted BMMSCs, wherein the BMMSCs are in a long fusiform shape and uniform in shape, and the cells are observed in a three-dimensional manner under an optical microscope. With the increase of culture time and the increase of passage times, BMMSCs have the problems of large and widened cell size, enlarged cell nucleus, increased pseudopodia and diversified forms, and the three-dimensional structure of BMMSCs is difficult to observe under an optical microscope. Since it is found that the morphology change of the mesenchymal stem cells has a certain reference value for the aging of the cells, researchers have utilized the concept of topological Reaction (Topographic Reaction) to control the morphology and pluripotency of the mesenchymal stem cells by controlling the microstructure of the surface of the culture substrate, and have obtained some preliminary research results (McMurray R J, Gadegaard N, Tsmibouri P M, et al. Nanoscale surface for the long-term architecture of the sensory stem cell photosystem and multipotency [ J ] Nature materials,2011,10(8):637 644.).
The methods for preparing surface microstructures, which have been reported to be more studied, mainly include conventional photolithography, micro-contact printing, inkjet printing, self-assembly methods, electrostatic spinning, etc., but these techniques are applied to basic research, and there are still many problems to be solved when they are applied to clinical research. Compared with other methods, the traditional photoetching technology has low requirements on instruments and equipment, but has lower resolution; although the resolution of the microstructure prepared by methods such as micro-contact printing and the like is improved compared with that of a photoetching method, the large-area preparation is difficult to realize; the electrostatic spinning technology not only needs high-voltage power supply equipment and has higher requirements on materials and environment, but also has poorer controllability of the prepared surface microstructure; the surface microstructure prepared by the self-assembly method has large adjustable range of resolution, but generally has poor stability and is difficult to prepare in a large area. Therefore, in order to achieve the purpose of promoting the BMMSCs to maintain pluripotency in vitro by controlling the surface microstructure of the culture substrate, and the culture substrate can be prepared in large quantities at low cost so as to meet the requirements of clinical application, a new method needs to be explored.
Therefore, it is required to provide a method for maintaining the multipotentiality of the mesenchymal stem cells during the in vitro amplification culture of the mesenchymal stem cells by controlling the orientation microstructure of the substrate surface.
Disclosure of Invention
The invention aims to provide a method for maintaining the pluripotency of mesenchymal stem cells in the in-vitro proliferation culture process of the mesenchymal stem cells.
In order to achieve the purpose, the invention adopts the following technical scheme:
the invention relates to a method for maintaining the pluripotency of mesenchymal stem cells in vitro, which comprises the following steps:
1) extracting bone marrow mesenchymal stem cells, primary culture and subculturing;
2) preparing a composite membrane culture substrate with an oriented microstructure on the surface;
3) culturing the mesenchymal stem cells on the composite membrane culture substrate prepared in the step 2).
Further, the mesenchymal stem cells can be derived from bone marrow of mice, rabbits, humans and other various animals.
Furthermore, the extraction of the mesenchymal stem cells can adopt a whole bone marrow culture method, a gradient centrifugation method and other methods.
Further, the primary culture is performed at 35-40 deg.C with 5% CO2Culturing in a cell culture box, half replacing liquid for the first time within 24-48 hours, completely replacing liquid for the first time within 36-72 hours, and replacing liquid once every 24-48 hours.
The subculture is carried out until the cell fusion ratio reaches 80% or more, and trypsin is used according to the proportion of every 105-106The ratio of trypsin to trypsin is 0.5-1 ml per cell and is 25-40 deg.CDissolving for 1-2 min until cell contraction becomes round, neutralizing with culture medium, blowing off cells, centrifuging at 750-1000rpm for 3-10 min, passaging at 1: 2-1: 4, and placing at 35-40 deg.C with 5% CO2Culturing in a cell culture box, and changing the culture solution every 36-48 hours.
Further, the subculture is subcultured to generations 2-5.
Further, the extraction, primary culture and subculture of the mesenchymal stem cells are performed in α -MEM, DMEM or DMEM/F12, wherein the culture medium contains 5-15% Fetal Bovine Serum (FBS) and 0.5-2% penicillin-streptomycin double antibody (P/S).
