Method for detecting cell invasion by coating cell cells with matrigel
By using diluted matrix gel in cell chambers and optimizing the coating process, the stability and cost problems of the existing matrix gel coating chamber technology are solved, and high-accuracy and low-cost cell invasion detection are achieved.
Patent Information
- Application Number
- CN202411835684.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-12-13
- Publication Date
- 2025-03-11
AI Technical Summary
The existing matrix gel coated chamber technology has problems such as structural instability, impurity pollution, lack of consistency, limited adhesion ability, limited growth environment, optical interference and high prices, and it is difficult to meet different experimental needs.
Matrigel was diluted with DMEM/F12 culture medium without phenol red to form a matrix gel solution, and cured in the upper chamber of the cell chamber. Combined with an optimized coating process and medium formula, a new matrix gel coating method was constructed.
The stability and uniformity of the matrix gel coat layer is achieved, background noise is reduced, and the accuracy and cost-effectiveness of cell invasion detection is improved. It is suitable for cell invasion and drug screening research.
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Figure CN119592656A_ABST
Abstract
Description
Technical Field
[0001] The invention relates to a method for detecting cell invasion by using a matrix gel-coated cell chamber, and belongs to the technical field of cell culture. Background Art
[0002] Matrigel is a basement membrane component extracted from animal tissues (such as mouse tumor tissues). Its main components are laminin, type IV collagen, nestin, etc., and it contains a variety of growth factors. It can provide support, tensile strength and scaffold support for tissues and cells, and at the same time serve as a three-dimensional substructure for cell adhesion and movement, as well as a reservoir for growth factors, chemokines and cytokines. Matrigel-coated chamber technology utilizes these characteristics of matrigel to coat it at the bottom or inside of the cell culture chamber to simulate the microenvironment of cell growth in the body. With the continuous development of biotechnology and materials science, matrigel-coated chamber technology is also constantly improving. For example, by optimizing the preparation process and formula of matrigel, its stability and biocompatibility have been improved; at the same time, a variety of new chamber designs and materials have been developed to meet different experimental needs.
[0003] Matrigel-coated chamber technology plays an important role in the fields of cell culture, tissue engineering and drug screening. In the field of matrigel-coated chamber technology, there are already a variety of mature existing technologies. These technologies mainly include: Traditional matrigel coating technology: directly apply matrigel on the bottom or inside of the cell culture chamber, and then inoculate cells for culture. This method is simple to operate, but there are problems such as uneven distribution of matrigel and easy shedding due to different people's operation. Improved matrigel coating technology: By improving the coating process and using new matrigel materials, the adhesion and stability of matrigel on the surface of the chamber are improved. For example, pre-cooled pipettes and cryopreservation tubes are used to operate matrigel to avoid its premature solidification; at the same time, matrigel products that do not contain LDEV (lactate dehydrogenase-enhancing virus) and low endotoxin have also been developed. Scaffold-based 3D cell culture technology: Use scaffold materials such as matrigel to construct a three-dimensional cell culture system to simulate the three-dimensional environment of cell growth in vivo. This technology can more realistically reflect the growth and differentiation process of cells in vivo, but the operation is relatively complicated and the cost is high.
[0004] Although the prior art has made some progress in the field of Matrigel-coated chamber technology, there are still some disadvantages and limitations:
[0005] (1) Structural instability: The structure of matrix gel is complex and unstable, and it is prone to dehydration, shrinkage or decomposition. This may cause its physical properties to change and become unsuitable for cell growth and attachment. (2) Impurity contamination: Chemical substances such as cross-linking agents and solvents may remain during the preparation of matrix gel, and these impurities may have a negative impact on cell growth and function. (3) Lack of consistency: There may be differences between different batches of matrix gel, resulting in a lack of stability and reproducibility in experimental results. This increases the difficulty of experimental verification and confirmation. (4) Limited adhesion capacity: The adhesion capacity of matrix gel to cells is limited and may not meet the needs of some special types of cells. This may cause cells to be unable to grow stably on the matrix gel or lose function. (5) Restricted growth environment: The chemical and physical properties of matrix gel may restrict the growth environment of cells, such as the delivery of oxygen and nutrients, the excretion of metabolites, etc. This may affect the growth rate and metabolic activity of cells. (6) Optical interference: The opacity of matrix gel may interfere with optical microscopy observation and imaging, limiting the accurate observation of cell behavior and subcellular structure. (7) High price: There are almost no mature matrix gel-coated cell chambers available for sale in China. The country has to rely on imports from abroad, so the price is very high. In addition, the delivery time is very long and the response speed is very slow.
