Cell collection method capable of simultaneously collecting three cells in co-culture model

Abstract

The invention relates to a cell collection method capable of simultaneously collecting three cells in a co-culture model, and belongs to the technical field of biology. The invention provides a cell collection method capable of simultaneously collecting three cells in a co-culture model, and the cell collection method comprises the following steps: after a three-cell co-culture model is constructed, taking out a Transwell chamber, retaining cells in the lower chamber, and collecting the cells in the lower chamber; respectively digesting the cells on the two sides of the Transwell cell membrane by using a trypsin solution with the concentration of 0.5 g / 100mL so as to respectively collect the cells on the two sides of the Transwell cell membrane. According to the cell collection method disclosed by the invention, the three cells in the three-cell co-culture model are simultaneously collected in a manner of digesting the cells on the two sides of the Transwell membrane step by step by using pancreatin, so that not only is the cell and consumable cost saved, but also the experimental synchronism of the three cells is ensured, and convenience is provided for optimizing the experimental process.

Description

TECHNICAL FIELD

[0001] The present application relates to a cell collection method for simultaneously collecting three cells in a co-culture model, belonging to the field of biotechnology. BACKGROUND

[0002] Cell co-culture is a technique that simulates the interaction of cells in the body in vitro, which involves the co-culture of two or more different types of cells in the same culture environment. This method can better simulate the in vivo environment, help understand the complex interactions between cells, and how these interactions affect the behavior and function of cells. Currently, cell co-culture can be divided into direct contact co-culture and indirect contact co-culture according to whether the co-cultured cells are in direct contact. Among them, direct contact co-culture refers to mixing two or more cells in a certain proportion and culturing them in the same culture dish. This method is suitable for studying the interaction between adjacent tissue cells, etc. In indirect contact co-culture, different cells are physically isolated, but can still exchange signal molecules through cell culture medium, which can effectively avoid the influence of direct contact between cells on their normal interaction, and obtain more accurate research results.

[0003] Transwell three-cell indirect co-culture is a kind of indirect contact co-culture, which can effectively simulate the in vivo microenvironment and is mainly used in the construction of in vitro models of brain neurovascular units, retinal neurovascular units, tumor and immune microenvironment, etc. In recent years, many studies have shown that single-cell or double-cell co-culture cannot meet the interaction of multiple types of cells and the influence of spatial structure in vitro. For example, single-cell or double-cell co-culture cannot realize the research of multiple cells acting on the same pathway. Using Transwell three-cell co-culture not only ensures the simultaneous culture and interaction of three cells, but also ensures the independent growth and indirect interaction of three cells. Therefore, at present, Transwell three-cell indirect co-culture is the best method for simulating the pathological and physiological state of the target structure in vitro.

[0004] When using Transwell three-cell indirect co-culture to construct a three-cell co-culture model in vitro, it is necessary to collect the cells in the co-culture model for the next step of research. The existing cell collection method for Transwell three-cell indirect co-culture mainly involves directly placing the target cells to be studied in the lower chamber and retaining the cells in the lower chamber separately. Specifically, first, three cells are cultured in vitro, then the target cells are seeded in the lower chamber of the cell culture plate at an appropriate density, and the other two cells are seeded on the outside and inside of the Transwell chamber at an appropriate density for indirect co-culture. After the three cells grow to an appropriate density, the Transwell chamber is discarded, and the target cells in the lower chamber are retained for the next step of research (see the operation flow chart in FIG. 1). Figure 1), in this mode of operation, if the pathophysiological state of three cells needs to be studied simultaneously, multiple three-cell co-culture models must be cultured simultaneously with three cells as target cells to complete the subsequent experiments of target cells, which not only requires more reagent consumables, but also needs more cell quantity, seriously limits the flexibility of the experiment, increases the resource consumption of the experiment, and in addition, due to the complexity of the three-cell co-culture model, if multiple three-cell co-culture models are constructed simultaneously, the complexity and time cost of the experimental process will be greatly increased, which is not conducive to repeated experiments. SUMMARY

[0005] To solve the above-mentioned defects, the present application provides a cell collection method for simultaneously collecting three cells in a co-culture model, which comprises: after the construction of the three-cell co-culture model is completed, the Transwell chamber is taken out, the lower chamber cells are retained, and trypsin solution with a concentration of 0.5 g / 100 mL is used to digest the cells on both sides of the Transwell chamber membrane to collect the cells on both sides of the Transwell chamber membrane, respectively.

