Method for in vitro isolation and culture of limulus hemacyte amebocyte

Abstract

The application discloses a method for in vitro isolation and culture of limulus blood ameba-like cells, and belongs to the field of marine biological cell engineering. The method realizes high-purity (≥95%) and high-activity isolation of cells by minimally invasive blood sampling, composite anticoagulant protection, double-mode gradient centrifugal purification, full-chemical-defined serum-free culture, non-enzymatic subculture and GMP quality control. The cells can be stably subcultured for more than 12 generations, the survival rate is ≥65% in 120 days, and the endotoxin response function does not attenuate. The method is suitable for all limulus species in the world, the damage of blood sampling to the limulus body is extremely low, the sustainable utilization of resources can be realized, the culture system is free of animal-derived components, meets the GMP requirements, is suitable for large-scale production in a bioreactor, effectively solves the bottleneck problems of low purity, short survival, difficult amplification and damage to the limulus body in the prior art, and can be applied to the fields of limulus reagent preparation and natural immune research.

Description

Technical Field

[0001] This invention belongs to the field of marine biological cell engineering and in vitro cell culture technology, specifically relating to an in vitro isolation and culture method for horseshoe crab blood amoeboid cells. It is particularly suitable for the efficient isolation, high activity maintenance, phenotypic fidelity preservation, and long-term stable culture of amoeboid cells (also known as amoeboid cells) from all extant horseshoe crab species worldwide, including the Chinese horseshoe crab, Indian horseshoe crab, American horseshoe crab, and roundtail horseshoe crab. It can be widely applied in biomedical, biopharmaceutical, and ecological conservation fields such as GMP-level large-scale preparation of horseshoe crab reagents, research on innate immune mechanisms, drug toxicity detection, screening of bioactive substances, and protection of endangered marine species. Background Technology

[0002] Horseshoe crabs are ancient arthropods originating over 400 million years ago, often referred to as "living fossils." Only three genera and four species exist today, with the Chinese horseshoe crab and the round-tailed horseshoe crab being Class II protected animals in my country and both listed in the IUCN Red List of Threatened Species. Horseshoe crab blood contains only one type of circulating cell: amoebocyte-like cells. These cells are a core component of the horseshoe crab's innate immune system and contain numerous granules highly sensitive to bacterial endotoxins (lipopolysaccharide, LPS). These granules are the core raw material for preparing Limulus Amoebocyte Lysate (LAL / TAL). Based on its specific cascade coagulation reaction to endotoxins, LAL / TAL has become the gold standard for bacterial endotoxin detection mandated by global pharmacopoeias. It is widely used in the quality control of injectables, medical devices, vaccines, and other products, and has irreplaceable application value in clinical diagnosis and environmental monitoring.

[0003] Currently, obtaining horseshoe crab blood amoeboid cells relies heavily on live cardiac puncture blood collection from wild horseshoe crabs. This method not only causes irreversible damage to the horseshoe crabs, with a mortality rate as high as 10%-30% after blood collection, exacerbating the endangered status of wild horseshoe crab populations, but also results in a limited amount of blood collected per collection, which cannot meet the needs of large-scale industrial production. With increasing global efforts to protect horseshoe crab resources and increasingly stringent regulations on live blood collection, establishing an efficient, stable, and scalable system for the in vitro isolation and long-term culture of horseshoe crab blood amoeboid cells has become a core key to resolving the contradiction between resource scarcity and market demand.

[0004] In recent years, researchers both domestically and internationally have conducted preliminary explorations into the in vitro culture of horseshoe crab blood amoeboid cells, but existing technologies still suffer from the following insurmountable systemic technical defects:

[0005] 1. Imperfect separation methods, resulting in low purity and activity: Existing technologies mostly use single density gradient centrifugation or low-speed centrifugation, which cannot effectively separate amoeboid cells from impurities such as plasma proteins, red blood cell fragments, and connective tissue cells, with separation purity usually below 85%; the centrifugation process does not have a cell protection mechanism, and mechanical damage leads to a cell activity loss rate of over 20%; traditional anticoagulants can only achieve basic anticoagulation and cannot inhibit premature cell degranulation and activation, further reducing separation efficiency and cell activity.