[0006] In summary, although some progress has been made in the field of Matrigel-coated chamber technology, there are still many challenges and problems to be solved. Future research should focus on developing more stable, cost-effective, biocompatible and easy-to-operate Matrigel materials and technologies to meet different experimental needs and promote the development of related fields. Summary of the invention
[0007] In order to solve the above problems, the present invention provides a method for detecting cell invasion by using a matrix gel-coated cell chamber, and the detection effect is accurate.
[0008] The first object of the present invention is to provide a method for detecting cell invasion using a matrix gel-coated cell chamber, comprising the following steps:
[0009] S1, diluting the matrigel with DMEM / F12 medium without phenol red to obtain a matrigel solution;
[0010] S2, adding the matrix gel solution into the upper chamber of the cell chamber so that it covers the bottom of the upper chamber, and solidifying the matrix gel;
[0011] S3, adding cells to be detected and a first culture medium to the upper chamber of the cell chamber, adding a second culture medium to the lower chamber of the cell chamber, culturing, and detecting cell invasion through the cells in the lower chamber after the culturing;
[0012] The first culture medium is a culture medium without fetal bovine serum, and the second culture medium is a culture medium containing fetal bovine serum.
[0013] Furthermore, the first culture medium is a DMEM culture medium without fetal bovine serum, and the second culture medium is a DMEM culture medium containing 5%-20% fetal bovine serum.
[0014] Furthermore, the concentration of the matrix gel solution is 0.3125-1.25 mg / mL.
[0015] Furthermore, the ratio of the matrix gel solution to the surface area of the upper chamber bottom was 1-10 μg / cm 2 Add Matrigel solution.
[0016] Furthermore, the matrix glue is the matrix glue of Zhejiang Saining Biotechnology Co., Ltd. (8101310).
[0017] Furthermore, the coating time is 2-10 hours.
[0018] Furthermore, the coating temperature is 35-38°C.
[0019] Furthermore, after the coating step, the method further includes removing excess collagen solution and drying the solution.
[0020] The second object of the present invention is to provide application of the method in drug screening.
[0021] Furthermore, the drug includes an anti-tumor drug.
[0022] Furthermore, when anti-tumor drug screening is performed, the drugs to be screened are added to the upper chamber respectively, and the effects of different drugs on the invasive ability of tumor cells are detected to screen out potential anti-tumor drugs.
[0023] Furthermore, the tumor cells include human fibrosarcoma cells; and the anti-tumor drugs include fibrosarcoma therapeutic drugs.
[0024] Beneficial effects of the present invention:
[0025] The invention provides a new matrix gel coating method and constructs a new cell invasion experimental route based on the method, optimizes the coating process, including matrix gel dilution solvent, dilution concentration, gelling amount, gelling temperature and time, etc., and combines the optimization of culture medium in a cell chamber to obtain a matrix gel coated chamber with low cost, simple operation, stable coating and uniform coating layer, which has broad application prospects in cell invasion and related research, and it has been verified that the invasion experiment background noise under the optimal route is low and the invasion detection effect is excellent. BRIEF DESCRIPTION OF THE DRAWINGS
[0026] Figure 1 The results of the invasion experiment of HT1080 cells were shown in Figure 2. DETAILED DESCRIPTION
[0027] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.
