CN110272860B - Construction method and application of three-dimensional cell culture microenvironment - Google Patents
Construction method and application of three-dimensional cell culture microenvironment Download PDFInfo
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Abstract
The invention discloses a method for constructing a cell three-dimensional culture microenvironment based on a specific extracellular matrix and application thereof, which combines an extraction method of the specific extracellular matrix, and application of the specific extracellular matrix and a photocuring material which are compounded to prepare hydrogel in vitro three-dimensional culture, particularly in an organ chip technology. The technical scheme of the invention selects surfactants with different cell removing capacities aiming at tissues or organs with different densities, and selects enzyme treatment and ammonia water as a solvent combination mode to treat particularly compact tissues or organs, wherein the preferable combination of the enzyme and the surfactant comprises pancreatin solution, Triton X-100 solution, and 0.05-0.5% ammonia water as the solvent. The hydrogel prepared by compounding the extracellular matrix and the photoinitiated gel forming material is used for constructing a cell culture microenvironment, and the hydrogel has the advantages of high gel forming speed, simple gel forming conditions, controllable mechanical properties of the hydrogel and the like.
Description
Technical Field
The invention relates to the field of biological materials and tissue engineering, in particular to a method for extracting specific extracellular matrix and constructing a three-dimensional cell culture microenvironment, and particularly relates to application of the specific extracellular matrix in an organ chip.
Background
In 1993, L anger and Vacanti conceptually propose the definition of tissue engineering, which applies the principles and techniques of life science and engineering to develop biological substitutes for repairing, maintaining and promoting tissue functions.
Chinese patent 201410145791.7 discloses a acellular matrix repair gel and a novel preparation method thereof, which is used for carrying out virus inactivation on animal tissues and organs by using peroxyacetic acid, degreasing by using a surfactant, carrying out acellular by using enzyme, and carrying out nuclease removal. And (3) carrying out enzyme digestion on the obtained matrix, and then adjusting pH to form gel, which is applied to specific repair of human damaged tissues. The invention is mainly applied to the repair of damaged tissues of a human body, and the damaged tissues are degreased by using a surfactant and are decellularized by using enzyme.
The Chinese invention patent 201410566192.2 discloses a kit for obtaining extracellular matrix of multiple organs and tissues and a using method thereof, wherein a surfactant is used for perfusing the organs to obtain a decellularized scaffold of the whole organ for regenerative medicine and tissue engineering research and application. The reagent kit uses a surfactant for decellularization, but the reagent kit lacks an amphoteric surfactant, is the mildest decellularization reagent at present, and has less damage to effective components when the tissue or organ with lower density is decellularized; the whole organ decellularization is carried out by using the perfusion method, and the complete decellularized organ scaffold is obtained.
The Chinese patent application 201810734419.8 discloses an application of extracellular matrix lyophilized powder, which is prepared by enzyme extraction, urea extraction, acid extraction or water extraction, and is applied to beauty products, cell culture, medicines for treating wound repair and medical instruments for treating wound repair.
The Chinese patent application 201611262064.4 discloses a decellularized extracellular matrix and a preparation method and application thereof, which is to inactivate tissue viruses, remove DNA by acid and alkali, perform decellularization treatment by protease, perform degreasing treatment by organic matters, perform dehydration treatment on the obtained extracellular matrix, and perform high-temperature vacuum crosslinking or chemical crosslinking. It has problems that the time for removing DNA is long due to acid and alkali treatment, and the damage to the effective components of extracellular matrix is large; organic matter treatment also has great damage to the effective components of extracellular matrix.
Chinese patent 201710126703.2 discloses a biological tissue matrix material, its preparation method and its application, and uses a combination of enzyme protease and ultrasound to perform cell removal. The ultrasonic is a physical decellularization method, the physical method has small damage to the active ingredients of the extracellular matrix, but the ultrasonic equipment is needed, particularly the device which needs the double-frequency ultrasonic wave is high in price, and the ultrasonic is not beneficial to implementation unless special production is carried out.
Chinese invention patent 201410229506.X discloses a two-layer cell culture system organ chip and a preparation method thereof, wherein culture matrix glue of the cell culture chamber is gelatin, chitosan, silk fibroin and the like. The three-dimensional cell culture medium can be one or more of the existing natural biological materials and high polymers.
One of the main methods for constructing the in vitro three-dimensional cell culture microenvironment is to prepare various forms such as hydrogel, sponge, film, particle and the like by using the existing materials including one or more of natural matrixes, natural biomaterials, synthetic polymers and the like.
