CN116333977A - Stem cell three-dimensional differentiation model, construction method and application thereof - Google Patents
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Abstract
The invention discloses a stem cell three-dimensional differentiation model, a construction method and application thereof. The construction method comprises the following steps: and separating and two-dimensionally culturing stem cells from a sample, culturing and passaging to P3 generation, mixing the stem cells with a stem cell culture medium to prepare stem cell suspension, and co-culturing the 3D printed porous titanium alloy bracket and the stem cell suspension to obtain the three-dimensional stem cell differentiation model. According to the invention, scaffolds with different apertures and porosities are constructed by adopting a 3D printing technology, more primitive cells-cells interaction is adopted to promote a more real cell physiological environment, so that cells maintain better growth activity and biological function, the optimal promotion of physical properties and cell adhesion is realized, the yield of stem cell exosomes is further improved, and the immunoregulation of stem cells is promoted.
Description
Technical Field
The invention belongs to the technical field of biomedical materials, and relates to a stem cell three-dimensional differentiation model, a construction method and application thereof.
Background
Stem cells are a class of undifferentiated pluripotent cells with a high self-renewal and rapid proliferation capacity. Under specific conditions, stem cells can induce differentiation into other tissue cells of the human body, such as skeletal muscle cells, cardiac muscle cells, osteoblasts, neural cells, etc. In recent years, with the development of tissue engineering and regenerative medicine, paracrine action of cells has been receiving great attention since being discovered, and more evidence supports the view that stem cells are active in paracrine manner, especially that secretion of exosomes plays a vital role in their biological functions. Currently, numerous studies have demonstrated that stem cells mediate the biological functions of stem cells and induce differentiation into various cell types mainly through paracrine action, particularly exosomes. Exosomes are extracellular vesicles that contain a large amount of genetic information and abundant RNA species, including DNA, proteins, mRNA, miRNA, rRNA, etc. The stem cell derived exosome has similar function to stem cells, and can promote tissue repair and regeneration through angiogenesis, and play an important role in treating acute injury of organs. Meanwhile, the exosome treatment can avoid the risks of stem cell transplantation, such as immune rejection, reduced stem cell regeneration capability, tumorigenicity and the like. More and more studies have demonstrated that non-coding RNAs carried by exosomes, particularly mirnas, can be transported across cells, regulating key signaling pathways such as cell differentiation, proliferation and apoptosis at posttranscriptional levels. Therefore, the stem cell exosome is possible to be a replacement therapy for the stem cell therapy in the research fields of tissue repair and regenerative medicine, but the problems of low separation purity, high separation cost, low production efficiency and the like of the stem cell exosome limit the potential of clinical application. The problem of the adherence mode culture, namely the two-dimensional (2D) culture, is mainly represented by the adherence growth of two-dimensional cells, which are flat or long fusiform, the single growth of most cells with small nuclei, the rare occurrence of balling aggregation, the original three-dimensional structure and morphology of the cells are destroyed after a plurality of generations of culture, the interaction among the cells is reduced, and the properties of the cells are difficult to maintain; the structure, proliferation and differentiation ability of the two-dimensional environmental cells are impaired and the cell-specific properties are gradually lost; the surface area for cell adhesion growth in a two-dimensional environment is limited, and the oxygen and nutrient exchange and metabolic transport capacity among cells is limited. The survival rate of the cells cultured for a long time is reduced, and the biological activity is reduced.
CN108148811a discloses a method for establishing xenograft tumor model of colorectal cancer patient source based on a temperature-sensitive biogel three-dimensional culture system, which consists of temperature-sensitive biogel, digestive juice required for separating and culturing tumor cells, basal medium and human intestinal stem cell medium. Colorectal cancer cells can be further amplified and grown in the temperature-sensitive type biological gel three-dimensional culture system, the tumorigenic capacity of the colorectal cancer cells in an immunodeficiency mouse is enhanced, the preparation method is simple, the process is mature, but the cell sphere is limited in size, the cell sphere is too large to influence the material exchange of the cell sphere, the activity of the cells is influenced, and a large number of exosomes are not beneficial to obtaining.