[0028] The scheme involved in the present invention is as follows:
[0029] Cell migration refers to the property of cells to migrate from one place to another after being stimulated by external signals. Cell invasion refers to the ability of cells to migrate from one area to another through the extracellular matrix or basement membrane matrix. Cell migration is the movement of cells without resistance, but cell invasion refers to the ability of cells to move only after breaking off "resistance" (such as extracellular matrix ECM). Therefore, not all migrating cells are invasive.
[0030] In tumor treatment, it is generally believed that inhibiting tumor cell migration and invasion is an effective treatment method. Therefore, after intervention (overexpression, knockdown, knockout, point mutation, drug treatment) of tumor cells, testing the migration and invasion ability of cells can be used as an effective indicator to evaluate the effect of intervention.
[0031] The commonly used experimental method to detect cell invasion ability is the Transwell experiment. The Transwell experiment is a perforation experiment, which is to place a small chamber in a 24-well plate with culture medium added, and divide it into an upper chamber and a lower chamber. The small chamber contains densely packed small holes. When the cell suspension is added to the small chamber, the cells can deform through the holes in the upper chamber and run to the lower chamber and stick to the outside. By counting the cells in the lower chamber, the strength of the cell invasion ability can be judged. For cell invasion, a layer of matrix gel needs to be laid on the micropores to simulate the extracellular matrix. Sometimes the cell invasion efficiency is not as expected. This may be due to unsuitable culture conditions, the thickness and composition of the mechanism are not suitable for studying cell invasion, and the experimental environment is not conducive to cell invasion.
[0032] Therefore, it is very important to provide a method that can effectively detect cell invasion. The present invention provides a method for detecting cell invasion by using a matrix gel-coated cell chamber, comprising the following steps:
[0033] S1, diluting the matrigel with DMEM / F12 medium without phenol red to obtain a matrigel solution;
[0034] S2, adding the matrix gel solution into the upper chamber of the cell chamber so that it covers the bottom of the upper chamber, and solidifying the matrix gel;
[0035] S3, adding cells to be detected and a first culture medium to the upper chamber of the cell chamber, adding a second culture medium to the lower chamber of the cell chamber, culturing, and detecting cell invasion through the cells in the lower chamber after the culturing;
[0036] The first culture medium is a culture medium without fetal bovine serum, and the second culture medium is a culture medium containing fetal bovine serum.
[0037] Preferably, the first culture medium is a DMEM culture medium without fetal bovine serum, and the second culture medium is a DMEM culture medium containing 5%-20% fetal bovine serum. The preferred embodiment of the present invention can reduce background noise. How to reduce background noise during the experiment is a challenge. From the experimental results, in the method provided by the present invention, the background noise of the blank group is smaller, especially when the dilution multiple increases, the advantage of using domestic matrix gel is more obvious.
[0038] Preferably, the bottom of the upper chamber contains micropores for cells to pass through, and the diameter of the micropores is 6-10 μm.
[0039] Preferably, the concentration of the matrix gel solution is 0.3125-1.25 mg / mL.
[0040] Preferably, the ratio of the matrix gel solution to the surface area of the upper chamber bottom is 1-10 μg / cm 2 Add Matrigel solution. For example, 1.5 μg / cm 2 , 2μg / cm 2 , 2.5μg / cm 2 , 3μg / cm 2 , 3.5μg / cm 2 , 4μg / cm 2 , 4.5μg / cm 2 , 5μg / cm 2 , 5.5μg / cm 2 , 6μg / cm 2 、6.5μg / cm 2 , 7μg / cm 2 , 7.5μg / cm 2 , 8μg / cm 2 , 8.5μg / cm 2 , 9μg / cm 2 , 9.5μg / cm 2 Etc., including but not limited to the ratios listed above.
