CN113817663A

CN113817663A - Method for extracting matrix gel from skin of cattle and donkey

Info

Publication number: CN113817663A
Application number: CN202110969176.8A
Authority: CN
Inventors: 张志平; 贺来增; 张君涛; 邓立新; 赵英博; 石亚珂; 王新卫; 董海聚
Original assignee: Henan Agricultural University
Current assignee: Henan Agricultural University
Priority date: 2021-08-23
Filing date: 2021-08-23
Publication date: 2021-12-21

Abstract

The invention discloses a method for extracting matrix gel from skin of cattle and donkey, and relates to the technical field of biological materials. The matrix gel is a soluble basement membrane matrix extracted from extracellular matrix, can be used as a biological scaffold material for three-dimensional cell culture, and can better simulate the in-vivo growth environment of cells so that the cells can better exert the functions and characteristics of the cells. The matrix gel is characterized by being an aqueous solution at 4 ℃ and a gel at 37 ℃. The invention belongs to the field of biotechnology, and aims to establish and verify the effect of extracted gel in three-dimensional cell culture and screen out more excellent biological materials for three-dimensional cell and organoid culture by taking the skin of 3-6-month-old fetal cows as a research object in experiments.

Description

Method for extracting matrix gel from skin of cattle and donkey

Technical Field

The invention relates to the technical field of biological materials, in particular to a method for extracting matrix gel from skin of cattle and donkey.

Background

In vitro culture of animal cells provides a clear platform for studying cells and tissues in vitro in physiology and pathophysiology studies, and culture replacement tissues for regenerative medicine. Traditionally, this has been done by culturing single cell populations on a two-dimensional (2D) substrate, however, it has been demonstrated that cells behave more naturally when cultured in a three-dimensional environment. Cell biology studies usually rely on tissue culture, however artificial substrates such as plastic or glass may force cells to conform to flat and hard surfaces, distorting the results. In contrast, the true matrix of most cells in a living organism is an extracellular matrix, whose molecular composition is three-dimensional, complex and dynamic, and whose flexibility is variable. In the natural 3D environment of cells, the structural scaffold surrounding the cells is the basement membrane, a particular heterogeneous entity in the extracellular matrix. The microenvironment affects gene expression, and thus the behavior of a cell depends largely on its interaction with the extracellular matrix, neighboring cells, and soluble local and systemic signals. Integrins in various types of membrane-associated structures, called cell matrix adhesion, transmit information between the extracellular matrix and the cytoplasm in a bidirectional manner. Cell-matrix adhesion mediates physiological responses that regulate cell growth, migration, differentiation, survival, tissue architecture, and matrix remodeling. The type of cell-matrix adhesion of tissues in vitro by integrins and the signals they transmit are strongly influenced by the flat, hard surface of the tissue culture dish; therefore, the in vivo environment should be brought closer by culturing the cells in a three-dimensional gel or matrix.

Synthetic hydrogels and natural hydrogels have become popular three-dimensional cell culture platforms. The native biomaterials used to develop scaffolds may be composed of components in the extracellular matrix (ECM), such as collagen, fibrinogen, hyaluronic acid, glycosaminoglycans, hydroxyapatite, etc., and thus have the advantages of bioactivity, biocompatibility, and mechanical properties similar to those of native tissues. Other natural materials include materials derived from plant, insect or animal components (e.g., cellulose, chitosan, silk fibroin, alginate, etc.). The matrix gel provides a favorable microenvironment for cell culture. Due to the presence of a myriad of endogenous factors, they also promote many functions of the cell, which is beneficial for the viability, proliferation, and development of many types of cells. The matrigel is a biological scaffold extracted from animal extracellular matrix and used for three-dimensional culture, and is widely used for three-dimensional culture of cells. Basement membrane is an important extracellular matrix, present in all epithelial and endothelial tissues. The matrigel consists of laminin, iv type collagen, heparan sulfate, proteoglycan, pentapeptide and nidogen. After polymerization at room temperature, the matrix gel produces a bioactive matrix resembling the extracellular matrix produced by mammalian cells. Depending on the number and type of components of the commercially available tumor-derived matrix, the composition of the material is more embryo-like, which may explain why so many cell types (including stem cells and tissue explants) respond to this.