Further, the step 2) is a method combining rubbing orientation and epiphytic crystallization: first, a polymer rod is rubbed at a constant speed and force on a substrate on a hot stage at a temperature below the melting point of the polymer; then cooling to room temperature, and pulling the substrate loaded with the polymer in the biopolymer solution to obtain a biopolymer/polymer composite membrane; and finally, heating the prepared composite membrane substrate at the temperature of 5-15 ℃ lower than the melting point temperature of the biopolymer material to realize epiphytic crystallization, thereby obtaining the composite membrane culture substrate with the large-area surface having the oriented microstructure. The depth of the groove of the surface microstructure of the composite film substrate prepared by the method can reach 50nm or more, and the large-area oriented growth of the bone marrow mesenchymal stem cells can be induced.
The substrate can be glass material such as glass slide, cover glass and the like, and before use, the piranha washing liquid and the like are used for soaking and cleaning.
The polymer can be polytetrafluoroethylene, polyvinylidene fluoride, polycarbonate, polyethylene, polystyrene and the like, and the heating temperature of the hot stage in the friction process is lower than the melting point of the polymer and is specifically determined according to the type of the polymer.
The biological polymer is a high polymer material with good biocompatibility such as polycaprolactone, polylactic acid and the like, and the concentration of the high polymer solution and the used solvent in the film drawing process are determined according to the types of polymers; the concentration of the polycaprolactone is 0.1-0.01g/ml, and the solvent is chloroform, dichloromethane, ethyl acetate, tetrahydrofuran and dimethyl sulfoxide; the concentration of the polylactic acid is 0.2-0.01g/ml, and the solvent is chloroform, dichloromethane, acetone, ethyl acetate and tetrahydrofuran; the temperature of the heating table in the post-treatment heating process is 5-15 ℃ lower than the melting point temperature of the high polymer material, and is determined according to the type of the high polymer material.
Further, the seeding density of the mesenchymal stem cells in the step 3) is 2000-10,000/square centimeter; the culture conditions are 35-40 deg.C, 5% CO2Culturing in a cell culture box, and replacing the fresh culture medium once every 36-48 hours.
In the process of culturing and growing the mesenchymal stem cells on the composite orientation membrane substrate, the mesenchymal stem cells still keep the typical morphology of the mesenchymal stem cells in the form of 4 weeks of culture, still keep the multidirectional differentiation potential in the function and have better cell proliferation activity due to the combined action of the surface orientation microstructure, the mechanical hardness and other factors of the composite membrane substrate.
The invention has the following beneficial effects:
the method has the advantages of simple operation, cheap and easily obtained raw materials, no need of large-scale equipment, low economic investment and capability of achieving the purpose of obtaining the mesenchymal stem cells with large area and good proliferation activity.
The invention can provide more basis for tissue engineering research and bone marrow mesenchymal stem cell research, and provides more possibility for clinical application of the bone marrow mesenchymal stem cells.
Drawings
The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
Fig. 1 is a schematic diagram showing a preparation process of a culture substrate having an oriented microstructure on a surface thereof and a culture process of mesenchymal stem cells in example 1.
FIG. 2a shows a polarization microscope image of the composite culture substrate having an alignment microstructure on the surface prepared in example 1 (rubbing-induced alignment moiety in the upper right corner); 2b shows the height image of the AFM prepared in example 1 to obtain a composite culture substrate with an oriented microstructure on the surface.
FIG. 3a shows fluorescence confocal images of mesenchymal stem cells grown on the culture substrate with the microstructure oriented on the surface in example 1, and 3b is stem cells on the culture substrate without the microstructure oriented on the surface (control group), (blue: nucleus; green: actin).
FIG. 4 shows qRT-PCR data of the mesenchymal stem cells of example 1 after culturing for 4 weeks on a culture substrate with an oriented microstructure on the surface, and the stem cells cultured for 4 weeks on a substrate without an oriented microstructure on the surface are used as a control (Nanog, Sox-2, Pou5f1 is a stem cell pluripotency marker gene, and a housekeeping gene GAPDH is used as a reference).