For example, chinese patent 201310057768.8 discloses a method for constructing a three-dimensional neural stem cell model in two steps based on microfluidic technology, which uses collagen as a main material and mixes a suitable culture medium to prepare a three-dimensional complex for the culture of neural stem cells. The method can realize the in vitro three-dimensional culture of the neural stem cells, but mainly adjusts and controls according to culture medium substances such as growth factors and the like, and does not form a supporting environment which is beneficial to the growth, proliferation and differentiation of cells.
For example, chinese patent 201510045823.0 discloses a method for preparing a three-dimensional scaffold attached to cells and its application, which comprises mixing one or more of collagen, gelatin, hyaluronic acid, chitosan or sodium alginate with cells, crosslinking to form microspheres, mixing the microspheres with the matrix material, and performing 3D printing to form the three-dimensional scaffold. The biological material used by the method has good biocompatibility and universality, but can not provide adaptive support for specific cells, and the 3D forming method is complex and needs special equipment.
The hydrogel prepared by compounding the specific extracellular matrix and the modified gelatin and the application of the hydrogel as a three-dimensional cell culture microenvironment construction, particularly as an organ chip are not reported.
Disclosure of Invention
The invention provides a method for constructing a three-dimensional cell culture microenvironment based on a specific extracellular matrix and application thereof, and aims to obtain an active extracellular matrix as a biomaterial, combine the obtained extracellular matrix with a photocuring material to prepare hydrogel, and have the advantages of fast time and controllable mechanical properties of the gel compared with the prior art of directly preparing the gel. The other purpose of the invention is to take the material as one of the raw materials, finally mix the material with the light-cured material, greatly shorten the gelling time, conveniently regulate and control the mechanical properties of the prepared hydrogel and be beneficial to meeting the requirements of three-dimensional culture of different cells. The invention is applied to specific cell three-dimensional culture, realizes the in-vitro culture of human miniature organs, further constructs organ chips with different functions, and the implementation of the invention is beneficial to constructing a more bionic specific cell three-dimensional culture system.
The invention provides a method for constructing a three-dimensional cell culture microenvironment based on a specific extracellular matrix, which comprises the steps of preparing a decellularized matrix, preparing hydrogel by compounding the specific extracellular matrix and a photoinitiated gel-forming material, and constructing the cell culture microenvironment. The step of preparing the acellular matrix comprises the steps of pretreating, acellular treating and post-treating the tissues or organs of the mammal; the steps of preparing the hydrogel by compounding the specific extracellular matrix and the photoinitiated gel forming material and constructing a cell culture microenvironment comprise dissolving the specific extracellular matrix, dissolving the photoinitiated gel forming material, mixing and photoinitiating gel forming and using the gel forming material to construct the cell culture microenvironment.
According to one aspect of the invention, the pretreatment of the mammalian tissue or organ in the step of preparing the acellular matrix comprises selecting a mammalian tissue or organ, washing, freezing, and cutting; the cell removing treatment comprises washing the pretreated tissue or organ by PBS buffer solution, then using a cell removing solution of surfactant or combination of enzyme and surfactant for cell removing treatment, and then using PBS mixed solution of combined enzyme for treatment; the post-treatment comprises freeze drying treatment and crushing treatment after cleaning.
Alternatively, the specific operation of the pretreatment of the tissue or organ of the mammal comprises:
(i) selecting a tissue or organ of a mammal, said mammal including pig, cow, mouse, etc., preferably pig, said tissue or organ including heart, liver, lung, spleen, kidney, intestine, brain, nerve, blood vessel, skin, etc.;
(ii) cleaning the tissue and organ to remove attachments such as fat, blood and the like;
(iii) (iii) freezing said tissue or organ of (ii) and then mechanically cutting into 100-500 μm slices, which is performed on ice.
As an option, the specific operation of the decellularization treatment comprises:
(iv) washing the pretreated tissue or organ slice with 0.01M PBS buffer solution (phosphate buffer) under stirring for 5-30min, preferably 15min, and repeating for 1-3 times;
(v) (iv) subjecting the washed sheet of (iv) to a stirring treatment in a cell-removing solution; cleaning with 0.01M PBS buffer solution under stirring for 5-30min, preferably 15min every 2h, replacing the cell-removing solution, and repeating for 2-6 times; the decellularization solution comprises a surfactant or a combination of an enzyme and a surfactant; preferably, the surfactant comprises nonionic surfactant, ionic surfactant and amphoteric surfactant, and particularly preferably comprises Triton X-100 (0.5-5% by volume solution), sodium deoxycholate (1-5% by mass solution), sodium dodecyl sulfate (0.1-2% by mass solution), benzalkonium bromide (0.1-0.5% by volume solution), CHAPS (0.1-3% by mass solution) and the like, wherein the enzyme is selected from pancreatin (0.01-0.25% by mass solution), and the solvent of the decellularized solution comprises deionized water or ammonia water (0.05-0.5% by volume solution);
(vi) (vi) subjecting the tissue or organ slice treated with the decellularization solution in (v) to a stirring treatment for 1-2h with a 0.01M PBS mixed solution containing 2000Ku ribonuclease and 2000Ku deoxyribonuclease.
