CN111621475A - Umbilical cord mesenchymal stem cell membrane and preparation method thereof - Google Patents
Umbilical cord mesenchymal stem cell membrane and preparation method thereof Download PDFInfo
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Abstract
The invention provides an umbilical cord mesenchymal stem cell membrane and a preparation method thereof, wherein the umbilical cord mesenchymal stem cell membrane contains umbilical cord mesenchymal stem cells and all extracellular matrixes and growth factors secreted by the umbilical cord mesenchymal stem cells in a proliferation process. The method completely retains umbilical cord mesenchymal stem cells and extracellular matrix and growth factors secreted by the umbilical cord mesenchymal stem cells in the proliferation process and separates the umbilical cord mesenchymal stem cells from the surface of a culture dish under the conditions of no digestion by enzyme and the like and no physical stripping to obtain the lamellar cell membrane.
Description
Technical Field
The invention relates to the field of regenerative medicine and cell biology, in particular to an umbilical cord mesenchymal stem cell membrane and a preparation method thereof.
Background
The cell sheet (cell sheet) technology is a new technology for stem cell transplantation and application. In a certain sense, the cell patch engineering can simulate the process of embryonic development tissue formation to the maximum extent. Since 1993 Okano et al invented cell membrane sheet technology, cell membrane sheet technology has been widely used in tissue engineering research.
The umbilical cord mesenchymal stem cells exist in Wharton's jelly of the umbilical cord of the mammal, and have good in-vitro amplification capacity, multidirectional differentiation capacity and immunoregulation capacity.
At present, umbilical cord mesenchymal stem cells are mainly used as cell suspension for direct injection or transplantation after being combined with a tissue engineering scaffold material in the basic research and clinical application of self tissue repair, and both have certain limitations. The direct injection of stem cell suspension can cause the loss of a large amount of stem cells, the utilization rate of the cells is low, and the function of the stem cells for tissue repair is limited; the problem of cell loss is solved by transplanting after the cells are combined with the tissue engineering scaffold, but the scaffold material may cause inflammatory reactions of different degrees in a living body, and degradation products of the material may change the microenvironment of local tissues to cause more serious pathological changes.
Disclosure of Invention
The invention aims to provide an umbilical cord mesenchymal stem cell membrane and a preparation method thereof.
In a first aspect, the present invention provides an umbilical cord mesenchymal stem cell patch, which contains umbilical cord mesenchymal stem cells and all their extracellular matrix and growth factors secreted during proliferation. The umbilical cord mesenchymal stem cell membrane has the advantages of high umbilical cord mesenchymal stem cell density, uniform membrane thickness and regular edges. The umbilical cord mesenchymal stem cell membrane can secrete various cell factors including angiogenesis and immunoregulation, and participates in the repair of tissues and organs.
In a specific embodiment, the umbilical cord mesenchymal stem cell membrane sheet of the present invention may be used to regenerate cornea, cardiac muscle tissue, liver tissue or bone tissue. In a specific embodiment, the umbilical cord mesenchymal stem cell membrane sheet of the present invention can be used for treating various diseases such as nerve injury, myocardial injury. Without being bound by theory, the umbilical cord mesenchymal stem cell membrane of the invention is believed to achieve the treatment effect by secreting cytokines to repair the damaged tissue and organ or promoting the self-repair of the damaged tissue and organ. These factors mainly include interleukin-6 (IL-6), transforming growth factor-beta (TGF-beta), prostaglandin E2(prostaglandin E2, PGE2), hepatocyte growth factor, epidermal growth factor, fibroblast growth factor, platelet-derived growth factor, vascular endothelial growth factor, insulin growth factor, stromal cell-derived growth factor-1, tryptophan metabolizing enzyme (indole 2,3-dioxygenase, IDO), nitric oxide synthase (iNOS), and the like.
In a second aspect, the present invention provides a method for preparing an umbilical cord mesenchymal stem cell membrane, comprising: umbilical cord mesenchymal stem cells and extracellular matrix and growth factors secreted by the umbilical cord mesenchymal stem cells in the proliferation process are completely retained and separated from the surface of a culture dish without enzyme and analog digestion to form an umbilical cord mesenchymal stem cell membrane. Specifically, the method for preparing the umbilical cord mesenchymal stem cell membrane comprises the following steps: (1) coating a layer of substrate on the surface of the temperature-sensitive culture dish; (2) adding the single cell suspension of the umbilical cord mesenchymal stem cells into a temperature-sensitive culture dish for culture; (3) reducing the temperature, and separating the umbilical cord mesenchymal stem cells and the extracellular matrix secreted by the umbilical cord mesenchymal stem cells into slices to obtain the umbilical cord mesenchymal stem cell membrane.