[0006] In an embodiment of the present application, the cell collection method comprises: after the construction of the three-cell co-culture model is completed, the Transwell chamber is taken out, the lower chamber cells are retained, and the collection of the lower chamber cells is completed;

[0007] After the outer side of the membrane of the Transwell chamber is soaked in the trypsin solution, it is placed at 36.0-37.0℃ for 2-3 min to obtain a digestion product; after the placement is completed, the outer side of the membrane of the Transwell chamber is washed with a washing reagent to obtain a washing product; the digestion product and the washing product are centrifuged to obtain a precipitate, and the collection of the cells on the outer side of the membrane of the Transwell chamber is completed;

[0008] After the inner side of the membrane of the Transwell chamber is added with the trypsin solution, it is placed at 36.0-37.0℃ for 5-6 min to obtain a digestion product; after the placement is completed, the inner side of the membrane of the Transwell chamber is washed with a washing reagent to obtain a washing product; the digestion product and the washing product are centrifuged to obtain a precipitate, and the collection of the cells on the inner side of the membrane of the Transwell chamber is completed.

[0009] In an embodiment of the present application, the washing reagent comprises a cell culture medium, physiological saline and / or a buffer.

[0010] In an embodiment of the present application, the cell culture medium comprises a complete culture medium; and the buffer comprises a PBS buffer and / or a DPBS buffer.

[0011] The present application also provides the use of the above-mentioned cell collection method in the preparation of a three-cell co-culture model.

[0012] The application further provides a method for screening drugs, which comprises: constructing a three-cell co-culture model by in-vitro Transwell indirect co-culture, and modeling a disease by physical or chemical intervention means to the three-cell co-culture model during or after the construction, to obtain a disease model; administering a drug to be screened to the disease model, and collecting target cells by using the above-mentioned cell collection method during or after the administration, and judging whether the drug to be screened is effective for treating the disease by detecting the biological behavior change of the target cells.

[0013] In an embodiment of the application, the disease model comprises a cerebral vascular disease model and / or a retinal vascular disease model; the retinal vascular disease model comprises a diabetic retinopathy (DR) model and / or a retinal vascular obstruction model; the cerebral vascular disease model comprises an ischemic stroke model and / or a hemorrhagic stroke model.

[0014] In an embodiment of the application, when the disease model is a diabetic retinopathy model, the three-cell co-culture model is a three-cell co-culture model of retinal microvascular endothelial cells, retinal ganglion cells and retinal Muller cells.

[0015] In an embodiment of the application, when the disease model is a diabetic retinopathy model, the disease modeling comprises: culturing the three-cell co-culture model of retinal microvascular endothelial cells, retinal ganglion cells and retinal Muller cells by using a high-glucose medium.

[0016] The application further provides an application of the above-mentioned cell collection method or the above-mentioned method for screening drugs in screening drugs.

[0017] In an embodiment of the application, the drug is a disease treatment drug; the disease comprises a cerebral vascular disease and / or a retinal vascular disease; the retinal vascular disease comprises diabetic retinopathy (DR) and / or retinal vascular obstruction; and the cerebral vascular disease comprises ischemic stroke and / or hemorrhagic stroke.

