A method for separating and culturing high-activity primary cells of Apis cerana ovary

Abstract

The application discloses a kind of high activity Apis mellifera ovary primary cell separation culture method, including selecting the virgin queen of Apis mellifera that hatches 48~72h, and the ovary tube middle segment ovicell formation area tissue fragment is used as experimental material;Dissociate cell using the composite mode of collagenase IV combination mechanical stripping, complete cell purification with filtration, low-speed centrifugation.The cell is inoculated, and high-oxygen short-time pretreatment is carried out within 24h after adhering, preferably 40%O2, 5%CO2 Condition processing 2h, can promote cell antioxidant capacity and survival length.Follow-up can be sorted by CFSE fluorescent labeling combined with Fas3, Tj antibody flow, and the primary seed cell of high proliferation activity is enriched.The application is based on APIS culture medium suitable for physiological characteristics of bees, and cell release efficiency can reach more than 70%, cell activity is higher than 85%, and effective survival period is extended to more than 30 days.The application does not introduce exogenous gene and mutagenic reagent, and is high in safety, and the prepared cell can be used for honeybee cell immortalization induction, virology and breeding mechanism and other related research.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, and more specifically to a method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee. Background Technology

[0002] The Chinese honeybee is a unique and superior native pollinating insect in my country, playing an irreplaceable role in maintaining ecosystem homeostasis, pollinating agricultural and forestry crops, and protecting biodiversity. The ovarian cells of the Chinese honeybee can maintain continuous proliferative activity in vivo for 3-5 years. Due to its unique long-term proliferative characteristics, it has become an ideal in vitro research model for exploring the mechanisms of insect cell immortalization, analyzing the pathogenic mechanisms of bee-derived viruses, and protecting and innovatively utilizing bee germplasm resources, possessing extremely high scientific research value and application prospects.

[0003] However, the current in vitro isolation and culture techniques for primary ovarian cells of the Chinese honeybee still have the following shortcomings: On the one hand, cell release efficiency is low. The densely structured brood membrane surrounding the ovarian tissue of the Chinese honeybee forms a physical barrier, preventing the release of functional cells within the ovarian duct. Current conventional preparation methods, often employing simple mechanical tearing or single enzymatic digestion, struggle to overcome this physical barrier and completely dissect the ovarian tissue, thus failing to effectively release the functional cells from the ovarian duct. Existing technologies generally achieve cell release efficiencies below 30%, leaving a large number of viable cells trapped within the ovarian duct tissue, resulting in extremely low cell yields and increasing the risk of cell damage and decreased viability, making it difficult to obtain a sufficient quantity of intact primary cells.

[0004] On the other hand, the mismatch between the in vitro oxygen environment and the ovarian environment triggers rapid oxidative apoptosis in cells. The ovarian tissue of the Chinese honeybee is in a hypoxic microenvironment, with a dissolved oxygen content of approximately 0.28 mg / L, corresponding to an oxygen concentration of about 2% O2. However, existing in vitro culture systems use a conventional atmospheric oxygen environment (21% O2, dissolved oxygen 4.82 mg / L). This significant difference in oxygen environment before and after in vitro culture triggers severe oxidative stress. Within 72 hours of cell in vitro culture, a large surge of reactive oxygen species (ROS) occurs, causing disordered expression of core antioxidant enzymes such as superoxide dismutase (SOD) and catalase (CAT). Simultaneously, the HIF-1α hypoxia adaptation pathway cannot be effectively activated, ultimately leading to rapid senescence and apoptosis of primary cells. This makes it difficult to stably obtain sufficient quantities of highly active primary ovarian cells from the Chinese honeybee, severely limiting the smooth progress of subsequent experiments such as cell immortalization induction, viral mechanism research, and germplasm resource conservation.

[0005] In summary, existing techniques for isolating and culturing primary cells from the ovaries of the Chinese honeybee are inadequate. Therefore, this invention is proposed. Summary of the Invention

[0006] The purpose of this invention is to address the problems mentioned in the background section by providing a method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee. This invention utilizes precise material selection, enzymatic hydrolysis, combined mechanical cell release, and high-oxygen pre-adaptation activation processes to efficiently obtain high-proliferative-potential, highly stable primary ovarian cells of honeybees with high safety.

[0007] A method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee includes the following steps.