A. Separation and culture of umbilical cord mesenchymal stem cells
The method for separating the umbilical cord mesenchymal stem cells mainly comprises a wall pasting method and an enzyme digestion method. The enzyme digestion method can obtain umbilical cord mesenchymal stem cells with higher purity, but the obtaining efficiency is lower, the cost is higher, the single cell adherence capability is weak, and the cells are easy to be damaged. The tissue block wall pasting method is simple and convenient to operate, low in cost, small in damage to cells and suitable for clinical application.
In addition, mesenchymal stem cell culture medium is also critical to cell yield and cell quality. In the currently known sources, the content of mesenchymal stem cells is low, so that in order to obtain a sufficient number of mesenchymal stem cells, not only the separation method needs to be optimized and the cell yield is increased, but also the obtained mesenchymal stem cells need to be cultured in vitro and subjected to passage amplification.
Specifically, the invention provides a separation culture method of umbilical cord mesenchymal stem cells, which combines subculture on the basis of a tissue block adherence method and comprises the following steps:
isolating Wharton's jelly from umbilical cord tissue;
cutting up the Wharton's jelly to obtain a tissue mass;
spreading the tissue block in a culture container for culture;
adding a proper amount of complete culture medium to cover the tissue blocks, and continuing culturing;
when cells attached to the culture vessel appear around the tissue mass and the cells grow to 70-100% confluence, the tissue mass is removed and the cells are passaged.
In a specific embodiment, the method comprises the steps of: healthy umbilical cord tissue of the newborn was thoroughly washed with PBS buffer or normal saline to remove blood stains. Mechanically separating umbilical cord, removing Wharton's jelly, and removing umbilical artery and umbilical vein; uniformly shearing the peeled Wharton's jelly into tissue blocks by using sterile scissors. And then uniformly paving the tissue blocks in a culture container coated with a matrix, placing the culture container in an incubator, adding a proper amount of complete culture medium to cover the tissue blocks after 2-7 days, and allowing the cells to climb out of the Wharton jelly tissue blocks and grow around the tissue blocks in an adherent manner in 8-21 days. The cells are in the form of fibers and have uniform shape. And removing the tissue block when the crawled cells grow to 70-100% confluence, and carrying out passage operation on the cells.
In a specific embodiment, the umbilical cord tissue is washed with PBS buffer or normal saline without the green chain double antibody.
In a particular embodiment, the Wharton's jelly is sheared into tissue pieces having a diameter of 0.1 to 2.5mm (preferably 1 to 2 mm).
In a specific embodiment, the tissue blocks are uniformly paved in a culture container coated with a matrix according to the interval of 2-30 mm.
In a specific embodiment, the conditions of the incubator are 37 ℃ and 5% CO2。
In a specific embodiment, the mesenchymal stem cell culture medium is a basal medium + a nutritional additive + a growth factor for culturing mesenchymal stem cells. Specifically, the basal medium may be 1640, DMEM, alpha-MEM, DMEM/F12, F12, or the like. Nutritional additives may include, but are not limited to, fetal bovine serum, calf serum, newborn bovine serum, human serum, at concentrations of 2% to 20%. The growth factor may include, but is not limited to, human fibroblast growth factor, human epidermal growth factor, human hepatocyte growth factor, and the like.
The passaging of the cells may include: the cells are separated from the culture vessel and uniformly dispersed in a medium, and then inoculated in the culture vessel. Adding a proper amount of culture medium, replacing a proper amount of fresh culture medium every 1-5 days according to the growth state of the cells, and repeating the passage operation when the cells grow to 70-100% and are converged. The passage number of cells increases by 1 each time the cells are passaged. The umbilical cord mesenchymal stem cells grow adherently, are in a fibroid shape and are uniform in shape.
In particular embodiments, methods of separating the cells from the culture vessel include, but are not limited to, digestion with pancreatin and the like, use of cell scraping, and the like.