[0018] The technical scheme of the application has the following advantages:

[0019] The application provides a cell collection method for simultaneously collecting three cells in a co-culture model, which comprises the following steps: after the construction of a three-cell co-culture model is completed, a Transwell chamber is taken out, the lower chamber cells are reserved, and a trypsin solution with a concentration of 0.5 g / 100 mL is used to digest the cells on both sides of the Transwell chamber membrane to collect the cells on both sides of the Transwell chamber membrane, respectively. The cell collection method of the application is based on trypsin digestion, and compared with the cell collection method used in the prior art (i.e., directly placing the target cells to be studied in the lower chamber and reserving the lower chamber cells alone), the cell collection method based on trypsin digestion used in the application has the following advantages:

[0020] Firstly, the cell collection methods used in the prior art all need to construct multiple models or increase the use amount of cell consumables, while the cell collection method based on trypsin digestion used in the application simultaneously collects the three cells in the three-cell co-culture model by using trypsin to stepwise digest the cells on both sides of the Transwell membrane, which not only ensures the saving of cell and consumable costs, but also ensures the experimental synchronicity of the three cells and provides convenience for optimizing the experimental process.

[0021] Secondly, in addition to the cell collection method based on trypsin digestion, the application also attempts a cell collection method based on a cell scraper, but the experimental results show that this method is easy to damage the cell structure and cause the cells to die and gather into a mass, and the cell survival rate is low, which cannot be used for subsequent experiments, while the cell collection method based on trypsin digestion used in the application degrades the proteins on the cell membrane and its contact surface by trypsin digestion, separates the cells to achieve a catalytic process, and the cells do not change in structure or function in this process, which maximizes the maintenance of the original state of the cells, and more cell strains can be reserved for other research for cells that are difficult to separate or have complex culture conditions.

[0022] Thirdly, scientific experiments need to be repeated more than three times to ensure the authenticity and effectiveness of the results, but the cell collection method based on a cell scraper cannot uniformly scrape the number and survival rate of cells each time, while the cell collection method based on trypsin digestion used in the application can meet the repeatability of experiments and ensure the reliability of experimental results by uniformly controlling the liquid amount and digestion time of trypsin and sufficient liquid blowing and washing.

[0023] Fourthly, the Transwell chamber is currently mainly adapted to 6-well, 12-well and 24-well cell culture plates, wherein the pore diameter of the Transwell chamber adapted to the 12-well and 24-well cell culture plates is only 12mm and 6.5mm, and the smallest cell scraper width is 13mm, so it is difficult to collect cells in the Transwell chamber adapted to the 12-well and 24-well cell culture plates, and therefore, the cell collection method based on the cell scraper which has been tried by the present application is only applicable to the Transwell chamber adapted to the 6-well cell culture plate, and the cell collection method based on trypsin digestion used in the present application is applicable to all pore diameters of the Transwell chamber, which is beneficial to the development of more kinds of research.

[0024] In summary, the cell collection method based on trypsin digestion of the present application has great advantages in experimental cost and cell survival rate compared with the cell collection method used in the prior art and the cell collection method based on the cell scraper which has been tried by the present application, and can not only be used for evaluating the change of cell state in the disease model, but also provide an in vitro platform for the subsequent disease mechanism research of model simulation, judgment of whether the drug to be screened is effective for the treatment of the disease and the like.

[0025] Further, the cell collection method based on trypsin digestion of the present application uses 0.5% trypsin, which not only ensures that the biological characteristics of the cells are not affected and meets the 100% survival rate, but also ensures that more target cells are collected. BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 The operation flow diagram of the cell collection method used in the prior art (i.e. directly placing the target cells to be studied in the lower chamber and retaining the cells in the lower chamber alone).

[0027] Figure 2 The operation flow diagram of the cell collection method based on the cell scraper.

[0028] Figure 3 The construction flow diagram of the Transwell three-cell indirect co-culture model.

[0029] Figure 4 The structure comparison diagram of the cells collected by the cell scraper and normal cells.

[0030] Figure 5 The operation flow diagram of the cell collection method based on trypsin digestion.