Alternatively, the post-processing comprises the following specific operations:
(vii) stirring and cleaning the matrix subjected to decellularization by using deionized water, replacing the deionized water every 15min, repeating the steps for 3 times, and detecting that a decellularization reagent is removed, wherein the detection method comprises but is not limited to conductivity detection, high performance liquid chromatography, scanning electron microscope detection and the like;
(viii) (vii) draining the substrate, and freeze-drying;
(ix) (viii) mechanically pulverizing said lyophilized matrix of (viii) into a powder.
According to another aspect of the invention, the steps of preparing the hydrogel by compounding the specific extracellular matrix with the photoinitiated gel-forming material for constructing the cell culture microenvironment are dissolving the specific extracellular matrix, dissolving the photoinitiated gel-forming material, mixing the photoinitiated gel-forming material and using the mixed photoinitiated gel-forming material for constructing the cell culture microenvironment.
Alternatively, the specific operation of lysing the specific extracellular matrix comprises:
(i) selecting specific extracellular matrix according to cultured cell species, preparing matrix mother liquor of 5-10mg/M L with solvent of 0.01-0.1M hydrochloric acid solution containing 1-5mg/M L pepsin, and shaking at room temperature for 48-120 h;
(ii) and (c) centrifuging the matrix mother liquor in the step (i), wherein the rotating speed is more than 10000rpm/min, and taking supernatant.
As an option, the specific operation of dissolving the photo-initiation gel-forming material comprises:
(iii) dissolving a photoinitiated gel-forming material by using 0.01M PBS buffer solution, preparing a photoinitiated gel-forming material mother solution of 50-100mg/M L, adding a photoinitiator in a water bath at 50 ℃ for half an hour, wherein the photoinitiator comprises methacrylated gelatin, methacrylated hyaluronic acid, methacrylated chitosan and the like, and comprises L AP (phenyl-2, 4, 6-trimethylbenzoyllithium phosphonate), 2959 (2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone) and the like.
Alternatively, the specific operations of mixing and photoinitiating gelling and using it to construct a cell culture microenvironment comprise:
(iv) diluting the extracellular matrix-containing clear liquid to 2-5mg/m L with culture medium including DMEM, RPMI1640, L15, F12, Mccoy' 5A, EGM2, etc., adjusting pH to 7-8 with sodium hydroxide solution, mixing with photoinitiator-containing photoinitiated gel-forming material mother liquid in proportion, and filtering with 0.22 μm filter membrane;
(v) digesting and centrifuging the cells to be cultured, and resuspending the cells in the mixed solution obtained by the mixing of (iv) to adjust the cell concentration to 1 × 104~1×106Cell/m L, including various primary cells, cell line cells, stem cells, etc.;
(vi) and (v) adding the cell suspension prepared in the step (v) into a culture carrier, wherein the culture carrier comprises a standard cell culture plate, a culture dish, microfluidic chambers of various organ chips and the like, and irradiating by using 405nm blue light for 1-5min to form hydrogel.
(vii) Adding a culture medium containing conventional serum, double antibody and growth factors into the solidified hydrogel in the step (vi), and culturing at 37 ℃ in a 5% carbon dioxide environment.
The invention also provides application of the cell three-dimensional culture microenvironment based on the specific extracellular matrix, namely the constructed microenvironment is used for cell culture, namely the three-dimensional culture of specific cells on different culture carriers.
Having thus described the invention, it will be apparent that the invention can be varied in many ways which do not depart from the spirit and scope of the method and all such modifications will be obvious to those skilled in the art and are intended to be included within the scope of the patent claims.
Compared with the prior art, the invention has the beneficial technical effects that:
1. the technical scheme provided by the invention combines the method for extracting the specific extracellular matrix (I); (II) compounding the specific extracellular matrix with a photocuring material to prepare hydrogel; (III) use in three-dimensional culture in vitro, in particular in organ chip technology.
2. The invention can prepare specific matrix materials according to the culture requirements of different cells, provide a three-dimensional culture microenvironment adapted to the specific matrix materials, and promote the proliferation, migration, differentiation and other behaviors of the cells.
3. The invention adopts a physical, chemical and biological combined method to prepare the specific extracellular matrix, greatly shortens the preparation time, is beneficial to retaining the effective components, has relatively low price of the used reagent, does not need additional special equipment, and utilizes the expanded production.
4. The invention compounds the specific extracellular matrix with the light-cured material, can quickly form hydrogel in a short time, is beneficial to maintaining the three-dimensional cell culture structure, can adjust the mechanical property of the formed hydrogel through material compounding, and provides a structural environment suitable for cell growth.