In a specific embodiment, the cells are passaged throughUniformly dispersing in the culture medium by stirring, vortexing, etc., and inoculating. Preferably, the inoculation density is 500-100000/cm2。
Optionally, after obtaining umbilical cord mesenchymal stem cells by culturing, a cell growth curve may be measured by MTT method, WST method, DNA content detection method, ATP detection method, or the like to evaluate growth activity of umbilical cord mesenchymal stem cells. In addition, the isolated and cultured umbilical cord mesenchymal stem cells can be identified by detecting cell surface markers by flow cytometry, detecting cell expression genes by a three-way differentiation assay and a PCR method.
B. Preparation of umbilical cord mesenchymal stem cell membrane
The umbilical cord mesenchymal stem cells after separation, culture and identification can be further prepared into the umbilical cord mesenchymal stem cell membrane according to the method of the invention. In particular, the invention utilizes the temperature-sensitive culture dish to prepare the cell membrane, and does not use enzyme and analogue digestion nor physical stripping in the stripping process of the cell membrane. The method can completely reserve the umbilical cord mesenchymal stem cells and extracellular matrixes and growth factors secreted by the umbilical cord mesenchymal stem cells in the proliferation process and separate the umbilical cord mesenchymal stem cells from the culture dish ground, so that the obtained umbilical cord mesenchymal stem cell membrane has high cell density, uniform membrane thickness and regular edges, can secrete various cell factors including angiogenesis and immunoregulation, and participates in the repair of tissues and organs. In addition, the living cell ratio in the umbilical cord mesenchymal stem cell membrane prepared by the method is high.
Specifically, the invention provides a method for preparing an umbilical cord mesenchymal stem cell membrane, which comprises the following steps:
coating a layer of substrate on the surface of the temperature-sensitive culture dish;
adding the umbilical cord mesenchymal stem cell suspension into the temperature-sensitive culture dish for culture;
reducing the temperature, and separating the umbilical cord mesenchymal stem cells and the extracellular matrix secreted by the umbilical cord mesenchymal stem cells into slices to obtain the umbilical cord mesenchymal stem cell membrane.
In a specific embodiment, the substrate used to coat the temperature sensitive culture dish is selected from the group consisting of serum, collagen, gelatin, fibronectin, and vitronectinWhite, laminin, polyornithine, polylysine. Preferably, the substrate is Fetal Bovine Serum (FBS). In particular embodiments, the coating temperature is 20-40 ℃ (e.g., 20 ℃, 25 ℃, 30 ℃, 35 ℃, or 40 ℃; e.g., 37 ℃). In a specific embodiment, the coating time is 0.5 to 48 hours, such as 2 to 48 hours, 2 to 24 hours. In a specific embodiment, the coating ratio is not less than 0.1ml FBS/cm2。
Specifically, 30-100% (v/v) FBS diluted with phosphate buffer solution or physiological saline can be coated at 20-40 deg.C (e.g. 20 deg.C, 25 deg.C, 30 deg.C, 35 deg.C, or 40 deg.C; e.g. 37 deg.C) for 0.5-48 h (e.g. 2-48 h, 2-24 h, e.g. overnight) at a coating ratio of not less than 0.1ml FBS/cm2. When the cell membrane is not coated or is coated by less than 30 percent of serum, the prepared cell membrane is very thin, and part of the cell membrane spontaneously grows in advance when the temperature is not changed, so that the cell membrane is torn from the part without growing the cell membrane, the cell membrane is broken, the part with growing the cell membrane in advance is also shrunk and grown together, and the cell membrane cannot be unfolded even if subsequent treatments such as blowing and beating are carried out.
In some embodiments, the single cell suspension of mesenchymal stem cells is at 5 × 105-2×106Individual cell/cm2The cell culture density is added into the temperature-sensitive culture dish and cultured for about 4-24 h, so that the inoculated mesenchymal stem cells form a lamellar sheet.
In other embodiments, a single cell suspension of mesenchymal stem cells is at 1 × 103-1×106Individual cell/cm2The cell culture density is added into the temperature-sensitive culture dish and cultured for about 24-168 hours, so that the inoculated mesenchymal stem cells proliferate and form a lamellar sheet. In a specific embodiment, the medium is replaced every other day during the above-described culturing process.