[0031] Figure 6 The observation result of the cells collected by the cell collection method based on trypsin digestion.

[0032] Figure 7: Comparison of the structure of the cells collected by trypsin digestion with the cells collected by using a cell scraper.

[0033] Figure 8 : Effect of trypsin concentration on the Transwell three-cell indirect co-culture model.

[0034] Figure 9 : Effect of digestion time and washing times on the Transwell three-cell indirect co-culture model. DETAILED DESCRIPTION

[0035] The following examples are provided to better further understand the present application and are not limited to the best mode contemplated, do not constitute a limitation of the content and scope of the present application, and any person skilled in the art under the guidance of the present application or the combination of the present application with other prior art features will fall within the scope of the present application.

[0036] The following examples are provided to better further understand the present application and are not limited to the best mode contemplated, do not constitute a limitation of the content and scope of the present application, and any person skilled in the art under the guidance of the present application or the combination of the present application with other prior art features will fall within the scope of the present application.

[0037] Experimental Example 1: Effect of cell collection method on the Transwell three-cell indirect co-culture model

[0038] This experimental example provides an experiment of the effect of cell collection method on the Transwell three-cell indirect co-culture model, and the experimental process is as follows:

[0039] 1. Experimental materials

[0040] Rat retinal microvascular endothelial cells (rRMECs, purchased from Shanghai Xiyaxia Biological Company), rat retinal ganglion cells (rRGCs, purchased from Shanghai Xiyaxia Biological Company), rat retinal Muller cells (purchased from Guangzhou Jinieuo Biological Technology Co., Ltd.), Dulbecco's Modified Eagle Medium (DMEM medium, purchased from Gibco), fetal bovine serum (FBS, purchased from Gibco), complete medium (90% DMEM medium + 10% FBS, % refers to the volume ratio), 0.5% trypsin (% refers to the mass volume ratio, g / 100 mL, purchased from Gibco, containing EDTA), 0.25% trypsin (% refers to the mass volume ratio, g / 100 mL, purchased from Gibco, containing EDTA), 24-well cell culture plates and suitable Transwell chambers (purchased from Corning), culture dishes (purchased from Corning), PBS buffer (purchased from Gibco Life Technologies).

[0041] 2. Experimental method

[0042] 2.1. The influence of scraper collection on the Transwell three-cell indirect co-culture model

[0043] The cell collection method based on the cell scraper is as follows: first, three kinds of cells are cultured in vitro, then the three kinds of cells are inoculated in the lower chamber of the cell culture plate and the outside and inside of the Transwell chamber membrane at appropriate densities for indirect co-culture, after the three kinds of cells grow to an appropriate density, the Transwell chamber is not discarded, but is taken out and placed in a new cell culture plate, and the cells on both sides of the Transwell membrane are scraped with a cell scraper, and the scraped cells are collected in a centrifuge tube, resuspended after centrifugation, and inoculated in a new cell culture plate for culture, after the two kinds of cells grow to an appropriate cell density, the next step of the experiment is performed (see Figure 2 ).

[0044] The specific process is as follows:

[0045] Take rat retinal ganglion cells as target cell 1, rat retinal microvascular endothelial cells as target cell 2, and rat retinal Muller cells as target cell 3; invert the Transwell chamber, and take 1×10 5Cell suspension of target cells 1 (obtained by resuspending target cells in complete culture medium) was added dropwise at a rate of 0.1 mL / well to the outer membrane of the Transwell chamber. The Transwell chambers were then placed in a sterile cell culture incubator at 37°C and 5% (v / v) CO2 for cell culture. After 3 hours of culture, target cells 1 adhered to the outer membrane of the Transwell chamber. At this point, the culture medium on the outer membrane of the Transwell chamber was discarded, and complete culture medium was added at a rate of 0.5 mL / well to the lower chamber of a 24-well cell culture plate (No. 1). The Transwell chamber was then inverted and placed in the 24-well cell culture plate (No. 1). The 24-well cell culture plate was then placed in a sterile cell culture incubator at 37°C and 5% (v / v) CO2 for cell culture. After 24 hours of culture, target cells 1 adhered and grew on the outer membrane of the Transwell chamber. At this point, cells containing 1×10⁶ cells were cultured. 5 Cell suspension of target cells 2 (obtained by resuspending target cells in complete culture medium) was seeded at a rate of 0.1 mL / well into the inner membrane of the Transwell chamber. A sample containing 6 × 10⁶ cells / mL was taken. 4 Cell suspension of target cells 3 (obtained by resuspending target cells in complete culture medium) was inoculated at 0.5 mL / well into the lower chamber of a 24-well cell culture plate (No. 2). Both 24-well cell culture plates (No. 1 and No. 2) were placed in a sterile cell culture incubator at 37°C and 5% (v / v) CO2 for cell culture. After 24 hours of culture, target cells 2 adhered to the inner wall of the Transwell chamber and grew, while target cells 3 adhered to the inner wall of the 24-well cell culture plate (No. 2). At this point, the Transwell chamber with target cells growing on both sides of the membrane was placed into the 24-well cell culture plate (No. 2), and the 24-well cell culture plate was placed in a sterile cell culture incubator at 37°C and 5% (v / v) CO2 for cell culture. After 24 hours of culture, the Transwell three-cell indirect co-culture model was obtained (the construction process of the Transwell three-cell indirect co-culture model is described in [link to documentation]). Figure 3 (Image drawn by Figdraw)

[0046] After obtaining three Transwell cell indirect co-culture models, the culture medium was discarded, and the Transwell chambers with target cells growing on both sides of the membrane were removed. Target cells 1 and 2 were scraped off from both sides of the Transwell chamber membrane using a cell scraper. After scraping, the cells collected by the scraper were observed using an inverted microscope and compared with normal cell structures. The comparison results are shown in [Figure number missing]. Figure 4 .

[0047] Depend on Figure 4It can be seen that the cell scraping method based on cell scraper can easily damage the cell structure and cause cell death to gather into a group, and cannot be used for subsequent experiments of Transwell three-cell indirect co-culture model. It can be seen that compared with the cell collection method used in the prior art (i.e. directly placing the target cells to be studied in the lower chamber and retaining the lower chamber cells alone), the cell collection method based on cell scraper is effective in simplifying the operation steps of the experiment, but it also has great limitations. First, the filter membrane used in the Transwell chamber is mainly composed of polycarbonate, polyester or polyethylene terephthalate and polytetrafluoroethylene, and the thickness is only 10 μm, which is easily punctured during sample addition, so the cell scraper cannot guarantee multiple and same force operations, and it is not easy to collect all cells and subsequent repeated experiments; second, the main component of the scraper is thermoplastic elastomer, which can easily damage the cell structure and reduce the survival rate of the scraped cells; third, the cells are tightly attached to the filter membrane, making separation and collection extremely difficult, especially for those complex separation or harsh culture condition cell types; in addition, the Transwell chamber is currently mainly adapted to 6-well, 12-well and 24-well cell culture plates, among which the pore diameter of the Transwell chamber adapted to 12-well and 24-well cell culture plates is only 12 mm and 6.5 mm, respectively, and the smallest width of the cell scraper is 13 mm, which is difficult to collect in the Transwell chamber adapted to 12-well and 24-well cell culture plates, so this method is only suitable for the Transwell chamber adapted to 6-well cell culture plates, further increasing the waste of cost.