[0041] In the present invention, if cells cannot effectively pass through the matrix in the Transwell experiment, possible reasons include too high concentration of matrix gel, lack of factors that promote cell invasion, etc. Therefore, the concentration of matrix gel solution directly affects the coating thickness. Higher concentrations (higher than 10 μg / cm 2 ) will lead to the formation of a thicker matrix gel layer, resulting in excessive matrix rigidity, which will affect the cell invasion rate; if the concentration is too low, non-target cells will migrate to the lower chamber, resulting in inaccurate detection of invasion ability.
[0042] The ratio of Matrigel to surface area also affects the thickness and uniformity of the coating. Different culture vessels require different volumes of collagen solution to ensure uniform coating. There are many different Matrigel coating methods, such as thin gel method and thick gel method. The Matrigel-coated invasion chamber provides cells with conditions that allow their invasive ability to be evaluated in vitro. Therefore, the coating method and amount of Matrigel directly determine whether invasive cells can pass through specific barriers and whether non-targeted migration of non-invasive cells can be prevented.
[0043] Another purpose of the present invention is to reduce the cost of cell invasion experiments. As is known to all, the price of foreign-purchased matrix is very high, while the price of domestic matrix is generally only 40%-60% of the imported matrix. If a method can be found to make the invasion experiment effect of domestic matrix glue equivalent to that of imported matrix glue, the experimental cost will be greatly reduced. The present invention compares domestic Senin matrix glue and imported Corning matrix glue, and optimizes the dilution solvent and coating process, and finally finds an optimal route, which not only retains the function and simple coating of matrix glue, but also achieves the effect equivalent to that of foreign matrix glue.
[0044] Preferably, the DMEM / F12 medium without phenol red, the cell chamber and other experimental equipment in contact with the matrix gel should be pre-cooled at 0-5°C. The matrix gel coating product usually needs to be carried out at a low temperature to prevent the matrix gel from gelling and solidifying prematurely. When spreading the gel, try to add it vertically in the middle of the bottom of the chamber to avoid bubbles.
[0045] Preferably, the coating temperature is 35-38° C., such as 35.5° C., 36° C., 36.5° C., 37° C., 37.5° C., 38° C., etc., including but not limited to the temperatures listed above. Most preferably, it is 37° C. Matrigel exists in a polymeric form in a frozen state. Matrigel can gel at a temperature above 10° C., and at a temperature above 37° C., its components will gradually unravel the cross-linked structure due to thermal motion between molecules, the fluidity will be enhanced, and substances such as growth factors and extracellular matrix proteins will be released. Therefore, temperature is an important parameter in the gel formation of Matrigel.
[0046] Preferably, the coating time is 2-10h. For example, 2.5h, 3h, 3.5h, 4h, 4.5h, 5h, 5.5h, 6h, 6.5h, 7h, 7.5h, 8h, 9h, 10h, etc., including but not limited to the durations listed above. Matrigel can be gelled by standing at 37°C for about 30 minutes, and the time required for temperature increase may be shorter, but the present invention not only ensures sufficient gelation by extending the gelation time, but more importantly, it is to promote the uniformity of the gel, and can reduce the inhomogeneity caused by rapid gelation, thereby also enhancing the mechanical properties and adhesion properties of the matrigel. Moreover, the integrity of the matrigel usually affects the in vitro behavior of cells, and extending the gelation time can improve the attachment, proliferation and migration behavior of cells. However, the present invention also finds that if the gelation time is too long, it will reduce the mechanical properties, etc., and form a non-colloidal layer at the same time, which is not conducive to the study of cell invasion.
[0047] Preferably, after the coating step, the method further comprises the steps of removing excess matrix gel solution and drying.
[0048] The present invention also lies in that the above method is used to screen drugs, such as anti-tumor drugs.
[0049] In particular, different drugs can be added to the upper chamber to detect their effects on the invasive ability of tumor cells, and potential anti-tumor drugs can be screened.