The "temperature-sensitive culture dish" refers to a culture dish coated with a layer of temperature-sensitive polymer on the surface, wherein the polymer has different molecular chain segments extending at different temperatures, so that the polymer shows hydrophilicity or hydrophobicity, and the hydrophilicity and hydrophobicity of the polymer can be changed along with the change of external temperature. When the surface of the temperature-sensitive culture dish is hydrophilic, the adhesion with cells and extracellular matrix secreted by the cells is poor, and the cells are exfoliated in layers. In one embodiment, when the temperature is lowered below the Lower Critical Solution Temperature (LCST) of the polymer, the surface of the temperature sensitive petri dish is hydrophilic, so that cells will be exfoliated.
The invention successfully realizes the preparation of the umbilical cord mesenchymal stem cells by using the temperature-sensitive culture dish under the conditions of not using enzyme and analogue digestion nor physical stripping. Therefore, in a specific embodiment, the temperature is reduced to below 32 ℃ (e.g., 4-32 ℃ or 4-30 ℃), and the umbilical cord mesenchymal stem cells are detached from the bottom of the temperature-sensitive culture dish in a sheet-like shape to form a cell sheet with intact connection of extracellular matrix.
In a specific embodiment, the umbilical cord mesenchymal stem cell is prepared by the isolated culture method of umbilical cord mesenchymal stem cell provided in the above a.
C. Characterization of umbilical cord mesenchymal stem cell membranes
The umbilical cord mesenchymal stem cell membrane prepared by the method contains umbilical cord mesenchymal stem cells and all extracellular matrix and growth factors secreted by the umbilical cord mesenchymal stem cells in the proliferation process. In addition, because enzymes and analogues are not used for digestion, and a physical method is not used for stripping, the umbilical cord mesenchymal stem cells in the umbilical cord mesenchymal stem cell diaphragm are high in density, the thickness of the diaphragm is uniform, and the edge is neat. In addition, the living cell ratio in the umbilical cord mesenchymal stem cell membrane is high.
Optionally, after the umbilical cord mesenchymal stem cell membrane is prepared, the surface structure of the cell membrane can be observed by a scanning electron microscope. In addition, the amount of cytokines secreted from the cell membrane, proteins contained in the extracellular matrix in the cell membrane, and the like can be detected.
In a specific embodiment, the cell density within the umbilical cord mesenchymal stem cell membrane sheet of the present invention is about 0.025 × 106-25×106。
In a specific embodiment, the umbilical cord mesenchymal stem cell patch of the present invention comprises 1-120 layers of cells. In a specific embodiment, the umbilical cord mesenchymal stem cell patch comprises 1-120 layers of umbilical cord mesenchymal stem cells. In a specific embodiment, the umbilical cord mesenchymal stem cell patch comprises at least 2 (e.g., 2-120) umbilical cord mesenchymal stem cell layers stacked.
In a specific embodiment, the umbilical cord mesenchymal stem cell membrane of the present invention has a thickness of about 20-5000 μm.
In a specific embodiment, the umbilical cord mesenchymal stem cell membrane is gray white, compact in structure and smooth and flat in surface.
In a specific embodiment, the umbilical cord mesenchymal stem cell membrane of the present invention is capable of secreting at least interleukin-6 (IL-6), Hepatocyte Growth Factor (HGF), Vascular Endothelial Growth Factor (VEGF), and the like. In a specific embodiment, the umbilical cord mesenchymal stem cell sheet of the present invention is capable of secreting IL-6, HGF and VEGF in large amounts.
In a specific embodiment, the extracellular matrix of the umbilical cord mesenchymal stem cell membrane of the present invention comprises at least Fibronectin (Fibronectin), integrin family, vitronectin, and the like. In a specific embodiment, the extracellular matrix in the umbilical cord mesenchymal stem cell membrane of the invention comprises a large amount of Fibronectin (Fibronectin), integrin family, vitronectin.
The invention has the advantages of
1. Compared with the traditional injection method, the umbilical cord mesenchymal stem cell membrane avoids the loss of umbilical cord mesenchymal stem cells and improves the utilization rate of the umbilical cord mesenchymal stem cells. The umbilical cord mesenchymal stem cell membrane can directly secrete cell factors to the tissues or organs at the attachment position, and the factors can achieve the effects of inhibiting inflammatory reaction, repairing damaged tissues and organs and promoting self-repair of the damaged tissues and organs.
