CN115261302A - Matrigel and preparation method and application thereof - Google Patents
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Abstract
The invention relates to a matrigel and a preparation method and application thereof, and the matrigel comprises the following steps: preparing a culture medium: adding collagen into 1640 culture medium containing 10% fetal calf serum; resuspending the sarcoma cells, and then inoculating; placing the cell culture bottle in a shaking table, and placing the whole shaking table in a cell culture box for culture at 37 ℃; in the process of cultivation, liquid changing is carried out every 3-5 days, during liquid changing, centrifugation is carried out for 5-10 minutes at 1000rpm, supernatant is removed, and then the culture medium is used for re-suspension; fifthly, collecting cells after culturing for 14-28 days; centrifuging at 1000rpm for 5min, and removing supernatant; sixthly, adding a cell removal solution, resuspending and precipitating, and processing overnight at 2-8 ℃; centrifuging at high speed for 30min, and collecting supernatant; after adding cell removal liquid for heavy suspension washing, centrifuging at high speed for 30min, collecting supernatant, and repeating the step for washing for 3 times; mixing the collected supernatant liquid, and discarding the precipitate; repeatedly dialyzing the combined liquid for 3-5 times by using dialysate; collecting dialyzed liquid, and concentrating the liquid until the total concentration of protein is 10-15mg/ml to obtain the matrigel. The method of the invention reserves the main components of extracellular matrix and is beneficial to organoid culture.
Description
Technical Field
The invention relates to the technical field of biological medicines, and particularly relates to matrigel and a preparation method and application thereof.
Background
Organoids are a three-dimensional cell culture technique that can mimic the tissue structure and gene expression of in vivo organs in vitro. Organoids are three-dimensional aggregates formed by self-differentiation and self-assembly of various cells including stem cells in vitro, and the cell types, the cell proportions, the spatial structures and the arrangement of the cells have higher similarity with those of internal organs, and can simulate partial functions of the internal organs. Furthermore, organoids have gene expression much closer to that in vivo than in conventional two-dimensional cell culture. Therefore, organoid-based assays of biological activity are more relevant in vivo. Therefore, organoids are now widely used in biological research, pharmaceutical research, medical research, transformation applications for precision medicine, and the like.
Currently, due to high cell requirements, organoid culture still relies mainly on the support and space provided by biomaterials such as extracellular matrix (ECM) gels. Although ECM gels have high biocompatibility, the problems of batch stability, complexity and uncontrollable components limit organoid research and applications. First, the major ECM gels are currently biological preparations obtained by tissue or cell extraction from animals, but the animals have large individual differences, and the cell preparations are also extremely unstable and uncertain. In addition, fully synthetic hydrogels have also recently been extensively tried for three-dimensional cell culture including organoids, which have advantages in the stability of synthetic materials, as well as the uniformity of structure. However, fully synthetic materials often lack cytokines required for cell growth and are poorly biocompatible, and therefore, the cell culture efficiency of fully synthetic hydrogels is far inferior to that of matrigel of biological origin.
Disclosure of Invention
In view of the above, there is a need to provide a matrigel, a preparation method and applications thereof.
In order to solve the problems in the prior art, the technical scheme adopted by the invention is as follows:
in a first aspect, the present invention provides a method for preparing matrigel, comprising the following steps:
preparing a culture medium: adding collagen into 1640 culture medium containing 10% fetal calf serum;
secondly, suspending the sarcoma cells by using the culture medium, and then inoculating the sarcoma cells into a cell culture device;
culturing the three fruits in an environment with the rotating speed of 20-100rpm and the temperature of 37 ℃;
in the process of cultivation, liquid changing is carried out every 3-5 days, during liquid changing, centrifugation is carried out for 5-10 minutes at 1000rpm, supernatant is removed, and then the culture medium is used for re-suspension;
fifthly, collecting cells after culturing for 14-28 days; centrifuging at 1000rpm for 5min, and removing supernatant;
sixthly, adding a cell removal solution, resuspending and precipitating, and processing overnight at 2-8 ℃;
centrifuging at high speed for 30min, and collecting supernatant to a new centrifuge tube;
after the cell removal liquid is added in the step sixteenth, heavy suspension washing is carried out, the cell removal liquid is centrifuged at a high speed for 30min, the supernatant is collected to a new centrifugal tube, and the step is repeated for 3 times;
mixing the collected supernatant liquid, and discarding the precipitate;
the combined liquid is repeatedly dialyzed for 3 to 5 times by using dialysate, and each time is dialyzed for 2 to 4 hours;
collecting dialyzed liquid, measuring total protein concentration, and concentrating the liquid until the total protein concentration is 10-15mg/ml to obtain matrigel.