[0006] 2. The culture system has a core bottleneck and cannot achieve long-term functional maintenance: Existing culture systems mostly rely on traditional culture media supplemented with fetal bovine serum and horseshoe crab serum. The serum composition is complex, and there are large batch variations, which can easily introduce exogenous pathogens. Moreover, they lack specific nutrient factors for cell growth, resulting in cell proliferation arrest, degranulation, and lysis after 2-4 weeks of in vitro culture. The cell survival rate after 60 days is only 1.7%-6.8%, which cannot achieve long-term passage. A few serum-free systems have not achieved full chemical limitation and still contain animal-derived components, which cannot meet the GMP production requirements of biopharmaceuticals. In addition, the intracellular toxin response function declines rapidly with passage.

[0007] 3. Passage process is highly damaging and has poor phenotypic fidelity: Existing technologies mostly use trypsin-EDTA for cell digestion and passage. The potent protease will irreversibly damage cell surface receptors and extracellular matrix proteins, resulting in low cell adhesion rate and loss of function after passage, making it impossible to achieve continuous and stable passage.

[0008] 4. Lack of standardization and scalability of processes, and poor industrial adaptability: Existing methods are mostly small-scale laboratory explorations, and no standardized operating procedures have been established for the entire process. The reproducibility of results from different laboratories is poor. The processes have not been designed to adapt to industrial production and cannot be adapted to large-scale production modes such as bioreactors and microcarrier culture, making it difficult to meet the mass production needs of the horseshoe crab reagent industry.

[0009] 5. Incomplete quality control system and insufficient compliance: The existing technology only uses basic trypan blue staining and morphological observation for quality control, and lacks a quantitative detection system for cell viability, purity and functional integrity. It does not set pharmaceutical-grade quality control indicators such as exogenous factors and endotoxin residues, and cannot meet the requirements of pharmacopoeia and regulatory authorities.

[0010] 6. Narrow species applicability and insufficient resource protection: Existing methods are mostly optimized only for a single horseshoe crab species and cannot be adapted to all existing horseshoe crab species in the world; the blood collection method causes great damage to the horseshoe crab body, and no system for the recovery and reuse of horseshoe crab bodies after blood collection has been established, which cannot fundamentally alleviate the protection pressure on wild horseshoe crab resources.

[0011] For example, while PubMed's method for culturing Indian horseshoe crab gill valves can achieve long-term release of amoeboid cells, it relies on horseshoe crab serum, and the purity of the isolated cells is unclear, failing to address the core issues of maintaining cell purity and viability. The primary culture method for Chinese horseshoe crab blood cells reported in the *Acta Hydrobiologica Sinica* uses 2×L⁻¹⁵ medium supplemented with fetal bovine serum, which suffers from short cell survival time and low survival rate. Existing serum-free culture protocols have not achieved full chemical confinement and lack industrial-scale avenues, thus failing to meet the needs of industrial application. Therefore, developing a simple, highly pure, long-term stable cell viability and function method for the in vitro isolation and culture of horseshoe crab blood amoeboid cells, with minimal damage to the horseshoe crab body and compatibility with multiple species, has significant practical and industrial value. Summary of the Invention

[0012] The purpose of this invention is to overcome the systemic defects of existing technologies and provide a method for the in vitro isolation and culture of horseshoe crab blood amoeboid cells. This method achieves high-purity isolation, long-term maintenance of high activity, faithful endotoxin response, stable continuous passage, and large-scale production of amoeboid cells through a comprehensive anticoagulation protection system, a dual-mode precise separation process, a fully chemically defined serum-free culture system, a non-enzymatic hydrolysis and gentle passage scheme, a complete GMP-level quality control system, an industrial scale-up pathway, and a sustainable utilization scheme for horseshoe crab resources. Simultaneously, it minimizes damage to wild horseshoe crabs, balancing industrial development and ecological protection, and completely overcomes the core bottlenecks of existing technologies.