5. The invention has great potential for three-dimensional culture of cells in vitro, particularly application in organ chips, and is beneficial to promoting the development of organ chip technology in aspects of drug screening, toxicological evaluation, disease model construction and the like.
6. The invention adopts the method of freezing and slicing for pretreatment, selects the surfactants with different cell removing capacities aiming at tissues or organs with different densities, and selects the mode of combining enzyme treatment and ammonia water as a solvent for treating particularly compact tissues or organs, thereby greatly shortening the preparation time.
7. The preferred combination of enzyme and surfactant of the present invention (0.01% to 0.25% pancreatin, 0.5% to 5% Triton X-100, solvent 0.05% to 0.5% ammonia).
8. The hydrogel prepared by compounding the extracellular matrix and the photoinitiated gel-forming material is used for constructing a cell culture microenvironment, and the hydrogel has the advantages of high gel-forming speed, simple gel-forming conditions, controllable mechanical properties of the hydrogel and the like.
9. The hydrogel has the structural characteristics of a cross-linked network structure, has the pore size and porosity suitable for cell growth, is convenient for exchanging nutrient substances and metabolites, and can promote cell proliferation, migration and differentiation.
Drawings
FIG. 1: cell three-dimensional culture microenvironment construction preparation flow chart based on specific extracellular matrix
FIG. 2: a freeze-dried sample of extracellular matrix of pig liver
b porcine liver extracellular matrix powder sample
FIG. 3: pig liver extracellular matrix scanning electron microscope picture
FIG. 4 shows the results of activity assays on days 3, 7 and 14 of three-dimensional cell culture (wherein: extracellular matrix complex methacrylic acid gelatin; ○: methacrylic acid gelatin; blank control without addition of gelatin)
FIG. 5: the cells were cultured in an extracellular matrix complex methacrylic acid gelatin gel environment for three-dimensional fluorescence staining patterns on days 3, 7 and 14 (a: day 3, b: day 7 and c: day 14).
Detailed Description
The present invention will be further illustrated with reference to the following specific examples, but the present invention is not limited to the following examples. The method is a conventional method unless otherwise specified. The reactants are commercially available from the open literature unless otherwise specified.
Example one
(1) Selecting fresh heart of pig, cleaning to remove fat and blood, freezing at-20 deg.C, cutting into slices of about 500 μ M on ice with a scalpel, stirring and cleaning the slices with 0.01M PBS buffer solution for 15min, and repeating for 3 times; draining the sheet, stirring with 0.1% pancreatin for 2 hr, treating with 3% Triton X-100 for 2 hr, treating with 1% SDS for 4 hr, stirring and washing with 0.01M PBS buffer solution for 15min every 2 hr, stirring and treating with 0.01MPBS mixed solution containing 2000Ku ribonuclease and 2000Ku deoxyribonuclease for 1 hr, removing cells, stirring and washing with deionized waterChanging deionized water once every 15min, repeating for 3 times, detecting conductivity less than 10 μ s/cm, draining the matrix, freeze drying, pulverizing into powder, preparing the powder into 10mg/M L matrix mother liquor with 0.1M hydrochloric acid solution containing 5mg/M L pepsin, shaking at room temperature for 48H, centrifuging at 10000rpm/min, collecting supernatant, preparing 50mg/M L methacrylic acid gelatin solution with 0.01M PBS buffer solution, adding 10% photoinitiator L AP in 50 deg.C water bath for half an hour, diluting the matrix supernatant with DMEM complete culture medium to 5mg/M L, adjusting pH to 7-8 with sodium hydroxide solution, mixing with methacrylic acid gelatin solution containing photoinitiator according to a ratio of 1: 2, filtering with 0.22 μ M filter membrane, digesting human cardiac muscle cell H9C2, centrifuging, precipitating, adjusting cell concentration to 1 × 10 heavy suspension with the above mixed solution4And m L, adding the cell suspension into a 96-well cell culture plate, irradiating by using 405nm blue light for 1min to form hydrogel, adding DMEM complete culture medium, and culturing at 37 ℃ in a 5% carbon dioxide environment.