2. The umbilical cord mesenchymal stem cell membrane avoids the use of exogenous biological scaffold materials and avoids inflammatory reaction possibly caused by the degradation of exogenous biological materials in vivo.
3. According to the invention, by using a temperature-sensitive intelligent culture dish, umbilical cord mesenchymal stem cells and extracellular matrix and growth factors secreted by the umbilical cord mesenchymal stem cells in the proliferation process are completely retained and separated from the surface of the culture dish under the conditions of not using enzyme and analogue for digestion and not through physical stripping, so that the lamellar cell membrane is obtained. The cell patch obtained by the method has high cell density, high living cell ratio, uniform thickness and complete structure. The cell membrane prepared by the method does not lose extracellular matrix, and the fibronectin in the cell membrane can enable the cell membrane to be attached to the surface of a target tissue or organ without an additional fixing method such as suturing.
4. Compared with adult-derived mesenchymal stem cells such as bone marrow mesenchymal stem cells and adipose mesenchymal stem cells, the umbilical cord mesenchymal stem cells are younger, have higher proliferation capacity and low immunogenicity, and secrete relevant factors for tissue repair and immunoregulation, so that the umbilical cord mesenchymal stem cells are ideal seed resource cells for regenerative medicine.
Drawings
Representative pictures of umbilical cord mesenchymal stem cells of P0 (tissue mass), P3 and P6 are shown in fig. 1, respectively.
Figure 2 shows an exemplary growth curve of umbilical cord mesenchymal stem cells.
Figure 3 shows the cord mesenchymal stem cell adipogenic, osteogenic, chondrogenic three-way differentiation immunofluorescence staining.
FIG. 4 shows a patch of umbilical cord mesenchymal stem cells prepared using a temperature-sensitive intelligent culture dish and correctly coating serum (FIG. 4A), a patch of umbilical cord mesenchymal stem cells obtained when FBS concentration of uncoated or coating solution is insufficient (FIG. 4B), and a patch of cells prepared using a common culture dish (FIG. 4C).
Figure 5 shows the secretion of factors by umbilical cord mesenchymal stem cell patch.
FIG. 6 expression of umbilical cord mesenchymal stem cell membrane cell surface matrix Fibronectin and Integrin-beta 1.
Fig. 7-8 show umbilical cord mesenchymal stem cell micro-topography.
Detailed Description
The present invention will be described below based on examples, but the present invention is not limited to only these examples.
Example 1 isolation and culture of umbilical cord mesenchymal Stem cells
Healthy neonatal umbilical cord tissue was thoroughly washed with a 1xPBS buffer solution containing no cyan double antibody to remove blood stains. Mechanically separating umbilical cord, removing Wharton's jelly, and removing umbilical artery and umbilical vein; uniformly shearing the peeled Wharton's jelly into 2mm pieces by using sterile scissors3The tissue mass of (1). The tissue blocks were then spread evenly at 30mm intervals on FBS (from Gibco) coated 100mm diameter petri dishes (from Corning), the culture vessel was placed in a 37 ℃ 5% CO2 incubator, and after 2 days an appropriate amount of DMEM complete medium (formulation 90% DMEM + 10% FBS, DMEM from Corning) was added to cover the tissue blocks, and the cells were allowed to crawl out of the Wharton's jelly tissue blocks and grow adherently around the tissue blocks on day 21. . When the cells had grown to 85% confluence, the tissue mass was removed, the cells were digested from the culture vessel with pancreatin (purchased from Sigma) and uniformly dispersed in the above DMEM complete medium, followed by incubation at 10000/cm2Is inoculated in a culture vessel. Adding appropriate amount of culture medium, changing 50% fresh culture medium every 3 days according to cell growth state, and repeating passage operation when cell growth reaches 85% confluence. The passage number of cells increases by 1 each time the cells are passaged. The umbilical cord mesenchymal stem cells grow adherently, are in a fibroid shape and are uniform in shape. Representative pictures of umbilical cord mesenchymal stem cells at P3 and P6 generations are shown in fig. 1.