Further, the collagen is one of type I collagen, type II collagen, type III collagen or type IV collagen.
Further, the collagen is added in an amount of 1-10% by volume of the culture medium.
Further, the steps are 10 in the second step5-106The sarcoma cells were resuspended at a concentration of/ml.
Further, the sarcoma cell is a fibrosarcoma cell or an osteosarcoma cell.
Further, the cell removal solution is composed of 1-4M urea, 0.2-2M ammonium sulfate, 1-20mM EDTA and 10-200mM acetic acid.
Further, the conditions of the high-speed centrifugation are: 12000rpm at 4 ℃.
Further, the dialysate is 1-20mM Tris-HCl (PH = 7.0).
In a second aspect, the present invention provides a matrigel prepared by the method of any one of claims 1 to 8.
In a third aspect, the present invention provides the use of matrigel in organoid culture.
The invention has the beneficial effects that:
the preparation method is simple in preparation process, good in repeatability, suitable for large-scale operation under laboratory conditions, and capable of preparing the matrix hydrogel from stable cell sources.
Compared with tissue acellular matrigel, the matrigel prepared by the method has simple and controllable components and few impurities, the acellular liquid adopted by the method is used for gently dissociating cells without damaging the extracellular matrix structure, the adopted dialysate is neutral salt ions, is suitable for preserving protein components and is suitable for cell culture, and the method retains the main components of the extracellular matrix and is beneficial to organoid culture. The preparation method can use the human cell strain to prepare the matrigel, and the obtained matrigel is suitable for the growth of human organs, avoids source difference among species, and lays a foundation for in vivo experiments in the future.
Drawings
FIG. 1 is a morphology of matrigel obtained in example 1;
FIG. 2 is a morphology diagram of matrigel obtained in example 2 after solidification at 37 ℃;
FIG. 3 is a morphology of matrigel obtained in example 3;
FIG. 4 is a morphology diagram of matrigel obtained in example 4 after solidification at 37 ℃;
FIG. 5 is a morphological structure diagram of a lung cancer organoid obtained in application example 1 under a normal light microscope;
FIG. 6 is a morphological structure diagram of an organoid obtained in application example 2 under a normal optical microscope;
FIG. 7 is a configuration diagram of an organoid obtained in application example 3 under a normal optical microscope.
Detailed Description
Tris-HCl (pH = 7.0) used in the examples of the present invention was purchased from Biotechnology engineering (Shanghai) Inc.
In the description of the present invention, it is to be noted that those whose specific conditions are not specified in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturers. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The present invention will now be described in further detail with reference to the following figures and specific examples, which are intended to be illustrative, but not limiting, of the invention.
Example 1:
the preparation method of the matrigel mainly comprises the following steps:
preparing a culture medium: to 1640 medium containing 10% fetal bovine serum, 5% type I collagen was added.
The culture medium is used according to the formula 106Resuspend human fibrosarcoma cells at a concentration of/ml, then inoculate into cell culture flasks.
Thirdly, placing the cell culture bottle in a shaking table at a set rotating speed of 50rpm, and then placing the whole shaking table in a cell culture box for culture at 37 ℃.
During the culturing process, liquid change is performed every 4 days. When the medium was changed, the medium was centrifuged at 1000rpm for 7 minutes, and the supernatant was removed and then resuspended using the medium of step 1.