Example two
(1) Selecting fresh lung of pig, cleaning to remove fat and blood, freezing at-20 deg.C, cutting into slices of about 500 μ M on ice with a scalpel, stirring and cleaning the slices with 0.01M PBS buffer solution for 15min, and repeating for 3 times; the flakes were drained and then treated with 1% CHAPS (3- [3- (cholamidopropyl) dimethylamino)]Propanesulfonic acid inner salt) for 2 hours, 2 percent sodium deoxycholate for 4 hours, stirring and cleaning for 15 minutes by 0.01M PBS buffer solution every 2 hours, stirring and treating for 1 hour by 0.01M PBS mixed solution containing 2000Ku ribonuclease and 2000Ku deoxyribonuclease, finishing the cell removing treatment, stirring and cleaning by deionized water, replacing the deionized water every 15 minutes, repeating for 3 times, detecting no surfactant in the high performance liquid chromatography, draining the matrix, freeze-drying, crushing into powder by a grinder, preparing the powder into 10mg/M L matrix mother solution by 0.01M hydrochloric acid solution containing 5mg/M L pepsin, oscillating for 48 hours at room temperature, centrifuging at 10000rpm/min, taking supernatant, preparing 50mg/M L methacrylic acid gelatin solution by 0.01M PBS buffer solution, adding 5 percent photoinitiator L AP into water bath at 50 ℃, diluting the matrix supernatant by RP1640 MI L, adjusting the pH of sodium hydroxide solution to half an hour to 5mg/M7-8, mixing with methacrylic acid gelatin solution containing photoinitiator at a ratio of 3: 1, filtering with 0.22 μm filter membrane, digesting human lung cancer cell NCI-H23, centrifuging, precipitating, re-suspending the cells with the above mixed solution, and adjusting cell concentration to 1 × 104And m L, adding the cell suspension into a 96-well cell culture plate, irradiating by using 405nm blue light for 1min to form hydrogel, adding RPMI1640 complete culture medium, and culturing at 37 ℃ in a 5% carbon dioxide environment.
EXAMPLE III
(1) Selecting fresh pig lung, washing to remove fat and blood, freezing at-20 deg.C, cutting into slices of about 500 μ M on ice with scalpel, washing the slices with 0.01M PBS buffer solution under stirring for 15min, repeating for 3 times, draining, treating with 1% SDS for 4h, treating for 2h, washing with 0.01M PBS buffer solution under stirring for 15min, treating with 0.01M PBS mixed solution containing 2000Ku ribonuclease and 2000Ku deoxyribonuclease under stirring for 1h, washing with deionized water under stirring, replacing deionized water every 15min, repeating for 3 times, detecting conductivity less than 10 μ s/cm, draining, freeze drying, pulverizing into powder, preparing with 0.01M hydrochloric acid solution containing 5mg/M L pepsin to obtain 10mg/M L matrix mother solution, shaking for 48h at room temperature, 10000rpm/min, centrifuging supernatant, preparing with 0.01M PBS buffer solution, preparing 50mg/M PBS buffer solution, adding 677 mg/M gelatin solution containing gelatin, precipitating with 80% light, digesting with 368% gelatin solution, precipitating with 80% gelatin solution, adding gelatin solution containing 80% formaldehyde, precipitating with 367-20 mg/M gelatin solution, mixing with ammonium hydroxide solution, adding gelatin solution, dissolving initiator, adding gelatin solution, dissolving and pH adjusting pH to 367, adding gelatin solution, mixing4And m L, adding the cell suspension into a 96-well cell culture plate, irradiating by using 405nm blue light for 1min to form hydrogel, adding RPMI1640 complete culture medium, and culturing at 37 ℃ in a 5% carbon dioxide environment.
Example four
(1) Selecting fresh liver of pig, cleaning to remove fat and blood, freezing at-20 deg.C, cutting into slices of about 500 μ M on ice with a scalpel, and slowly adding 0.01M PBSWashing with water under stirring for 15min, repeating for 3 times, draining, washing with 3% Triton X-100 containing 0.2% ammonia water for 2h, washing with 0.5% benzalkonium bromide for 4h, washing with 0.01M PBS buffer solution for 2h, washing with 0.01MPBS mixed solution containing 2000Ku ribonuclease and 2000Ku deoxyribonuclease for 1h, washing with deionized water under stirring, changing deionized water every 15min, repeating for 3 times, detecting conductivity less than 10 μ s/cm, draining, freeze drying, grinding with a grinder to obtain powder, preparing with 0.1M hydrochloric acid solution containing 5mg/M pepsin L pepsin to obtain 10mg/M L matrix mother solution, shaking for 48h at room temperature, centrifuging at 10000rpm/min, collecting supernatant, preparing with 0.01M PBS buffer solution to obtain 50mg/M L methacrylic acid gelatin solution, precipitating at 50 deg.C, adding 15% light-water, filtering with 2959 light-water-bath to obtain supernatant, mixing with 0.7 mg PBS buffer solution containing 5mg of 5mg/M pepsin, precipitating with 3 μ M gelatin, filtering, mixing with 3M PBS buffer solution, precipitating with 3 g 3-7 g gelatin solution, adding sodium hydroxide, filtering, mixing4And m L, adding the cell suspension into a 96-well cell culture plate, irradiating by using 405nm blue light for 1min to form hydrogel, adding DMEM complete culture medium, and culturing at 37 ℃ in a 5% carbon dioxide environment.