CN111925982a discloses a process for culturing human bone marrow mesenchymal stem cells by using three-dimensional cells, which comprises a three-dimensional cell culture carrier and human bone marrow mesenchymal stem cells, wherein the formula of the three-dimensional cell culture carrier is that 1.5% of sulfhydrylation hyaluronic acid is dissolved in DMEM low-sugar basic medium and 4% of polyethylene glycol diacrylate is dissolved in DMEM low-sugar basic medium, and the two solutions are mixed uniformly to form in-situ crosslinking hyaluronic acid hydrogel at room temperature.
In summary, the existing two-dimensional stem cell culture has the problems of difficult maintenance of cell properties, impaired structure, proliferation and differentiation, limited surface area, low survival rate, low bioactivity and the like, and the three-dimensional gel culture operation is complex, so that accurate micropore size is difficult to obtain, and the exosomes are not easy to obtain. How to provide a three-dimensional differentiation model of stem cells, improve the cell death phenomenon inside cell aggregates, better maintain the functional characteristics of cells and better maintain the function of stem cells for regulating immunity, and is one of the problems to be solved in the technical field of biomedical materials at present.
Disclosure of Invention
Aiming at the defects and actual demands of the prior art, the invention provides a three-dimensional differentiation model of stem cells, a construction method and application thereof, which solve the problems that the cell characteristics are difficult to maintain, the structure, proliferation and differentiation capacity are damaged, the surface area is limited, the survival rate is low, the biological activity is low and the like in the two-dimensional culture of the stem cells, and the three-dimensional gel culture has complex operation, is difficult to realize the accurate micropore size, is not beneficial to obtaining exosomes and the like, better maintains the functional characteristics of the stem cells, and better maintains the function of regulating the immunity of the stem cells.
In order to achieve the aim of the invention, the invention adopts the following technical scheme:
in a first aspect, the present invention provides a method for constructing a three-dimensional differentiation model of stem cells, the method comprising: and separating and two-dimensionally culturing stem cells from a sample, culturing and passaging to P3 generation, mixing the stem cells with a stem cell culture medium to prepare stem cell suspension, and co-culturing the 3D printed porous titanium alloy bracket and the stem cell suspension to obtain the three-dimensional stem cell differentiation model.
According to the invention, scaffolds with different apertures and porosities are constructed by adopting a 3D printing technology, more primitive cells-cells interaction is adopted to promote a more real cell physiological environment, so that cells maintain better growth activity and biological function, the optimal promotion of physical properties and cell adhesion is realized, the yield of stem cell exosomes is further improved, and the immunoregulation of stem cells is promoted.
Preferably, the method of 3D printing comprises selective laser melting.
Preferably, the laser power of the selective laser melting is 180-240W, the scanning speed is 180-240nm/s, the layer thickness is 10-30 μm, the laser spot size is 50-100 μm, the lap joint distance is 20-50 μm, and the layer-by-layer rotation angle is 50-67 degrees.
Specific point values in the above 180-240 may be selected from 180, 190, 200, 210, 220, 240, etc.
Specific point values of 10 to 30 may be selected from 10, 15, 20, 25, 30, etc.
Specific point values among the above 50 to 100 may be selected from 50, 60, 70, 80, 90, 100, etc.
Specific point values of 20 to 50 may be selected from 20, 30, 40, 50, etc.
The specific point values 50-67 may be selected from 50, 55, 60, 67.
Preferably, the 3D printing porous titanium alloy bracket is disc-shaped, and the diameter range is phi (1-4) multiplied by L (8-35) mm.
The specific point values of 1-4 can be 1, 2, 3, 4.
Specific point values in the above 8 to 35 may be selected from 8, 9, 10, 15, 18, 20, 22, 25, 30, 31, 32, 33, 35, etc.
Preferably, the pore diameter of the 3D printing porous titanium alloy bracket is 200-1000 mu m, and the porosity is 30-70%.
