CN114807005B - Method for preparing matrigel by using animal carcasses - Google Patents
Method for preparing matrigel by using animal carcasses Download PDFInfo
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- CN114807005B CN114807005B CN202210378740.3A CN202210378740A CN114807005B CN 114807005 B CN114807005 B CN 114807005B CN 202210378740 A CN202210378740 A CN 202210378740A CN 114807005 B CN114807005 B CN 114807005B
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- 108010082117 matrigel Proteins 0.000 title claims abstract description 49
- 239000010868 animal carcass Substances 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims abstract description 23
- 239000006228 supernatant Substances 0.000 claims abstract description 21
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 18
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims abstract description 16
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims abstract description 16
- 210000002744 extracellular matrix Anatomy 0.000 claims abstract description 16
- 241001465754 Metazoa Species 0.000 claims abstract description 12
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims abstract description 12
- 239000007788 liquid Substances 0.000 claims abstract description 9
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 9
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims abstract description 8
- 235000011130 ammonium sulphate Nutrition 0.000 claims abstract description 8
- 210000001519 tissue Anatomy 0.000 claims abstract description 7
- 238000007710 freezing Methods 0.000 claims abstract description 5
- 230000008014 freezing Effects 0.000 claims abstract description 5
- 210000001835 viscera Anatomy 0.000 claims abstract description 5
- 230000001376 precipitating effect Effects 0.000 claims abstract description 4
- 238000010298 pulverizing process Methods 0.000 claims abstract description 3
- 230000001954 sterilising effect Effects 0.000 claims abstract description 3
- 239000002244 precipitate Substances 0.000 claims description 8
- 238000000502 dialysis Methods 0.000 claims description 6
- 102000004169 proteins and genes Human genes 0.000 claims description 6
- 108090000623 proteins and genes Proteins 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 4
- 239000012535 impurity Substances 0.000 claims description 2
- 238000011002 quantification Methods 0.000 claims description 2
- 239000000126 substance Substances 0.000 claims description 2
- 238000003756 stirring Methods 0.000 claims 1
- 238000002474 experimental method Methods 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract description 4
- 238000012604 3D cell culture Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 28
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 8
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 8
- 239000002609 medium Substances 0.000 description 7
- 239000006285 cell suspension Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 229910002092 carbon dioxide Inorganic materials 0.000 description 4
- 238000004113 cell culture Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 229920006395 saturated elastomer Polymers 0.000 description 3
- 206010003497 Asphyxia Diseases 0.000 description 2
- 108060003393 Granulin Proteins 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 239000001963 growth medium Substances 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 238000001556 precipitation Methods 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 229920000742 Cotton Polymers 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- 241000577979 Peromyscus spicilegus Species 0.000 description 1
- 238000012870 ammonium sulfate precipitation Methods 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000004709 cell invasion Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 230000036571 hydration Effects 0.000 description 1
- 238000006703 hydration reaction Methods 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 108010008217 nidogen Proteins 0.000 description 1
- 201000008968 osteosarcoma Diseases 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000004017 serum-free culture medium Substances 0.000 description 1
- 210000003491 skin Anatomy 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000037351 starvation Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 210000004881 tumor cell Anatomy 0.000 description 1
- 208000025421 tumor of uterus Diseases 0.000 description 1
- 206010046766 uterine cancer Diseases 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/0062—General methods for three-dimensional culture
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue
Abstract
The application discloses a method for preparing matrigel by using animal carcasses, which comprises the following steps: sterilizing the animal carcasses; removing skin and viscera of animal carcasses, freezing the rest animal tissues with liquid nitrogen, and pulverizing; extracting the extracellular matrix component, precipitating the extracellular matrix component using ammonium sulfate; redissolving extracellular matrix components, centrifuging, and collecting supernatant, wherein the supernatant is matrigel. The method can prepare the matrigel by using the animal carcasses left in animal experiments, thereby realizing the reutilization of the animal carcasses. When the matrigel yield is high, animals can be raised to prepare matrigel. The method has the advantages of convenient material obtaining, high matrigel yield and capability of meeting the 3D cell culture requirement. Using the method of the present application, one mouse can obtain about 10mL matrigel and one rat can prepare 50mL matrigel.
Description
Technical Field
The application relates to a method for preparing matrigel by using animal carcasses, belonging to the technical field of biochemistry.
Background
Matrigel has wide application in biological experiments, and can be used for treating cell culture plates to promote cell adhesion; can be used for paving a Transwell chamber to study the invasion capacity of tumor cells; can be used for 3D culture of cell lines or cells derived from patients, and can be used for screening antitumor drugs.
Matrigel is a compound composed of soluble extracellular matrix, and mainly comprises collagen, laminin, nidogen, etc. Currently, the main source of matrigel is EHS (Engelbreth-Holm-Swarm) matrigel. EHS matrigel is extracted from EHS cells. EHS cells are cells that cannot be cultured in vitro and are therefore inconvenient to expand on a large scale, which brings great inconvenience to the production of matrigel. Meanwhile, the preparation of the EHS matrigel needs to inoculate osteosarcoma in a mouse body, which is a disadvantageous factor for animal welfare.
