Preparation method of Sf9 cell feeder layer

Abstract

The invention belongs to the technical field of insect cell culture, and discloses a preparation method of an Sf9 cell feeder layer, which mainly comprises the following two steps: S1, subculturing Sf9 cells: culturing the Sf9 cells in a suitable culture medium to a suitable cell confluence degree; and S2, mitomycin C treatment of the Sf9 cells: treating the subcultured Sf9 cells at specific time and temperature by using mitomycin C with specific concentration to inhibit the cell division ability of the Sf9 cells, then washing to remove the mitomycin C, and replacing a fresh culture medium. The obtained feeder layer cells can effectively inhibit self proliferation, maintain good cell activity and structural integrity, can stably survive and provide necessary support for a co-culture system, and can be used for culture of insect symbiotic bacteria and other insect cells difficult to culture; the method is easy and convenient to operate, mild in condition and good in repeatability, the prepared feeding layer is stable in effect, and a powerful tool is provided for the related research field.

Description

Technical Field

[0001] The present invention belongs to the fields of cell biology and biotechnology, and particularly relates to a method for preparing an insect cell feeder layer, especially a method for preparing a Spodoptera frugiperda Sf9 cell feeder layer. Background Art

[0002] Feeder layer cells usually refer to a type of cells that have lost their proliferative ability but still maintain metabolic activity after special treatment (such as irradiation or chemical drug treatment). They spread on the bottom of the culture vessel during in vitro culture, forming a monolayer structure, and can provide structural support, secrete necessary cytokines or nutrients for the target cells co-cultured, simulate the in vivo microenvironment, thereby promoting the survival, growth, and / or differentiation of target cells (such as stem cells, primary cells, cells in certain specific differentiation states) that are difficult to grow, proliferate, or maintain specific functions under conventional culture conditions.

[0003] For some insect cells that are difficult to culture in vitro, or symbionts that have a close symbiotic relationship with insects and rely on host cells to provide a specific environment or nutrients, culturing them using a feeder layer prepared from insect cells is a potential and feasible method. This method is expected to solve problems such as slow growth, difficulty in maintenance, and easy death encountered when directly culturing these cells or microorganisms.

[0004] Sf9 cells are a cell line derived from the ovarian tissue of the Spodoptera frugiperda pupal stage, with advantages such as fast growth rate, relatively uniform cell size, easy suspension or semi-adherent growth in serum-free medium, and simple subculture operation. They are commonly used tool cells in insect cell research and recombinant protein expression. Using Sf9 cells as a basis to prepare a feeder layer has obvious advantages such as easy availability of material sources, mature culture conditions, and stable cell state.

[0005] However, there are already many research reports on the preparation and application of mammalian cell feeder layers (such as using MEF cells as a feeder layer for embryonic stem cells), but there are relatively few research reports on the preparation method of insect cell (especially Sf9 cell) feeder layers and their application in the culture of insect symbionts or difficult-to-culture insect cells. Existing methods are either unvalidated or have unstable effects, lacking a standardized, highly reliable insect cell feeder layer preparation technology.

[0006] Therefore, developing a stable and effective method for preparing insect cell feeder layers is of great significance for promoting insect cell culture, research on insect symbiotic microorganisms, and related biotechnological applications (such as the production of virus pesticides and the optimization of recombinant protein expression). Summary of the Invention

[0007] The object of the present invention is to overcome the deficiency in the prior art of lacking an effective and stable method for preparing insect cell (especially Sf9 cell) feeder layers, and to provide a method for preparing Sf9 cell feeder layers that is simple to operate, has optimized conditions, and reliable effects. The feeder layer prepared by this method can effectively support the in vitro culture of insect symbiotic bacteria or difficult-to-culture insect cells.

[0008] To achieve the above object, the present invention provides a method for preparing an Sf9 cell feeder layer, including the following core steps: Step S1: Subculture of Sf9 cells.

[0009] Resuspend Sf9 cells in the logarithmic growth phase with a suitable serum-free medium (such as Sf-900™ IISFM cell culture medium) and dilute to an appropriate cell density (for example, 0.6 - 1.0×10 6 cells / mL), then inoculate into a cell culture dish and culture at the optimal growth temperature (for example, 27°C) until the cell confluence reaches a level suitable for subsequent treatment (for example, about 70%). This step aims to obtain a sufficient number of Sf9 cells with good status as the basis for the feeder layer.

[0010] Step S2: Treatment of Sf9 cells with Mitomycin C (MMC).