Fig. 5a-b show osteogenesis and adipogenesis induction optical images of the 3 rd generation bone marrow mesenchymal stem cells in example 1 (a. osteogenesis, b. adipogenesis); fig. 5c-d show bone marrow mesenchymal stem cell osteogenesis and adipogenesis induced optical images (c. osteogenesis, d. adipogenesis) cultured for 4 weeks on a culture substrate having an oriented microstructure on the surface in example 1.
Detailed Description
In order to more clearly illustrate the invention, the invention is further described below with reference to preferred embodiments and the accompanying drawings. Similar parts in the figures are denoted by the same reference numerals. It is to be understood by persons skilled in the art that the following detailed description is illustrative and not restrictive, and is not to be taken as limiting the scope of the invention.
Example 1A method for maintaining the pluripotency of mesenchymal stem cells in vitro
A method for maintaining the pluripotency of mesenchymal stem cells in vitro mainly comprises the following steps:
1) the femur and tibia were separated by 3-week SD rats with broken neck, adherent muscle tissue was further removed in PBS (pH 7.4) in a sterile operating table, both ends of the bone were removed, a sufficient amount of α -MEM (10% FBS, 1% P/S) medium was aspirated with a 5-ml syringe to blow out the whole bone marrow until the marrow cavity became white, the whole bone marrow was blown out with a 2-ml syringe, and the bone marrow was transferred to 37 ℃ with 5% CO2Culturing in a cell culture box, carrying out half liquid change for the first time after 48 hours, carrying out full liquid change for the first time after 72 hours, and then carrying out liquid change once every 48 hours; when the cells are fused to 80-90%, trypsin is used according to 10% of each6Cell 1ml pancreatic eggDigestion with white enzyme at 37 deg.C for 1 min, neutralization with α -MEM (containing 10% FBS, 1% P/S) medium, blowing off cells, centrifuging at 800rpm for 5 min at room temperature, passaging at 1:3 ratio, and placing at 37 deg.C and 5% CO2And (5) culturing in a cell culture box, changing liquid every 48 hours, repeating the passage operation until the fusion reaches 80-90 percent, and reaching the third generation of the mesenchymal stem cells.
2) Rubbing a Polytetrafluoroethylene (PTFE) rod on a clean glass slide treated by piranha washing liquid on a 280 ℃ hot bench to obtain a glass slide substrate loaded with an oriented PTFE layer, quickly pulling the glass slide substrate loaded with the oriented PTFE layer in a trichloromethane solution of polycaprolactone (PC L) with the number average molecular weight of 80,000 and the concentration of 0.02g/ml to obtain a PC L/PTFE oriented composite membrane, heating the membrane at 45 ℃ for 2 hours after solvent evaporation to promote further crystallization of a high molecular polymer, and finally obtaining a PC L/PTFE oriented composite culture substrate (the specific steps are shown in figure 1). As a comparison, the unoriented substrate is directly pulled in the trichloromethane solution with the same concentration of PC L to pull the clean glass slide treated by piranha washing liquid, and is treated on the 45 ℃ hot bench for 2 hours after the solvent is volatilized to finally obtain the PC L membrane substrate without the oriented microstructure on the surface.
Comparing the structure of the composite culture substrate with the oriented microstructure on the surface with the structure of the culture substrate without the oriented microstructure on the surface, as shown in fig. 2, as can be seen from the polarization microscope image of fig. 2a, compared with the spherulite structure of PC L, the PC L forms plate crystals along the PTFE orientation direction under the induction of the oriented polytetrafluoroethylene molecules, the crystallization is relatively uniform, the film-forming property of the high polymer material is good, and as can be seen from the atomic force microscope height image of the composite culture substrate with the oriented microstructure on the surface of fig. 2b, the groove depth of the oriented microstructure on the surface of the prepared composite membrane substrate can reach 50nm, and the cells can be completely induced to be oriented along the groove direction.