Example 2 identification of umbilical cord mesenchymal Stem cells
2.1 measurement of growth Activity of umbilical cord mesenchymal Stem cells
The umbilical cord mesenchymal stem cells prepared in example 1 were dispersed, inoculated into a culture well plate at a certain density with reference to the WST kit instructions, and subjected to a liquid change operation according to normal culture conditions. Cell viability was measured at fixed times of day over a period of time according to the instructions to obtain data on cell viability or quantity. As shown in figure 2, the activity of umbilical cord mesenchymal stem cells is detected by WST reagent at a fixed time every day within 7 days, the WST reagent is added into a cell pore plate which is being cultured according to the proportion recommended by the instruction, after the cell is incubated for a fixed time in a cell culture box, the absorbance value of the cell plate cell liquid at the wavelength of 450 nm is detected by a microplate reader or an ultraviolet spectrophotometer, and the value reflects that the cell activity is totally and positively correlated with the cell number. From the results, it was found that the cell activity in the chamber was increased with the increase of the culture time, and it was concluded that the number of cells was increased with the increase of the culture time.
2.2. Identification of umbilical cord mesenchymal stem cell surface marker
The umbilical cord mesenchymal stem cells are dispersed in a culture medium and then centrifuged, and the cells are stained with cell surface marker proteins including but not limited to CD73, CD90, CD105, CD34, CD11B, CD19, CD45 and HLA-DR in an isotonic physiological solution with the content of serum or serum protein of 1-20%. Wherein the phenotype of CD73, CD90 and CD105 is positive, the ratio is not less than 95%, the phenotype of CD34, CD11B, CD19, CD45 and HLA-DR is negative, the ratio is not more than 2%. Table 1 shows the flow cytometry detection results of certain P5 umbilical cord mesenchymal stem cells.
Table 1: cell surface marker expression of Umbilical Cord Mesenchymal Stem Cells (UCMSC)
2.3 three-dimensional induced differentiation of umbilical cord mesenchymal Stem cells
The umbilical cord mesenchymal stem cells prepared in the example 1 are inoculated in a proper culture vessel according to the proportion of the three-dimensional induced differentiation reagent specification, and when the cells detected by osteogenesis are grown to 50-90% confluence and the cells detected by adipogenesis are grown to more than 90% confluence, osteogenesis and adipogenesis induction culture media are respectively added. When in chondrogenesis induction, a certain amount of cells are centrifuged to the bottom of a centrifuge tube, then chondrogenesis induction culture medium is added, and after the cells are agglomerated into small balls, the small balls of the cells are separated from the tube bottom to ensure that the cells are completely contacted with the induction culture medium.
The cells were tested after induction culture for more than 7 days. Osteogenic induction may be stained with, but not limited to, alizarin red, anti-osteooncocin, adipogenic induction may be stained with, but not limited to, oil red O, anti-mFABP4, and chondrogenic induction may be stained with, but not limited to, alcian blue, safranin O, anti-Aggrecan.
As shown in fig. 3, the fluorescence microscope photograph shows that the P5 generation umbilical cord mesenchymal stem cells can be stained by anti-mFABP4 (red) after adipogenic induced differentiation, by anti-haggarcan (red) after osteogenic induced differentiation, by anti-Aggrecan (red) after chondrogenic induced differentiation, and the cell nucleus is stained by DAPI (blue).
Example 3 preparation of umbilical cord mesenchymal Stem cell Membrane
Coating FBS on the surface of a temperature-sensitive intelligent culture dish in advance under the condition that FBS (30-100% (v/v)) diluted by phosphate buffer solution or normal saline is used for coating at 37 ℃ overnight, wherein the coating proportion is not less than 0.1ml of FBS/cm2Then single cell suspension of mesenchymal stem cells was washed with 1 × 106Individual cell/cm2Inoculating the strain in a coated temperature-sensitive intelligent culture dish, and culturing for 16 h; alternatively, at 26700 cells/cm2The density of (A) was inoculated in a 35mm coated temperature-sensitive intelligent petri dish and cultured for 3 days. When the temperature is reduced to be below 32 ℃, the umbilical cord mesenchymal stem cells are separated into slices from the bottom of the temperature-sensitive intelligent culture dish to form a cell membrane which is completely connected with the extracellular matrix, the macroscopic morphology of the cell membrane is shown in figure 4A, and the umbilical cord mesenchymal stem cell membrane is grey white, compact in structure, smooth and flat in surface.