[0011] After the Sf9 cells reach the predetermined confluence, discard the old medium and replace it with fresh cell culture medium. Add Mitomycin C solution to the medium so that its final concentration reaches a level that can effectively inhibit the DNA replication and cell division of Sf9 cells but does not cause a large number of cell deaths (for example, 100 μg / mL). Incubate the cells at the same temperature as the culture (for example, 27°C) for a precisely controlled time (for example, 2 hours) to ensure that Mitomycin C acts fully. After the incubation, the cells must be thoroughly rinsed multiple times (for example, 3 times) with a sterile buffer solution (such as 1×PBS) to completely remove the residual Mitomycin C and avoid its toxicity to the target cells or microorganisms in the subsequent co-culture. Finally, add fresh cell culture medium. The Sf9 cells obtained at this time constitute a feeder layer that has lost the ability to proliferate but retains its activity.

[0012] By precisely controlling the treatment concentration and time of mitomycin C, and subsequent thorough washing steps, the present invention achieves effective inhibition of the proliferation of Sf9 cells while maximizing the retention of cell viability and structural integrity, enabling it to serve as a feeder layer to provide a necessary growth support environment for target cells or microorganisms.

[0013] Further, in step S1, the Sf9 cells are diluted to 0.5 - 1.5×10 6 cells / mL, preferably 0.6 - 1.0×10 6 cells / mL.

[0014] Further, in step S1, the cell confluence preferably reaches 60% - 80%, more preferably 70%.

[0015] Further, the culture and incubation temperature is preferably 26 - 28°C, more preferably 27°C.

[0016] Further, in step S2, the final concentration of the mitomycin C solution is preferably 80 - 120 μg / mL, more preferably 100 μg / mL.

[0017] Further, in step S2, the incubation time is preferably 1.5 - 2.5 hours, more preferably 2 hours.

[0018] Further, in step S2, the sterile buffer solution used is phosphate buffered saline (PBS).

[0019] Further, in step S2, it is preferably rinsed 3 times with 1×PBS.

[0020] Further, the cell culture medium used is a commercially available or self - prepared serum - free medium suitable for the growth of Sf9 cells, such as the Sf - 900™ IISFM cell culture medium of Thermo Fisher Scientific.

[0021] A method for preparing an Sf9 cell feeder layer according to the present invention has the following significant advantages compared with the prior art: 1) The method is simple and reliable: The steps provided by the present invention are clear, the operation is relatively simple, and the reagents (mitomycin C) and equipment (incubator, pipette, etc.) used are all conventional laboratory configurations, which are easy to implement and standardize. By optimizing the key parameters (MMC concentration and treatment time), the stability of the preparation process and the repeatability of the results are ensured.

[0022] 2) Stable feeder layer effect: For Sf9 cells treated by the method of the present invention, their proliferation ability is effectively inhibited, but they can maintain cell viability and structural integrity for a long time (for example, at least 9 days) during subsequent culture (verified by MTT assay and morphological observation). This ensures that the feeder layer can continuously and stably play its supporting role.

[0023] 3) Wide application potential: The Sf9 cell feeder layer prepared by the present invention can be directly used to culture insect cells that are difficult to culture alone (such as certain primary insect cells, genetically modified insect cell lines) or microorganisms that are closely related to the symbiotic relationship of insects (such as certain endosymbiotic bacteria, fungi), providing important technical support for research in these fields.

[0024] 4) Solved the technical gap: The present invention specifically solves the problem of insufficient research on the preparation method of the feeder layer of insect cells (especially Sf9 cells), and provides a verified and effective solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings, where: Figure 1 It is a schematic diagram of morphological observation of the proliferation of Sf9 cells after treatment with mitomycin C; Figure 2 It is a bar chart comparing the viability of Sf9 cells after treatment with different concentrations of mitomycin C for 2 h and then cultured for different days. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0026] The following will specifically describe the implementation details of a method for preparing an Sf9 cell feeder layer according to the present invention in combination with specific embodiments, so that those of ordinary skill in the art can implement the present invention.

[0027] Example 1: Preparation and verification of the Sf9 cell feeder layer 1. Step S1: Subculture of Sf9 cells After quickly thawing the cryopreserved Sf9 cells, add an appropriate amount of Sf-900™ IISFM cell culture medium, purchased from Thermo Fisher Scientific (company website: https: / / www.thermofisher.cn), and gently pipette to make a cell suspension. Use a cell counting chamber to count the cells and adjust the cell density to 0.6 - 1.0×10 6Inoculate cells at a density of cells / mL into a cell culture dish with a diameter of 30 mm. Place the culture dish in a constant-temperature cell incubator at 27°C for culture. Observe the cell growth status daily. After culturing for about 3 - 5 days, when the cell confluence reaches about 70%, proceed to the next step. The entire culturing process is carried out under standard aseptic operation conditions.

