A method for preparing an Sf9 cell feeder layer

Abstract

The present invention belongs to the field of insect cell culture technology and discloses a method for preparing an Sf9 cell feeder layer. The method mainly comprises two steps: step S1 is subculture of Sf9 cells, in which Sf9 cells are cultured in a suitable culture medium to a suitable cell confluence; step S2 is mitomycin C treatment of the Sf9 cells, in which a specific concentration of mitomycin C is used to treat the subcultured Sf9 cells at a specific time and temperature to inhibit their cell division ability, followed by washing to remove mitomycin C and replacing with fresh culture medium. The feeder layer cells obtained by the present invention can effectively inhibit their own proliferation while maintaining good cell activity and structural integrity. They can survive stably and provide necessary support for the co-culture system. They can be used for the cultivation of insect symbiotic bacteria and other difficult-to-culture insect cells. The method is simple to operate, has mild conditions, and good reproducibility. The prepared feeder layer has a stable effect, providing a powerful tool for related research fields.

Description

Technical Field

[0001] The present invention belongs to the field of cell biology and biotechnology, and specifically relates to a method for preparing an insect cell feeder layer, in particular to a method for preparing a Spodoptera frugiperda (Spodoptera frugiperda) Sf9 cell feeder layer. Background Art

[0002] Feeder layer cells generally refer to cells that have lost their proliferative capacity but still retain metabolic activity after undergoing special treatments (such as radiation or chemical treatment). During in vitro culture, they spread out on the bottom of the culture vessel, forming a monolayer structure. They provide structural support for the co-cultured target cells, secrete essential cytokines or nutrients, and mimic the in vivo microenvironment, thereby promoting the survival, growth, and / or differentiation of target cells (such as stem cells, primary cells, and cells in certain differentiated states) that have difficulty growing, proliferating, or maintaining specific functions under conventional culture conditions.

[0003] For some difficult-to-culture insect cells in vitro, or for symbiotic bacteria that have a close symbiotic relationship with insects and rely on host cells for specific environments or nutrients, using insect cell-derived feeder layers to cultivate these cells is a potential and feasible method. This approach is expected to overcome the problems encountered when directly culturing these cells or microorganisms, such as slow growth, difficulty in maintenance, and easy death.

[0004] Sf9 cells, a cell line derived from the ovarian tissue of the pupal stage of the fall armyworm (Spodoptera frugiperda), have the advantages of rapid growth, relatively uniform cell size, ease of suspension or semi-adherent growth in serum-free culture medium, and simple passaging. They are a commonly used tool cell in insect cell research and recombinant protein expression. Using Sf9 cells as a base for feeder layer preparation offers significant advantages, including readily available material, mature culture conditions, and stable cell status.

[0005] While there are numerous reports on the preparation and application of mammalian cell feeder layers (e.g., the use of MEF cells as a feeder layer for embryonic stem cells), relatively few studies have focused on the preparation of insect cell (particularly Sf9) feeder layers and their application in the cultivation of insect symbiotic bacteria or difficult-to-cultivate insect cells. Existing methods are either unvalidated or inconsistent in their effectiveness, resulting in a lack of a standardized, efficient, and reliable technique for preparing insect cell feeder layers.

[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 biotechnology applications (such as viral insecticide production, recombinant protein expression optimization, etc.). Summary of the Invention

[0007] The present invention aims to overcome the deficiency in the prior art of the lack of effective and stable methods for preparing feeder layers of insect cells (especially Sf9 cells), and to provide a method for preparing Sf9 cell feeder layers that is simple to operate, has optimized conditions, and has reliable effects. The feeder layers 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 objectives, the present invention provides a method for preparing an Sf9 cell feeder layer, comprising the following core steps:

[0009] Step S1: Subculture of Sf9 cells.

[0010] Resuspend Sf9 cells in the logarithmic growth phase in a suitable serum-free medium (such as Sf-900™ IISFM cell culture medium) and dilute to an appropriate cell density (e.g., 0.6-1.0×10 6 cells / mL), then seeded into cell culture dishes and cultured at an optimal growth temperature (e.g., 27°C) until the cell confluence reaches a level suitable for subsequent processing (e.g., approximately 70%). This step aims to obtain a sufficient number of healthy Sf9 cells as the basis for the feeder layer.

