CN115261302B

CN115261302B - Matrigel and preparation method and application thereof

Info

Publication number: CN115261302B
Application number: CN202210861333.8A
Authority: CN
Inventors: 廖传荣; 朱宇; 邱培; 黄敏
Original assignee: Accurate International Biotechnology Guangzhou Co ltd
Current assignee: Accurate International Biotechnology Guangzhou Co ltd
Priority date: 2022-07-20
Filing date: 2022-07-20
Publication date: 2023-06-06
Anticipated expiration: 2042-07-20
Also published as: CN115261302A

Abstract

The invention relates to matrigel and a preparation method and application thereof, comprising the following steps: preparing a culture medium: adding collagen into 1640 culture medium containing 10% of fetal bovine serum; resuspension of sarcoma cells, followed by inoculation; placing the cell culture bottle in a shaking table, and placing the whole shaking table in a cell culture box for culturing at 37 ℃; in the process of culturing, liquid is changed every 3-5 days, when the liquid is changed, centrifugation is carried out at 1000rpm for 5-10 minutes, the supernatant is removed, and then the culture medium is used for re-suspending; fifthly, culturing for 14-28 days, and collecting cells; centrifuging at 1000rpm for 5 minutes, and removing the supernatant; adding the decellularized liquid to resuspend and precipitate, and processing at 2-8 ℃ overnight; centrifuging at high speed for 30min, and collecting supernatant; adding the decellularized liquid to carry out resuspension washing, carrying out high-speed centrifugation for 30min, collecting supernatant, and repeating the step for 3 times; combining the collected supernatant and discarding the precipitate; repeatedly dialyzing the mixed liquid with dialyzate for 3-5 times; and (3) collecting the liquid after dialysis, and concentrating the liquid until the total concentration of protein is 10-15mg/ml to obtain matrigel. The method of the invention retains the main components of extracellular matrix, and is beneficial to organoid culture.

Description

Matrigel and preparation method and application thereof

Technical Field

The invention relates to the technical field of biological medicine, in particular to matrigel and a preparation method and application thereof.

Background

Organoids are a three-dimensional cell culture technique that mimics tissue structure and gene expression of organs in vivo in vitro. Organoids are three-dimensional aggregates formed by in vitro self-differentiation and self-assembly of various cells including stem cells, and have higher similarity to in vivo organs in terms of cell types, ratios and spatial structures and arrangements of cells, and can simulate part of functions of in vivo organs. In addition, organoid gene expression is more closely related to in vivo than in traditional two-dimensional cell culture. Thus, organoid-based biological activity assays are more relevant in vivo. Therefore, organoids are currently widely used in biological research, pharmaceutical research, medical research, transformation applications in precision medicine, and the like.

At present, due to the high cell requirements, organoid culture still depends mainly on the support and space provided by biomaterials such as extracellular matrix (ECM) gels. ECM gels, although highly biocompatible, have limited organoid studies and applications due to problems of stability between batches, complexity and uncontrollable composition. First, the major ECM gels at present are all biological preparation products, which are obtained by extraction from animal tissues or cells, but animals have large individual differences, and cell preparations are also extremely unstable and uncertain. In addition, total synthetic hydrogels have also been widely tried recently for three-dimensional cell culture including organoids, which has advantages in terms of stability of synthetic materials, and uniformity of structure. However, total synthetic materials often lack cytokines required for cell growth and are poorly biocompatible, and therefore, the cell culture efficiency of total synthetic hydrogels is far less than that of biogenic matrigel.

Disclosure of Invention

In view of the foregoing, it is desirable to provide a matrigel, and a preparation method and application thereof.

In order to solve the problems existing in the prior art, the invention adopts the following technical scheme:

in a first aspect, the present invention provides a method for preparing matrigel, comprising the steps of:

preparing a culture medium: adding collagen into 1640 culture medium containing 10% of fetal bovine serum;

resuspension of sarcoma cells using the above media, followed by inoculation into a cell culture device;

culturing under the environment with the rotating speed of 20-100rpm and the temperature of 37 ℃;

in the process of culturing, liquid is changed every 3-5 days, when the liquid is changed, centrifugation is carried out at 1000rpm for 5-10 minutes, the supernatant is removed, and then the culture medium is used for re-suspending;

fifthly, culturing for 14-28 days, and collecting cells; centrifuging at 1000rpm for 5 minutes, and removing the supernatant;

adding the decellularized liquid to resuspend and precipitate, and processing at 2-8 ℃ overnight;

centrifuging at high speed for 30min, and collecting supernatant to a new centrifuge tube;

adding step II, namely, after the cell-free liquid is subjected to resuspension washing, centrifuging at a high speed for 30min, collecting supernatant to a new centrifuge tube, and repeating the step III for washing for 3 times;

combining the collected supernatant and discarding the precipitate;

repeatedly dialyzing the combined liquid with dialysate for 3-5 times, each time for 2-4 hours;

and (3) collecting the liquid after dialysis, measuring the total concentration of protein, and concentrating the liquid until the total concentration of protein 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 addition amount of the collagen is 1-10% of the volume of the culture medium.