After culturing for 20 days, collecting cells. Centrifuge at 1000rpm for 5 minutes and remove the supernatant.
Sixthly, adding the cell removal liquid into the cell sediment until the sediment is completely mixed, re-suspending the sediment, and processing the sediment at 5 ℃ overnight.
Centrifuging at 12000rpm for 30min at 4 deg.C, precipitating impurities, and collecting supernatant to new centrifuge tube.
And after the sixteenth step of adding the sixteenth acellular liquid, resuspending and washing, centrifuging at 12000rpm for 30min at 4 ℃, precipitating impurities, and collecting a supernatant to a new centrifuge tube. This step was repeated 3 washes.
The resulting supernatant is combined and the precipitate is discarded.
The combined liquid was repeatedly dialyzed 4 times against 10mM Tris-HCl (pH = 7.0) as a dialysis solution. Each dialysis was performed for 3 hours.
Collecting dialyzed liquid, measuring total protein concentration to be 0.84mg/ml, and concentrating the liquid until the total protein concentration is 12mg/ml to obtain the matrigel as shown in figure 1.
The cell removal solution described in this example was prepared from 2M urea, 1M ammonium sulfate, 10mM EDTA and 150mM acetic acid by a conventional method, and was sterilized by high-temperature treatment at 121 ℃ for 20 minutes.
Example 2:
the preparation method of the matrigel mainly comprises the following steps:
preparing a culture medium: 1% type II collagen was added to 1640 medium containing 10% fetal bovine serum.
The culture medium is used according to the formula 105The human osteosarcoma cells were resuspended in a concentration of/ml and then inoculated into the bioreactor.
Thirdly, setting the rotating speed of the bioreactor to be 100rpm, and culturing at the temperature of 37 ℃.
And fourthly, in the process of culturing, liquid is changed every 3 days. In the case of changing the medium, the medium was centrifuged at 1000rpm for 5 minutes, and the supernatant was removed and then resuspended using the medium of step.
After culturing for 28 days, collecting cells. Centrifuge at 1000rpm for 5 minutes and remove the supernatant.
Sixthly, adding the cell removal liquid into the cell sediment until the sediment is completely mixed, re-suspending the sediment, and processing the sediment at 8 ℃ overnight.
Centrifuging at 12000rpm for 30min at 4 deg.C, precipitating impurities, and collecting supernatant to new centrifuge tube.
And after the sixteenth step of adding the sixteenth acellular liquid, resuspending and washing, centrifuging at 12000rpm for 30min at 4 ℃, precipitating impurities, and collecting a supernatant to a new centrifuge tube. This step was repeated 3 washes.
The resulting supernatant is combined and the precipitate is discarded.
The combined liquid was repeatedly dialyzed 3 times against 20mM Tris-HCl (pH = 7.0) as a dialysis solution. Each dialysis was performed for 2 hours.
Collecting dialyzed liquid, determining the total concentration of protein to be 1.14mg/ml, and concentrating the liquid until the total concentration of protein is 15mg/ml to obtain the matrigel. Matrigel was dropped on a petri dish and solidified at 37 ℃ as shown in FIG. 2.
The cell removal solution described in this example was prepared from 1M urea, 2M ammonium sulfate, 20mM EDTA and 200mM acetic acid by a conventional method, and was sterilized at high temperature.
Example 3:
the preparation method of the matrigel mainly comprises the following steps:
preparing a culture medium: 10% type III collagen was added to 1640 medium containing 10% fetal bovine serum.
The culture medium is used according to the formula 106The EHS cells were resuspended at a concentration of/ml and then seeded into cell culture flasks.
Thirdly, placing the cell culture bottle in a shaking table at a set rotating speed of 20rpm, and then placing the whole shaking table in a cell culture box for culture at 37 ℃.
And fourthly, in the process of culturing, liquid is changed every 5 days. At the time of fluid change, centrifugation was carried out at 1000rpm for 10 minutes, and the supernatant was removed and then resuspended using the medium of step 1.
After culturing for 28 days, collecting cells. Centrifuge at 1000rpm for 5 minutes and remove the supernatant.