EXAMPLE five
(1) Selecting fresh large intestine of pig, cleaning to remove fat and blood, freezing at-20 deg.C, cutting into slices of about 500 μ M on ice with scalpel, stirring and cleaning the slices with 0.01M PBS buffer solution for 15min, repeating for 3 times, draining, treating with 0.5% benzalkonium bromide for 2h, treating with 0.5% sodium deoxycholate for 4h, stirring and cleaning with 0.01M PBS buffer solution for 15min every 2h, stirring and treating with 0.01M PBS mixed solution containing 2000Ku ribonuclease and 2000Ku deoxyribonuclease for 1h, removing cells, stirring and cleaning with deionized water, replacing deionized water every 15min, repeating for 3 times, detecting conductivity less than 10 μ s/cm, draining, freeze drying, pulverizing with grinder into powder, and preparing into matrix with 5mg/M L pepsin-containing 0.05M hydrochloric acid solution of 10mg/M LShaking the mother liquor at room temperature for 48h, centrifuging at 10000rpm/min, collecting supernatant, preparing 50mg/M L methacrylic acid gelatin solution with 0.01M PBS buffer solution, water-bathing at 50 deg.C for half an hour, adding 5% photoinitiator L AP, diluting the matrix supernatant with Mccoy' 5A complete culture medium to 5mg/M L, adjusting pH to 7-8 with sodium hydroxide solution, mixing with methacrylic acid gelatin solution containing photoinitiator at a ratio of 1: 1, digesting human colon cancer cells HT-29, centrifuging, precipitating, re-suspending the cells with the above mixed solution, adjusting cell concentration to 1 × 104And m L, adding the cell suspension into a 96-well cell culture plate, irradiating by using 405nm blue light for 1min to form hydrogel, adding Mccoy' 5A complete culture medium, and culturing at 37 ℃ in a 5% carbon dioxide environment.
EXAMPLE six
(1) Selecting fresh thoracic aorta of pig, washing to remove fat and blood, freezing at-20 deg.C, cutting into slices of about 500 μ M on ice with scalpel, washing the slices with 0.01M PBS buffer solution under stirring for 15min, repeating for 3 times, draining, stirring with 0.25% pancreatin for 2h, treating with 0.5% benzalkonium bromide for 2h, treating with 1% SDS for 2h, washing with 0.01M PBS buffer solution under stirring for 2 min each time, treating with 0.01MPBS mixed solution containing 2000Ku ribonuclease and 2000Ku deoxyribonuclease for 1h, washing with deionized water under stirring, replacing deionized water every 15min for 3 times, detecting conductivity less than 10 μ s/cm, draining, freeze drying, pulverizing into powder, mixing with 0.05M hydrochloric acid solution containing 5mg/M pepsin to obtain 10mg/M L pepsin solution, stirring for 3 times, treating with PBS buffer solution containing 10 μ s/cm, washing with 0.05M PBS buffer solution containing 5mg/M pepsin, centrifuging at room temperature to obtain supernatant, precipitating with 397 mg/M solution containing gelatin, centrifuging at room temperature to obtain supernatant, precipitating with 397 mg/M, adding gelatin, centrifuging, precipitating with 397, adding gelatin solution containing 0.7, precipitating with 0.7, centrifuging, precipitating with 0.7 g, precipitating with 0.7 g, centrifuging, precipitating with 3 mg/M gelatin, precipitating with 0.74At L/m, adding the cell suspension into 96-well cell culture plate, irradiating with 405nm blue light for 1min to form hydrogel,adding EGM2 complete medium, and culturing at 37 deg.C under 5% carbon dioxide atmosphere.