[0008] S1. Select virgin queen bees of the Chinese honeybee 48-72 hours after emergence, and dissect and remove tissue fragments from the oocyte formation area in the middle segment of the ovarian duct.

[0009] Specifically, the queen bee ovary is dissected and obtained under a dissecting microscope, and a tissue fragment from the oviduct formation area is excised. Further, the length of the tissue fragment in S1 is 2-3 mm. The tissue fragment consists of stage 5 follicular epithelial cells from the oviduct formation area, and is cubic in shape with an EdU incorporation rate >15%. Compared to the cell proliferation activity of the ovaries of larvae, pupae, virgin queens, first-time queens, and high-producing queens, the stage 5 follicular epithelial cells from the oviduct formation area of ​​a virgin Chinese honeybee 48-72 hours after emergence are in an active state of mitotic proliferation.

[0010] S2. The tissue fragments were placed in APIS medium containing collagenase IV for isothermal enzymatic hydrolysis. After hydrolysis, the perinatal membrane was peeled off to allow the cells to migrate out and obtain a cell suspension. The cell suspension was filtered, centrifuged and the supernatant was discarded. The cell pellet was resuspended in APIS medium to obtain primary ovarian cells.

[0011] Specifically, the enclosing membrane is mechanically peeled off under a dissecting microscope using micro-tweezers, and cells are gently blown out to obtain a cell suspension.

[0012] Furthermore, in S2, the concentration of collagenase IV in APIS medium is 0.5~2 mg / mL, and the enzymatic hydrolysis temperature is 28~32℃ and the time is 20~40 min.

[0013] Furthermore, in S2, the concentration of collagenase IV in APIS medium is 1 mg / mL, and the enzymatic hydrolysis temperature is 32℃ and the time is 30 min.

[0014] Furthermore, in S2, the cell suspension is filtered through a 200-mesh filter, and the centrifugation parameters are 300×g and 5min.

[0015] Furthermore, the APIS medium has a pH of 4.8–5.0 and an osmotic pressure of 450–500 mOsm / kg, using InsectXPRESS or Leibovitz's L-15 as the basal medium, and is supplemented with 10% fetal bovine serum, 3 g / L NaCl, 20 mmol / L glucose, 5 mmol / L trehalose, 0.1 mg / mL royal jelly water extract, 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0016] Furthermore, the pH of the APIS culture medium is adjusted using an HCl solution.

[0017] S3. Primary ovarian cells were seeded in APIS medium and cultured adherently.

[0018] Furthermore, in S3, primary ovarian cells are expressed at a concentration of 1×10⁻⁶. 5 ~5×10 5 cells / cm 2 The inoculation density was determined by inoculating the culture medium with APIS.

[0019] Specifically, the cells were cultured under normoxic conditions of 28-32℃, 5% CO2, and saturated humidity to allow them to adhere fully and resume growth.

[0020] S4. Within 24 hours of primary ovarian cells adhering to the culture vessel, transfer the primary ovarian cells to a three-gas incubator with gas conditions of 38-42% O2, 4-6% CO2 and the remainder N2 for 1-4 hours of pretreatment. After pretreatment, transfer them back to a normal oxygen environment for continued culture.

[0021] Specifically, S4 is a hyperoxia pre-adaptation activation treatment; primary ovarian cells are transferred to a three-gas incubator and subjected to short-term antioxidant pretreatment with a hyperoxia mixed gas. After the pretreatment, the cells are returned to the original normoxic culture environment for continued culture to enhance the cells' antioxidant capacity and in vitro culture stability.

[0022] Furthermore, the isolation and culture method further includes step S5, which is as follows: After pretreatment, primary ovarian cells were fluorescently labeled and incubated with antibodies. Primary seed cells of bee ovaries were obtained by flow cytometry and used for subsequent immortalization induction.

[0023] Furthermore, S5 specifically includes: S51. Pretreated cells were labeled with 5 μmol / L carboxyfluorescein diacetate succinimide, incubated at 32°C for 15 min, washed and resuspended to obtain cell suspension. Among them, carboxyfluorescein diacetate succinimide ester, or CFSE, is a cell proliferation-specific fluorescent dye.