[0048] 2.2, Effect of trypsin digestion on Transwell three-cell indirect co-culture model

[0049] The cell collection method based on trypsin digestion is as follows: first, three cells are cultured in vitro, then the three cells are inoculated on the cell culture plate lower chamber and the outer and inner sides of the Transwell chamber membrane at appropriate densities for indirect co-culture, and after the three cells grow to an appropriate density, the Transwell chamber is not discarded, but the cells on both sides of the Transwell chamber membrane are digested with trypsin, the cells obtained by digestion are collected in a centrifuge tube, and after centrifugation and resuspension, they are inoculated in a new cell culture plate for culture. After the two cells grow to an appropriate cell density, the next step of the experiment is performed (see operation process Figure 5 ).

[0050] The specific process is as follows:

[0051] The Transwell three-cell indirect co-culture model is constructed according to the method of 2.1.

[0052] After obtaining the Transwell three kinds of intercellular indirect co-culture model, the culture medium was discarded, and the target cell 1 and target cell 2 on both sides of the Transwell chamber membrane were collected by trypsin digestion. The cell collection process was as follows:

[0053] Lower chamber (cell 3): Take out the Transwell chamber, and keep the lower chamber cells for standby in the sterile cell incubator at 37°C, 5% (v / v) CO2.

[0054] Outside the chamber membrane (cell 1): Take a new 24-well cell culture plate, and add 200 μL of 0.5% trypsin to each well as a digestion well. After transferring the Transwell chamber to the digestion well, transfer the 24-well cell culture plate to the sterile cell incubator at 37°C, 5% (v / v) CO2 and stand for 2 min. After standing, add 400 μL of complete culture medium to each well of the 24-well cell culture plate to terminate digestion. After digestion is complete, rinse the bottom of the Transwell chamber (i.e. the outside of the membrane) with complete culture medium, and rinse the bottom cells to a clean culture dish with a volume of 1 mL each time, for a total of 3 times. After rinsing, transfer the liquid in the culture dish and the liquid in the digestion well to a centrifuge tube, and centrifuge at 22°C, 1000 r / min for 3 min. After centrifugation, discard the supernatant, and take the cell pellet. Add 3 mL of PBS buffer to the cell pellet to resuspend it, and centrifuge at 22°C, 1000 r / min for 3 min. After centrifugation, discard the supernatant, and take the cell pellet. Repeat this PBS buffer washing process twice to collect the cells (see the operation process of the trypsin digestion-based cell collection method in Figure 5 ). Add 6000 μL (calculated based on the total number of wells, 12 wells) of complete culture medium to the collected cells to resuspend them, and obtain a cell resuspension. After inoculating the cell resuspension into the 24-well cell culture plate at a seeding amount of 500 μL / well, continue to place the 24-well cell culture plate in the sterile cell incubator at 37°C, 5% (v / v) CO2 for cell culture standby.

[0055] Inner membrane of the transwell chamber (cell 2): ​​Aspirate the liquid from the transwell chamber and add 100 μL of 0.5% trypsin to each well. Transfer the transwell chamber to a sterile cell culture incubator at 37°C and 5% (v / v) CO2 and incubate for 5 min. After incubation, add 200 μL of complete culture medium to each well of the transwell chamber to terminate digestion. After digestion, transfer the liquid inside the transwell chamber to a centrifuge tube and rinse the inner wall of the transwell chamber (i.e., the inner membrane) with 200 μL of complete culture medium each time, for a total of 3 rinses. After rinsing, transfer the rinse fluid obtained from rinsing the transwell chamber to a centrifuge tube. Centrifuge the tube at 22°C and 1000 rpm for 3 min. After centrifugation, discard the supernatant and collect the cell pellet. Resuspend the cell pellet in 3 mL of PBS buffer and centrifuge at 22°C and 1000 rpm for 3 min. After centrifugation, discard the supernatant and collect the cell pellet. Repeat this PBS buffer washing process twice to collect the cells. The collected cells were resuspended in 6000 μL of complete culture medium (based on a total of 12 wells) to obtain a cell resuspension. The cell resuspension was then inoculated into 24-well cell culture plates at a seeding rate of 500 μL / well. The 24-well cell culture plates were then placed in a sterile cell culture incubator at 37°C and 5% (v / v) CO2 for cell culture.