[0050] Preferably, the tumor cells include but are not limited to human fibrosarcoma cells. Different types of cells have different adhesion and penetration activities to matrix gel. When selecting matrix gel coating consumables, it is necessary to carefully select according to specific experimental requirements and cell types. Therefore, the anti-tumor drug of the present invention is most preferably a fibrosarcoma therapeutic drug, and the matrix gel is preferably Cynin 8101310 matrix gel.
[0051] The materials involved in the following embodiments are as follows:
[0052] Matrigel: Corning (354234) Matrigel and Zenin (8101310) Matrigel.
[0053] DMEM / F12 culture medium: purchased from Zhejiang Saining Biotechnology Co., Ltd. (8013000500mL)
[0054] DMEM medium with or without fetal bovine serum: purchased from Zhejiang Saining Biotechnology Co., Ltd. (8011100500 mL).
[0055] Sai Ning PET8.0 cell chamber (1102130): A cell culture chamber made of polyethylene terephthalate (PET) membrane material with a membrane pore size of 8.0 microns (μm).
[0056] Example 1
[0057] The specific steps of the Matrigel coating experiment are as follows:
[0058] 1. Matrigel coating:
[0059] (1) The day before coating, take out the -20℃ Senin (8101310) matrix gel and slowly thaw it in an ice box containing ice until it is in a liquid state. Also, pre-cool the diluted and matrix gel-contacted DMEM / F12 culture medium, pipette tips, and pipettes in a 4℃ refrigerator.
[0060] (2) After the ultra-clean bench is sterilized for 30 minutes in the production workshop, the 24-well 8.0 μm PET cell chamber is placed in the ultra-clean bench and an ice box filled with ice.
[0061] (3) Take Matrigel and determine its concentration to be 10 mg / mL, then dilute the Matrigel with culture medium at a certain dilution ratio.
[0062] (4) According to 0.1mg / cm 2 Coating with the concentration of matrix gel: add 100μl volume to each 24-well chamber and then place the 24-well 8.0μm PET cell chamber in a 37℃, 5% CO2 cell culture incubator for 2-6 hours to allow it to fully gel and solidify. After the gel solidifies, a thin layer will form on the membrane. After removing the upper liquid, seal the plate with an aluminum foil bag, and then store the cell chamber at -20℃.
[0063] Any of the above consumables that come into contact with matrix gel must be pre-cooled to 4°C before use, and the packaging process must be done on ice.
[0064] 2. Cell culture
[0065] (1) HT-1080 (human fibrosarcoma cell line) cells were revived and cultured in DMEM medium supplemented with 10% FBS.
[0066] (2) When the cells reach a sufficient density (80-90%), subsequent experiments can be performed, the culture medium is removed, and the cells are rinsed twice with 5 mL of PBS;
[0067] (3) Add 1-2 mL of 0.25% trypsin / EDTA to the cells. Make sure the trypsin covers the entire cell culture surface.
[0068] (4) Incubate at 37°C for 3-5 minutes to allow the cells to detach from the plate surface;
[0069] (5) Add 5 mL of medium containing FBS to terminate digestion;
[0070] (6) Harvest the cells and transfer the cell suspension into a 15 mL centrifuge tube and centrifuge at 1500 RPM for 3 minutes;
[0071] (7) Discard the supernatant and resuspend the cells in 2 mL of serum-free DMEM medium. Pipette up and down several times to resuspend the cells. (Note: The cell concentration needs to be counted).
[0072] (8) Use DMEM medium without FBS and adjust the cell density to 5 x 10 5 150uL of cell suspension was added to each Transwell A chamber (the final number of cells per well was 75,000 / well) for cell migration assay;
[0073] (9) In the experimental group, 800 uL of DMEM medium containing 10% FBS was added to chamber B of the 24-well plate, where FBS played a chemotactic role;
[0074] (10) Culture the cells overnight in a 37°C, 5% CO2 humidified incubator.
