Bee cell culture medium and preparation method thereof

Abstract

The invention discloses a bee cell culture medium and a preparation method thereof, and relates to the technical field of cell culture, and the survival rate (50.2%) and the activity period (90 days) of bee primary cells are remarkably improved by simulating the osmotic pressure (450-500mOsm / kg), pH (4.5-5.0) and disaccharide metabolism characteristics (glucose: trehalose = 1: 4) of bee hemolymph. The APIS culture medium greatly improves the adherence efficiency of primary cells, establishes a novel platform for in-vitro study of virus-ovary interaction, is suitable for in-vitro culture, immortalized induction and virology model construction of bee ovary cells, solves the problem of rapid cell aging caused by mismatch of physiological parameters of traditional culture genes, and has a good application prospect. The method provides key technical support for swarm health regulation and control, and has important scientific significance and application prospects.

Description

Technical Field

[0001] The present invention relates to the technical field of cell culture, and more particularly to a honeybee cell culture medium and a preparation method thereof. Background Art

[0002] Honey bees are key pollinators that maintain a balanced agricultural ecosystem. The development of their queen bee's ovaries directly impacts the colony's reproductive capacity. When a honey bee in a colony is infected with a virus, it not only harms the health of individual bees but also affects the development of the queen bee's ovaries, further impacting reproductive capacity. Honey bee cell culture medium, a nutrient mixture specifically designed to support the growth and reproduction of honey bee cells in vitro, plays a crucial role in honey bee research.

[0003] However, current bee cell culture still has the following problems:

[0004] 1. The survival rate of cells in vitro culture is low (less than 50%), which not only affects the reliability of research data, but also leads to poor cell adhesion ability, which in turn affects the cell cycle division and ultimately leads to a decrease in cell proliferation rate;

[0005] 2. The functional activity window is short, and the cell survival time is usually less than 7 days, which makes it difficult to meet the requirements of long-term experiments such as gene editing and viral infection, and also affects the accuracy and reproducibility of experimental results;

[0006] 3. Traditional insect culture media are not adapted to the unique metabolic characteristics of bees and cannot supply energy based on the unique metabolic characteristics of honey, which will lead to an imbalance in cellular energy homeostasis. Among them, the trehalose (a disaccharide composed of two molecules of glucose) in the bee hemolymph is about 50-100mM (accounting for 9.6%-16.4% of soluble sugars), while the trehalose content in the hemolymph of most insects (such as desert locusts and silkworms) can reach 80%-90%, with a concentration as high as 150-200mM. This provides bee cells with an energy supply mechanism that is different from directly using glucose.

[0007] Therefore, it is necessary to propose a honeybee cell culture medium and a preparation method thereof to solve the above problems. Summary of the Invention

[0008] The present invention aims to solve the problems existing in the existing honeybee cell culture technology mentioned in the background technology. To this end, the present invention provides a honeybee cell culture medium and a preparation method thereof.

[0009] In order to achieve the above-mentioned purpose, the present invention specifically adopts the following technical solutions:

[0010] A honey bee cell culture medium and a preparation method thereof, wherein the culture medium comprises the following components: 10% fetal bovine serum, 3g / L NaCl, 20mM glucose, 5mM trehalose, 0.1mg / ml royal jelly water extract, 100U / mL penicillin, and 100μg / mL streptomycin;

[0011] The parameters of the culture medium are: osmotic pressure of 450-500mOsm / kg, pH of 4.5-5.0.

[0012] Preferably, the culture medium is one of Insect-XPRESS, Sf900 II SFM, DMEM, and Leibovitz's L-15.

[0013] Preferably, the osmotic pressure of the culture medium is preferably 480 mOsm / kg; and the pH is preferably 4.8.

[0014] Preferably, the molar ratio of glucose to trehalose is 1:4.

[0015] A method for preparing honey bee cells comprises the following steps:

[0016] S1. Collection: Ovaries, fat body tissue, and blood cells were collected from healthy queen bees or worker bees. The cells were separated and inoculated into cell culture flasks with breathable caps.

[0017] S2. Culture: Freshly isolated tissue cells are placed in a special insect cell culture medium containing specific nutrients for adherent culture at a temperature of 25°C-32°C. The culture medium is replaced regularly every 7-10 days during the culture process. It has been measured that the tissue cells can survive for 90 days.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1. By simulating the physiological environment parameters of bees, adjusting the osmotic pressure and pH value, and combining it with bionic disaccharide culture medium, not only did we solve the problems of low primary cell adhesion efficiency and short survival period, extending the survival period from the original 7 days to 90 days, but we also established a new platform for in vitro research on virus-ovary interactions, providing key technical support for the regulation of bee colony health.