EXAMPLE seven
(1) Selecting fresh skin of pig, washing to remove attachments such as fat and blood, collecting dermal tissue, freezing at-20 deg.C, cutting into slices of about 500 μ M on ice with a scalpel, washing the slices with 0.01M PBS buffer solution under stirring for 15min, repeating for 3 times, draining, stirring with 0.25% pancreatin for 2h, treating with 1% Triton X-100 for 2h, treating with 1% SDS for 4h, washing with 0.01M PBS buffer solution under stirring for 2h, treating with 0.01M PBS buffer solution containing 2000Ku ribonuclease and 2000Ku deoxyribonuclease under stirring for 1h, washing with deionized water under stirring, changing deionized water every 15min for 3 times, detecting conductivity less than 10 μ s/cm, draining, freeze drying, pulverizing into powder, mixing with 0.05M hydrochloric acid solution containing 5mg/M L pepsin, preparing 10mg/M matrix, L, adding 10mg/M PBS buffer solution containing 10 μ s/cm, washing with 0.01M PBS buffer solution containing 10 mg/cm, centrifuging, adding gelatin buffer solution containing 5 mg/7 mg of gelatin buffer solution, adding gelatin buffer solution containing gelatin, centrifuging at room temperature to 3 rpm, adding gelatin buffer solution containing normal pH of 1-80 mg gelatin buffer solution, adding gelatin buffer solution, mixing, stirring to 3 rpm, preparing gelatin buffer solution, adding gelatin buffer solution, mixing, stirring to 3, stirring to obtain gelatin buffer solution containing normal gelatin buffer solution, mixing, adding gelatin buffer solution containing normal gelatin buffer solution, stirring to obtain gelatin buffer solution, stirring to obtain gelatin buffer solution containing 10 mg/8, stirring, mixing, stirring, dissolving4And m L, adding the cell suspension into a 96-well cell culture plate, irradiating by using 405nm blue light for 1min to form hydrogel, adding DMEM complete culture medium, and culturing at 37 ℃ in a 5% carbon dioxide environment.
Example eight
The porcine liver extracellular matrix and methacrylic acid gelatin are compounded according to the above examples to carry out HepG2 cell three-dimensional culture, and cell activity detection and cell state fluorescent staining observation are carried out on days 3, 7 and 14. And (3) detecting the activity of the cells: the detection time point is advanced by 12h, the Amara blue staining solution is added, and after incubation for 12h at 37 ℃ in a 5% carbon dioxide environment in the dark, the detection is carried out by an enzyme-linked immunosorbent assay (ELISA) instrument, wherein the wavelength is 570nm, and the reference wavelength is 600 nm. And (3) cell fluorescent staining observation: the detection time point is advanced by 24h, a cell tracing dye CMTPX is added, incubation is carried out for 24h at 37 ℃ in a 5% carbon dioxide environment in a dark place, then a fluorescent microscope is used for photographing, the excitation wavelength is 577nm, and the emission wavelength is 6022 nm.
Taking the hydrogel prepared in the fourth embodiment as an example, data results shown in figures 4 and 5 in the specification are obtained through tests, wherein the results of activity detection of cells in three-dimensional culture on days 3, 7 and 14 show that the cells can be remarkably proliferated in the gel environment and the cell proliferation promoting effect of the extracellular matrix composite methacrylic gelatin is better than that of single methacrylic gelatin compared with the common three-dimensional culture (the extracellular matrix composite methacrylic gelatin; ○: the methacrylic gelatin; blank control is no gelatin added), the results show that the fluorescence is remarkably enhanced along with the increase of culture time and shows that the cells are remarkably proliferated in the three-dimensional culture on days 3, 7 and 14, and the experimental number after the culture shows that the hydrogel supports the growth of the cells through activity detection and fluorescence detection.
Although the preferred embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and can be embodied in other specific forms without departing from the spirit or essential characteristics thereof, within the knowledge of one skilled in the art.
Claims (5)
1. A method for constructing a three-dimensional cell culture microenvironment based on a specific extracellular matrix is characterized by comprising the steps of preparing a decellularized matrix, preparing hydrogel by compounding the specific extracellular matrix and a photoinitiated gel-forming material, and constructing the cell culture microenvironment; wherein the step of preparing the acellular matrix comprises the steps of pretreating, acellular treating and post-treating tissues or organs of mammals to obtain an extracellular matrix; the pretreatment of the mammalian tissue or organ in the step of preparing the acellular matrix comprises selecting the mammalian tissue or organ, washing, freezing and cutting; the cell removing treatment comprises washing the pretreated tissue or organ by PBS buffer solution, then using a cell removing solution of surfactant or combination of enzyme and surfactant for cell removing treatment, and then using PBS mixed solution of combined enzyme for treatment; the post-treatment comprises freeze drying treatment and crushing treatment after cleaning;
the steps of preparing the hydrogel by compounding the specific extracellular matrix and the photoinitiated gel forming material and constructing a cell culture microenvironment comprise dissolving the specific extracellular matrix, dissolving the photoinitiated gel forming material, mixing and photoinitiating gel forming, and using the gel forming material to construct the cell culture microenvironment; wherein the content of the first and second substances,