Sixthly, adding the cell removal liquid into the cell sediment until the sediment is completely mixed, re-suspending the sediment, and processing the sediment at the temperature of 2 ℃ overnight.
Centrifuging at 12000rpm for 30min at 4 deg.C, precipitating impurities, and collecting supernatant to new centrifuge tube.
And after the step sixteenth of adding the cell removal liquid, resuspending and washing, centrifuging at 12000rpm at 4 ℃ for 30min at a high speed, precipitating impurities, and collecting a supernatant to a new centrifugal tube. This step was repeated 3 washes.
The resulting supernatant is combined and the precipitate is discarded.
The combined liquid was repeatedly dialyzed 5 times against 1mM Tris-HCl (pH = 7.0) as a dialysis solution. Dialysis was performed for 4 hours each time.
Collecting dialyzed liquid, measuring total protein concentration to be 0.72mg/ml, and concentrating the liquid until the total protein concentration is 10mg/ml to obtain the matrigel as shown in figure 3.
The cell removal solution described in this example was prepared from 4M urea, 0.2M ammonium sulfate, 1mM EDTA and 10mM acetic acid by a conventional method, and was treated at 121 ℃ for 20 minutes.
Example 4:
the preparation method of the matrigel mainly comprises the following steps:
preparing a culture medium: 5% type IV collagen was added to 1640 medium containing 10% fetal bovine serum.
The culture medium is used according to the formula 106The EHS cells were resuspended at a concentration of/ml and then seeded into cell culture flasks.
Thirdly, placing the cell culture bottle in a shaking table at a set rotating speed of 50rpm, and then placing the whole shaking table in a cell culture box for culture at 37 ℃.
And fourthly, in the process of culturing, liquid is changed every 4 days. At the time of fluid change, centrifugation was carried out at 1000rpm for 7 minutes, and the supernatant was removed and then resuspended using the medium of step 1.
After culturing for 20 days, collecting cells. Centrifuge at 1000rpm for 5 minutes and remove the supernatant.
Sixthly, adding the cell removal liquid into the cell sediment until the sediment is completely mixed, re-suspending the sediment, and processing the sediment at 5 ℃ overnight.
Centrifuging at 12000rpm for 30min at 4 deg.C, precipitating impurities, and collecting supernatant to new centrifuge tube.
And after adding the cell removal liquid in the step 6, carrying out heavy suspension washing, centrifuging at 12000rpm for 30min at 4 ℃, precipitating impurities, and collecting a supernatant to a new centrifuge tube. This step was repeated 3 washes.
The self-skin combines the collected supernatant and removes the precipitate.
The combined liquid was repeatedly dialyzed 4 times against 10mM Tris-HCl (pH = 7.0) as a dialysis solution. Each dialysis was performed for 3 hours.
Collecting dialyzed liquid, determining the total concentration of protein to be 0.84mg/ml, and concentrating the liquid until the total concentration of protein is 12mg/ml to obtain the matrigel. Matrigel was dropped on a petri dish and solidified at 37 ℃ as shown in FIG. 4.
The cell removal solution described in this example was prepared from 2M urea, 1M ammonium sulfate, 10mM EDTA and 150mM acetic acid by a conventional method, and was treated at 121 ℃ for 20 minutes.
Comparative example 1:
the comparison example is only different from the embodiment 1 in 10 in the steps of two steps6Concentration of/ml resuspended Lung cancer cells H1975, as in example 1, and the results after dialysis were determined to be a total protein concentration of 0.24ng/ml, which failed to gel.
Comparative example 2:
the comparative example differs from example 1 only in the composition of the decellularization solution, and the composition of the decellularization solution added in the comparative example is as follows: 1% SDS,2M NaCl, and the rest of the same as in example 1, the liquid after dialysis was collected, and the total protein concentration was measured to be 0.19mg/ml. The protein concentration obtained is much lower than in example 1, which indicates that the cell-free fluid of example 1 facilitates the harvest of more matrigel protein.