[0028] Step S2: Treatment of Sf9 cells with mitomycin C (1) Preparation: Prepare a stock solution of mitomycin C (MMC, purchased from Inalco, USA, full company name is Inalco Pharmaceuticals, company website is https: / / www.inalcopharm.com / capabilities / ). For example, dissolve MMC powder in sterile ultrapure water and make the volume up to 1 mg / mL. After filtering and sterilizing through a 0.22 μm filter membrane, aliquot and store in the dark at -20°C. Dilute it to the required working concentration with fresh medium before use.

[0029] (2) MMC treatment: Aspirate the old medium in the culture dish, and gently rinse the cells once with sterile 1×PBS buffer. Add an appropriate amount of fresh Sf-900™ IISFM cell medium. Add the MMC working solution to the medium and gently mix well to make the final concentration of MMC 100 μg / mL. Return the culture dish to the 27°C incubator and incubate for 2 hours.

[0030] (3) Washing: After incubation, carefully aspirate the medium containing MMC. Add sufficient sterile 1×PBS, gently shake the culture dish to rinse the cells, and then aspirate the PBS. Repeat this washing step 3 times in total. The washing operation should be gentle to avoid a large number of cells falling off.

[0031] (4) Completion of preparation: After washing, add an appropriate amount of fresh Sf-900™ IISFM cell medium to the culture dish. At this time, the Sf9 cells at the bottom of the culture dish are the prepared feeder layer cells. The prepared feeder layer can be used immediately or maintained in the incubator for short-term (such as several days) standby.

[0032] 3. Verification of treatment effect (1) Corresponding Figure 1 Observation of proliferation: To determine the optimal MMC treatment conditions, different treatment groups were set: the final concentrations of MMC were 50 μg / mL, 100 μg / mL, and 150 μg / mL respectively, and the treatment times were 1 hour and 2 hours respectively. At the same time, a control group without MMC (0 μg / mL) was set. Each group had 3 replicates. After treatment and washing, add fresh medium. On the 0th, 3rd, 5th, 7th, and 9th days after treatment, use an inverted microscope to observe the cell morphology and density of each group.

[0033] like Figure 1 As shown, the figure shows the morphology and density changes of Sf9 cells under a microscope after being treated with different concentrations of MMC for 1 hour or 2 hours and then continued to be cultured for different days (0d, 3d, 5d, 7d, 9d).

[0034] Results: The cells in the control group continued to proliferate during the culture period, and the density continued to increase. In the groups treated with MMC for 1 hour, obvious cell proliferation was still observed in the early stage of culture (3 days), indicating that 1 hour of treatment was not enough to completely inhibit proliferation. In the groups treated with MMC for 2 hours, the 50μg / mL group still had slow proliferation in the later stage (7 days-9 days); while in the 100μg / mL and 150μg / mL groups, the number of cells did not increase significantly during the entire observation period (3 days-9 days), and the morphology remained good, indicating that cell proliferation was effectively inhibited.

[0035] (2) Correspondence Figure 2 1. Cell viability detection: The relative viability of cells in each group on the 3rd, 5th, 7th and 9th days after the above-mentioned MMC treatment for 2 hours was detected by the MTT method. The brief steps are as follows: at the detection time point, MTT solution was added to the culture medium for incubation, and then the dissolving solution was added to dissolve the formazan crystals, and the absorbance (OD value) at a specific wavelength was measured by a microplate reader. The OD value is proportional to the number of living cells.

[0036] like Figure 2 As shown in the figure, the relative viability (expressed as OD value) of Sf9 cells detected by MTT method after being treated with 0μg / mL (control group), 50μg / mL, 100μg / mL, and 150μg / mL MMC for 2 hours and then cultured for 3 days, 5 days, 7 days and 9 days, respectively, and statistical analysis was performed (different lowercase letters indicate significant differences with p<0.05).

