Osmotic pressure-nutrition dual-adaptive marine invertebrate primary cell culture method

Abstract

The invention establishes an osmotic pressure-nutrition dual-adaptive marine invertebrate primary cell culture method, and systematically reveals and solves two key technical problems of osmotic pressure adaptation mismatch and nutrition supply mismatch generally existing in a marine invertebrate primary cell culture process for the first time. A dynamic osmotic pressure compensation mechanism with universality and adjustability and a species-specific nutrition supply system are constructed. By introducing a gradient osmotic pressure regulation and control module, the osmotic pressure of a culture system can be continuously adjusted within the range of 600-1100 mOsm / L, and the technical bottleneck that the osmotic pressure of a traditional culture medium is fixed and the adaptation range is limited is broken through; meanwhile, by combining a nutrition compound formula taking homologous hemolymph as a core, the metabolic requirements of marine invertebrate cells are accurately matched, and cell damage and death caused by osmotic stress and insufficient nutrition are effectively relieved. Therefore, the survival rate and stability of primary cell culture are remarkably improved, and a primary cell culture method capable of maintaining cell activity and biological functions for a long time is established.

Description

Technical Field

[0001] This invention belongs to the interdisciplinary field of marine biotechnology and cell engineering, and specifically relates to a method for primary cell culture of marine invertebrates with dual osmotic pressure-nutrient adaptation. Background Technology

[0002] Marine invertebrates are a core group of marine biodiversity, encompassing more than 30 phyla including mollusks, arthropods, and cnidarians, accounting for over 95% of all marine organisms. Long-term evolutionary adaptation has led to highly diverse and species-specific physiological regulatory mechanisms to cope with complex and ever-changing marine environmental conditions. From the frequent and dramatic salinity fluctuations in the intertidal zone to the long-term stability of osmotic pressure in the deep sea, marine invertebrates have evolved adaptive strategies in osmotic pressure regulation and nutrient metabolism that are significantly different from those of terrestrial and freshwater organisms.

[0003] At the level of osmotic regulation, marine invertebrates generally rely on sophisticated ion transport systems and compatible solute dynamic balance mechanisms to maintain cellular homeostasis, and their tolerable osmotic pressure range typically spans 200–1200 mOsm / L. Species in different ecological niches exhibit significant differences in the molecular composition of osmotic regulation. For example, intertidal mussels buffer changes in external osmotic pressure by accumulating large amounts of taurine (with intracellular concentrations reaching up to 450 mM), while deep-sea cephalopods rely on trimethylamine N-oxide (TMAO) to stabilize protein conformation and the intracellular environment.

[0004] In terms of nutritional metabolism, marine invertebrates also exhibit highly species-specific requirements. Filter-feeding bivalves (such as oysters) are highly dependent on polyunsaturated fatty acids, with docosahexaenoic acid (DHA) typically accounting for over 22% of their cell membrane lipid composition; while carnivorous cephalopods rely on amino acids such as arginine and proline as important energy metabolic substrates. These differences in essential amino acid profiles, lipid metabolic pathways, and energy supply methods make it difficult for universal culture systems to meet the physiological needs of diverse marine invertebrate cells.

[0005] Due to the factors mentioned above, the precise matching of osmotic pressure conditions and essential nutrients has become a key bottleneck restricting the primary cell culture of marine invertebrates. Although there have been sporadic research reports on the primary cell culture of marine invertebrates in recent years, a comprehensive culture method with broad adaptability and systematic optimization strategies is still lacking overall. The osmotic pressure of existing commercial or commonly used cell culture media is usually stable at 300–320 mOsm / L, and their ionic composition and nutrient ratios are mainly designed based on the needs of mammalian cells, which is seriously mismatched with the physiological characteristics of marine invertebrates, resulting in low primary cell adhesion, short survival time, and poor functional maintenance.

[0006] In conclusion, constructing a highly adaptable culture system that can simultaneously address osmotic pressure regulation and species-specific nutritional needs is a crucial prerequisite for overcoming the bottleneck in long-term primary cell culture technology for marine invertebrates. Solving this problem will provide important technical support for cell biology research, functional gene validation, and the efficient development of marine bioactive substances in marine invertebrates. Summary of the Invention

[0007] Based on the above reasons, the purpose of this invention is to provide a primary cell culture method for marine invertebrates with dual osmotic pressure and nutrient adaptation. This method can provide a standardized primary cell culture method for marine invertebrates with high osmotic pressure range and interspecies nutritional heterogeneity, and provide a cell-level verification method for the analysis of economic traits of marine invertebrates and the development of new marine drugs.