At present, the main preparation material of matrigel is EHS cytoma, and tissue such as muscle, skin, liver, uterine tumor and the like is also reported to be used as a starting material for preparing matrigel. No report was made before that matrigel was prepared using whole animal carcasses as starting materials. In many animal experiments, only partial tissues or blood of animals are needed, and the rest parts of animal carcasses are required to be subjected to harmless treatment, which is labor-intensive, so that the search for a better method for treating animal carcasses is of great significance.
Disclosure of Invention
The application aims to provide a method for preparing matrigel by using animal carcasses, which has the advantages of convenient material acquisition and high matrigel yield.
The application adopts the technical means that:
a method for preparing matrigel by using animal carcasses, comprising the steps of:
(1) Sterilizing the animal carcasses;
(2) Removing skin and viscera of animal carcasses, freezing the rest animal tissues with liquid nitrogen, and pulverizing;
(3) Extracting the extracellular matrix component, precipitating the extracellular matrix component using ammonium sulfate;
(4) Redissolving extracellular matrix components, centrifuging, and collecting supernatant, wherein the supernatant is matrigel.
Preferably, the extraction of extracellular matrix components in step (3) specifically means that after liquid nitrogen is completely volatilized, a precooled PBS solution is added into crushed animal tissues, stirred overnight at 4 ℃, centrifuged, and the precipitate is discarded, and the supernatant is taken.
Preferably, the step (3) of removing the impurities of the extracellular matrix component by using ammonium sulfate precipitation is to add a saturated ammonium sulfate solution into the supernatant slowly until the concentration of ammonium sulfate in the system is 10-30%, and centrifuging to obtain a precipitate, namely the extracellular matrix component. Wherein the concentration of ammonium sulfate of 10% means that 10g of ammonium sulfate is contained in 100mL of the solution.
Preferably, the step (4) specifically comprises: dissolving the precipitate with PBS, centrifuging to remove insoluble substances, and collecting supernatant as matrigel.
Preferably, the supernatant in step (4) is subjected to a further dialysis treatment.
Preferably, the step (1) is specifically to soak the animal carcasses with 75% ethanol for more than 1 minute.
Preferably, the method further comprises the step (5): protein quantification is carried out on the supernatant, and the protein concentration is controlled to be 2-30 mg/mL.
The method can prepare the matrigel by using the animal carcasses left in animal experiments, thereby realizing the reutilization of the animal carcasses. When the matrigel yield is high, animals can be raised to prepare matrigel. The method has the advantages of convenient material obtaining, high matrigel yield and capability of meeting the 3D cell culture requirement. Using the method of the present application, one mouse can obtain about 10mL matrigel and one rat can prepare 50mL matrigel.
Drawings
FIG. 1 is a graph showing the results of cell invasion experiments using matrigel prepared according to the present application.
FIG. 2 is an experimental result of matrigel prepared according to the present application for 3D cell culture.
Detailed Description
Example 1
An 8-week-old Balb/c mouse was weighed to 23 g, and after carbon dioxide asphyxiation, the skin and viscera were removed by peeling, and the remaining portion was about 16 g. The mouse carcasses were crushed using surgical scissors, snap frozen in liquid nitrogen and carefully ground into powder using a mortar. After the liquid nitrogen had evaporated, 32mL of pre-chilled PBS solution was added and stirred overnight at 4 ℃ to extract the cell matrix proteins. After centrifugation at 8000g, the pellet was discarded. The supernatant was slowly added with saturated ammonium sulfate to 20% saturation, allowed to stand at 4℃for 30 minutes for precipitation, and the precipitate was collected by centrifugation at 8000 g. The pellet was dissolved with 8ml pbs and dialyzed overnight using a 7kD dialysis bag. The dialysate was changed and dialysis continued overnight using DMEM high sugar medium. Packing matrigel on ice in an ultra clean bench, loading into a centrifuge tube, measuring protein concentration by BCA method to 8.7mg/mL, and freezing matrigel at-20deg.C for use.
Example 2
An SD mouse of 8 weeks old was weighed 232 g, and after carbon dioxide asphyxiation treatment, the skin and hair were peeled off, and the viscera were cleaned, with the remainder being about 137 g. Mouse carcasses were crushed using a liquid nitrogen crusher. After the liquid nitrogen had evaporated, 270mL of pre-chilled PBS was added and stirred overnight at 4℃to extract the cell matrix proteins. After centrifugation at 8000g, the pellet was discarded. The supernatant was slowly added with saturated ammonium sulfate to 20% saturation, allowed to stand at 4℃for 30 minutes for precipitation, and the precipitate was collected by centrifugation at 8000 g. The pellet was dissolved with 40ml pbs and dialyzed overnight using a 7kD dialysis bag. The dialysate was changed and dialysis continued overnight using DMEM/F12 medium. Packing matrigel into centrifuge tube on ice in super clean bench, and freezing at-20deg.C for use with protein concentration of 15.3mg/mL measured by BCA method.