3) Sterilizing the obtained culture substrate with surface orientation or non-orientation by ultraviolet irradiation under ultraviolet lamp for 24 hr, soaking the sterilized culture substrate in PBS (pH 7.4) and α -MEM (containing 10% FBS and 1% P/S) for 24 hr, inoculating the extracted mesenchymal stem cells of generation 3 onto the culture substrate with surface orientation or non-orientation at a density of 4000/sq cm,at 37 ℃ with 5% CO2The culture was carried out in a cell incubator, and the fresh medium was replaced every 48 hours.
4) Extracting total RNA of mesenchymal stem cells cultured on an oriented composite membrane substrate after four weeks of culture, then carrying out reverse transcription to obtain cDNA, then carrying out relative quantification on the expression of Nanog, Sox-2 and Pou5f1 pluripotency marker genes in the mesenchymal stem cells by using qRT-PCR (taking housekeeping gene GAPDH as reference), meanwhile, taking the mesenchymal stem cells cultured on a substrate without a surface oriented microstructure for 4 weeks as comparison, further, digesting the mesenchymal stem cells cultured on a PC L/PTFE substrate for 4 weeks by using trypsin, planting the mesenchymal stem cells in a 12-well plate according to 5000/square centimeter density, carrying out osteogenesis or adipogenesis induction by using an osteogenesis inducer when the cell fusion proportion reaches 80%, then dyeing by using alizarin red S or oil red O, and comparing with the mesenchymal stem cells of the third generation to determine whether the mesenchymal stem cells have pluripotency.
FIG. 3 shows fluorescence confocal images of 5 days after the growth of mesenchymal stem cells on PC L/PTFE or PC L, from which it can be known that the mesenchymal stem cells realize large area orientation along the groove direction of the surface orientation microstructure of the PC L/PTFE composite membrane, FIG. 4 shows qRT-PCR data (Nanog, Sox-2, Pou5f1 are stem cell pluripotency marker genes, referenced to housekeeping gene GAPDH) after the mesenchymal stem cells are cultured on a PC L/PTFE or PC L substrate for 4 weeks, FIGS. 5a-b show osteogenesis and adipogenesis induced optical images (a. osteogenesis, b. adipogenesis) of the mesenchymal stem cells of generation 3, FIGS. 5c-d show osteogenesis and adipogenesis induced optical images (c. osteogenesis, d. adipogenesis) of the mesenchymal stem cells cultured on a substrate with the surface orientation structure for 4 weeks, and it can be known from FIGS. 4-5 that the mesenchymal stem cells can still maintain the pluripotency of the stem cells after being cultured by the method of the present invention.
Example 2 method for maintaining multipotentiality of mesenchymal stem cells in vitro
The steps are the same as the embodiment 1, and the difference is that the orientation-loaded polytetrafluoroethylene glass slide is obtained by using a rubbing orientation method in the step 2), the glass slide is directly used for cell culture after ultraviolet sterilization, the step of drawing and film forming by polycaprolactone is not carried out, and the clean glass slide subjected to ultraviolet sterilization is used as a contrast.
The result shows that the oriented composite culture substrate obtained by pulling and filming polycaprolactone is more beneficial to maintaining the pluripotency of the mesenchymal stem cells.
Example 3A method for maintaining the multipotentiality of mesenchymal stem cells in vitro
The steps are the same as example 1, except that in step 1), the source of the bone marrow mesenchymal stem cells is replaced by 2-week SD rats, 3-week KM mice and 12-week New Zealand rabbits, and the bone marrow mesenchymal stem cells are selected from 3 rd generation to 5 th generation.
Example 4A method for maintaining the multipotentiality of mesenchymal stem cells in vitro
The steps are the same as the example 1, except that the mesenchymal stem cells in the step 1) are extracted by a gradient centrifugation method to obtain the mesenchymal stem cells from generation 2 to generation 5 for culture.
Example 5A method for maintaining the multipotentiality of mesenchymal stem cells in vitro
The procedure was the same as in example 1, except that α -MEM (containing 10% FBS, 1% P/S) medium was replaced with DMEM/F12 (containing 8% FBS, 1% P/S) medium or DMEM (containing 15% FBS, 2% P/S) medium.