[0008] To achieve the above objectives, the present invention discloses a method for primary cell culture of marine invertebrates with osmotic pressure-nutrient dual adaptation, comprising the following steps:

[0009] Determination of osmotic pressure in marine invertebrates: Using an osmotic pressure measuring device, the osmotic pressure values ​​of the external environment and internal environment (tissue fluid) of marine invertebrates were measured to provide a basis for setting the osmotic pressure of primary cell culture systems.

[0010] Preparation of primary cell culture solutions for marine invertebrates: An optimized basic cell culture solution (MBSS) of approximately 1100 mOsm / L was prepared, comprising: sodium chloride 26.22 mg / mL, potassium chloride 1.08 mg / mL, magnesium chloride hexahydrate 4.7 mg / mL, magnesium sulfate heptahydrate 6.52 mg / mL, sodium dihydrogen phosphate dihydrate 0.058 mg / mL, calcium chloride dihydrate 1.48 mg / mL, glucose 0.3 mg / mL, and a mixture of L15 and M199 culture media at a volume ratio of 3:1 and concentrated to 600 mOsm / L. Based on this, by controlling the mixing ratio of the two solutions, an isotonic primary cell culture medium for marine invertebrates with an osmotic pressure range of 600–1100 mOsm / L was obtained to meet the osmotic pressure adaptation requirements of different species and tissues.

[0011] Preparation of essential nutrients for marine invertebrates: Tissue fluid was drawn from the open circulatory system of marine invertebrates using a 1 mL sterile syringe and immediately placed on ice to prevent coagulation. The mixture was centrifuged at 600 g for 5 minutes at 4 °C, the cell pellet was discarded, and the supernatant was collected. The supernatant was then heat-inactivated in a 56 °C constant temperature water bath, filtered through a 0.22 μm filter membrane, and stored at −20 °C as an essential nutrient for primary cell culture.

[0012] Primary cell culture of marine invertebrates: Under aseptic conditions, the marine invertebrates to be dissected were washed and disinfected by immersion in 70% ethanol, then air-dried in a sterile fume hood. The tissues to be cultured were placed in MBSS solution containing 200 U / mL penicillin-streptomycin and 2.5 μg / mL amphotericin B. The tissues were minced and collected using sterilized dissecting tools. At room temperature, the tissues were processed in a constant temperature shaker at 50 rpm, followed by centrifugation at 600 g for 5 minutes. The supernatant was discarded, and 750 μL of disodium EDTA solution containing 0.25% trypsin was added. The cells were dissociated at room temperature and 50 rpm for 15 minutes. Then, an equal volume of isotonic primary cell culture medium and 10% fetal bovine serum were added to terminate the enzymatic digestion. The cells were allowed to stand for another 10 minutes, and after centrifugation at 600 g, the supernatant was discarded. The MBSS solution containing 7.5% fetal bovine serum and 2.5% homologous hemolymph was cultured. The solution was used as the growth medium. After adding 200 U / mL penicillin-streptomycin, it was inoculated into T flasks or cell culture plates together with the primary cell culture medium containing explants for culture. During the culture process, the growth medium was replaced every 10 days or when the medium showed obvious discoloration (pH decrease).

[0013] The beneficial effects of this invention are as follows:

[0014] This invention addresses two major technical bottlenecks in primary cell culture of marine invertebrates: osmotic pressure mismatch and nutrient supply mismatch. It innovatively constructs a dynamic osmotic pressure compensation mechanism and a species-specific nutrient supply system. By overcoming the limitations of traditional culture medium osmotic pressure ranges (continuously adjustable from 600-1100 mOsm / L) through a gradient osmotic pressure regulation module, and combining this with a homologous nutrient compound formula to precisely match cell metabolic characteristics, it solves the problem of low cell viability and activity caused by osmotic stress and nutrient deficiency, establishing a culture system that can stably maintain cell function. This technical system forms a standardized operating framework, significantly improving the reliability of cell experimental data. It provides a scalable technical platform for research on the cell mechanisms of marine invertebrates, drug screening, and germplasm resource conservation, filling the gap in systematic culture protocols in this field. Attached Figure Description

[0015] Figure 1 Osmotic pressure determination of oyster tissue fluid, artificial seawater, and mixed culture medium.

