CN113456891A - Method for extracting extracellular matrix layer from cell layer - Google Patents
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Abstract
The invention discloses a method for extracting an extracellular matrix layer from a cell layer, which comprises the following steps: adding fibronectin or gelatin into buffer solution, and stirring at a temperature of not lower than 37 deg.C to obtain mixed liquid; the concentration of the fibronectin or the gelatin in the mixed liquid is 50-200 mg/mL; filtering the mixed liquid by adopting a membrane filter, and placing the filtered mixed liquid in a mould (at the temperature of 25-40 ℃); freezing (<25 ℃ C.) until the solution gels, and mixing the liquid to obtain a solution that solidifies to form a gel; taking the gel out of the mold, covering the gel on a cover glass with cultured cells, and pressing the gel to make the gel fully contact with the cells; the gel was torn off and the coverslip was rinsed with 37 ℃ buffer to obtain extracellular matrix of the substrate. Through the scheme, the method has the advantages of simple process, reliable extraction, low cost, reutilization and the like.
Description
Technical Field
The invention relates to the technical field of medical biomaterials, in particular to a method for extracting an extracellular matrix layer from a cell layer.
Background
Extracellular matrix (ECM) is a complex mixture of structural functional proteins, glycoproteins and proteoglycans arranged in a unique, tissue-specific three-dimensional ultrastructure. These proteins have many functions, for example, they can provide structural support, tonicity strength and attachment sites for cell surface receptors, and serve as reservoirs for signaling factors that regulate various host processes, such as angiogenesis, cell migration, cell proliferation, inflammatory responses, immune responses and wound healing. In other words, the ECM is an important, dynamic, indispensable component of all tissues and organs, a natural scaffold in nature for tissue and organ morphogenesis, maintenance and reconstruction after injury. ECM has been used as a biological scaffold for the reconstruction of many different tissue types in preclinical and human clinical studies.
To date, the methods for decellularizing and extracting ECM that have been applied in the market are mainly chemical methods (treatment with acid, alkali, nonionic detergent, tributyl phosphate, etc.) and biological methods (enzymes, chelating agents, toxins, etc.). At present, the ECM extraction technology in the prior art has the following problems:
first, the removed cells cannot be recovered: the above described decellularization methods mostly employ different techniques to disrupt cells in conjunction with other methods to elute cells and residual components within cells, which results in the inability to use the above methods to obtain ECM for some precious cells.
Second, the adverse consequences of residual reagents: the above described decellularisation methods involve the use of a large proportion of biochemical reagents which have a potent effect in disrupting cellular structures. However, if the residual concentration of the biochemical agent in the ECM is high, it may cause strong toxic effects on host cells during transplantation into the body, and some chemical agents may also affect physicochemical properties of the ECM;
thirdly, the operation is complicated or the cost is high, the required materials are more, and the process is complex.
Therefore, it is urgently needed to provide a method for extracting an extracellular matrix layer from a cell layer, which has a simple process and reliable extraction.
Disclosure of Invention
In view of the above problems, the present invention provides a method for extracting an extracellular matrix layer from a cell layer, which adopts the following technical scheme:
a method of extracting a layer of extracellular matrix from a layer of cells, comprising the steps of:
adding fibronectin or gelatin into buffer solution, and stirring at a temperature of not lower than 37 deg.C to obtain mixed liquid; the concentration of the fibronectin or the gelatin in the mixed liquid is 50-200 mg/mL;
filtering the mixed liquid by adopting a membrane filter, and placing the filtered mixed liquid into a mould;
freezing at a temperature below 25 deg.C until the solution gels to obtain a gel formed by the coagulation of the solution;
taking the gel out of the mold, covering the gel on a cover glass with cultured cells, and pressing the gel to make the gel fully contact with the cells;
the gel was torn off and the coverslip was rinsed with 37 ℃ buffer to obtain extracellular matrix of the substrate.
Preferably, the buffer is a phosphate buffer.
Further, the concentration of the buffer is 50-250 mM.
Preferably, the pH of the buffer is 6 to 8.
Preferably, the membrane filter has a filtration pore size of 0.22 μm or 0.45 μm.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention adopts the temperature-sensitive gel with sol-gel property as the supporting material of the adhesion cells, adheres the cells in a gel state, adheres the cells from the substrate and the ECM layer by utilizing the property of dissolving the gel at higher temperature and adhering the cells from the substrate and the ECM layer by physical tension and specificity, thereby realizing the extraction of the ECM and the recovery and reutilization of the cells;
(2) the invention adopts temperature-sensitive gel (mainly fibronectin or gelatin gel) with sol-gel property as raw material to prepare gel, and uses the gel as the media of the adhesion cells to extract ECM, and has low cost and simple process. In addition, the cells are removed by a physical method, no harmful biochemical reagent is added, the later-stage removal of the residual reagent is avoided, and the strong toxic and side effects on the host cells in the process of transplanting the cells into the body due to the high adsorption residual concentration of the cells in the ECM are avoided;
(3) the method adopts fibronectin or gelatin as a raw material, utilizes the tension of fibronectin or gelatin gel, abundant integrin ligands and the property of melting at a higher temperature and solidifying at a lower temperature to tightly contact with cells at the lower temperature, utilizes the tension and specific adhesion to remove the cells from an interface, and then dissolves the gel at 37 ℃, so that the cells can be recovered and continuously cultured, and the extraction of ECM of precious cells is facilitated;
(4) the invention adopts the ECM extracted by physically removing the cells from the gel as a natural scaffold for the morphogenesis, maintenance and reconstruction after injury of tissues and organs in the nature, and has the characteristics of closer approximation to the original tissue morphology and internal microstructure compared with the artificially synthesized scaffold material, so the biological scaffold material formed by the ECM has better function reconstruction effect, is easier to realize the regeneration of blood vessels and nerve tissues and the reconstruction of tissue biological functions, is easier to re-implant cells or cell factors and plays better role;
in conclusion, the invention has the advantages of simple process, reliable extraction, low cost, reutilization and the like, and has high practical value and popularization value in the technical field of medical biomaterials.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention, and therefore should not be considered as limiting the scope of protection, and it is obvious for those skilled in the art that other related drawings can be obtained according to these drawings without inventive efforts.