[0024] S52. Add Fas3 antibody and Tj antibody at a volume ratio of 1:200 to the cell suspension and incubate at 4°C in the dark for 30 min. S53, Fas3 was sorted by flow cytometry. + / Tj + / Carboxyfluorescein diacetate succinimide ester strong fluorescent cell population, obtained primary seed cells of bee ovaries.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. This invention limits the sampling target to virgin queen bees of the Chinese honeybee, which have emerged 48-72 hours prior to birth, and specifically selects the oocyte formation area in the middle segment of the ovarian duct as the optimal sampling site. This region has strong cell proliferation capacity and an EdU positivity rate higher than 15%, which can provide high-quality original seed cells for subsequent cell immortalization induction, ensuring the basic performance of cells from the source; 2. This invention employs a combined treatment method of collagenase IV enzymatic hydrolysis and mechanical dissection, replacing traditional tissue block culture or single dissociation methods. Cell release efficiency is increased from less than 30% using traditional methods to over 70%. Trypan blue staining shows that overall cell viability can reach over 85%. The dissociation process causes minimal cell damage, resulting in highly efficient acquisition of highly active single-cell suspensions. 3. This invention is the first to apply high-oxygen short-term preconditioning technology to primary honeybee ovarian cell culture. Experimental results show that after pretreatment with 40% O2 for 2 hours, the total antioxidant capacity of the cells increased by 2.3 times, and the effective survival period of the cells under conventional culture conditions was extended from less than 14 days to more than 30 days, significantly improving the in vitro survival status of the cells. 4. The entire isolation and culture process of this invention does not use viral vectors, exogenous oncogenes, or chemical mutagens. It does not require genetic modification of cells, can completely preserve the natural genetic traits of cells, and effectively avoids phenotypic abnormalities and biosafety risks caused by gene modification. Attached Figure Description

[0026] Figure 1 This is due to the deviation between the traditional culture system and the physiological parameters of the hemolymphocytes of the Chinese honeybee; Figure 2 The effects of different cell types, developmental stages, and culture media on primary honeybee ovarian cell culture; Figure 3 Comparison of queen bee ovarian development stages, cell types, and methods of brood membrane removal; Figure 4 Screening for high oxygen pretreatment concentrations. Detailed Implementation

[0027] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. The materials and instruments used in the following embodiments are all commercially available.

[0028] A method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee includes the following steps.

[0029] S1. Select virgin queen bees of the Chinese honeybee 48-72 hours after emergence, and dissect and remove tissue fragments from the oocyte formation area in the middle segment of the ovarian duct.

[0030] Furthermore, the length of the tissue fragment in S1 is 2-3 mm.

[0031] S2. The tissue fragments were placed in APIS medium containing collagenase IV for isothermal enzymatic hydrolysis. After hydrolysis, the perinatal membrane was peeled off to allow the cells to migrate out and obtain a cell suspension. The cell suspension was filtered, centrifuged and the supernatant was discarded. The cell pellet was resuspended in APIS medium to obtain primary ovarian cells.

[0032] Furthermore, the concentration of collagenase IV in S2 in APIS medium was 0.5~2 mg / mL, and the enzymatic hydrolysis temperature was 28~32℃ and the time was 20~40 min.

[0033] Furthermore, the concentration of collagenase IV in S2 in APIS medium was 1 mg / mL, and the enzymatic hydrolysis temperature was 32℃ and the time was 30 min.

[0034] Furthermore, the cell suspension in S2 was filtered through a 200-mesh filter, and the centrifugation parameters were 300×g and 5min.

[0035] Furthermore, the APIS medium has a pH of 4.8–5.0 and an osmotic pressure of 450–500 mOsm / kg. InsectXPRESS or Leibovitz's L-15 is used as the basal medium, and 10% fetal bovine serum, 3 g / L NaCl, 20 mmol / L glucose, 5 mmol / L trehalose, 0.1 mg / mL royal jelly water extract, 100 U / mL penicillin, and 100 μg / mL streptomycin are added.

[0036] Furthermore, the pH of the APIS medium was adjusted using HCl solution.

[0037] S3. Primary ovarian cells were seeded in APIS medium and cultured adherently.

[0038] Furthermore, primary ovarian cells in S3 were expressed at a rate of 1×10⁻⁶. 5 ~5×10 5 cells / cm 2 The inoculation density was determined by inoculating the culture medium with APIS.

[0039] S4. Within 24 hours of primary ovarian cells adhering to the culture vessel, transfer the primary ovarian cells to a three-gas incubator with gas conditions of 38-42% O2, 4-6% CO2 and the remainder N2 for 1-4 hours of pretreatment. After pretreatment, transfer them back to a normal oxygen environment for continued culture.