[0016] Figure 2 Image of primary cell culture of oyster heart

[0017] Figure 3 A shows the survival rate of oyster heart cells before optimization of nutritional conditions and osmotic pressure, analyzed by flow cytometry; B shows the survival rate of oyster heart cells under 900 mOsm / L conditions, analyzed by flow cytometry with 7.5% fetal bovine serum and 2.5% oyster hemolymph.

[0018] Figure 4 Survival rate of oyster heart cells under different nutrient formulations and densities at 900 mOsm / L Detailed Implementation

[0019] The present invention will be explained below with reference to embodiments. Those skilled in the art will understand that the following embodiments are only used to further illustrate the present invention and should not be construed as limiting the scope of protection of the present invention.

[0020] The first embodiment of the present invention, taking the primary cell culture method of heart tissue of Pacific oyster as an example, discloses a method for primary cell culture of marine invertebrates with dual osmotic pressure-nutrient adaptation, including the following steps:

[0021] Preparation of isotonic primary cell culture medium for Pacific oysters: The osmotic pressure of oyster tissue fluid, artificial seawater, and L15 and M199 mixed culture medium was measured using a Fiske Associates 210 osmometer. Figure 1The results showed that the osmotic pressure of the oyster tissue fluid was 897 mOsm / L, while that of artificial seawater was 828 mOsm / L. Based on this, a cell culture solution (MBSS) containing sodium chloride (26.22 mg / mL), potassium chloride (1.08 mg / mL), magnesium chloride hexahydrate (4.7 mg / mL), magnesium sulfate heptahydrate (6.52 mg / mL), sodium dihydrogen phosphate dihydrate (0.058 mg / mL), calcium chloride dihydrate (1.48 mg / mL), and glucose (0.3 mg / mL) was prepared, with an osmotic pressure of 1097 mOsm / L. Simultaneously, L15 medium and M199 medium were mixed at a 1:1 volume ratio and concentrated by one-fold to obtain a culture medium with an osmotic pressure of 607 mOsm / L. Subsequently, the MBSS solution was mixed with the concentrated L15 / M199 mixed culture medium at a volume ratio of 3:2 to obtain an isotonic primary cell culture medium of approximately 900 mOsm / L that is suitable for the osmotic pressure characteristics of oyster tissue.

[0022] Preparation of homologous nutrients from Pacific oysters: Homologous hemolymph from Pacific oysters was extracted from the pericardial cavity using a 24-gauge sterile syringe and immediately placed on ice to prevent coagulation. The samples were centrifuged at 600g for 5 minutes using a pre-cooled centrifuge at 4℃, cell particles were discarded, and the supernatant was inactivated in a 56℃ water bath for 40 minutes. After filtration through a 0.22 μm filter, the samples were stored at -20℃ for later use.

[0023] Primary cell culture of Pacific oyster: Pacific oysters used for primary cell culture were thoroughly washed in artificial seawater, then disinfected by immersion in 70% ethanol for 30 seconds, and air-dried naturally in a sterile fume hood. Heart tissue from Pacific oysters was collected and placed in MBSS solution containing 200 U / mL penicillin-streptomycin and 2.5 μg / mL amphotericin B. The tissue was separated, collected, and minced using ethanol-sterilized dissecting tools. The treated tissue was placed in a constant-temperature shaker and sterilized at 50 rpm at room temperature. Subsequently, it was centrifuged at 600g for 5 min at room temperature, and the supernatant was discarded. 750 μL of EDTA disodium solution containing 0.25% trypsin was added to the precipitate, and the tissue was dissociated for 15 min at room temperature and 50 rpm in a constant-temperature shaker. After dissociation, 750 μL of isotonic primary cell culture medium containing 10% fetal bovine serum was added to terminate the enzymatic reaction, and the mixture was allowed to stand for another 10 min. The cells were then centrifuged at 600g at room temperature, and the supernatant was discarded. During the culture phase, fetal bovine serum and homologous hemolymph were mixed and added to MBSS at different volume ratios, making the total added amount 10% of the MBSS volume. 200 U / mL penicillin-streptomycin was added to create experimental groups with different cell densities and nutrient levels. Each experimental group was replicated three times and, together with the explants from the heart of the Pacific oyster, was colonized in 12-well cell culture plates and cultured at 20°C. Figure 2 ).