Comparative example 3:
this comparative example is different from example 1 only in that physiological saline was used as a dialysate in the step of extraction, and the rest is the same as example 1. The matrigel obtained was concentrated and stored overnight, and precipitation was observed, whereas example 1 did not. This indicates that Tris-HCl (PH = 7.0) as dialysate is advantageous for matrigel preservation.
Comparative example 4:
the comparative example differs from example 1 only in the second step 103Resuspend fibrosarcoma cells at a concentration of/ml, as in example 1. The cells were observed to be small during the cell culture, and the total concentration of protein was determined to be 0.07mg/ml by collecting the dialyzed liquid. The protein concentration obtained is much lower than in example 1, which indicates that the cell concentration in example 1 favors the harvest of more matrigel protein.
Comparative example 5:
the comparative example differs from example 1 only in the second step 108Resuspend fibrosarcoma cells at a concentration of/ml, as in example 1. After 3 days of culture in step 4, a large amount of apoptosis was observed, and the dialyzed liquid was finally collected and the total protein concentration was measured to be 0.79ng/ml. The protein concentration obtained is much lower than in example 1, which indicates that the cells of example 1 are concentratedThe cell concentration favors the harvest of more matrigel protein.
Application example 1:
the matrigel is applied to lung cancer organoid culture.
1) An appropriate amount of lung cancer organoid culture medium was mixed with matrigel of example 1 at an equal ratio, then the lung cancer cells were resuspended in the mixed solution, and the cell-mixed gel was dropped into a 60mm petri dish using a pipette, approximately 50ul per drop.
2) Putting the culture dish after the glue dripping into CO2Standing in the incubator for 2min, carefully reversing after no obvious flow of light shaking glue drops, and fully solidifying for 30min.
3) Adding lung cancer organoid medium to the petri dish, and then placing in a constant temperature incubator at 37 deg.C, 5%2Culturing under the concentration.
4) The culture medium is replaced every 2 days, lung cancer organoids can be obtained after 6 days of culture, the tissue morphology structure observed under a common optical microscope is shown in figure 5, the average size of the organoids is more than 100 mu M, and the cell activity is good.
Application example 2:
the matrigel is applied to culturing intestinal cancer organoid.
1) Pretreating an intestinal cancer sample to obtain a cell mass with the cell number of 50 cells, and centrifuging to remove a supernatant for later use;
2) Animal liver organoid medium was mixed with matrigel as described in example 2 at a ratio of 1:1.2 mixing to obtain a mixed gel culture solution, placing the mixed gel culture solution on ice, then re-suspending the cell mass precipitate obtained in the step 1) by using the mixed gel culture solution, inoculating the gel drops, standing for 2-3 min at 25 ℃, then inverting the gel drops, continuing to keep the temperature and standing until the gel drops are fully solidified;
3) Further allowing the gel drops obtained in step 2) to stand at room temperature for 15min, adding a cell culture medium preheated at 37 ℃ and culturing at 37 ℃ and 5% CO2 concentration, replacing the cell culture medium every 2 days, and culturing for 4 days to obtain organoids having an average size diameter of more than 50 μ M in the intestinal cancer organoids, as shown in FIG. 6.
Application example 3:
the matrigel is applied to the culture of mouse liver organoid.
1) Pretreating a fresh sample of a mouse liver to obtain a cell mass with the cell number of 50 cells, and centrifuging to remove a supernatant for later use;
2) Animal liver organoid medium was mixed with matrigel as described in example 3 at a ratio of 1:2 mixing to obtain a mixed gel culture solution, placing the mixed gel culture solution on ice, then re-suspending the cell mass precipitate obtained in the step 1) by using the mixed gel culture solution, inoculating the gel drops, standing for 2-3 min at 25 ℃, then inverting the gel drops, continuing to keep the temperature and standing until the gel drops are fully solidified;
3) Further standing the gel drops obtained in step 2) at room temperature for 15min, adding a cell culture medium preheated at 37 deg.C, culturing at 37 deg.C and 5% CO2 concentration, replacing the cell culture medium every 2 days, and culturing for 7 days to obtain organoids in the shape of cavity of mouse liver organoids as shown in FIG. 7.