Specific point values in the above 200-1000 may be selected from 200, 300, 400, 500, 600, 700, 800, 900, 930, 950, 1000, etc.
Specific point values in the above 30 to 70 may be selected from 30, 35, 40, 45, 50, 55, 60, 65, 68, 69, 70, etc.
Preferably, the pretreatment of the 3D printed porous titanium alloy scaffold includes a lotion soak and an autoclave.
Preferably, the washing liquid comprises an acid washing liquid or an alkali washing liquid.
Preferably, the alkaline wash comprises a potassium hydroxide-isopropanol solution.
Preferably, the concentration of the potassium hydroxide in the potassium hydroxide-isopropanol solution is 0.05-0.1mol/L.
Specific point values of 0.05 to 0.1 may be selected from 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, etc.
Preferably, the soaking time is greater than 24 hours (e.g., 25 hours, 26 hours, 27 hours, 30 hours, 48 hours, 60 hours, etc.).
Preferably, the pressure of the autoclave is 104.0-137.3kPa, the temperature is 121-126 ℃ and the time is 20-30min.
Specific point values of 104.0-137.3 may be selected from 104.0, 105.0, 106.0, 110, 120, 125, 130, 131, 131.1, 131.2, 131.3, etc.
Specific point values among 121-126 described above may be selected from 121, 122, 123, 124, 125, 126, etc.
Specific point values among the above 20 to 30 may be selected from 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, etc.
Preferably, the stem cells include any one or a combination of at least two of menstrual blood-derived mesenchymal stem cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells or adipose mesenchymal stem cells.
In a second aspect, the invention provides a three-dimensional stem cell differentiation model, which is constructed by the method for constructing the three-dimensional stem cell differentiation model in the first aspect.
In a third aspect, the present invention provides a stem cell culture system comprising a stem cell exosome and the three-dimensional differentiation model of stem cells of the first aspect.
Preferably, the stem cell exosomes comprise any one or a combination of at least two of Rabs protein, transmembrane protein, ribosomal protein, signal transduction factor or adhesion factor.
In a fourth aspect, the invention provides the use of the stem cell three-dimensional differentiation model of the second aspect or the stem cell culture system of the third aspect in drug screening, physiopathological research, cell therapy and tissue engineering.
Compared with the prior art, the invention has the following beneficial effects:
(1) The 3D printing porous titanium alloy bracket can form a 3D cell aggregate to better maintain a cell microenvironment, increase cell adhesion capacity, better maintain cell characteristics in an in-vitro cell culture process and increase cell biological activity;
(2) 3D printing of porous titanium alloy bracket co-culture stem cells, changing exosome components, and 3D culturing cells can obtain a large number of exosomes and keep the function of stem cells for regulating immunity;
(3) The 3D porous bracket structure increases the surface area for cell adhesion growth, expands the gaps among cells, provides a channel for exchange and transportation of oxygen, nutrient substances and metabolites in the aggregate, reduces the death condition in the cell aggregate, and better maintains the functional characteristics of the cells;
(4) The 3D printing porous titanium alloy bracket has better biocompatibility, and can effectively solve the problems of insufficient non-physical elastic modulus and biocompatibility of the traditional solid titanium alloy bracket;
(5) The cells do not need to be passaged, and can be continuously cultured to obtain a large number of exosomes;
(6) The stem cell exosome is obtained by constructing a three-dimensional environment, the preparation process is simple, the cost is low, the expansion production is easy, and the method is suitable for clinical application;
(7) The culture system provided by the invention is environment-friendly, can be repeatedly utilized, can be cleaned, disinfected and recycled for multiple times after cell enzymolysis, has strong plasticity and good stability of the titanium alloy bracket, can not rust, deform and wear, can be used as metal for recycling after the bracket is abandoned, and is more environment-friendly compared with the existing common plastic production system, and the long-term culture cost is reduced. Compared with the traditional 2D disc/bottle culture, the three-dimensional culture reduces the incubation space, and compared with the stirrer culture, the three-dimensional culture does not need complex machinery.