Example 3
HepG2 cells grow to 60% -80% of confluence, PBS is gently rinsed for 3 times, a serum-free culture medium is replaced, and the cells are placed in a cell culture box with 37 ℃ and 5% CO2 saturated humidity for continuous culture for 24 hours (serum starvation); the mouse-derived matrigel prepared in example 1 was thawed at 4 degrees. Transwell upper chamber add 1:8 dilution ofMatrigel (diluted with serum-free DMEM medium) 70 μl was placed at 37deg.C in 5% CO 2 After 8h incubation in a saturated humidity cell incubator, the supernatant was discarded and rinsed gently with PBS for 2 times; 100 μl of serum-free DMEM is added to each well for hydration for 3-4 hours at 37deg.C; the culture medium is discarded by HepG2 cells, and after PBS is slightly and flexibly washed for 3 times, the cells are digested by trypsin; after stopping digestion, single cell suspensions were prepared and collected and centrifuged at 1500rpm for 3min; the supernatant was discarded, the cells were resuspended in serum-free DMEM medium, and after counting the cells by a hemocytometer, the cell concentration was adjusted to 2.5X10 s with serum-free DMEM medium 4 Individual cells/ml. 200. Mu.l of the cell suspension was added to the upper Transwell chamber, and the mixture was placed in 600. Mu.l of the lower Transwell chamber containing 20% serum medium, and the culture was continued in a cell incubator at 37℃and 5% CO2 saturation humidity for 24 hours; taking out the Transwell upper chamber, rinsing with PBS, gently wiping off non-migrated cells in the chamber with a cotton swab, fixing in 4% paraformaldehyde for 15min, staining with 0.1% crystal violet dye solution at room temperature for 30min, and rinsing with PBS. Each group of cells was observed under a 200 x inverted phase contrast microscope, photographed randomly, and the number of migrated cells counted (fig. 1). The average number of migrated cells was 137.+ -.22.
Example 4
Pre-cooling a 24-hole cell culture plate and a pipette tip on ice, slowly thawing a matrigel on ice or at 4 ℃, taking HepG2 cells in a logarithmic phase, after PBS is gently rinsed, digesting the cells with trypsin, preparing and collecting single cell suspension after stopping digestion, and centrifuging at 1500rpm for 3min; the supernatant was discarded, and after the cells were resuspended in DMEM complete medium and counted by a hemocytometer, the cell concentration was adjusted to 1.5X10 5 Individual cells/ml. Matrigel prepared in example 2 and single cell suspension 1 with adjusted concentration were taken: 1 are gently mixed on ice, 40 to 50 mu l of the mixed single cell suspension is taken by a precooled 200 mu l liquid-transferring gun head and vertically dripped into a precooled 24-pore plate, so that arch cell droplets are formed, and the temperature is 37 ℃ and the concentration is 5 percent of CO 2 After the cell culture box with saturated humidity is stabilized for 30min, 1-2 ml of DMEM complete culture medium is added into each hole for continuous culture, and the cells are observed and photographed every day for recording. FIG. 2 shows the results of the 3D culture on the fourth day.
The embodiments of the present application have been further described above with reference to the accompanying drawings and specific embodiments, and it is not to be construed that the embodiments of the present application are limited to the descriptions. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the application, and these should be considered to be within the scope of the application.
Claims (7)
1. A method for preparing matrigel by using animal carcasses, which is characterized by comprising the following steps:
(1) Sterilizing the animal carcasses;
(2) Removing skin and viscera of animal carcasses, freezing the rest animal tissues with liquid nitrogen, and pulverizing;
(3) Extracting the extracellular matrix component, precipitating the extracellular matrix component using ammonium sulfate;
(4) Redissolving extracellular matrix components, centrifuging, and collecting supernatant, wherein the supernatant is matrigel.
2. The method for preparing matrigel using animal carcasses according to claim 1, wherein the extracting of extracellular matrix components in step (3) specifically means adding pre-chilled PBS solution to crushed animal tissues after the liquid nitrogen is completely volatilized, stirring overnight at 4 ℃, centrifuging, discarding the precipitate, and taking the supernatant.
3. The method for preparing matrigel using animal carcasses according to claim 2, wherein: in the step (3), ammonium sulfate is used for precipitating and removing impurities of extracellular matrix components, specifically, saturated ammonium sulfate solution is slowly added into supernatant until the concentration of ammonium sulfate in a system is 10-30%, and the extracellular matrix components are obtained by centrifuging and taking the precipitate.
4. A method of preparing matrigel using animal carcasses according to claim 3, wherein: the step (4) is specifically as follows: dissolving the precipitate with PBS, centrifuging to remove insoluble substances, and collecting supernatant as matrigel.
5. The method for preparing matrigel using animal carcasses according to claim 4, wherein: and (3) carrying out dialysis treatment on the supernatant in the step (4).
6. The method for preparing matrigel using animal carcasses according to claim 1, wherein: the step (1) is specifically to soak the animal carcasses for more than 1 minute by using 75% ethanol.
7. The method for preparing matrigel using animal carcasses according to claim 1, wherein: further comprising the step (5): protein quantification is carried out on the supernatant, and the protein concentration is controlled to be 2-30 mg/mL.
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| CN114807005B true CN114807005B (en) | 2023-11-21 |
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