[0013] To achieve the above-mentioned objectives, the present invention adopts the following technical solution: a method for in vitro isolation and culture of horseshoe crab blood amoeboid cells, comprising the following steps:

[0014] Step 1: Horseshoe crab pretreatment and minimally invasive blood collection

[0015] Select healthy adult horseshoe crabs. Chinese horseshoe crabs / round-tailed horseshoe crabs should weigh 450-750g, and Indian horseshoe crabs / American horseshoe crabs should weigh 2-5kg. Both males and females are acceptable, but females are preferred (as they produce more cells). Place the horseshoe crabs in an artificial rearing environment for 24-48 hours to acclimatize. The rearing environment conditions are: water temperature 24-27℃, salinity 25-30‰, pH 7.8-8.2, continuous aeration, and do not feed them during the rearing period to avoid external stimulation that could activate cells.

[0016] After the temporary holding period, the horseshoe crabs are sterilized: after wiping the blood collection site with povidone-iodine, the blood collection sites such as the junction of the cephalothorax and abdominal carapace and joints are wiped with 75% medical alcohol. Then, the horseshoe crabs are rinsed 2-3 times with pyrogen-free sterile saline to remove surface impurities and microorganisms and avoid introducing contamination during the blood collection process.

[0017] A minimally invasive, non-invasive blood collection method was adopted. Using an 18-gauge sterile, endotoxin-free syringe, the needle was precisely inserted into the pericardial cavity through the joint space at the junction of the cephalothorax and plastron of the horseshoe crab. Blood was slowly drawn from the horseshoe crab at a rate of 0.5-1 mL / min. The blood volume collected from each horseshoe crab did not exceed 20% of its total blood volume or 5% of its body weight. Immediately after blood collection, the horseshoe crab blood was injected into a sterile, pyrogen-free anticoagulant tube pre-cooled to 4°C. A modified caffeine-EDTA-mannitol composite anticoagulant solution was added to the anticoagulant tube beforehand. The volume ratio of the anticoagulant to the horseshoe crab blood was 1:4-1:5. The anticoagulant tube was gently inverted 3-5 times to ensure thorough mixing and prevent blood coagulation and cell activation and degranulation.

[0018] The modified caffeine-EDTA-mannitol composite anticoagulant protective solution is formulated as follows: NaCl 8.0g, KCl 0.2g, Na2HPO4・12H2O 2.9g, KH2PO4 0.2g, caffeine 0.1-0.2g, EDTA-Na2 0.5-1.0g, mannitol 0.1-0.2mol / L, heparin sodium 2-5U / mL, and deionized water is added to bring the volume to 1000mL. The pH is adjusted to 7.4-7.6, and the osmotic pressure is 1020-1080mOsm / kg. After filtration and sterilization through a 0.22μm filter membrane, it is stored at 4℃ for later use.

[0019] After blood collection, rinse the blood collection site with pyrogen-free sterile saline, apply sterile petroleum jelly to the blood collection site to seal the wound, and place the horseshoe crab back into the temporary holding environment for 7-10 days of further culture. After the physiological indicators have fully recovered, the horseshoe crab can be temporarily held for another 3 months before a second blood collection, or it can be directly released back into its natural habitat to achieve the sustainable use of horseshoe crab resources.