Further, according to 10 in the steps ⁵ -10 ⁶ The sarcoma cells were resuspended at a concentration of/ml.

Further, the sarcoma cell is a fibrosarcoma cell or a osteosarcoma cell.

Further, the decellularized liquid consists 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 was 1-20mM Tris-HCl (pH=7.0).

In a second aspect, the present invention provides a matrigel prepared by the preparation method according to any one of claims 1 to 8.

In a third aspect, the invention provides the use of matrigel in organoid culture.

The beneficial effects of the invention are as follows:

the preparation method is simple in preparation process, good in repeatability, suitable for large-scale operation under laboratory conditions, and is a method for preparing the matrix hydrogel from stable cell sources.

Compared with tissue acellular matrix glue, the matrix glue prepared by the method has the advantages of simple and controllable components and few impurities, the temperature of the acellular liquid adopted by the method is low, the cells are dissociated, the extracellular matrix structure is not damaged, the adopted dialysate is neutral salt ions, the protein component is suitable to be preserved, the method is suitable to be applied to cell culture, the main components of the extracellular matrix are reserved by the method, and the organoid culture is facilitated. The preparation method can use the human cell strain to prepare the matrigel, and the matrigel is suitable for human organ growth, 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 of the 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 of the 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 common optical microscope;

FIG. 6 is a morphological structure diagram under a common optical microscope of the organoids obtained in application example 2;

FIG. 7 is a morphological structure of the organoids obtained in application example 3 under a common optical microscope.

Detailed Description

Tris-HCl (pH=7.0) used in the examples of the present invention was purchased from the division of biological engineering (Shanghai).

In the description of the present invention, it is to be noted that the specific conditions are not specified in the examples, and the description is performed under the conventional conditions or the conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The invention will now be described in further detail with reference to the drawings and to specific examples, which are given by way of illustration and not limitation.

Example 1:

the preparation method of the matrigel mainly comprises the following steps:

preparing a culture medium: 5% type I collagen was added to 1640 medium containing 10% fetal bovine serum.

The culture medium was used according to 10 ⁶ The human fibrosarcoma cells were resuspended at a concentration of/ml and then inoculated into cell culture flasks.

Third, the cell culture flask was placed in a shaker at 50rpm, and then the whole shaker was placed in a cell incubator at 37 ℃.

In the culture process, liquid exchange is carried out every 4 days. In the liquid change, 1000rpm centrifugal 7 minutes, remove the supernatant, then using step 1 medium heavy suspension.

And after 20 days of culture, collecting the cells. Centrifugation at 1000rpm for 5 minutes, the supernatant was removed.

Adding the decellularized liquid into the cell sediment until the sediment is completely mixed, re-suspending the sediment, and processing at 5 ℃ overnight.

And (3) centrifuging at 12000rpm for 30min at the temperature of 4 ℃, precipitating impurities, and collecting the supernatant to a new centrifuge tube.

And (3) adding step (d) after re-suspending and washing the decellularized liquid, centrifuging at 12000rpm for 30min at 4 ℃, precipitating impurities, and collecting the supernatant to a new centrifuge tube. This step was repeated 3 times.

The collected supernatant was pooled and the pellet was discarded.

The pooled liquids were dialyzed repeatedly 4 times against 10mM Tris-HCl (pH=7.0) as dialysate. Each dialysis was performed for 3 hours.

And collecting the dialyzed liquid, measuring the total concentration of protein to be 0.84mg/ml, and concentrating the liquid to the total concentration of protein to be 12mg/ml to obtain matrigel as shown in figure 1.

The decellularized liquid in this example is prepared from 2M urea, 1M ammonium sulfate, 10mM EDTA and 150mM acetic acid according to a conventional method, and is 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 was used according to 10 ⁵ Per mlHuman osteosarcoma cells were resuspended in concentration and then inoculated into a bioreactor.

Third, the bioreactor was set to 100rpm and incubated at 37 ℃.

In the culture process, liquid exchange is carried out every 3 days. When changing the liquid, centrifuging at 1000rpm for 5 minutes, removing the supernatant, and then using the step to obtain the culture medium for re-suspension.

After 28 days of culture, the cells were collected. Centrifugation at 1000rpm for 5 minutes, the supernatant was removed.

Adding the decellularized liquid into the cell sediment until the sediment is completely mixed, re-suspending the sediment, and processing at 8 ℃ overnight.

The collected supernatant was pooled and the pellet was discarded.