[0037] Results: The OD value of the cells in the control group (0 μg / mL) continued to increase with the extension of culture time, indicating that the cells proliferated in large numbers. In the 50 μg / mL MMC treatment group, the OD value increased slowly within 7 days and increased significantly on the 9th day, indicating that the cells still had some proliferation ability. In the 100 μg / mL MMC treatment group, the OD value remained relatively stable from 3 to 9 days, and was significantly lower than that of the control group and the 50 μg / mL group (p<0.05), indicating that cell proliferation was effectively inhibited and cell viability was maintained at a relatively stable level. In the 150 μg / mL MMC treatment group, the OD value was not significantly different from that of the 100 μg / mL group, but there was a slight downward trend on the 9th day, suggesting that there may be a certain amount of cytotoxicity leading to a small number of cell deaths.

[0038] (3) Conclusion: Based on the results of cell proliferation observation and MTT viability assay, it was determined that treating Sf9 cells with mitomycin C at a final concentration of 100 μg / mL for 2 hours was the preferred implementation method of the present invention. Under this condition, the proliferation of Sf9 cells was effectively inhibited, and the cells maintained good activity and stability during subsequent culture (at least 9 days), making them very suitable for use as feeder layers.

[0039] Example 2: Application of Sf9 Feeder Layer (Anticipatory / Guiding) Prepare the Sf9 cell feeder layer culture dish prepared according to the method of Example 1. Inoculate the target insect cells that are difficult to culture alone (for example, a certain specific primary insect cell) or insect symbiotic microorganisms (for example, symbiotic bacteria isolated and purified from insects) into the culture dish containing the feeder layer at an appropriate density, and perform co-culture using a medium suitable for the growth of the target cells / microorganisms.

[0040] In the control group, the target cells / microorganisms were inoculated into a culture dish without a feeder layer. Cultivate under suitable conditions and regularly observe the growth status, quantity change or specific biological indicators of the target cells / microorganisms.

[0041] The expected result is that, with the support of the Sf9 feeder layer, the growth conditions of the target cells / microorganisms (such as survival rate, proliferation rate, maintenance time, etc.) will be significantly better than those of the control group. This indicates that the Sf9 feeder layer prepared by the present invention can effectively support the in vitro culture of these difficult-to-culture objects.

[0042] Although the specific implementation manners of the present invention have been described above, those skilled in the art should understand that these specific implementation manners are only illustrative. Without departing from the principles and essence of the present invention, those skilled in the art can make various omissions, substitutions and changes to the details of the above methods and systems. For example, combining the above method steps so as to perform substantially the same function in a substantially the same way to achieve substantially the same result belongs to the scope of the present invention. Therefore, the scope of the present invention is only defined by the appended claims.

Claims

1. A method for preparing a feeder layer of Sf9 cells, characterized in that, Including the following steps: Step S1, subculture of Sf9 cells: After resuspending and diluting Sf9 cells with an appropriate cell culture medium, inoculate them into a cell culture dish and culture at an appropriate temperature until the cell confluence reaches a predetermined value; Step S2, treatment of Sf9 cells with mitomycin C: Replace the cell culture medium in the cell culture dish after the culture in step S1 with a fresh cell culture medium, add a mitomycin C solution to make its final concentration in the medium reach a concentration that can effectively inhibit cell proliferation, incubate at the same or similar temperature as in step S1 for a preset time sufficient to inhibit cell division, then rinse with a sterile buffer solution at least 2 times to remove mitomycin C, and then add a fresh cell culture medium to prepare an Sf9 cell feeder layer.

2. The method for preparing a feeder layer of Sf9 cells according to claim 1, characterized in that, In the step S1, the Sf9 cells are diluted to 0.5 - 1.5×10 6 cells / mL.

3. A method for preparing a Sf9 cell feeder layer according to claim 1, characterized in that, In step S1, the cell confluence reaches 60% - 80%.

4. A method for preparing a Sf9 cell feeder layer according to claim 1, characterized in that, The culture temperature or incubation temperature in step S1 and step S2 is 26 - 28 °C.

5. A method for preparing a Sf9 cell feeder layer according to claim 1, wherein, In step S2, the final concentration of the mitomycin C solution is 80 - 120 μg / mL.

6. A method for preparing a Sf9 cell feeder layer according to claim 1, characterized in that, The incubation time in step S2 is 1.5 - 2.5 hours.

7. A method for preparing a Sf9 cell feeder layer according to claim 1, characterized in that, In step S2, the sterile buffer solution is phosphate buffered saline (PBS).

8. A method for preparing a Sf9 cell feeder layer according to claim 7, characterized in that, In step S2, rinse 3 times with 1×PBS.

9. A method for preparing a Sf9 cell feeder layer according to claim 1, wherein, The cell culture medium is a serum-free medium suitable for the growth of Sf9 cells, such as Sf-900™ IISFM cell culture medium.

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