[0024] Optimal conditions for primary oyster cell culture were investigated: Cells cultured for 7 days were removed from the incubator, and suspended and adherent cells were collected and mixed. After centrifugation, the supernatant was discarded. 750 μL of Calcein / PI cytotoxicity assay reagent was added to each cell group, and the cells were incubated at 37 ℃ for 40 min. After incubation, the cells were washed twice with PBS buffer to remove unbound fluorescent reagent. After single-channel gain correction, flow cytometry was performed on each sample using FITC-A and PE channels. Figure 3 The study also analyzed the cell mortality rates in different experimental groups. Results showed that under the tested conditions, the optimal cell death rate was [missing information - likely related to cell death rates]. The culture system osmotic pressure was approximately 900 mOsm / L, the culture medium consisted of 7.5% fetal bovine serum and 2.5% oyster homologous hemolymph, and the cell seeding density was 1 × 10⁻⁶ cells / L. 6 At a cell count of 1,000 cells, primary oyster cells exhibited the lowest mortality rate and optimal survival, thus determining the above conditions as the optimal parameter combination for primary oyster cell culture. Figure 4 ).

[0025] This invention establishes a dual-adaptive osmotic pressure and nutrient supply method for primary marine invertebrate cell culture. It systematically reveals and solves for the first time two key technical problems commonly encountered in primary marine invertebrate cell culture: osmotic pressure mismatch and nutrient supply mismatch. A dynamic osmotic pressure compensation mechanism and a species-specific nutrient supply system with both universality and adjustability are constructed. By introducing a gradient osmotic pressure regulation module, the osmotic pressure of the culture system can be continuously adjusted within the range of 600–1100 mOsm / L, overcoming the technical bottleneck of fixed osmotic pressure and limited adaptation range in traditional culture media. Simultaneously, combined with a nutrient complex formula centered on homologous hemolymph, the metabolic needs of primary marine invertebrate cells are precisely matched, effectively alleviating cell damage and death caused by osmotic stress and nutrient deficiency. Therefore, this invention significantly improves the survival rate and stability of primary cells, establishing a primary cell culture method capable of maintaining cell viability and biological function over the long term.

[0026] The paradigmatic primary cell culture framework formed by this technology is applicable to most marine invertebrates, which can significantly improve the exploration of marine invertebrate diversity. It provides a scalable technical platform for cell mechanism, drug screening and germplasm resource protection, and fills the limitations of systematic culture protocols in this field.