[0075] (11) After overnight culture, remove the Transwell A chamber from the incubator and rinse the A chamber twice with PBS;
[0076] (12) Use a moistened cotton swab to gently wipe the cells on the inside of the membrane (Note: Make sure to wipe off all cells, especially those on the edge of the membrane;
[0077] (13) Stain the cells on the lower surface of the membrane with crystal violet for 10-15 min;
[0078] (14) Rinse the membrane surface twice with PBS to remove unbound crystal violet;
[0079] (15) Ventilate and dry chamber A at room temperature;
[0080] (16) To determine the total number of cells in the Transwell, the average number of cells per region was divided by the area of the microscopic observation area and then multiplied by the total area of the Transwell insert (0.33 cm). 2 ).
[0081] 3. Conditional testing
[0082] Follow the steps 1-2 above to make the following groups, and keep the rest the same:
[0083] (1) The culture medium for diluting Matrigel is DMEM / F12 medium (without phenol red) or DMEM medium containing phenol red.
[0084] (2) The matrix gel was Corning (354234) matrix gel or Senin (8101310) matrix gel, and the dilution ratios were 1 / 8, 1 / 16, and 1 / 32, respectively. The specific group settings are as follows:
[0085]
[0086] 4. Test results
[0087] (1) After comparing different dilution solvents, we found that different solvents would also affect the coating marks and cell adhesion. Specifically, we used two different culture media, DMEM containing phenol red and DMEM / F12, to dissolve the matrix gel. The comparison found that DMEM containing phenol red was not as good as DMEM / F12 in dissolving the cell adhesion, and DMEM containing phenol red would affect the coating marks after dissolving the matrix gel. Therefore, we decided to use DMEM / F12 to dissolve the matrix gel.
[0088] (2) The results of the invasion experiment of HT1080 cells inoculated with the same concentration under different dilution ratios and different sources of matrix gel are as follows Figure 1 from Figure 1 It can be seen that after dilution with DMEM / F12, the invasion effect of PET8.0 cell chambers coated with Senin Matrigel is comparable to that of Corning Matrigel, but the cost of Senin Matrigel is lower.
[0089] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.
Claims
1. A method for detecting cell invasion using a matrix gel-coated cell chamber, characterized in that: The following steps are involved: S1, diluting the matrigel with DMEM / F12 medium without phenol red to obtain a matrigel solution; S2, adding the matrix gel solution into the upper chamber of the cell chamber so that it covers the bottom of the upper chamber, and solidifying the matrix gel; S3, adding cells to be detected and a first culture medium to the upper chamber of the cell chamber, adding a second culture medium to the lower chamber of the cell chamber, culturing, and detecting cell invasion through the cells in the lower chamber after the culturing; The first culture medium is a culture medium without fetal bovine serum, and the second culture medium is a culture medium containing fetal bovine serum.
2. The method according to claim 1, characterized in that The first culture medium is a DMEM culture medium without fetal bovine serum, and the second culture medium is a DMEM culture medium containing 5%-20% fetal bovine serum.
3. The method according to claim 1, characterized in that The concentration of the matrix gel solution is 0.3125-1.25 mg / mL; the ratio of the matrix gel solution to the surface area of the upper chamber bottom is 1-10 μg / cm 2 Add the matrix gel solution; the matrix gel is Senin 8101310 matrix gel.
4. The method according to claim 1, characterized in that: The coating time is 2-10 hours.
5. The method according to claim 1, characterized in that The coating temperature is 35-38°C.
6. The method according to claim 1, characterized in that After the coating step, the steps of removing excess collagen solution and drying are also included.
7. Use of the method according to any one of claims 1 to 6 in drug screening.
8. The use according to claim 7, characterized in that: The drugs include anti-tumor drugs.
9. The use according to claim 8, characterized in that: When screening anti-tumor drugs, the drugs to be screened are added to the upper chamber respectively, and the effects of different drugs on the invasive ability of tumor cells are tested to screen out potential anti-tumor drugs.
10. The use according to claim 9, characterized in that: The tumor cells include human fibrosarcoma cells; the anti-tumor drugs include fibrosarcoma therapeutic drugs.