the specific operation of the pretreatment of the tissue or organ of the mammal includes: selecting a tissue or organ of a mammal; washing the tissue or organ to remove appendages, the appendages comprising fat and/or blood; freezing the cleaned tissue or organ and then mechanically cutting the tissue or organ into pieces of 100-500 μm, which is performed on ice;
the specific operation of the cell removing treatment comprises the following steps: stirring and washing the pretreated tissue or organ slice of mammal with 0.01M PBS buffer solution for 5-30min, and repeating for 1-3 times; stirring the washed tissue or organ slices in a cell removing solution, stirring and washing the slices for 5-30min by using 0.01M PBS buffer solution after each 1-3h of treatment, replacing the cell removing solution, and repeating for 2-6 times, wherein the cell removing solution contains a surfactant or a cell removing solution of enzyme and surfactant combination; stirring and treating the tissue or organ slice treated by the cell removing solution for 1-2h by using a 0.01MPBS mixed solution containing 2000Ku ribonuclease and 2000Ku deoxyribonuclease;
the surfactant is selected from a nonionic surfactant, an ionic surfactant or an amphoteric surfactant;
the specific operation of post-processing the extracellular matrix comprises the following steps: stirring and cleaning the matrix subjected to decellularization with deionized water, replacing the deionized water every 15min, repeating the steps for 3 times, and detecting that a decellularization reagent is removed; draining the washed substrate, and freeze drying; mechanically pulverizing the lyophilized matrix into powder to obtain extracellular matrix;
selecting specific extracellular matrix according to the type of cultured cells, preparing the extracellular matrix into matrix mother liquor of 5-10mg/M L by using a solvent, shaking the matrix mother liquor for 48-120h at room temperature, centrifuging the matrix mother liquor at the rotating speed of more than 10000rpm, and taking supernatant, wherein the solvent is 0.01-0.1M hydrochloric acid solution containing 1-5mg/M L pepsin;
dissolving a photoinitiated gel-forming material by using 0.01M PBS buffer solution, preparing a photoinitiated gel-forming material mother solution with the concentration of 50-100mg/M L, carrying out water bath at 50 ℃ for half an hour, and adding a photoinitiator with the mass percent of 5-15%, wherein the photoinitiator is selected from phenyl-2, 4, 6-trimethylbenzoyl lithium phosphonate or 2-hydroxy-4' - (2-hydroxyethoxy) -2-methyl propiophenone;
the photoinitiated gel-forming material is selected from methacrylated gelatin, methacrylated hyaluronic acid or methacrylated chitosan;
the specific operation of mixing and photoinitiating gel formation and using it to construct cell culture microenvironment includes diluting the clear liquid containing extracellular matrix with culture medium to 2-5mg/m L, regulating pH to 7-8 with sodium hydroxide solution, mixing with photoinitiating gel-forming material mother liquor in proportion, filtering with 0.22 micron filtering film, digesting and centrifugally precipitating the cells to be cultured, re-suspending the cells with the mixed filtrate obtained by mixing and filtering, and regulating cell concentration to 1 × 104～1×106M L, adding the prepared cell suspension into a culture carrier, irradiating with 405nm blue light for 1-5min to form hydrogel, adding culture medium containing conventional serum, double antibody and growth factor into the cured hydrogel, and culturing at 37 deg.C in 5% carbon dioxide environment.
2. The method for constructing a three-dimensional culture microenvironment for cells based on specific extracellular matrix according to claim 1, wherein the mammal is selected from pig, cow or mouse; the tissue or organ is selected from heart, liver, lung, spleen, kidney, intestine, brain, nerve, blood vessel or skin.
3. The method for constructing a three-dimensional culture microenvironment for cells based on a specific extracellular matrix, according to claim 1, wherein the decellularization solution containing the surfactant comprises one or more of 1-5% by mass of sodium deoxycholate solution, 0.1-2% by mass of sodium dodecyl sulfate solution, 0.1-3% by mass of CHAPS solution, 0.5-5% by volume of Triton X-100 solution, and 0.1-0.5% by volume of benzalkonium bromide solution; the cell-removing solution containing the enzyme and the surfactant contains pancreatin solution with the mass concentration of 0.01-0.25%; the solvent is selected from deionized water or ammonia water with volume concentration of 0.05% -0.5%.
4. The method for constructing the three-dimensional culture microenvironment of the cells based on the specific extracellular matrix according to claim 1, wherein the culture medium is DMEM, RPMI1640, L15, F12, Mccoy' 5A or EGM2, the cells are primary cells, cell line cells or stem cells, and the culture carrier is a microfluidic chamber of a standard cell culture plate, a culture dish or an organ chip.
5. Use of a specific extracellular matrix-based three-dimensional culture microenvironment for cells, characterized in that the microenvironment constructed by the method according to any one of claims 1 to 4 is used for the three-dimensional culture of specific cells on different culture carriers.
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| CN114457018A (en) * | 2022-02-17 | 2022-05-10 | 安徽骆华生物科技有限公司 | Three-dimensional breast cancer organoid model and culture method and application thereof |
| CN114796615B (en) * | 2022-04-20 | 2023-08-25 | 诺一迈尔（苏州）医学科技有限公司 | Cartilage acellular matrix and preparation method thereof |
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