Example 6A method for maintaining the multipotentiality of mesenchymal stem cells in vitro
The procedure is the same as in example 1, except that polytetrafluoroethylene is replaced with polyethylene, polypropylene, polystyrene, polyvinylidene fluoride or polycarbonate; the temperature of the hot stage in the rubbing orientation process in the step 2) is respectively polyethylene (75 ℃), polypropylene (140 ℃), polystyrene (200 ℃), polyvinylidene fluoride (130 ℃) and polycarbonate (190 ℃).
Example 7A method for maintaining the multipotentiality of mesenchymal stem cells in vitro
The procedure was as in example 1 except that the chloroform solution of polycaprolactone (PC L) at a concentration of 0.02g/ml was replaced with a dichloromethane solution of polycaprolactone at a concentration of 0.01g/ml to 0.02 g/ml.
Example 8A method for maintaining the multipotentiality of mesenchymal stem cells in vitro
The steps are the same as the embodiment 1, except that the biological macromolecule is replaced by the polylactic acid from the polycaprolactone, and the temperature of the hot stage in the heat treatment process after the drawing film forming in the step 2) is 90 ℃.
The experimental results of examples 3-8 show that the same as example 1 can maintain the pluripotency of the mesenchymal stem cells after being cultured for four weeks.
It should be understood that the above-mentioned embodiments of the present invention are only examples for clearly illustrating the present invention, and are not intended to limit the embodiments of the present invention, and it will be obvious to those skilled in the art that other variations or modifications may be made on the basis of the above description, and all embodiments may not be exhaustive, and all obvious variations or modifications may be included within the scope of the present invention.
Claims (5)
1. A method for maintaining the pluripotency of mesenchymal stem cells in vitro comprises the following steps:
1) taking the mesenchymal stem cells which are subcultured to the 2 nd-5 th generation;
2) preparing a composite membrane culture substrate with an oriented microstructure on the surface; the step 2) comprises the following steps:
(1) rubbing the polymer rod at a constant speed and a constant force on a heated platen having a temperature below the melting point of the polymer and provided with a substrate; (2) cooling the substrate to room temperature, then immersing the substrate into a biopolymer solution, and pulling the polymer-loaded substrate to obtain a biopolymer/polymer composite film; (3) the method comprises the following steps of heating a prepared composite membrane substrate at a temperature 5-15 ℃ lower than the melting point temperature of a biopolymer material to realize epiphytic crystallization, wherein the depth of a groove of a surface microstructure of the composite membrane culture substrate is 50nm or more;
3) culturing mesenchymal stem cells on the composite membrane culture substrate prepared in the step 2); the seeding density of the mesenchymal stem cells is 2000-10,000/square centimeter; the culture conditions are 35-40 deg.C, 5% CO2Culturing in a cell culture box, and replacing the fresh culture medium once every 36-48 hours.
2. The method of claim 1, wherein: the substrate is a glass slide or a cover slip.
3. The method of claim 1, wherein: the polymer is polytetrafluoroethylene, polyvinylidene fluoride, polycarbonate, polyethylene or polystyrene; the biological polymer is polycaprolactone or polylactic acid.
4. The method of claim 1, wherein: the subculture is carried out until the cell fusion ratio reaches 80% or more, and trypsin is used according to the proportion of every 105-106Digesting the cells at a trypsin ratio of 0.5ml to 1ml at 25 ℃ to 40 ℃ for 1 to 2 minutes until the cells shrink and become round, neutralizing the cells with a culture medium, blowing the cells, centrifuging the cells at 750-1000rpm for 3 to 10 minutes, passaging the cells at a ratio of 1:2 to 1:4, placing the cells at 35 ℃ to 40 ℃ and 5% CO2Culturing in a cell culture box, and changing the culture solution every 36-48 hours.
5. The method according to claim 4, wherein the medium for subculturing the mesenchymal stem cells is α -MEM, DMEM or DMEM/F12.
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