Drawings
FIG. 1 is a schematic illustration of a 3D printed porous titanium alloy stent;
FIG. 2 is a graph showing the phenotype identification result of mesenchymal stem cells;
FIG. 3A is 2X 10 8 A statistical plot of total exosomes cultured in 2D and 3D culture systems for individual MSCs;
FIG. 3B is a statistical plot of the number of 3D-exos particles and protein content harvested at different time points by the 3D culture system;
FIG. 4A is a diagram showing morphology and size of exosomes in two-dimensional culture;
FIG. 4B is a diagram showing morphology and size of exosomes in three-dimensional culture;
FIG. 4C is a graph showing the average distribution of exosome particle numbers in two-dimensional culture;
FIG. 4D is a graph showing the average distribution of exosome particle numbers in three-dimensional culture;
FIG. 4E is a graph showing the expression of CD81 and TSG101 proteins in exosomes.
Detailed Description
The technical means adopted by the invention and the effects thereof are further described below with reference to the examples and the attached drawings. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting thereof.
The specific techniques or conditions are not identified in the examples and are described in the literature in this field or are carried out in accordance with the product specifications. The reagents or apparatus used were conventional products commercially available through regular channels, with no manufacturer noted.
Example 1
This example prepares a 3D printed porous titanium alloy stent.
(1) The porous titanium alloy scaffold was obtained by EBM technique (EOS M280, germany) and its composition was Ti6Al4V. The porosity and pore size of the porous scaffolds were 70% and 300 μm, respectively. Porosity= (apparent volume of material in natural state-absolute volume of material)/apparent volume of material in natural state. The scaffold used for the cell experiments was disc-shaped (phi 4 XL 35 mm). Converting the 3D model of the designed titanium alloy bracket into a universal language file, inputting the universal language file into a 3D printer, and synthesizing the bracket by using 55 mu m Ti6Al4V powder by using an EBM machine, as shown in figure 1;
(2) Completely soaking the titanium alloy bracket with attachments removed in an acid-base jar containing acid-base washing liquid (3% potassium hydroxide-isopropanol solution) for 48 hours, and after the soaking is finished, washing under ultrapure water and drying;
(3) After drying, the pH value is measured and stabilized at pH8, high-pressure steam sterilization is carried out, and the completely digested tissue passes through a 120-mesh copper mesh cell sieve.
Example 2
The present example performs a menstrual Mesenchymal Stem Cell (MSC) culture.
(1) Menstrual cup for collecting menstrual fluid, monocyte separation of the menstrual blood sample using sample density separating liquid, washing the cells with PBS for 2 times to obtain stem cell pellet, re-suspending the cells with 10% FBS-containing medium, inoculating into culture flask, culturing in 5% CO 2 Culturing in an incubator at 37 ℃ until the cells grow to 85%, carrying out pancreatin digestion and passage, and taking P3 generation stem cells;
(2) Observing the morphology of stem cells under an inverted microscope, taking P3 generation cells to be evenly divided into 4 groups, setting the cell number of 6E 5/tube, adding mouse anti-human CD44, CD90, CD105, CD73, CD45, CD38, CD11b, CD19, CD34, CDHLA and the peer control, incubating for 30min at 25 ℃ in a dark place, detecting by a flow cytometer after centrifugal resuspension, respectively adding antibodies after stem cell characteristic identification, putting the cells into a low-adsorptivity 6-hole plate containing a culture medium and placing the 3D titanium alloy bracket prepared in the embodiment 1, continuously culturing, and determining the cell phenotype identification result as shown in figure 2 according to the phenotype result, wherein the obtained mesenchymal stem cells are obtained.
Example 3
In this example, the menstrual mesenchymal stem cells obtained in example 2 were subjected to exosome extraction and identification and protein quantification.