In conclusion, the method of the invention retains the main components of the extracellular matrix and is beneficial to organoid culture.
The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the present invention. It should be noted that various changes and modifications can be made by those skilled in the art without departing from the spirit of the invention, and these changes and modifications are all within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
Claims (10)
1. The preparation method of the matrigel is characterized by comprising the following steps:
preparing a culture medium: adding collagen into 1640 culture medium containing 10% fetal calf serum;
secondly, suspending the sarcoma cells by using the culture medium, and then inoculating the sarcoma cells into a cell culture device;
culturing the three fruits in an environment with the rotating speed of 20-100rpm and the temperature of 37 ℃;
in the process of culturing, liquid changing is carried out every 3-5 days, centrifugation is carried out for 5-10 minutes at 1000rpm when liquid changing is carried out, supernatant is removed, and then the culture medium is used for resuspension;
fifthly, collecting cells after culturing for 14-28 days; centrifuging at 1000rpm for 5min, and removing supernatant;
sixthly, adding a cell removal solution, resuspending and precipitating, and processing overnight at 2-8 ℃;
centrifuging at high speed for 30min, and collecting supernatant to a new centrifuge tube;
after the cell removal liquid is added in the step sixteenth, heavy suspension washing is carried out, the cell removal liquid is centrifuged at a high speed for 30min, the supernatant is collected to a new centrifugal tube, and the step is repeated for 3 times;
mixing the collected supernatant liquid, and discarding the precipitate;
the combined liquid is repeatedly dialyzed for 3 to 5 times by using dialysate, and each time is dialyzed for 2 to 4 hours;
collecting dialyzed liquid, measuring total protein concentration, and concentrating the liquid until the total protein concentration is 10-15mg/ml to obtain matrigel.
2. The method of claim 1, wherein the matrigel is prepared by the following steps: the collagen is one of type I collagen, type II collagen, type III collagen or type IV collagen.
3. The method for preparing matrigel according to claim 1 or 2, characterized in that: the addition amount of the collagen is 1-10% of the volume of the culture medium.
4. The method for preparing matrigel according to claim 1, wherein: the steps are as follows 105-106Resuspend sarcoma cells at a concentration of/ml.
5. The method for preparing matrigel according to claim 1 or 4, wherein: the sarcoma cell is a fibrosarcoma cell or an osteosarcoma cell.
6. The method for preparing matrigel according to claim 1, wherein: the cell removal solution consists of 1-4M urea, 0.2-2M ammonium sulfate, 1-20mM EDTA and 10-200mM acetic acid.
7. The method for preparing matrigel according to claim 1, wherein: the conditions of the high-speed centrifugation are as follows: 12000rpm at 4 ℃.
8. The method for preparing matrigel according to claim 1, wherein: the dialysate was 1-20mM Tris-HCl (pH = 7.0).
9. A matrigel characterized by: is prepared by the preparation method of any one of claims 1 to 8.
10. Use of a matrigel according to claim 9 in organoid culture.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114921413A (en) * | 2022-05-31 | 2022-08-19 | 创芯国际生物科技(广州)有限公司 | Osteosarcoma organoid culture medium and culture method |
| CN117586939A (en) * | 2023-11-23 | 2024-02-23 | 广州蕊特生物科技有限公司 | Matrigel for three-dimensional cell culture and preparation method thereof |
| CN117586939B (en) * | 2023-11-23 | 2024-05-31 | 广州蕊特生物科技有限公司 | Matrigel for three-dimensional cell culture and preparation method thereof |
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| CN117586939A (en) * | 2023-11-23 | 2024-02-23 | 广州蕊特生物科技有限公司 | Matrigel for three-dimensional cell culture and preparation method thereof |
| CN117586939B (en) * | 2023-11-23 | 2024-05-31 | 广州蕊特生物科技有限公司 | Matrigel for three-dimensional cell culture and preparation method thereof |
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