[0020] Step 2: In vitro isolation and purification of horseshoe crab blood amoeboid cells

[0021] The anticoagulated horseshoe crab blood collected in step 1 was placed in a 4°C environment and allowed to stand for 30-60 minutes to allow the plasma and blood cells to initially separate into layers. Then, a dual-mode gradient centrifugation method was used for separation and purification. The sucrose density gradient mode or iodixanol density gradient mode can be selected according to the horseshoe crab species and application scenario. The specific steps are as follows:

[0022] 1. Sample pretreatment

[0023] After the anticoagulated horseshoe crab blood has been allowed to stand, gently aspirate a small amount of plasma from the top layer using a sterile pipette to remove any floating impurities. Then, dilute the remaining blood 1-2 times with modified isotonic PBS buffer and gently pipette to mix the blood evenly to obtain diluted horseshoe crab blood. Avoid vigorous shaking during the dilution process to prevent mechanical damage to the cells.

[0024] The modified isotonic PBS buffer formulation is as follows: NaCl 8.0g, KCl 0.2g, Na2HPO4·12H2O 2.9g, KH2PO4 0.2g, CaCl2 0.1g, MgCl2 0.1g, add deionized water to a final volume of 1000mL, pH 7.4-7.6, osmotic pressure 1045-1065mOsm / kg, sterilize before use.

[0025] 2. Density gradient preparation

[0026] Sucrose density gradient mode (routine laboratory scenario): Take a sterile centrifuge tube and add 30%, 45%, and 60% sucrose density gradient solutions sequentially. The volume of each gradient solution is 3-5 mL. Use a slow dropwise addition method to avoid confusion of gradient layers. Pre-cool in an environment of 4°C for 10-15 min. The sucrose density gradient solutions are prepared using the above-mentioned modified isotonic PBS buffer as the solvent.

[0027] Iodixanol density gradient mode (high purity requirements / industrial application scenarios): Take a sterile centrifuge tube and layer iodixanol gradient solutions with densities of 1.070 g / mL, 1.055 g / mL, and 1.040 g / mL sequentially from bottom to top, with each layer having a volume of 3-5 mL. Pre-cool at 4°C for 10-15 min. The iodixanol gradient solutions are prepared by diluting the modified isotonic PBS buffer solution described above as a solvent.

[0028] 3. Gradient centrifugation

[0029] Slowly add the diluted horseshoe crab blood obtained in step 2.1 dropwise to a pre-cooled density gradient centrifuge tube. The volume ratio of diluted horseshoe crab blood to density gradient solution should be 1:2-1:3. When adding the solution, keep the tip of the pipette close to the inner wall of the centrifuge tube to avoid damaging the gradient layer. Place the centrifuge tube in a high-speed refrigerated centrifuge, set the centrifugation temperature to 4°C, the centrifugation speed to 1200-1500 r / min (centrifugal force 300-400×g), and the centrifugation time to 15-20 min. Use a slow acceleration and slow deceleration mode during centrifugation (5 min acceleration time, 5 min deceleration time) to reduce mechanical damage to cells.

[0030] 4. Cell collection

[0031] After centrifugation, clear layers formed in the centrifuge tube. Under sucrose gradient mode, from top to bottom, the layers were: plasma layer, 30% sucrose layer, amoeboid cell layer (located between the 30% and 45% sucrose layers, a milky white turbid band), 45% sucrose layer, impurity layer, 60% sucrose layer, and bottom sediment layer. Under iodixanol gradient mode, amoeboid cells were enriched at the 1.055-1.070 g / mL interface. The amoeboid cell layer was gently aspirated with a sterile pipette and transferred to a new sterile centrifuge tube. 3-5 volumes of modified isotonic PBS buffer were added, and the mixture was gently pipetted to obtain an amoeboid cell suspension.

[0032] 5. Washing and Purification

[0033] Put the amoeboid cell suspension into a high-speed refrigerated centrifuge, centrifuge at 4°C and 800 - 1000 r / min for 5 - 8 min, discard the supernatant, and collect the cell precipitate; add modified isotonic PBS buffer to resuspend the cells, and repeat the washing 2 - 3 times until the supernatant is clear to obtain purified horseshoe crab blood amoeboid cells.