When the cell membrane is not coated or the blood serum with the concentration less than 30% is used for coating the incubator, the prepared cell membrane is very thin, and part of the cell membrane spontaneously grows in advance when the temperature is not changed, so that the cell membrane is torn from the part without growing the cell membrane, the cell membrane is broken, the part with the film grown in advance is also shrunk and grows together, and the cell membrane cannot be unfolded even if subsequent treatments such as blowing and the like are carried out. An exemplary picture thereof is shown in fig. 4B.
For comparison, umbilical cord mesenchymal stem cell sheets were also prepared using a common culture dish under the same conditions. When the common culture dish is used, the connection between the bottom of the dish and cells cannot be changed when the temperature is changed, the scraper is used for processing, the cells are shoveled into a broken lamellar shape, and compared with a membrane prepared by using a temperature-sensitive culture dish, the membrane has poor integrity and is more fragile in texture. The cell sheets prepared using the general culture dish have cracks, creases, cracks, and unevenness, and an exemplary picture thereof is shown in FIG. 4C.
Example 4 characterization of umbilical cord mesenchymal Stem cell Membrane
4.1 enzyme-linked immunosorbent assay for detecting cell membrane secretion factor
The supernatant obtained in the preparation process of the cell membrane in example 3 is detected according to the specifications of an enzyme linked immunosorbent assay kit, and the content of interleukin-6 (interleukin-6, IL-6), Hepatocyte Growth Factor (HGF) and Vascular Endothelial Growth Factor (VEGF) secreted by the cell membrane is determined. The secretion factor content is shown in FIG. 5.
4.2. Tissue section observation of protein condition in cell patch
The cell membrane prepared in example 3 is fixed with paraformaldehyde or formalin fixing solution, and then the cell membrane is prepared into a tissue section with the thickness of 4-10 μm by a paraffin section method or a frozen section method to be stained, and the protein content of extracellular matrix in the cell membrane is observed, wherein the protein includes but is not limited to Fibronectin (Fibronectin), integrin, vitronectin and the like, and the cell nucleus is usually stained with fluorescent dye such as DAPI or Hoest 33258 and the like at the same time of staining to assist positioning. In addition to immunofluorescence methods, staining observations may also be made using methods including, but not limited to, immunohistochemistry methods and the like. Here, fibronectin and integrin- β 1 were used as examples, and as shown in fig. 6, both fibronectin and integrin- β 1 were stained with a fluorescein-labeled antibody dye, and the nucleus was stained with DAPI, and it was found that the prepared cell membrane contained a large amount of fibronectin and integrin- β 1.
4.3 scanning electronic fiberscope observation cell patch surface structure
And (3) fixing the obtained cell membrane with 2.5% glutaraldehyde stationary liquid, dehydrating by gradient alcohol, and naturally drying to obtain a dried cell membrane for scanning electron microscope analysis. And adhering the dried cell membrane on the surface of a sample table by using a conductive double-sided adhesive, and then carrying out surface gold spraying treatment by using a vacuum magnetron sputtering method to make the surface of the observed sample conductive. The sample stage was placed in a scanning electron microscope (Hitachi S-4800 used) for observation. The topography pictures are shown in fig. 7 and 8.
Wherein figure 7 is the one side of cell diaphragm contact intelligent culture dish, and surface appearance is more level and smooth, compares with other culture systems, and the cell diaphragm that has serum culture system to obtain is the film more easily, and the bottom is more smooth than the diaphragm that other culture systems obtained during the film-forming, and the picture exhibition red circular arch is mesenchymal stem cell, also can see intercellular protein and connect. FIG. 8 shows the side of the cell patch not contacting the smart culture dish, and the stacking of cells is visible.
While specific embodiments of the invention have been described above, it will be appreciated by those skilled in the art that these are by way of example only, and that the scope of the invention is defined by the appended claims. Various changes and modifications to these embodiments may be made by those skilled in the art without departing from the spirit and scope of the invention, and these changes and modifications are within the scope of the invention.