[0056] Repeat the above experimental procedure twice. Collect cells from both experiments and observe the cells collected after trypsin digestion using an inverted microscope. Compare the structure of the cells collected using a cell scraper with the structures of the cells collected after digestion. The results are shown in the table below. Figure 6 The comparison results are shown in Figure 7 .

[0057] Depend on Figure 6 It can be seen that there was no significant difference in the number of cells collected from both sides of the membrane in the two experiments, and the cells showed normal growth and morphology, indicating that the cell collection method based on trypsin digestion is very stable and can meet the requirements for experimental reproducibility. Figure 7 It can be seen that the cells collected by trypsin digestion have a high survival rate, maintain healthy physiological morphology and cell quantity, and can be further cultured for subsequent experiments in the three Transwell cell indirect co-culture models.

[0058] 2.3 Effects of trypsin concentration on three Transwell cell indirect co-culture models

[0059] Since 0.25% trypsin is the preferred choice for digesting most adherent cells, this experimental case also tried using 0.25% trypsin before determining to use 0.5% trypsin.

[0060] The specific process is as follows:

[0061] Construct the Transwell three kinds of cells indirect co-culture model according to the method of 2.1.

[0062] After obtaining the Transwell three kinds of cells indirect co-culture model, discard the culture medium, collect target cell 1 and target cell 2 on both sides of the Transwell chamber membrane by trypsin digestion, and the cell collection process is as follows: on the basis of 2.2, replace 0.5% trypsin with 0.25% trypsin.

[0063] After the cell collection process is completed, observe the cells collected by trypsin digestion under an inverted microscope, and the observation results are shown in Figure 8 .

[0064] In the prior art, 0.25% trypsin is often used for cell digestion in a single culture model, and the steps are usually as follows: 0.25% trypsin is used for digestion at 37°C for 1-2 min, and then the cells in the single culture model can be collected. However, as shown in Figure 8 , the cells on the inner side and the outer side of the Transwell chamber membrane are in good growth state and have appropriate cell density before digestion, and after 0.25% trypsin digestion at 37°C for 1-2 min, no obvious change is observed under a microscope, and the cells are tightly attached to the wall, so that the cells cannot be effectively collected. Moreover, the cells after digestion of 6 24-well chambers can only be plated in 1 12-well (the bottom area of a 24-well plate is 2 cm 2 , and can accommodate 5×10 5 cells; the bottom area of a 12-well plate is 4.5 cm 2 , and can accommodate 10×10 5 cells), so that the amount of cells collected by this method is extremely small, and the cells cannot be used for subsequent experiments of the Transwell three kinds of cells indirect co-culture model.

[0065] 2.4, Effect of digestion time and washing times on the Transwell three kinds of cells indirect co-culture model

[0066] Construct the Transwell three kinds of cells indirect co-culture model according to the method of 2.1.

[0067] After obtaining the Transwell three kinds of cells indirect co-culture model, discard the culture medium, collect target cell 1 and target cell 2 on both sides of the Transwell chamber membrane by trypsin digestion, and the cell collection process is as follows:

[0068] On the basis of 2.2, replace the digestion time of the inner side of the Transwell chamber membrane from 5 min to 8 min, and replace the digestion time of the outer side of the Transwell chamber membrane from 2 min to 5 min.

[0069] Alternatively, on the basis of 2.2, the number of times of washing the cells with PBS buffer is replaced from two times to zero times.

[0070] After the cell collection process is completed, the cells collected by trypsin digestion are observed by an inverted microscope, and the observation results are shown in Figure 9 .

[0071] As shown in Figure 9 , the cells on the inner side or the outer side of the Transwell chamber membrane present irregular shapes without the original spindle shape of the cells when the digestion time is too long or when there is no PBS washing step.