[0034] 6. Dual Inspection Quality Control of Cell Viability and Purity

[0035] Use the trypan blue staining method for primary screening, and cells with a viability of ≥90% are considered qualified; use the AO / PI fluorescence double staining method combined with flow cytometry for accurate quantitative detection, and cells with a viable cell ratio of ≥90% are considered qualified; use the Giemsa staining method combined with immunofluorescent markers to identify cell purity, and amoeboid cells accounting for ≥95% of the total cells are considered qualified. If the requirements are not met, repeat steps 2.4 - 2.5 until the quality control standards are satisfied.

[0036] Step 3: In Vitro Primary Culture of Horseshoe Crab Blood Amoeboid Cells

[0037] 1. Preparation of a Fully Chemically Defined Serum-Free Special Medium

[0038] The medium is a fully chemically defined system without any animal-derived components, based on the modified L-15 medium, and the following final concentration components are added:

[0039] Growth regulatory factors: recombinant human basic fibroblast growth factor (bFGF) 5 - 10 ng / mL, recombinant transforming growth factor β (TGF-β) 2 - 5 ng / mL, recombinant human insulin-like growth factor-1 (IGF-1) 5 - 10 ng / mL, insulin 5 - 10 μg / mL, transferrin 10 - 20 μg / mL;

[0040] Basic nutrient components: glutamine 2 - 4 mmol / L, non-essential amino acid mixture 40 - 60 mg / L, sodium pyruvate 0.1 - 0.2 g / L, glucose 5.0 mmol / L;

[0041] Vitamins and cofactors: vitamin C 5 - 15 mg / L, biotin 0.1 - 1.0 mg / L, nicotinamide 1.0 - 2.0 mg / L;

[0042] Lipids and membrane stabilizers: cholesterol-phospholipid complex 10 - 15 mg / L, taurine 1.0 - 2.0 mmol / L;

[0043] Antioxidant and anti-apoptosis components: reduced glutathione 0.5 - 1.0 mmol / L, N-acetylcysteine 0.1 - 0.3 mmol / L;

[0044] Buffering and antibacterial components: HEPES 10-15 mmol / L, penicillin 100 U / mL, streptomycin 100 μg / mL, gentamicin 50 μg / mL;

[0045] Osmotic pressure regulators: mannitol 0.1-0.15 mol / L, NaCl 6.0-6.5 g / L, with the total osmotic pressure of the system controlled at 1045-1065 mOsm / kg.

[0046] Adjust the pH of the culture medium to 7.4-7.6, sterilize it by filtration through a 0.22μm filter membrane, and store it at 4℃ in the dark. It has a shelf life of 30 days. Before use, restore the temperature to 25-27℃.

[0047] The modified L-15 culture medium formula is as follows: 1 bag of L-15 basic culture medium powder, 6.0g NaCl, 0.4g KCl, 1.0g NaHCO3, add deionized water to make up to 1000mL, sterilize and use for later use.

[0048] 2. Culture container coating

[0049] A coating solution of poly-L-lysine (50 μg / mL) and type IV collagen (10 μg / mL) was used to coat culture flasks / plates. After incubation at 37°C for 2 hours, the coating solution was aspirated, and the cells were washed three times with sterile PBS and air-dried for later use to improve cell adhesion rate and morphological stability.

[0050] 3. Stratified vaccination strategy

[0051] The purified horseshoe crab-like cells from step 2 were resuspended in a chemically defined, serum-free culture medium. The inoculation density was set according to the culture scenario: for laboratory-scale trials, the inoculation density was 1 × 10⁻⁶ cells / day. 4 -5×10 4 The inoculation density in pilot / large-scale scenarios is 1×10⁶ cells / cm². 6 -5×10 6 Cells per mL, gently pipette to mix evenly and avoid cell aggregation, then inoculate into the coated culture container. After inoculation, gently shake to distribute the cells evenly.

[0052] 4. Control of primary culture conditions

[0053] Place the inoculated culture container in a constant temperature incubator and set the culture conditions as follows: temperature 20-24℃, optimal 22℃; relative humidity 85-95%; normal air environment, no need to introduce additional CO2; culture in the dark throughout the process to avoid cell activation caused by light.