FIG. 1 is a flow chart of the extraction process of the present invention.
FIG. 2 is a microscopic pattern of the front and back cover slips of the present invention.
FIG. 3 is a fluorescent staining pattern of the growth state of cells on a decellularized slide and on a blank slide according to the present invention.
Detailed Description
To further clarify the objects, technical solutions and advantages of the present application, the present invention will be further described with reference to the accompanying drawings and examples, and embodiments of the present invention include, but are not limited to, the following examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present application.
Example 1
As shown in fig. 1 to 3, the present embodiment provides a method for extracting an extracellular matrix layer from a cell layer, which includes the steps of:
the method comprises the following steps: preparing 100mM PBS (phosphate) buffer solution, and adjusting the pH of the PBS (phosphate) buffer solution to 6-7 by using NaOH solution or 1M NaOH solution;
step two: adding gelatin or fibronectin to ensure that the mass concentration of the gelatin or fibronectin in the mixed solution is 150mg/mL, and uniformly stirring;
step three: filtering the mixed solution with 0.45 μm membrane filter, pouring into a culture dish with diameter of 35mm, and cooling in a refrigerator at 4 deg.C for 15 min;
step four: taking out the gel from the culture dish, covering the gel on a cover glass cultured with cells, and slightly pressing the gel to make the gel fully contact with the cells;
step five: the gel was removed and washed with 37 degrees buffer to obtain ECM.
Example 2:
as shown in fig. 1 to 3, the present embodiment provides a method for extracting an extracellular matrix layer from a cell layer, which includes the steps of:
the method comprises the following steps: a, B, C three solutions were prepared, solution A was mixed with 90mg fibrinogen and 3ml normal saline at 37 deg.C, and solution B was thrombin S (500U ml-1) dissolved in normal saline. For solution C, CaCl2(4.5mg ml-1) was dissolved in saline. Solutions B and C were sterilized through 0.22- μm membrane filters.
Step two: solutions B and C were sterilized through 0.22- μm membrane filters.
Step three: 0.45ml of physiological saline, 0.3ml of the B solution and 0.3ml of the C solution were mixed, and 1.2ml of the A solution was slowly added.
Step four: pouring the mixed solution into a culture dish with the diameter of 35mm, and then cooling for 15min in a refrigerator at 4 ℃;
step five: taking out the gel from the culture dish, covering the gel on a cover glass cultured with cells, and slightly pressing the gel to make the gel fully contact with the cells;
step six: after the gel was removed, the ECM was washed with 37 degrees buffer.
Test 1:
in examples 1 and 2, micrographs were taken of the slides before and after the myxocytes, respectively, as shown in FIG. 2.
As can be seen from FIG. 2, the cells grown on the slide were almost completely removed after the use of the gel-adhered cells, indicating that the present invention has a superior cell-removing ability.
And (3) testing 2:
the same number of cells were cultured on the blank slide and the decellularized slide, respectively, as shown in FIG. 3, from the decellularized slides of examples 1 and 2. As can be seen from FIG. 3, after four hours of cell culture, the number of cells on the decellularized slide, the spreading of cells, and the fluorescence intensity of myosin were all significantly better than those on the blank glass, and significant clumping of cells on the blank glass occurred. The results show that the adhesion of the cells is promoted by the presence of ECM on the decellularized slide, so that the cells have better spreading and proliferation; while poor adhesion of cells to the slide on the blank slide promoted clumping between cells, with unbelled single cells having poor spreading.
The above-mentioned embodiments are only preferred embodiments of the present invention, and do not limit the scope of the present invention, but all the modifications made by the principles of the present invention and the non-inventive efforts based on the above-mentioned embodiments shall fall within the scope of the present invention.
Claims (5)
1. A method of extracting a layer of extracellular matrix from a layer of cells, comprising the steps of:
adding fibronectin or gelatin into buffer solution, and stirring at a temperature of not lower than 37 deg.C to obtain mixed liquid; the concentration of the fibronectin or the gelatin in the mixed liquid is 50-200 mg/mL;
filtering the mixed liquid by adopting a membrane filter, and placing the filtered mixed liquid into a mould;
freezing at a temperature below 25 deg.C until the solution is gelled to obtain a gel;
taking the gel out of the mold, covering the gel on a cover glass with cultured cells, and pressing the gel to make the gel fully contact with the cells;
the gel was torn off and the coverslip was rinsed with 37 ℃ buffer to obtain extracellular matrix of the substrate.
2. The method of claim 1, wherein the buffer is phosphate buffer.
3. The method of claim 1 or 2, wherein the buffer is at a concentration of 50 to 250 mM.
4. The method of claim 1 or 2, wherein the buffer has a PH of 6 to 8.
5. The method of claim 1, wherein the membrane filter has a filter pore size of 0.22 μm or 0.45 μm.
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