[0040] Furthermore, the isolation and culture method also includes S5, which is as follows: After pretreatment, primary ovarian cells were fluorescently labeled and incubated with antibodies. Primary seed cells of bee ovaries were obtained by flow cytometry and used for subsequent immortalization induction.

[0041] Furthermore, S5 specifically refers to: S51. Pretreated cells were labeled with 5 μmol / L carboxyfluorescein diacetate succinimide, incubated at 32°C for 15 min, washed and resuspended to obtain cell suspension. Among them, carboxyfluorescein diacetate succinimide ester, or CFSE, is a cell proliferation-specific fluorescent dye.

[0042] S52. Add Fas3 antibody and Tj antibody at a volume ratio of 1:200 to the cell suspension and incubate at 4°C in the dark for 30 min. S53, Fas3 was sorted by flow cytometry. + / Tj + / Carboxyfluorescein diacetate succinimide ester strong fluorescent cell population, obtained primary seed cells of bee ovaries.

[0043] The present invention will be further described below with reference to specific embodiments and comparative examples.

[0044] Experimental sample: Virgin queen bees of the Chinese honeybee, 48 hours after emergence, provided by the Guangxin Chinese Honeybee Base in Shaoguan, Guangdong.

[0045] Example 1 1) Sample pretreatment: Anesthetize the virgin queen bee of the Chinese honeybee on ice for 5 minutes, disinfect the body surface with 75% ethanol for 2 minutes, and then rinse 3 times with sterile PBS solution to remove residual ethanol and impurities on the body surface. 2) Precise tissue sampling: Under a stereomicroscope, the ovarian tissue of the queen bee is aseptically dissected, and a tissue fragment of the oocyte formation area in the middle segment of the ovarian duct is excised. The tissue fragment is about 2 mm in length. Depend on Figure 3It can be seen that there are significant differences in cell activity in different regions of the ovary: Figure 3 (A) is the anterior reproductive area of ​​the ovarian duct, where the oviduct has not yet developed; Figure 3 (D) is the ovarian duct stalk, where the cell distribution and growth zone differ greatly, making both of these locations unsuitable for sampling. Figure 3 (C) is the anterior segment of the ovarian tube growth zone of a primordial bee, where the cells have entered the differentiation state; Figure 3 (B) The middle section of the ovarian duct growth zone of virgin bees is the optimal sampling site, as the egg chambers in this area are fully developed and rich in proliferative follicular epithelial cells. Therefore, this experiment uniformly harvested tissue fragments from this area, approximately 2 mm in length; 3) Isothermal enzymatic digestion: Immerse the above tissue fragments in APIS medium containing 1 mg / mL collagenase IV and digest at 32°C for 30 min. 4) Cell release: After enzymatic digestion, the tissue is transferred to a dissecting microscope, and the pericellular membrane on the surface of the tissue is mechanically peeled off using micro-forceps. The tissue block is gently blown to promote the full migration of cells and obtain a crude cell suspension. Different methods of removing the perihelion membrane have significantly different effects: such as Figure 3 As shown in (E), the tissue remains clumped after treatment with simple microdissection, and the amount of cells released is limited; this embodiment uses a combined enzymatic-mechanical method, corresponding to... Figure 3 (F) can induce a large number of cells to migrate out, increasing cell release efficiency by 40%; 5) Cell purification and seeding: Filter the crude cell suspension through a 200-mesh sterile filter to remove incompletely dissociated tissue residue; collect the filtrate, centrifuge at 300×g for 5 min, discard the supernatant after centrifugation, and resuspend the cell pellet in fresh APIS medium. Repeat at a rate of 2×10⁻⁶ cells / year. 5 cells / cm 2 At the appropriate seeding density, the cell suspension was seeded into T25 cell culture flasks; 6) High oxygen preconditioning: After 12 hours of routine cell culture, the culture flasks were transferred to a three-gas incubator for short-term preconditioning. The gas composition of the incubator was set to 40% O2, 5% CO2, and 55% N2, and the treatment time was 2 hours. 7) Continued culture: After pretreatment, transfer the culture flask to a standard ambient oxygen incubator (21% O2, 5% CO2) for continued culture.