[0072] Obviously, the above examples are only examples for clearly illustrating but not limiting the embodiments. Based on the above description, other different forms of changes or variations can be made by those skilled in the art. Here, all the embodiments are not required to be exhausted, and the obvious changes or variations derived therefrom are still within the protection scope of the present application.

Claims

1. A cell collection method capable of simultaneously collecting three types of cells in a co-culture model, characterized by, The cell collection method comprises the following steps: after the construction of the three-cell co-culture model is completed, the Transwell chamber is taken out, the cells in the lower chamber are reserved, and the cells on both sides of the membrane of the Transwell chamber are respectively digested by using a trypsin solution with a concentration of 0.5 g / 100 mL to collect the cells on both sides of the membrane of the Transwell chamber.

2. The cell collection method of claim 1, wherein, The cell collection method comprises the following steps: after the construction of the three-cell co-culture model is completed, the Transwell chamber is taken out, the cells in the lower chamber are reserved, and the collection of the cells in the lower chamber is completed. After the outer side of the membrane of the Transwell chamber is soaked in the trypsin solution, the digestion product is obtained by standing at 36.0-37.0 ℃ for 2-3 min; after the standing is completed, the outer side of the membrane of the Transwell chamber is flushed by using a flushing reagent to obtain a flushing product; and the digestion product and the flushing product are centrifuged to obtain a precipitate, and the collection of the cells on the outer side of the membrane of the Transwell chamber is completed. After the inner side of the membrane of the Transwell chamber is added with the trypsin solution, the digestion product is obtained by standing at 36.0-37.0 ℃ for 5-6 min; after the standing is completed, the inner side of the membrane of the Transwell chamber is flushed by using a flushing reagent to obtain a flushing product; and the digestion product and the flushing product are centrifuged to obtain a precipitate, and the collection of the cells on the inner side of the membrane of the Transwell chamber is completed.

3. The cell collection method of claim 2, wherein the cell collection container is a cell collection bag. The flushing reagent comprises a cell culture medium, physiological saline and / or a buffer solution.

4. Application of the cell collection method according to any one of claims 1-3 in the preparation of a three-cell co-culture model.

5. A method of screening for a drug, characterized by, The method comprises the following steps: a three-cell co-culture model is constructed by indirect co-culture between Transwells in vitro, and during the construction or after the construction is completed, a disease model is obtained by physically or chemically intervening in the three-cell co-culture model; a drug to be screened is administered to the disease model, and during the administration or after the administration is completed, the target cells are collected by using the cell collection method according to any one of claims 1-3, and the change in the biological behavior of the target cells is detected to determine whether the drug to be screened is effective in treating the disease.

6. The method of claim 5, wherein, The disease model comprises a cerebral vascular disease model and / or a retinal vascular disease model; the retinal vascular disease model comprises a diabetic retinopathy model and / or a retinal vascular obstruction model; the cerebral vascular disease model comprises an ischemic cerebral stroke model and / or a hemorrhagic cerebral stroke model.

7. The method of claim 6, wherein, When the disease model is a diabetic retinopathy model, the three-cell co-culture model is a three-cell co-culture model of retinal microvascular endothelial cells, retinal ganglion cells and retinal Muller cells.

8. The method of claim 7, wherein, When the disease model is a diabetic retinopathy model, the disease modeling comprises: cell culture of the three-cell co-culture model of retinal microvascular endothelial cells, retinal ganglion cells and retinal Muller cells by using a high-glucose culture medium.

9. Application of the cell collection method according to any one of claims 1-3 or the method for screening a drug according to any one of claims 5-8 in the screening of a drug.

10. Use according to claim 9, wherein The drug is a disease treatment drug; the disease includes cerebrovascular disease and / or retinal vascular disease; the retinal vascular disease includes diabetic retinopathy and / or retinal vascular obstruction; the cerebrovascular disease includes ischemic stroke and / or hemorrhagic stroke.

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