The matrix gel is an agent extracted from extracellular matrix, and retains the characteristics of extracellular matrix. The most abundant protein in the mammalian body is collagen, which is mainly deposited in the extracellular matrix and has the function of maintaining the shape, structure and mechanical properties of tissues. When an organism is damaged or a pathogen invades, activated monocytes and macrophages generate matrix metalloproteinase, regulate and control continuous remodeling of collagen in extracellular matrix, promote migration of immunocytes to disease parts, and play a role in immunoregulation. Laminin, a major adhesion protein in the extracellular matrix, not only constitutes an important component of the basement membrane of the extracellular matrix, but also promotes cell migration and adhesion of various factors by virtue of its specific structure as a specific receptor for recognizing the cell surface. Hyaluronic acid is the only glycosaminoglycan free of sulfate and not bound to core protein, synthesized intracellularly by hyaluronic acid synthase and released extracellularly to participate in the formation of extracellular matrix. In the extracellular matrix of healthy tissues, hyaluronic acid exists in the form of high molecular weight hyaluronic acid (>1000kD), has a stable structure and is proved to have a certain immune tolerance effect. Hyaluronic acid can be covalently bound to various proteins, which affects the immune process of the body. Extracellular matrix, which is a secreted product of cells in tissues and organs, is a complex of functional and structural proteins, including growth factors and various proteins (e.g., collagen, fibronectin, and proteoglycans), which together affect the growth of cells. The matrix gel is rich in laminin, collagen and the like, can simulate the structure, the composition, the physical characteristics and the functions of a cell basement membrane in vivo, and is beneficial to the culture and the differentiation of cells in vitro. The extracellular matrix is rich in cytokines with immunoregulatory activity, and growth factors, tumor necrosis factors, chemokines and the like are closely related to proliferation, differentiation and migration of immune cells. Because multiple functions of the cells are regulated and controlled through the combined action of the regulatory factors, the cell adhesion factors and the connexin, the matrix gel not only provides a three-dimensional space required by the cells for survival, but also plays an important role in the aspects of disease diagnosis and treatment, tissue engineering regeneration and repair and the like. However, the molecular complexity and tissue structure of synthetic materials composed of collagen, fibronectin, or hyaluronate are very different from those of natural extracellular matrices, so compared with conventional synthetic materials, the extracellular matrices have good biocompatibility and cell affinity, can provide an environment closest to that in vivo for seed cells, and are beneficial to the growth, differentiation, and maturation of the seed cells.

The skin, which is composed of dermis, epidermis and subcutaneous tissue, is an important organ of the animal body. The dermis contains a large amount of collagen and a matrix, and the epidermis contains proteins such as keratin. Soluble collagen in skin, adult cattle and fetal cattle have great difference in the aspect of formed reticular space structures, fetal cattle hide glue has more precise reticular structures, obvious porous water seepage structures and fine fibers with good orientation. The bovine collagen structure is looser than that of fetal bovine, because the cross-linking components in the collagen of animal skin gradually increase with age, and the original regular network structure of the collagen is destroyed. The fetal calf has tissues such as epidermal papillary layer and the like from the aspect of tissues, but the fetal calf is not rich in subcutaneous fat, hair follicles are also in the newborn and development stages and are convenient to remove, and the fetal calf is in the newborn and development states due to soft and tough tissues, most of newborn skin collagen is collagen fibril, the immunogenicity is lower than that of adult pig skin collagen, and cell adhesion, growth and proliferation can be promoted. The skin contains collagen, which is a protein and biomacromolecule compound, and has the characteristics of colloid property, precipitation, denaturation, coagulation and the like. The skin contains both matrix and collagen, so the fetal calf skin is suitable as an extraction material of skin gel, and the skin-extracted gel is extracted from normal tissues of animals, has good affinity and environment suitable for cell and tissue growth, and is a good medium for 3D culture of cells. Meanwhile, the matrix gel contains nutrient substances such as collagen and the like, and has great potential in the development of skin care products. And the matrix gel can be injected in vivo, is clinically used for repairing tissues and organs, and has high research value in the aspects of modern medicine and pharmacy.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention discloses a method for extracting matrix gel from skin of cattle and donkey, which aims to solve the problems in the background technology.

(II) technical scheme

The invention overcomes the defects of single component and single biological function of the synthetic hydrogel; the adherence inhibition of the traditional cell culture is overcome; the invention provides a matrix gel which is extracted from skin tissues of fetal cows and can be used for three-dimensional cell culture. The extract is rich in proteins and factors, and can be used for injection gel and three-dimensional culture of in vitro cells, and has application value in skin care.