Claims (18)
1. A method of preparing an umbilical cord mesenchymal stem cell patch, comprising:
coating a layer of substrate on the surface of the temperature-sensitive culture dish;
adding the cell suspension of the umbilical cord mesenchymal stem cells into a temperature-sensitive culture dish for culture;
reducing the temperature, and separating the umbilical cord mesenchymal stem cells and the extracellular matrix secreted by the umbilical cord mesenchymal stem cells into slices to obtain the umbilical cord mesenchymal stem cell membrane.
2. The method of claim 1, wherein the coating matrix is selected from the group consisting of serum, collagen, gelatin, fibronectin, vitronectin, laminin, polyornithine, and polylysine.
3. The method according to claim 1 or 2, wherein the coating substrate concentration is 30-100% (v/v).
4. A process according to any one of claims 1 to 3, wherein the coating temperature is 20-40 ℃.
5. The method according to any one of claims 1 to 4, wherein the coating time is 0.5 to 48 hours.
6. The method according to any one of claims 1 to 5, wherein the coating ratio is not less than 0.1ml FBS/cm2。
7. The method of any of claims 1-6, wherein:
subjecting single cell suspension of umbilical cord mesenchymal stem cells to extraction at 5 × 105-2×106Individual cell/cm2Adding the obtained mixture into the temperature-sensitive culture dish, and culturing for about 4-24 h; alternatively, the first and second electrodes may be,
single cell suspension of umbilical cord mesenchymal stem cells at 1 × 103-1×106Individual cell/cm2The density of the culture medium is added into the temperature-sensitive culture dish, and the culture is carried out for about 24-168 hours.
8. The method of any one of claims 1-7, wherein the cell patch is peeled from the culture dish without treatment with a dispersing agent.
9. The method of any one of claims 1-7, wherein the cell patch is peeled from the culture dish without scraping treatment.
10. The method according to any one of claims 1 to 9, wherein the surface of the temperature-sensitive culture dish is coated with a layer of temperature-sensitive polymeric substance, the hydrophilicity and hydrophobicity of which can change with the change of temperature.
11. An umbilical cord mesenchymal stem cell patch prepared by the method of any one of claims 1 to 10.
12. The umbilical cord mesenchymal stem cell membrane of claim 11, which retains substantially all of the extracellular matrix secreted by umbilical cord mesenchymal stem cells.
13. The umbilical cord mesenchymal stem cell patch according to claim 11 or 12, wherein the umbilical cord mesenchymal stem cell patch prepared has one or more of the following characteristics:
(i) the cell density in the umbilical cord mesenchymal stem cell membrane is about 0.025 × 106-25×106；
(ii) The umbilical cord mesenchymal stem cell patch comprises 1-120 layers of cells;
(iii) the thickness of the umbilical cord mesenchymal stem cell membrane is about 20-5000 μm.
14. The umbilical cord mesenchymal stem cell patch of any one of claims 11-13, wherein the umbilical cord mesenchymal stem cell patch is capable of secreting at least IL-6, HGF and VEGF.
15. The umbilical cord mesenchymal stem cell membrane of any one of claims 11-14, wherein the extracellular matrix in the umbilical cord mesenchymal stem cell membrane comprises at least Fibronectin (Fibronectin), integrin family, vitronectin.
16. A method for separating and culturing umbilical cord mesenchymal stem cells comprises the following steps:
(1) isolating Wharton's jelly from umbilical cord tissue;
(2) cutting up the Wharton's jelly to obtain a tissue mass;
(3) spreading the tissue block in a culture container for culture;
(4) adding a proper amount of complete culture medium to cover the tissue blocks, and continuing culturing;
(5) when cells attached to the culture vessel appear around the tissue mass and the cells grow to 70-100% confluence, the tissue mass is removed and the cells are passaged.
17. The method of claim 16, further comprising, prior to step (1), the steps of: healthy umbilical cord tissue of the newborn was thoroughly washed with PBS buffer or normal saline to remove blood stains.
18. The method of claim 16 or 17, which is characterized by one or more of the following features:
(i) in the step (2), the Wharton jelly is sheared into tissue blocks with the diameter of 0.1-2.5mm (preferably 1-2 mm);
(ii) in the step (3), uniformly paving the tissue blocks in a culture container coated with a matrix at intervals of 2-30 mm;
(iii) in steps (3) to (4), the culture conditions were 37 ℃ and 5% CO2。
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