The pooled liquids were dialyzed repeatedly 3 times against 20mM Tris-HCl (pH=7.0) as dialysate. Each dialysis was carried out for 2 hours.

And (3) collecting the liquid after dialysis, measuring 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 matrigel. The matrigel was dropped onto a petri dish and solidified at 37℃as shown in FIG. 2.

The decellularized liquid in this example was prepared from 1M urea, 2M ammonium sulfate, 20mM EDTA and 200mM acetic acid according to a conventional method, and the high temperature sterilization was completed.

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 was used according to 10 ⁶ EHS cells were resuspended at a concentration of/ml and then inoculated into cell culture flasks.

Third, the cell culture flask was placed in a shaker at 20rpm, and then the whole shaker was placed in a cell incubator at 37 ℃.

In the culture process, liquid exchange is carried out every 5 days. In the liquid change, 1000rpm centrifugal 10 minutes, remove the supernatant, then using step 1 medium heavy suspension.

Adding the decellularized liquid into the cell sediment until the sediment is completely mixed, re-suspending the sediment, and processing at 2 ℃ overnight.

The collected supernatant was pooled and the pellet was discarded.

The pooled liquids were dialyzed repeatedly 5 times against 1mM Tris-HCl (pH=7.0) as dialysate. Each dialysis was performed for 4 hours.

And collecting the dialyzed liquid, measuring the total concentration of protein to be 0.72mg/ml, and concentrating the liquid to the total concentration of protein to be 10mg/ml to obtain matrigel as shown in figure 3.

The decellularized liquid in this example was prepared from 4M urea, 0.2M ammonium sulfate, 1mM EDTA and 10mM acetic acid according to a conventional method, and was subjected to a high temperature treatment 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.

And (3) adding the cell-free liquid obtained in the step (6), re-suspending and washing, centrifuging at a high speed of 12000rpm for 30min at a temperature of 4 ℃, precipitating impurities, and collecting the supernatant to a new centrifuge tube. This step was repeated 3 times.

The collected supernatant was pooled and the pellet was discarded.

And (3) collecting the liquid after dialysis, measuring 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 matrigel. The matrigel was dropped onto a petri dish and solidified at 37℃as shown in FIG. 4.

The decellularized liquid in this example was prepared from 2M urea, 1M ammonium sulfate, 10mM EDTA and 150mM acetic acid according to a conventional method, and was subjected to a high temperature treatment at 121℃for 20 minutes.

Comparative example 1:

this comparative example differs from example 1 only in that in step 10 ⁶ The lung cancer cells H1975 were resuspended at a concentration of/ml, and the total protein concentration was 0.24ng/ml after dialysis as a result of the same as in example 1, and therefore, gelation was not possible.

Comparative example 2:

the comparative example differs from example 1 only in the components of the decellularized liquid, and the components of the decellularized liquid added in the comparative example are as follows: 1% SDS,2M NaCl, the other way round in example 1, the liquid after dialysis was collected and the total concentration of protein was determined to be 0.19mg/ml. The resulting protein concentration was much lower than in example 1, indicating that the decellularized fluid of example 1 is advantageous for harvesting more matrigel.

Comparative example 3:

this comparative example differs from example 1 only in that physiological saline was used as a dialysate in the step (ii), and the rest was the same as example 1. The matrigel obtained was stored overnight after concentration, and precipitation was found to occur, whereas example 1 did not. This demonstrates that Tris-HCl (ph=7.0) as a dialysate facilitates the preservation of matrigel.

Comparative example 4:

this comparative example differs from example 1 only in terms of steps 10 ³ The fibrosarcoma cells were resuspended at a concentration of/ml, the remainder being the same as in example 1. The cell count was observed to be small during cell culture, and the post-dialysis fluid was collected to determine a total protein concentration of 0.07mg/ml. The resulting protein concentration was much lower than in example 1, indicating that the cell concentration in example 1 favors the harvesting of more matrigel.

Comparative example 5:

this comparative example differs from example 1 only in terms of steps 10 ⁸ The fibrosarcoma cells were resuspended at a concentration of/ml, the remainder being the same as in example 1. After 3 days of incubation in step 4, a large number of cells were found to be apoptotic, and finally the post-dialysis fluid was collected and the total protein concentration was determined to be 0.79ng/ml. The resulting protein concentration was much lower than in example 1, indicating that the cell concentration in example 1 favors the harvesting of more matrigel.

Application example 1:

the matrigel of the present invention is applied to lung cancer organoid culture.

1) An appropriate amount of lung cancer organoid medium was mixed with the matrigel of example 1 in equal proportions, then lung cancer cells were resuspended in the mixture, and the gel with cells was then dropped into a 60mm dish with a pipette, approximately 50ul per drop.