The technical scheme of the invention is as follows: a method for extracting matrix gel for three-dimensional cell culture from skin of cattle (donkey) comprises the following steps:

(1) taking skin tissue of cattle or donkey, removing subcutaneous adipose tissue of skin at 4 deg.C, washing in distilled water for 5min, and placing the skin tissue in solution for decellularization to obtain decellularized skin matrix.

(2) Physically crushing skin at 4 ℃, adding urea buffer solution with the same volume mass, beating into homogenate in a crusher at 4 ℃, and shaking for a certain time in a shaking table at 4 ℃ to prepare matrix mixed liquor.

(3) And subpackaging the vibrated matrix mixed solution into 1ml centrifuge tubes, centrifuging for a certain time at low temperature and high speed, and taking supernatant to obtain a primary skin gel solution.

(4) Transferring the obtained primary solution of skin gel into a treated specific dialysis bag, placing in TBS buffer solution, performing low-speed shaking at 4 deg.C under aseptic condition, and dialyzing for a period of time to obtain skin gel solution.

Because the matrix contains abundant proteins and factors, during the formation of gel, each component can spontaneously form a nanofiber structure, and the microscopic nanofiber structure has been proved to have guiding and promoting effects on the growth, migration, differentiation and the like of cells in tissue repair. This property can be exploited as an infusion gel via a matrix gel and a desired specific factor or protein combination to achieve an effect.

The used cell-removing solution can be one of high concentration saline, SDS, NaOH mixed solution, etc.

The invention discloses a method for extracting matrix gel from skin of cattle and donkey, which has the following beneficial effects:

1. the skin matrix gel is obtained from fetal calf skin tissue, belongs to a natural matrix, is rich in protein and beneficial molecules, and can provide good biological functions.

2. The gel obtained by the invention can form gel under the condition of body temperature, accords with the physiological condition of in vivo injection, and can be used for in vivo experiments of animals.

3. The gel obtained by the invention can be used for three-dimensional culture of in vitro cells and organoids.

4. The matrix obtained by the invention is rich in protein and beneficial factors, and is beneficial to skin care and beauty.

5. The matrix of the invention has a promoting effect on tissue repair.

Drawings

FIG. 1 shows skin extracts under different conditions.

FIG. 2 shows gradient detection of skin extract.

FIG. 3 shows the measurement of OD value of each concentration of skin extract.

FIG. 4 shows a skin extract under a microscope.

FIG. 5 shows nuclear staining of skin extracts.

FIG. 6 shows adherent cultured jejunal crypt stem cells.

FIG. 7 is a jejunal crypt stem cell cultured in skin gel.

FIG. 8 shows the expression of genes functionally related to the jejunal crypt stem cells.

FIG. 9 shows the water content of the skin extract.

Detailed Description

1. Gel testing of extracts

The extract was transferred to a 1.5mL centrifuge tube, labeled, and frozen in a 20 ℃ freezer for 48 h. Taking out the frozen extract sample, and thawing in a refrigerator at 4 deg.C for 4 hr. And (3) after the sample is completely thawed, putting the sample into a constant-temperature water bath kettle at 37 ℃ for incubation for 2-5min, and observing whether the sample to be detected forms gel.

2. Gradient gel testing of extracts

The skin extract was placed on ice, the extract was observed for impurities (sediment that may be drawn when the supernatant was taken after centrifugation), and the extract was subjected to brief low-speed centrifugation. Diluting the extract with precooled DMEM culture medium in an ice box in a gradient manner to make the concentration gradient of the extract be five gradients of stock solution, 50%, 33.3%, 25% and DMEM culture medium, uniformly blowing and beating the extract, placing the extract in a water bath kettle at 37 ℃, and observing the time for forming gel, the state for forming gel at each time point and the visual definition of each concentration.

3. Detection of concentration gradient OD value of extracting solution

And (3) performing gradient dilution on the extract on ice, wherein the dilution ratio is 1, 50%, 33.3%, 25% and 0%, uniformly mixing, and adding the mixture into an enzyme label plate, wherein each gradient has three holes, and each hole has 100uL, so that bubbles are avoided. The absorbance at 405nm was measured in a microplate reader preheated at 37 ℃ once every 2min for 50 times.

4. Detection of cells and nucleic acids in extract

Firstly, dripping the gel on a glass slide, and observing whether impurities such as cells exist or not under a microscope; the gel was subjected to nuclear staining and observed under a fluorescent microscope.