[0046] The samples in this embodiment were subjected to multi-index detection, with three biological replicates (n=3) set up for each experiment. The detection results are as follows: Cell release efficiency: Single-cell suspensions after enzymatic hydrolysis were collected, the total number of cells was counted, and the cell release efficiency was calculated. The result was 72.5% ± 5.2%. Cell viability: After staining with trypan blue, microscopic observation showed that positively stained cells were dead cells, with the percentage of positive cells being <12%, and the overall cell viability being >88%. Total antioxidant capacity: The total antioxidant capacity of cells was determined by the Trolox equivalent antioxidant capacity method (TEAC method). In this example, the value was 0.68±0.05 mmol / g; the value of the control group without hyperoxia preconditioning was 0.29±0.03 mmol / g, and the antioxidant capacity of the treatment group increased by 2.34 times. Long-term survival ability: Cell morphology and growth status were observed under a microscope daily. Cells maintained normal physiological activity for more than 30 days under normoxic culture conditions.

[0047] Example 2 This embodiment, based on Example 1, involves fluorescent labeling, antibody incubation, and flow cytometry sorting of cultured cells to directionally enrich highly proliferative primary seed cells from bee ovaries. The specific steps are as follows: 8) Cell collection: Take the primary ovarian cells cultured in step 7) of Example 1 and collect the cell suspension for later use; 9) Fluorescent labeling: Add 5 μM of carboxyfluorescein diacetate succinimide to the cell suspension for fluorescent labeling; 10) Antibody incubation: After labeling, wash the cells, add Fas3 antibody and Tj antibody at a volume dilution ratio of 1:200, mix well, and incubate at 4°C in the dark for 30 min. 11) Flow cytometry sorting: The double-labeled cell suspension is loaded onto the flow cytometer to sort Fas3 cells. + / Tj+ / carboxyfluorescein diacetate succinimide ester strong fluorescent cell population was used to obtain primary seed cells from honeybee ovaries.

[0048] Experimental test results: Sorting purity: Target cell population sorting purity > 90%; Proliferation capacity: The primary seed cells of honeybee ovaries obtained by sorting were cultured in APIS medium, and the population doubling time was about 28 hours. Cell proliferation was detected by EdU staining. The EdU positive rate was >25%, indicating that the cells have excellent proliferative activity and can be directly used for subsequent cell immortalization induction experiments.

[0049] Example 3 This example is a process parameter screening experiment to investigate the effects of short-term pretreatment with different oxygen concentrations on the morphology, antioxidant capacity, and survival status of primary ovarian cells of the Chinese honeybee, thereby determining the optimal pretreatment oxygen concentration. Except for the different oxygen concentration settings, the material collection, enzymatic digestion, inoculation, pretreatment duration, and subsequent culture conditions were consistent with those in Example 1. The specific operating steps are as follows: Virgin queen bees of the same batch, 48 hours after emergence, were selected. Following the standardized procedure in Example 1, the cells were collected, enzymatically digested, purified, and inoculated. After 12 hours of cell culture, the cells were divided into four groups for short-term three-gas pretreatment. The pretreatment duration was uniformly 2 hours, with a fixed CO2 concentration of 5% and a nitrogen equilibrium phase as the remaining gas. The oxygen concentrations for the four groups were set to 21%, 30%, 40%, and 50%, respectively. After pretreatment, all cells were transferred to a standard normoxic environment of 21% O2 and 5% CO2 for continuous culture for 30 days.

[0050] For detailed experimental results, please refer to [link / reference needed]. Figure 4 : Cell morphology observation: such as Figure 4 As shown in (A) to (D), cells in the 21%, 30%, and 50% O2 treatment groups showed varying degrees of shrinkage and apoptosis; the cells in the 40% O2 group were plump and had the best growth status. Total antioxidant capacity: such as Figure 4 As shown in (E), the 21% O2 control group had the lowest antioxidant capacity, while the 40% O2 treatment group had a 2.3-fold increase in antioxidant capacity compared to the control group, and its antioxidant capacity was significantly better than the other groups. Cell viability statistics: such as Figure 4 As shown in (F), the 40% O2 pretreatment group had the highest cell survival rate and the longest duration of activity maintenance; In summary, considering cell morphology, total antioxidant capacity, and long-term survival rate, the optimal process conditions for high-oxygen pre-adaptation of this invention were determined to be 40% O2, 5% CO2, and 55% N2, with a pretreatment time of 2 hours.