[0020] 2. Physiological adaptability: Precisely matches the osmotic pressure and pH value of bee hemolymph, effectively alleviating osmotic stress damage; Metabolic optimization: The combination of glucose and trehalose simulates the unique sugar metabolism characteristics of bees, ensuring a stable supply of cellular energy; Functional enhancement: Royal jelly extract provides bee-derived growth factors (such as juvenile hormone analogs) to activate pathways related to ovarian development. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1This is a comparison chart of the physical and chemical parameters of the hemolymph of the Chinese honeybee and the APIS culture medium in the present invention;

[0022] Figure 2 This is a diagram of the state of the primary ovary cells of the Chinese honeybee in the present invention in APIS culture medium;

[0023] Figure 3 This is a diagram showing the state of the ovaries of the Italian honey bee queen pupa in the comparative example L-15 culture medium of the present invention;

[0024] Figure 4 This is a diagram showing the state of the ovary of the Chinese honey bee queen pupa in the embodiment of the present invention being located in the APIS culture medium. DETAILED DESCRIPTION

[0025] To better simulate the physiological environment of honeybee ovarian cells and ensure cell growth and survival, the culture medium's osmotic pressure, composition, and culture conditions must be optimized. This example describes how to adjust the composition of a specialized culture medium (APIS) based on osmotic pressure and select a culture medium formulation optimal for honeybee ovarian cell growth.

[0026] pH determination: At 25°C, the pH of the hemolymph of the Chinese honey bee was measured using a pH meter (Sartorius PB-10) and showed a significant acidity (pH = 4.38), which was significantly lower than that of cotton bollworm, commonly used insect culture medium Insect-XPRESS (6.51), and commonly used mammalian culture medium DMEM (pH = 8.97). Distilled water (pH = 7.01) was used as a control (reference Figure 1 B).

[0027] Osmotic pressure measurement: At 25°C, the osmotic pressure of honey bee hemolymph was measured using an osmometer (Beijing Yasenbo Science Instrument Co., Ltd., YASNOsmo210). The results showed that it was about 550mOsm / kg. This value is significantly higher than that of commonly used insect culture media (such as Insect-XPRESS) and mammalian culture media (such as DMEM) (reference Figure 1 D).

[0028] Adjustment of physiological saline concentration: In order to simulate the osmotic pressure environment in the bee body, Ringer's physiological saline with a concentration of 2 times was prepared (refer to Figure 1 C, Figure 2 B) to ensure that the osmotic pressure of the solution during dissection is close to 550 mOsm / kg.

[0029] Optimization of culture medium composition: We tried a variety of honey bee cell culture media, including Insect-XPRESS, L-15, and DMEM. By comparing the cell adhesion rate and survival rate (7 days) in different culture media, we found that the improved APIS was most suitable for the growth of honey bee ovary cells (refer to Figure 2 , where A: 1×Ringer's, 2h, bar=50μm; B: 2×Ringer's, 2h, bar=100μm; C: L-15, 10 days, bar=50μm; D: DMEM medium, bar=50μm; E: Insect-XPRESS medium, bar=50μm; F: APIS special medium, bar=50μm).

[0030] APIS formulation: Modified Insect-XPRESS medium was supplemented with the following ingredients: 10% fetal bovine serum (FBS); 3 g / L NaCl (to increase osmotic pressure), 20 mM glucose, 5 mM trehalose (to simulate the sugar metabolism characteristics of honey bee hemolymph, with a molar ratio of trehalose to glucose of 1:4), 0.1 mg / ml royal jelly extract, 100 U / mL penicillin, and 100 μg / mL streptomycin; pH 4.5-5.0 (refer to Figure 1 B).

[0031] Comparison of culture temperature: The culture temperature optimization experiment showed that, using the ovaries of the Italian honey bee pupae as culture materials, the conventional insect cell culture temperature (25℃-28℃) and the recommended temperature (32℃) in the literature had a significant effect on the ovarian tissue adhesion rate (24h) ( Figure 1 D) and primary cell survival rate (14 days) ( Figure 1 E) showed no significant difference.

[0032] Through the above optimization, this embodiment successfully improved the survival rate of honey bee ovarian cells ( Figure 1 D and E), providing a more efficient and stable cell culture platform for the study of bee-related diseases, drug screening and gene editing.

[0033] Figure 1Comparison of physicochemical parameters of honeybee hemolymph and APIS culture medium; A: The dissolved oxygen in insect hemolymph (25℃, YSI-DO200) is significantly lower than that in in vitro cell culture medium; B: The pH of honeybee hemolymph (25℃, SartoriusPB-10) is significantly lower than that of commonly used insect or mammalian culture medium, and the APIS culture medium designed for honeybees is closer to the pH of honeybee hemolymph in vivo; C: The osmotic pressure of honeybees (25℃, YASN Osmo210) is significantly higher than that of commonly used insect or mammalian culture medium, and the APIS culture medium designed for honeybees is closer to the osmotic pressure of honeybee hemolymph in vivo; D: The attachment rate of ovarian tissue of honeybees mellifera at different culture temperatures (24h); E: The survival rate of primary ovarian cells of honeybees mellifera at different temperatures (14 days); Apis cerana: honeybee worker hemolymph; Helicoverpa armigera: hemolymph of cotton bollworm larvae; APIS: honeybee-specific culture medium; Insect-XPRESS: commonly used honeybee cell culture medium (phosphate buffer system); L-15: mammalian culture medium (phosphate buffer system); DMEM: mammalian culture medium (carbonate buffer system); Ringer's: commonly used insect physiological saline; H2O: distilled water.