[0054] Do not change the culture medium for the first 24 hours of culture to allow the cells to fully adhere to the culture wall and adapt to the culture environment. After 24 hours of culture, replace half of the culture medium with fresh medium to remove dead cells and impurities that have not adhered to the culture wall. After that, replace half of the culture medium with fresh medium every 48-72 hours. Use a low flow rate when changing the medium to avoid mechanically disturbing the adhered cells.

[0055] During primary culture, cell morphology and growth status are observed daily under an inverted microscope: freshly inoculated cells are round or oval, with uniform cytoplasm, plump granules, and clear nuclei; after 24-48 hours of culture, cells begin to adhere to the wall, gradually becoming irregular in shape and extending pseudopodia; after 7-10 days of culture, the cell density reaches 80%-90%, forming a monolayer of adherent cells, which can then be passaged.

[0056] Step 4: In vitro passage culture of horseshoe crab blood amoeba-like cells

[0057] 1. Non-enzymatic hydrolysis and gentle cell dissociation

[0058] When the primary / passaged cell density reaches 80%-90% confluence, proceed with subculture. First, discard the old culture medium in the culture vessel and rinse the cell surface 2-3 times with modified isotonic PBS buffer to remove residual culture medium and impurities. Then, add EDTA-mannitol non-enzymatic dissociation solution, ensuring the cell surface is completely covered. Incubate at 20-24℃ for 3-8 minutes, observing under an inverted microscope. When the cells begin to round and detach, immediately add 2-3 volumes of fully chemically defined serum-free culture medium to terminate dissociation. Gently pipette the cell surface to completely detach the cells, forming a homogeneous cell suspension.

[0059] The EDTA-mannitol non-enzymatic dissociation solution is formulated as follows: EDTA 2-5 mmol / L, KCl 0.04-0.05%, mannitol 0.1-0.15 mol / L, HEPES 10 mmol / L, prepared with modified isotonic PBS buffer, pH 7.4-7.6, osmotic pressure 1045-1065 mOsm / kg.

[0060] For strongly adherent cells, a 0.25% trypsin-EDTA digestion solution can be retained as an alternative dissociation method, with the digestion time controlled at 2-3 minutes to avoid over-digestion and cell damage.

[0061] 2. Cell counting and inoculation

[0062] Transfer the cell suspension to a sterile centrifuge tube and centrifuge at 800-1000 rpm for 5-8 minutes. Discard the supernatant and collect the cell pellet. Resuspend the cells in a fully chemically defined serum-free culture medium, gently pipetting to mix. Take a small amount of the cell suspension for cell counting and adjust the cell concentration to 1×10⁻⁶. 5-5×10 5 Cells / mL; the adjusted cell suspension was inoculated into a new coated culture container at the same inoculation density as the primary culture, and the passage ratio was 1:2-1:4, which was dynamically adjusted according to the cell growth status.

[0063] 3. Subculture and Phenotype Maintenance

[0064] The subculture conditions are exactly the same as the primary culture conditions, with half of the culture medium replaced every 48-72 hours. Cell growth is observed daily. This method enables long-term stable subculturing of horseshoe crab-like cells, with more than 12 subcultures, maintaining cell viability above 85% and no significant decline in endotoxin response function.

[0065] During passage, a full functional verification is performed every three generations. If abnormal cell morphology, decreased activity, or functional decline are found, the concentration of growth factors in the culture medium or the culture temperature is adjusted in a timely manner to ensure the stability of cell phenotype and function.

[0066] Step 5: Large-scale cell culture (adapted for industrial applications)

[0067] To address the need for large-scale production of horseshoe crab reagent, a microcarrier-bioreactor suspension-adherent culture system was established. The specific steps are as follows:

[0068] 1. Microcarrier pretreatment: Cytodex3 microcarriers were selected, soaked in PBS, autoclaved, and then coated with a mixture of poly-L-lysine and type IV collagen. The mixture was incubated at 37°C with shaking for 2 hours and washed three times with PBS before use.