[0011] Step S2: Treatment of Sf9 cells with mitomycin C (MMC).

[0012] Once the Sf9 cells reach the desired confluence, discard the old culture medium and replace with fresh cell culture medium. Add mitomycin C solution to the culture medium to a final concentration that effectively inhibits DNA replication and cell division in Sf9 cells without causing extensive cell death (e.g., 100 μg / mL). Incubate the cells at the same temperature as the culture medium (e.g., 27°C) for a precisely controlled period of time (e.g., 2 hours) to ensure full efficacy of mitomycin C. Following incubation, thoroughly rinse the cells multiple times (e.g., three times) with a sterile buffer solution (e.g., 1x PBS) to completely remove any residual mitomycin C and prevent its toxicity to target cells or microorganisms in subsequent co-cultures. Finally, add fresh cell culture medium. The resulting Sf9 cells now constitute a feeder layer that has lost its proliferative capacity but remains viable.

[0013] The present invention achieves effective inhibition of Sf9 cell proliferation by precisely controlling the treatment concentration and time of mitomycin C, as well as a subsequent thorough washing step, while maximally retaining the activity and structural integrity of the cells, enabling them to serve as a feeder layer and provide the necessary growth support environment for target cells or microorganisms.

[0014] Furthermore, in step S1, Sf9 cells were diluted to 0.5-1.5×10 6 cells / mL, preferably 0.6-1.0×10 6 cells / mL.

[0015] Furthermore, in step S1, the cell confluence preferably reaches 60% to 80%, more preferably 70%.

[0016] Furthermore, the culture and incubation temperature is preferably 26-28°C, more preferably 27°C.

[0017] Furthermore, in step S2, the final concentration of the mitomycin C solution is preferably 80-120 μg / mL, more preferably 100 μg / mL.

[0018] Furthermore, in step S2, the incubation time is preferably 1.5 to 2.5 hours, more preferably 2 hours.

[0019] Furthermore, in step S2, the sterile buffer solution used is phosphate buffered saline (PBS).

[0020] Furthermore, in step S2, it is preferred to rinse three times with 1×PBS.

[0021] Furthermore, the cell culture medium used is a commercially available or self-prepared serum-free medium suitable for growing Sf9 cells, such as Sf-900™ IISFM cell culture medium from Thermo Fisher Scientific.

[0022] The method for preparing an Sf9 cell feeder layer of the present invention has the following significant advantages over the prior art:

[0023] 1) Simple and reliable method: The steps provided in this invention are clear and relatively simple to operate. The reagents (mitomycin C) and equipment (incubator, pipette, etc.) used are all standard laboratory equipment, making them easy to implement and standardize. By optimizing key parameters (MMC concentration and treatment time), the stability of the preparation process and the reproducibility of the results are ensured.

[0024] 2) Stable feeder layer effect: While the proliferation of Sf9 cells treated with the present method is effectively suppressed, they maintain cell viability and structural integrity (verified by MTT assay and morphological observation) for a prolonged period (e.g., at least 9 days) in subsequent culture. This ensures that the feeder layer can continue to stably perform its supportive role.

[0025] 3) Broad 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 and genetically modified insect cell lines) or microorganisms that have a close symbiotic relationship with insects (such as certain endosymbiotic bacteria and fungi), providing important technical support for research in these fields.

[0026] 4) Solve the technical gap: The present invention specifically solves the problem of insufficient research on the preparation method of insect cell (especially Sf9 cell) feeder layer, and provides a verified and effective solution. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. Those skilled in the art can also derive other drawings based on these drawings without inventive work, among which:

[0028] Figure 1 Schematic diagram of the morphological observation of the proliferation of Sf9 cells after treatment with mitomycin C;

[0029] Figure 2 The bar graph shows the comparison of the viability of Sf9 cells after being treated with different concentrations of mitomycin C for 2 hours and then cultured for different days. DETAILED DESCRIPTION

[0030] The following describes in detail the implementation details of a method for preparing an Sf9 cell feeder layer of the present invention in conjunction with specific embodiments, so that those skilled in the art can implement the present invention.