[0051] Comparative Example 1 The difference between this comparative example and Example 1 is that this comparative example uses a traditional culture system, APIS medium, and the source of experimental samples and sterilization procedures are consistent with Example 1; only the cell isolation and culture methods are changed. The specific procedures are as follows: Virgin queen bees of the same batch, 48 hours after emergence, were selected. Ovarian tissue was randomly cut after disinfection and dissection, and the tissue blocks were directly cultured in adherent form. Collagenase IV enzymatic digestion process, high-oxygen three-gas pretreatment, and flow cytometry were not used. The entire culture was carried out in a normal aerobic environment of 21% O2 and 5% CO2, and the APIS medium was changed every 7 days.

[0052] like Figure 1As shown, there are significant deviations between the conventional culture system and the physiological parameters of the hemolymph of the Chinese honeybee: the conventional culture medium corresponds to 21% O2 and dissolved oxygen of 4.82 mg / L, while the hemolymph of the Chinese honeybee has only about 2% O2 and dissolved oxygen of 0.28 mg / L; the pH of the conventional culture medium is 6.5, while the pH of the hemolymph of the Chinese honeybee is 4.98; the osmotic pressure of the conventional culture medium is about 398 mOsm / kg, while the osmotic pressure of the hemolymph of the Chinese honeybee reaches 553 mOsm / kg, with substantial differences in these three core parameters. Meanwhile... Figure 1 (DE) confirmed that culture temperatures of 28℃ and 32℃ had no significant effect on cell growth. In the figure, APIS is a honeybee-specific culture medium, A. cerana represents the hemolymph of the Chinese honeybee, and H. armigera represents the hemolymph of the cotton bollworm. Combined with... Figure 2 The results also confirmed that, compared with conventional culture media such as L-15, InsectXPRESS, and DMEM, cells cultured in APIS medium exhibited the best cell condition.

[0053] Combination Figure 2 It is evident that different cell types, developmental stages, and culture media have an impact on the culture of primary honeybee ovarian cells; (A) Hemocytes of Italian honeybee larvae, cultured for 120 days in L-15 medium, bar=50 μm; (B) Fat bodies of Italian honeybee pupae, cultured for 120 days in L-15 medium, bar=100 μm; (C) Ovary of a newly laid queen bee of the Chinese honeybee, L-15 medium, bar=50 μm; (D) Ovary of newly laid Chinese honeybee queen, cultured for 10 days in Insect XPRESS medium, bar=50 μm; (E) Ovaries of newly laid Chinese honeybee queens, cultured for 10 to 90 days and 1 day after subculturing, in L-15 medium, bar=100μm; (F) Ovary of the Chinese honeybee pupa, cultured for 10 days in DMEM medium, bar=50 μm; (G) Ovaries of Chinese honeybee pupae, cultured for 10 days in Insect XPRESS medium, bar=50 μm; (H) Ovaries of Apis chinensis pupae, cultured for 10 days in APIS medium, bar=50 μm. The results showed that the cells were in the best condition in APIS medium.

[0054] Experimental results: The culture system contained a large number of tissue clumps, with low single-cell release and a high proportion of contaminating cells; cell viability was significantly low, with trypan blue staining positivity rate >35% and cell viability less than 65%; the total antioxidant capacity of cells was only 0.29±0.03mmol / g; the cells maintained normal viability for less than 15 days, and it was impossible to obtain highly active single cells and proliferating seed cells.

[0055] The performance of primary ovarian cells of Honeybee chinensis under two different culture processes, Example 1 and Comparative Example 1, is compared and analyzed below. The performance data are shown in Table 1. Table 1 Performance Data Comparative Example 1 followed the traditional tissue block culture method, which resulted in highly random tissue sampling and the absence of enzymatic hydrolysis to dissociate tissues. This led to the presence of numerous tissue clumps in the system, making it difficult to obtain a sufficient number of single cells. Figure 1 It was found that the conventional normoxic culture environment deviated significantly from the physiological parameters of the hemolymph of the Chinese honeybee, such as dissolved oxygen, pH, and osmotic pressure. Combined with the defects in the culture method, this ultimately resulted in cell viability of less than 65%, a total antioxidant capacity of only 0.29±0.03 mmol / kg, and an effective cell survival time of less than 15 days. Although this group used APIS medium suitable for bees (…),… Figure 2 This culture medium has been proven to be superior to conventional media such as L-15 and DMEM, but due to limitations in the isolation and culture process, the overall cell growth status is poor.