(1) Extraction of exosomes
MSC and 3D titanium alloy stent co-culture, each stent is planted with 5 multiplied by 10 6 Shaking culture to make cells adhere to titanium alloy bracket, collecting culture medium every 48 hr, filtering with 0.22 μm net, centrifuging at 1000 Xg for 20min to obtain supernatant, and centrifuging the filtered supernatant (100000 Xg at 4deg.C for 70 min) to obtain exosome granule without Ca 2+ And Mg (magnesium) 2+ Is resuspended in 500mL of 0.9% sodium chloride solution, centrifuged at 100000 Xg for 70min at 4℃and stored at-80℃after quantification using BCA.
(2) Identification of exosomes
BCA method for detecting exosome eggWhite concentration, protein quantification is carried out by using a BCA protein assay kit, 10 mu L of exosomes are taken and evenly mixed with equivalent double distilled water, the mixture is added into a 96-well plate, standard proteins are taken and added into the 96-well plate according to the amounts of 0, 1, 2, 4, 8, 12, 16 and 20 mu L, 200 mu L of standard proteins are added into a standard substance and a sample to be detected in each hole, the mixture is put into a water bath pot at 30 ℃ for incubation for 30min, and then the mixture is placed into an enzyme-labeled instrument, so that absorbance value is measured. Drawing a standard curve according to the absorbance value of the standard substance, and calculating the protein concentration (mg/mL) of the exosome sample according to the standard curve, wherein the concentration is 2 multiplied by 10 8 The statistics of total exosome amount of each MSCs cultured in the 2D and 3D culture systems are shown in FIG. 3A, the statistics of 3D-exos (exosome) particle number and protein content obtained by the 3D culture systems at different time points are shown in FIG. 3B, and the results show that the total exosome amount obtained under the 3D culture conditions is 3 times of the total exosome amount obtained under the 2D culture conditions, and the results show that a large number of exosome can be obtained by culturing stem cells by adopting the three-dimensional system of the invention.
Example 4
The present example performs exosome form detection and nanoparticle characterization (Nanoparticle Tracking Analysis, NTA).
The exosomes obtained in example 3 were dropped on a copper mesh, after the excess liquid was sucked off, phosphotungstic acid was dropped on the copper mesh, after the excess dye liquid was sucked off, pure water was dropped on the copper mesh, and after drying, the exosomes were used for observing the morphology and the size of exosomes in different vision fields by an electron microscope, as shown in fig. 4A and fig. 4B. According to the particle number recorded in the Nanosight-NS500 operation step, the machine is used for carrying out nanoparticle characteristic analysis (Nanoparticle Tracking Analysis, NTA), collecting and analyzing reports, and the exosomes are in a cup-shaped disc-shaped double-membrane structure under an electron microscope, wherein the average distribution of the exosomes particle number is shown in fig. 4C and fig. 4D, most of the exosomes particle sizes are concentrated at 128nm, and the fact that the exosomes are mainly extracted in the experiment is explained.
Example 5
In this example, western Blot was performed to detect the expression levels of the exosome markers CD81 and TSG 101.
The dilution ratio of CD81 (Abcam, ab 109201) to TSG101 protein primary antibody (Abcam, ab 133586) is 1:1000, exosomes are added with ultrapure water and then are broken by ultrasound for 1min, 12% polyacrylamide electrophoresis separation gel and 80V constant voltage electrophoresis separation are adopted, after electrophoresis, electrotransport proteins are put on a PVDF film of 0.22 mu m, and TBST is used for rinsing for 5min multiplied by 3 times; 5% nonfat dry milk was blocked for 80min and TBST rinsed 5min 3 times. Preparing an anti-working solution by using an anti-dilution solution, wherein the dilution concentration of the antibody is 1:1000. The strips of the target proteins are respectively sheared according to the molecular size, the protein surface of the membrane is immersed into the primary antibody working solution corresponding to each target protein downwards, and the membrane is placed on a shaking table for gentle shaking for 30min, and then placed at 4 ℃ for overnight incubation. HRP fluorescent secondary antibody (Thermo Fisher scientific, 31470) was incubated for 30min and TBST rinsed 5min X3 times. ECL luminescence solution is added and incubated for 5min at 25 ℃ in dark place. The results were analyzed by exposure to LI-COR 3600 and by Image Studio Digits Ver 4.0.4.0 software, as shown in FIG. 4E, for the expression of both the exosome markers CD81 and TSG101, demonstrating the success of obtaining large numbers of exosomes by culturing stem cells using the three-dimensional system of the present invention.