[0034] Figure 2 Figure 3. Effect of APIS medium on the survival of primary cells from the ovary of the Chinese honeybee. The cell survival period in the APIS medium was extended to 60 days (compared to 21 days in the control group). A: 1× Ringer's medium, 2 hours, bar = 50 μm; B: 2× Ringer's medium, 2 hours, bar = 100 μm; C: L-15, 10 days, bar = 50 μm; D: DMEM medium, bar = 50 μm; E: Insect-XPRESS medium, bar = 50 μm; F: APIS-specific medium, bar = 50 μm).

[0035] Honey bee ovary cell culture

[0036] Example:

[0037] S1. Preparation of APIS medium

[0038] 1. Preparation of base liquid:

[0039] Dissolve glucose (5 mM) and trehalose (20 mM) in deionized water; add NaCl (8.5 g / L), KCl (0.4 g / L), CaCl2 (0.2 g / L), and MgCl2 (0.1 g / L); adjust the pH to 4.8 (HCl titration); and supplement with royal jelly aqueous extract (10% v / v, sterilized by centrifugal filtration).

[0040] 2. Osmotic pressure calibration:

[0041] The osmometer (YASN Osmo210) was used to adjust the pressure to 550 mOsm / kg.

[0042] S2. Cell culture validation

[0043] 1. Collection: Take healthy queen bees, anesthetize them, put them in 75% ethanol for surface disinfection, and dissect the ovarian tissue under a dissecting microscope; cut them into pieces with dissecting scissors and place them in cell culture bottles, 25cm 2 The cell culture flask contains 0.5 mL of primary culture medium, which is APIS. The control medium uses Insect-XPRESS or L-15 medium.

[0044] 2. Adherent culture was performed at 28°C. Cell morphology was observed daily and the culture medium was replaced every 7 days during the culture process.

[0045] 3. The survival period of primary cells reached 90 days (the control group Insect-XPRESS culture medium was 7 days); the 7-day survival rates were 33.6% for Insect-XPRESS and 50.2% for APIS.

[0046] Comparative Example:

[0047] The comparative example uses conventional Italian honey bee primary cells, uses L-15 culture medium, and the culture conditions refer to Figure 3 .

[0048] Figure 3 This image shows an ovary of an Apis mellifera queen pupa, cultured in L-15 medium for 10 to 90 days and one day after passage. (Scale bar: 100 μm) Cells gradually die and cease to proliferate after passage. A: One day after primary culture; B: 14 days after primary culture; C: 25 days after primary culture; D: 75 days after primary culture; E: 90 days after primary culture; F: Cells no longer proliferate after the first passage.

[0049] Figure 4 This is an image of an ovary of a Chinese honey bee queen, cultured in APIS medium for 1 to 60 days. (Scale bar: 100 μm). The cells remain viable throughout the entire culture period. A: 1 day of primary culture; B: 3 days of primary culture; C: 30 days of primary culture; D: 60 days of primary culture.

[0050] It can be seen that the innovative fusion of simulating the physiological environment parameters of bees, adjusting the osmotic pressure and pH, and combining them with bionic disaccharide culture medium has successfully simulated the unique oxygen-energy coupling metabolic mechanism during bee flight. It not only significantly improves the adhesion efficiency of primary cells during culture, effectively prolongs their survival cycle, and solves the inefficiency and instability of cell culture, but also can accurately simulate the complex process of how viruses infect, replicate and potentially affect ovarian function in the bee ovary environment, ensuring the continued prosperity and health of the bee population.

Claims

1. A honey bee cell culture medium, characterized in that The culture medium included the following components: 10% fetal bovine serum, 3 g / L NaCl, 20 mM glucose, 5 mM trehalose, 0.1 mg / ml royal jelly aqueous extract, 100 U / mL penicillin, and 100 μg / mL streptomycin; The parameters of the culture medium are: osmotic pressure of 450-500mOsm / kg, pH of 4.5-5.

0.

2. A honeybee cell culture medium according to claim 1, characterized in that The culture medium is one of Insect-XPRESS, Sf900 II SFM, DMEM, and Leibovitz's L-15.

3. The honey bee cell culture medium according to claim 1, characterized in that The osmotic pressure of the culture medium is preferably 480 mOsm / kg; and the pH is preferably 4.

8.

4. The honey bee cell culture medium according to claim 1, characterized in that The molar ratio of glucose to trehalose is 1:

4.

5. A method for preparing honeybee cells, using the honeybee cell culture medium according to claim 1, characterized in that: The following steps are involved: S1. Collection: Ovaries, fat body tissues and blood cells are collected from healthy queen bees or worker bees, the cells are separated and inoculated into cell culture flasks; S2. Culture: Freshly isolated tissue cells are placed in a special insect cell culture medium containing specific nutrients for adherent culture at a temperature of 25°C-32°C. The culture medium is replaced regularly every 7-10 days during the culture process. It has been measured that the tissue cells can survive for 90 days.

Images