5. Determination of water content of extract

Taking a sample of the skin matrix extracting solution, and unfreezing at 4 ℃. The ultrasonically cleaned coverslips were autoclaved and dried, labeled, and weighed individually as m 1. Thawed samples were dropped onto coverslips using a pipette gun and the mass was measured as m2, taking three sets of 3 replicates each. The samples were placed in a ventilated place and naturally air-dried for 48 hours, and then the cover slips with the samples added were weighed respectively as m 3. The water content of the sample to be tested is m4-m 2-m1, the dry weight of the sample is m 5-m 3-m1, the mass of water in the sample is m 6-m 4-m5, and the water content of the sample is P-m 6/m4 × 100%.

6. Three-dimensional cell culture of extract

Thawing the frozen skin gel at 4 deg.C in advance (about 4 h), centrifuging in palm centrifuge to remove small amount of impurities (small amount of skin substrate may be absorbed before dialysis), and diluting the gel part in ice box; diluting the gel with culture solution to a final concentration of 25% of original skin gel, blowing uniformly, adding into 6-well plate with 2mL per well to avoid bubbles, and incubating in a carbon dioxide incubator at 37 deg.C for 30 min; digesting and centrifuging intestinal crypt stem cells which are cultured in a 25cm2 cell culture bottle and reach passage concentration (the used jejunal crypt stem cells are provided by the laboratory), removing upper layer culture solution, adding new 8mL 10% serum high-sugar culture medium, uniformly blowing, adding 1mL cell suspension per hole into a gel-forming six-hole plate, continuously culturing, and repeating each group for three times (the used gun head and a centrifuge tube are all pre-cooled at 4 ℃ after autoclaving).

7. Detecting the functional gene expression of the stem cells.

RNA extraction was performed on the three-dimensionally cultured jejunal crypt stem cells, and then fluorescence quantitative PCR was performed to detect the expression levels of Lgr5, CD133, DCLK1, and CD166 genes.

The foregoing shows and describes the general principles and broad features of the present invention and advantages thereof. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Claims

1. A method for extracting matrix gel from skin of cattle and donkey is characterized by comprising the following steps:

A. washing skin tissue of cattle or donkey for 2-3 times at normal temperature, removing blood stain, removing subcutaneous tissue at 4 deg.C, and removing cells in NaCl buffer solution with pH of 7.4 under stirring.

B. And C, physically crushing the skin tissue treated in the step A at 4 ℃, adding 2M urea buffer solution with the pH of 7.4, beating into homogenate at 4 ℃, and shaking in a shaking table at 4 ℃ for 3 d.

C. Homogenizing the vibrated skin, centrifuging at 16000rpm at 4 deg.C for 5min, collecting supernatant, and dialyzing in TBS buffer solution and DMEM culture medium to obtain extract as skin gel.

2. The method of claim 1, wherein the aqueous decellularized solution is not limited to one or more of a high concentration NaCl solution, ethanol, or acetone. The base extract is not limited to the urea buffer.

3. A method of preparing a bovine or donkey skin gel according to claim 1, comprising the steps of:

(1) taking fresh skin tissue of cattle or donkey at room temperature, removing subcutaneous adipose tissue of skin at 4 deg.C, washing in distilled water for 5min, and placing the skin tissue in solution for decellularization to obtain decellularized skin matrix.

4) Transferring the obtained primary solution of skin gel into a treated specific dialysis bag, placing in TBS buffer solution, performing low-speed shaking at 4 deg.C under aseptic condition, and dialyzing for a period of time to obtain skin gel solution.

4. The method for extracting matrix gel from skin of cattle or donkey according to claim 3, wherein the skin tissue is subjected to acellular treatment by soaking the skin tissue in 3.4M NaCl buffer solution (pH 7.4) for 10min under stirring; the urea buffer solution is 2M urea buffer solution (pH is 7.4); the dialysis bag used had a molecular weight of 7000.

5. The method of claim 3, wherein the skin gel is aqueous at 4 ℃ and forms a gel at 37 ℃; the storage conditions were-20 ℃.

6. The method of claim 3, wherein the obtained skin gel is free of cells and nucleic acids.

7. A method of extracting a matrix gel from the skin of cattle or donkey hide as claimed in claim 3, wherein the obtained skin gel is diluted 1:1, 1:2 and 1:3 and remains the same in the form of aqueous solution at 4 ℃ and gel formation at 37 ℃.

8. A method of extracting a matrix gel from the skin of cattle or donkey according to claim 3, wherein the resulting skin gel allows for three-dimensional cell growth.

9. A method of extracting matrix gel from the skin of cattle or donkey as claimed in claim 3, wherein the obtained skin gel maintains the pluripotency of stem cells.