[0031] Example 1: Preparation and verification of Sf9 cell feeder layer

[0032] 1. Step S1: Subculture of Sf9 cells

[0033] After rapid recovery of frozen 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. Count the cells using a cell counting plate and adjust the cell density to 0.6-1.0×10 6 Cells were seeded at a density of 10 cells / mL into a 30 mm diameter cell culture dish. The dish was placed in a 27°C incubator. Cell growth was monitored daily. The cells were cultured for approximately 3-5 days. When the cell confluence reached approximately 70%, the next step was performed. All culture procedures were performed under standard aseptic conditions.

[0034] Step S2: Mitomycin C treatment of Sf9 cells

[0035] (1) Preparation: Prepare mitomycin C (MMC, purchased from Inalco Pharmaceuticals, USA; the company's full name is Inalco Pharmaceuticals, and the company's website is https: / / www.inalcopharm.com / capabilities / ) stock solution. For example, dissolve MMC powder in sterile ultrapure water and adjust the volume to 1 mg / mL. After sterilization by filtration through a 0.22 μm filter, aliquot and store at -20°C in the dark. Dilute to the desired working concentration with fresh culture medium before use.

[0036] (2) MMC treatment: Aspirate the old culture medium from the culture dish and gently rinse the cells once with sterile 1× PBS buffer. Add an appropriate amount of fresh Sf-900™ IISFM cell culture medium. Add MMC working solution to the culture medium and gently mix to a final concentration of 100 μg / mL. Return the culture dish to the 27°C incubator and incubate for 2 hours.

[0037] (3) Washing: After incubation, carefully aspirate the culture 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 three times. The washing operation should be gentle to avoid excessive cell shedding.

[0038] (4) Preparation: After washing, add an appropriate amount of fresh Sf-900™ IISFM cell culture medium to the culture dish. At this point, 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 an incubator for a short period of time (e.g., several days) for future use.

[0039] 3. Treatment effect verification

[0040] (1) Correspondence Figure 1Proliferation Observation: To determine optimal MMC treatment conditions, different treatment groups were established: final MMC concentrations of 50 μg / mL, 100 μg / mL, and 150 μg / mL, with treatment times of 1 hour and 2 hours, respectively. A control group without MMC (0 μg / mL) was also established. Three replicates were set up for each group. After treatment and washing, fresh culture medium was added. Cell morphology and density were observed using an inverted microscope on days 0, 3, 5, 7, and 9 after treatment.

[0041] 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 cultured for different days (0d, 3d, 5d, 7d, 9d).

[0042] Results: Cells in the control group continued to proliferate and increase in density during the culture period. Significant cell proliferation was still observed in the initial culture period (day 3) in all concentration groups treated with MMC for 1 hour, indicating that a 1-hour treatment was insufficient to completely inhibit proliferation. In the groups treated with MMC for 2 hours, the 50 μg / mL group continued to proliferate slowly in the later stages (days 7-9). However, the 100 μg / mL and 150 μg / mL groups showed little significant increase in cell number throughout the observation period (days 3-9), and their morphology remained well-maintained, indicating that cell proliferation was effectively inhibited.

[0043] (2) Correspondence Figure 2 Cell viability assay: The relative viability of cells in each group was assessed on days 3, 5, 7, and 9 after 2 hours of MMC treatment using the MTT assay. The procedure was as follows: At the assay time points, MTT solution was added to the culture medium for incubation. Dissolution buffer was then added to dissolve the formazan crystals, and the absorbance (OD) at a specific wavelength was measured using a microplate reader. The OD value is proportional to the number of viable cells.

[0044] like Figure 2 As shown, the figure shows the relative viability (expressed as OD value) of Sf9 cells detected by MTT assay after being treated with 0 μg / mL (control group), 50 μg / mL, 100 μg / mL, and 150 μg / 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).