2) Placing the culture dish inoculated with the glue drop into CO ₂ Standing in the incubator for 2min, carefully reversing after the light shaking glue drops have no obvious flow, and fully solidifying for 30min.

3) Adding lung cancer organoid culture medium into culture dish, and placing in constant temperature incubator at 37deg.C and 5% CO ₂ Culturing under concentration.

4) The lung cancer organoids can be obtained by changing the culture medium every 2 days and culturing for 6 days, and the tissue morphology and structure are observed under a common optical microscope as shown in figure 5, the organoids have the average size of more than 100 mu M, and the cell activity is good.

Application example 2:

the matrigel of the present invention is applied to intestinal cancer organoid culture.

1) Pretreating a 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 organoids medium was mixed with matrigel as described in example 2 at 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 sediment obtained in the step 1) by using the mixed gel culture solution, standing at 25 ℃ for 2-3 min after inoculating a gel drop, and then continuously preserving heat and standing after inverting the gel drop until the gel drop is fully solidified;

3) And (3) continuously standing the glue drop obtained in the step (2) at room temperature for 15min, further adding a cell culture medium preheated at 37 ℃, culturing at 37 ℃ and 5% CO2 concentration, replacing the cell culture medium every 2 days, and culturing for 4 days to obtain the organoids shown in figure 6, wherein the average diameter of the intestinal cancer organoids is more than 50 mu M.

Application example 3:

the matrigel of the present invention is applied to the culture of liver organoids of mice.

1) Pretreating a fresh sample of the mouse liver to obtain a cell mass with the cell number of 50 cells, and centrifuging to remove the supernatant for later use;

2) Animal liver organoids medium was mixed with matrigel as described in example 3 at 1:2 mixing to obtain a mixed gel culture solution, placing the mixed gel culture solution on ice, then re-suspending the cell mass sediment obtained in the step 1) by using the mixed gel culture solution, standing at 25 ℃ for 2-3 min after inoculating the gel drop, and then continuously preserving heat and standing after inverting the gel drop until the gel drop is fully solidified;

3) And (3) continuously standing the glue drop obtained in the step (2) at room temperature for 15min, further adding a cell culture medium preheated at 37 ℃, culturing at 37 ℃ and 5% CO2 concentration, replacing the cell culture medium every 2 days, and culturing for 7 days to obtain the organoid shown in figure 7, wherein the organoid of the liver of the mouse is in a cavity shape.

In conclusion, the method of the invention retains the main components of extracellular matrix, which is beneficial to organoid culture.

The foregoing examples illustrate only a few embodiments of the invention and are described in detail herein without thereby limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. The preparation method of the matrigel is characterized by comprising the following steps:

(1) Preparing a culture medium: adding collagen into 1640 culture medium containing 10% of fetal bovine serum;

(2) Resuspension of sarcoma cells using the above media, followed by inoculation into a cell culture device;

(3) Culturing at 37 deg.C under 20-100 rpm;

(4) In the culture process, liquid exchange is carried out every 3-5 days, when the liquid exchange is carried out, centrifugation is carried out at 1000rpm for 5-10 minutes, the supernatant is removed, and then the culture medium in the step (1) is used for re-suspension;

(5) After culturing for 14-28 days, collecting cells; centrifuging at 1000rpm for 5 minutes, and removing the supernatant;

(6) Adding the decellularized liquid to resuspend and precipitate, and processing at 2-8deg.C overnight;

(7) Centrifuging at high speed for 30min, and collecting supernatant to a new centrifuge tube;

(8) Adding the decellularized liquid obtained in the step (6) for resuspension washing, centrifuging at a high speed for 30min, collecting supernatant to a new centrifuge tube, and repeating the step for washing for 3 times;

(9) Combining the collected supernatant fluid and discarding the precipitate;

(10) Repeatedly dialyzing the combined liquid with dialysate for 3-5 times, each time for 2-4 hours;

(11) Collecting the liquid after dialysis, measuring the total concentration of protein, and concentrating the liquid until the total concentration of protein is 10-15mg/ml to obtain matrigel;

the decellularized liquid consists of 1-4M urea, 0.2-2M ammonium sulfate, 1-20mM EDTA and 10-200mM acetic acid;

the dialysate is 1-20mM Tris-HCl, and the PH=7.0;

the sarcoma cell is a fibrosarcoma cell or a osteosarcoma cell.

2. The method for preparing matrigel according to claim 1, wherein: the collagen is one of type I collagen, type II collagen, type III collagen or type IV collagen.

3. The method for preparing the matrigel according to claim 1 or 2, wherein: 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: in the step II, the method is according to 10 ⁵ -10 ⁶ The sarcoma cells were resuspended at a concentration of/ml.

5. The method for preparing matrigel according to claim 1, wherein: the conditions of the high-speed centrifugation are: 12000rpm at 4 ℃.