[0056] Embodiment 1 of the present invention is based on Figure 3 The validated sampling area, combined with an enzymatic and mechanical peeling method, along with a gentle collagenase IV enzymatic hydrolysis process, achieved highly efficient single-cell preparation. Simultaneously, a short-term high-oxygen pretreatment, screened in Example 3, optimized cell metabolism and stress resistance. Ultimately, the cell release efficiency reached 72.5±5.2%, cell viability exceeded 88%, total antioxidant capacity increased to 0.68±0.05 mmol / kg, and cells could stably survive for more than 30 days.

Claims

1. A method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee, characterized in that, Includes the following steps: S1. Select virgin queen bees of the Chinese honeybee 48-72 hours after emergence, and dissect and dissect them to obtain tissue fragments of the oocyte formation area in the middle segment of the ovarian duct. S2. The tissue fragments were placed in APIS medium containing collagenase IV for isothermal enzymatic hydrolysis. After the enzymatic hydrolysis was completed, the perinatal membrane was peeled off to allow the cells to migrate out and obtain a cell suspension. The cell suspension was filtered, centrifuged and the supernatant was discarded. The cell pellet was resuspended in APIS medium to obtain primary ovarian cells. S3. Primary ovarian cells were seeded in APIS medium and cultured adherently. S4. Within 24 hours of primary ovarian cells adhering to the culture vessel, transfer the primary ovarian cells to a three-gas incubator with gas conditions of 38-42% O2, 4-6% CO2 and the remainder N2 for 1-4 hours of pretreatment. After pretreatment, transfer them back to a normal oxygen environment for continued culture.

2. The method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee as described in claim 1, characterized in that, The length of the tissue fragment in S1 is 2~3mm.

3. The method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee as described in claim 1, characterized in that, In S2, the concentration of collagenase IV in APIS medium is 0.5~2 mg / mL, and the enzymatic hydrolysis temperature is 28~32℃ and the time is 20~40 min.

4. The method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee as described in claim 3, characterized in that, In S2, the concentration of collagenase IV in APIS medium was 1 mg / mL, and the enzymatic hydrolysis temperature was 32℃ and the time was 30 min.

5. The method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee as described in claim 1, characterized in that, In S3, primary ovarian cells were used at a concentration of 1×102 5 ~5×10 5 cells / cm 2 The inoculation density was determined by inoculating the culture medium with APIS.

6. The method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee as described in claim 1, characterized in that, The APIS medium has a pH of 4.8–5.0 and an osmotic pressure of 450–500 mOsm / kg. It uses Insect XPRESS or Leibovitz's L-15 as the basal medium and is supplemented with 10% fetal bovine serum, 3 g / L NaCl, 20 mmol / L glucose, 5 mmol / L trehalose, 0.1 mg / mL royal jelly water extract, 100 U / mL penicillin, and 100 μg / mL streptomycin.

7. The method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee as described in claim 6, characterized in that, The pH of the APIS culture medium was adjusted using an HCl solution.

8. The method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee as described in claim 1, characterized in that, The cell suspension in S2 was filtered through a 200-mesh filter, and the centrifugation parameters were 300×g and 5min.

9. The method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee as described in claim 1, characterized in that, The isolation and culture method further includes S5, which is as follows: After pretreatment, primary ovarian cells were fluorescently labeled and incubated with antibodies. Primary seed cells of bee ovaries were obtained by flow cytometry and used for subsequent immortalization induction.

10. The method for isolating and culturing highly active primary ovarian cells of the Chinese honeybee as described in claim 9, characterized in that, Specifically, S5 is: S51. Pretreated cells were labeled with 5 μmol / L carboxyfluorescein diacetate succinimide, incubated at 32°C for 15 min, washed and resuspended to obtain cell suspension. S52. Add Fas3 antibody and Tj antibody at a volume ratio of 1:200 to the cell suspension and incubate at 4°C in the dark for 30 min. S53, Fas3 was sorted by flow cytometry. + / Tj + / Carboxyfluorescein diacetate succinimide ester strong fluorescent cell population, obtained primary seed cells of bee ovaries.

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