[0045] Results: The OD value of cells in the control group (0 μg / mL) continued to increase with prolonged culture time, indicating extensive cell proliferation. In the 50 μg / mL MMC-treated group, the OD value slowly increased over 7 days and significantly increased on day 9, indicating that the cells still had some proliferation capacity. In the 100 μg / mL MMC-treated group, the OD value remained relatively stable from 3 to 9 days and was significantly lower than that in the control and 50 μg / mL groups (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-treated group, the OD value did not differ significantly from that in the 100 μg / mL group, but showed a slight downward trend on day 9, suggesting that some cytotoxicity may have led to a small amount of cell death.

[0046] (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 embodiment of the present invention. Under this condition, Sf9 cell proliferation was effectively inhibited, and the cells maintained good activity and stability during the subsequent culture period (at least 9 days), making them very suitable for use as a feeder layer.

[0047] Example 2: Application of Sf9 Feeder Layer (Prospective / Instructional)

[0048] Prepare a culture dish containing the Sf9 cell feeder layer prepared according to the method of Example 1. Target insect cells (e.g., specific primary insect cells) or insect symbiotic microorganisms (e.g., symbiotic bacteria isolated and purified from insects) that are difficult to culture alone are inoculated at an appropriate density into the culture dish containing the feeder layer and co-cultured using a culture medium suitable for the growth of the target cells / microorganisms.

[0049] In the control group, the target cells / microorganisms are inoculated into a culture dish without a feeder layer. Cultured under appropriate conditions, the growth status, population changes, or specific biological indicators of the target cells / microorganisms are regularly observed.

[0050] The expected results are that, with the support of the Sf9 feeder layer, the growth conditions of the target cells / microorganisms (such as survival rate, proliferation rate, and maintenance time) will be significantly better than those in the control group. This demonstrates that the Sf9 feeder layer prepared in this invention can effectively support the in vitro culture of these difficult-to-cultivate subjects.

[0051] Although specific embodiments of the present invention have been described above, those skilled in the art will appreciate that these specific embodiments are merely illustrative, and that those skilled in the art may omit, substitute, and modify the details of the methods and systems described above without departing from the principles and spirit of the present invention. For example, combining the above method steps to perform substantially the same functions in substantially the same manner to achieve substantially the same results falls within the scope of the present invention. Accordingly, the scope of the present invention is limited solely by the appended claims.

Claims

1. A method for preparing an Sf9 cell feeder layer, characterized in that: The following steps are involved: Step S1: Subculture of Sf9 cells: After resuspending and diluting Sf9 cells with appropriate cell culture medium, inoculate them into a cell culture dish and culture them at an appropriate temperature until the cell confluence reaches a predetermined value; Step S2: Treatment of Sf9 cells with mitomycin C: The cell culture medium in the cell culture dish after culturing in step S1 is replaced with fresh cell culture medium, and mitomycin C solution is added to a final concentration in the culture medium of 80 to 120 μg / mL. After incubation at a temperature that is the same as or similar to that in step S1 for a preset time sufficient to inhibit cell division, the cells are rinsed at least twice with a sterile buffer solution to remove mitomycin C, and fresh cell culture medium is added to prepare an Sf9 cell feeder layer.

2. The method for preparing an Sf9 cell feeder layer according to claim 1, wherein: In step S1, Sf9 cells were diluted to 0.5-1.5×10 6 cells / mL.

3. The method for preparing an Sf9 cell feeder layer according to claim 1, wherein: In step S1, the cell confluence reaches 60% to 80%.

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

5. The method for preparing an Sf9 cell feeder layer according to claim 1, wherein: The incubation time in step S2 is 1.5 to 2.5 hours.

6. The method for preparing an Sf9 cell feeder layer according to claim 1, wherein: In step S2, the sterile buffer solution is phosphate buffered saline (PBS).

7. The method for preparing an Sf9 cell feeder layer according to claim 6, characterized in that: In step S2, the cells were rinsed three times with 1×PBS.

8. The method for preparing an Sf9 cell feeder layer according to claim 1, wherein: The cell culture medium is Sf-900™ IISFM cell culture medium.

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