In summary, the invention adopts 3D printing technology to construct scaffolds with different pore diameters and porosities, promotes more real cell physiological environment through more primary cell-cell interactions, and enables cells to maintain better growth activity and biological function, so as to realize optimal promotion of physical properties and cell adhesion, further promote the yield of stem cell exosomes and promote the immunoregulation of stem cells.
The applicant states that the detailed method of the present invention is illustrated by the above examples, but the present invention is not limited to the detailed method described above, i.e. it does not mean that the present invention must be practiced in dependence upon the detailed method described above. It should be apparent to those skilled in the art that any modification of the present invention, equivalent substitution of raw materials for the product of the present invention, addition of auxiliary components, selection of specific modes, etc., falls within the scope of the present invention and the scope of disclosure.
Claims (10)
1. A method for constructing a three-dimensional differentiation model of stem cells, the method comprising:
and separating and two-dimensionally culturing stem cells from a sample, culturing and passaging to P3 generation, mixing the stem cells with a stem cell culture medium to prepare stem cell suspension, and co-culturing the 3D printed porous titanium alloy bracket and the stem cell suspension to obtain the three-dimensional stem cell differentiation model.
2. The method of building according to claim 1, wherein the method of 3D printing comprises selective laser melting;
preferably, the laser power of the selective laser melting is 180-240W, the scanning speed is 180-240nm/s, the layer thickness is 10-30 μm, the laser spot size is 50-100 μm, the lap joint distance is 20-50 μm, and the layer-by-layer rotation angle is 50-67 degrees.
3. The construction method according to claim 1 or 2, wherein the 3D printed porous titanium alloy scaffold has a disc shape with a diameter in the range of phi (1-4) x L (8-35) mm;
preferably, the pore diameter of the 3D printing porous titanium alloy bracket is 200-1000 mu m, and the porosity is 30-70%.
4. A method of construction according to any of claims 1-3, wherein the pre-treatment of the 3D printed porous titanium alloy scaffold comprises a wash dip and autoclaving.
5. The method of claim 4, wherein the washing solution comprises an acid washing solution or an alkali washing solution;
preferably, the alkaline wash comprises a potassium hydroxide-isopropanol solution;
preferably, the concentration of the potassium hydroxide in the potassium hydroxide-isopropanol solution is 0.05-0.1mol/L;
preferably, the soaking time is greater than 24 hours;
preferably, the pressure of the autoclave is 104.0-137.3kPa, the temperature is 121-126 ℃ and the time is 20-30min.
6. The method of any one of claims 1-5, wherein the stem cells comprise any one or a combination of at least two of menstrual blood-derived mesenchymal stem cells, umbilical cord mesenchymal stem cells, bone marrow mesenchymal stem cells, or adipose mesenchymal stem cells.
7. A three-dimensional differentiation model of stem cells, wherein the three-dimensional differentiation model of stem cells is constructed by the method for constructing a three-dimensional differentiation model of stem cells according to any one of claims 1 to 6.
8. A stem cell culture system comprising a stem cell exosome and the three-dimensional differentiation model of stem cells of claim 7.
9. The stem cell culture system of claim 8, wherein the stem cell exosomes comprise any one or a combination of at least two of Rabs protein, transmembrane protein, ribosomal protein, signal transduction factor or adhesion factor.
10. Use of the stem cell three-dimensional differentiation model of claim 7 or the stem cell culture system of claim 8 or 9 in drug screening, physiopathological research, cell therapy and tissue engineering.
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