[0027] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; the preparation of MBSS solutions, etc., has been proposed, and the present invention protects applicable solutions to the above-mentioned problems of osmotic pressure and nutrient formulation. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions of the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for primary cell culture of marine invertebrates with osmotic pressure-nutrient dual adaptation, characterized in that, Includes the following steps: (1) Determination of osmotic pressure of marine invertebrates: Using an osmotic pressure measuring device, the osmotic pressure values ​​of the external environment and internal environment (tissue fluid) of marine invertebrates were measured to provide a basis for setting the osmotic pressure of the primary cell culture system. (2) Preparation of primary cell culture solution for marine invertebrates: Prepare an optimized cell culture basal solution (MBSS) of approximately 1100 mOsm / L, which consists of: sodium chloride 26.22 mg / mL, potassium chloride 1.08 mg / mL, magnesium chloride hexahydrate 4.7 mg / mL, magnesium sulfate heptahydrate 6.52 mg / mL, sodium dihydrogen phosphate dihydrate 0.058 mg / mL, calcium chloride dihydrate 1.48 mg / mL, glucose 0.3 mg / mL, and L15 and M199 culture medium mixed at a volume ratio of 3:1 and concentrated to 600 mOsm / L. Based on this, by controlling the mixing ratio of the two solutions, an isotonic primary cell culture medium for marine invertebrates with an osmotic pressure range of 600–1100 mOsm / L is obtained to meet the osmotic pressure adaptation requirements of different species and tissues. (3) Preparation of essential nutrients for marine invertebrates: Tissue fluid was extracted from the open tubular circulation system of marine invertebrates using a 1 mL sterile syringe and immediately placed on ice to prevent coagulation. The mixture was centrifuged at 600 g for 5 minutes at 4 °C, the cell pellet was discarded, and the supernatant was collected. The supernatant was then heat-inactivated in a constant temperature water bath at 56 °C, filtered through a 0.22 μm filter membrane, and stored at −20 °C as an essential nutrient for primary cell culture. (4) Primary cell culture of marine invertebrates: Under aseptic conditions, the marine invertebrates to be dissected were cleaned and disinfected by soaking in 70% ethanol. After being air-dried in a sterile fume hood, the tissues to be cultured were placed in MBSS solution containing 200 U / mL penicillin-streptomycin and 2.5 μg / mL amphotericin B. The tissues were minced and collected using sterilized dissecting tools. At room temperature, the tissues were placed in a constant temperature shaker at 50 rpm and then centrifuged at 600 g for 5 minutes. The supernatant was discarded, and 750 μL of disodium EDTA solution containing 0.25% trypsin was added. The cells were dissociated at 50 rpm for 15 minutes at room temperature. Then, an equal volume of isotonic primary cell culture medium and 10% fetal bovine serum were added to terminate the enzymatic reaction. The cells were allowed to stand for another 10 minutes. After centrifugation at 600 g, the supernatant was discarded. MBSS solution containing 7.5% fetal bovine serum and 2.5% homologous hemolymph was used as the growth medium, and 100 μL of isotonic primary cell culture medium was added. After administering U / mL penicillin-streptomycin, the culture medium was inoculated together with the primary cell culture medium containing explants into T-flasks or cell culture plates for culture. During the culture process, the growth medium was replaced every 10 days or when the culture medium showed obvious discoloration (pH decrease).

2. The method for primary cell culture of marine invertebrates with osmotic pressure-nutrient dual adaptation according to claim 1, characterized in that: The isotonic primary cell culture medium described in step (2) achieves continuous adjustment of the osmotic pressure of the culture system within the range of 600–1100 mOsm / L by mixing 1100 mOsm / L MBSS with 600 mOsm / L.

3. The method for primary cell culture of marine invertebrates with osmotic pressure-nutrient dual adaptation according to claim 1, characterized in that: In step (2), the MBSS solution removes sodium bicarbonate, which is provided by M199 medium. This supplements a more diverse range of nutrients while weakening the typical bicarbonate-carbon dioxide buffer system of mammals. Furthermore, by increasing the proportion of L15 medium, the phosphate-organic buffer system specific to aquatic organisms is strengthened, thus better adapting to the carbon dioxide-free primary cell culture conditions for marine invertebrates.

4. The method for primary cell culture of marine invertebrates with osmotic pressure-nutrient dual adaptation according to claim 1, characterized in that: In step (3), the homologous tissue fluid from open-tube circulating marine invertebrates is used as a nutrient supplement in combination with the L15 and M199 mixed culture medium prepared in step (2). This can increase the diversity of nutrients in the culture system and precisely match the metabolic needs of primary marine invertebrate cells, thereby significantly improving the survival rate of primary cells.

5. The method for primary cell culture of marine invertebrates with osmotic pressure-nutrient dual adaptation according to claim 1, characterized in that: In step (2), the L15 and M199 mixed culture medium is concentrated by one-fold, which increases the specific gravity of nutrients in the mixture when mixed with MBSS with an osmotic pressure of approximately 1100 mOsm / L, while simultaneously extending the dynamic adjustment range of osmotic pressure to 600–1100 mOsm / L. When the osmotic pressure of the culture system is below 600 mOsm / L, precise compensation and dynamic adjustment of osmotic pressure can be achieved by adjusting the concentration factor of the L15 and M199 mixed culture medium.

6. The method for primary cell culture of marine invertebrates with osmotic pressure-nutrient dual adaptation according to claim 1, characterized in that: The 200 U / mL penicillin-streptomycin and 2.5 μg / mL amphotericin B mentioned in step (4) are used to inactivate bacteria and fungi before tissue dissociation; while in the mixed culture medium, only 100 U / mL penicillin-streptomycin is added as an antibacterial agent to reduce bacterial contamination, and at the same time removes amphotericin B, thereby reducing its inhibitory effect on cell proliferation.

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