[0069] 2. Inoculation and Culture: The purified amoeboid cells were inoculated at a rate of 1.5 × 10⁻⁶. 5 The cells / mL were seeded in a bioreactor containing a chemically defined serum-free medium, and the pretreated microcarriers were added. The stirring speed was set to 10-20 rpm, the culture temperature was 22℃, and the dissolved oxygen was maintained at 60% air saturation. No additional CO2 was required.

[0070] 3. Medium replacement and expansion: 48 hours after inoculation, add 50% fresh culture medium for the first time, and then add 20% volume every 72 hours thereafter. When the cell coverage on the microcarrier surface reaches 80%, stop stirring, let the microcarrier settle, discard the supernatant, add non-enzymatic dissociation solution, gently shake and incubate for 10 minutes to allow the cells to detach, collect them and re-inoculate them into a new microcarrier system at a ratio of 1:3 to achieve continuous scale-up culture.

[0071] 4. Harvesting and Quality Control: When the cells reach the target density, the cell suspension is collected, washed and purified, and then subjected to a full range of tests for viability, purity, and functional integrity. After passing the tests, the cells are used for the preparation of Limulus Amebocyte Lysate (LAL) reagents or other downstream applications.

[0072] Step 6: Cell cryopreservation and thawing (preservation of cell resources)

[0073] 1. Serum-free chemically defined cell cryopreservation

[0074] When passaged cells are in the logarithmic growth phase (cell density 60%-80%, viability ≥90%), they should be cryopreserved. Discard the old culture medium, wash the cell surface twice with modified isotonic PBS buffer, add non-enzymatic dissociation solution for digestion, terminate dissociation, and centrifuge to collect the cell pellet; resuspend the cells in serum-free, chemically defined cryopreservation solution and adjust the cell concentration to 1×10⁻⁶. 6 -5×10 6 Mix the cells per mL by gently pipetting until homogeneous, then dispense 1-2 mL into sterile cryovials. Label each cryovial with information such as cryopreservation date, cell passage number, and horseshoe crab species.

[0075] The serum-free chemically limited cryopreservation solution is formulated as follows: 70% chemically limited serum-free culture medium, 0.2-0.3 mol / L trehalose, 5-10 mg / L recombinant human serum albumin substitute peptide, and 10% dimethyl sulfoxide (DMSO). After sterilization by filtration through a 0.22 μm filter membrane, it is stored at 4°C for later use.

[0076] The cryopreservation process uses a programmed gradient cooling method: place at 4℃ for 30 minutes, at -20℃ for 2 hours, at -80℃ for 12-24 hours, and then transfer to a liquid nitrogen tank for long-term storage, which can last for more than 2 years.

[0077] 2. Cell resuscitation

[0078] When frozen cells are needed, remove the cryovials from the liquid nitrogen tank and quickly place them in a 37-40℃ water bath. Shake rapidly to thaw the cryopreservation solution completely within 1-2 minutes. Transfer the thawed cell suspension to a sterile centrifuge tube, add 3-5 times the volume of fully chemically defined serum-free culture medium, gently pipette to mix, centrifuge at 800-1000 rpm for 5-8 minutes, and discard the supernatant. Resuspend the cells in fresh culture medium, adjust to the target concentration, and then inoculate them into coated culture containers. Incubate in an incubator and replace with fresh culture medium after 24 hours. The viability of the revived cells should be ≥85%, and they can be cultured and passaged normally.

[0079] Step 7: Full-process GMP-level quality control

[0080] Standardized quality control procedures (SOPs) are established throughout the entire process of cell isolation, primary culture, passage culture, large-scale scale-up, and cryopreservation and thawing. Regular comprehensive quality testing is conducted to ensure cell purity, viability, functional integrity, and compliance. Specific testing items are as follows:

[0081] 1. Cell viability assay: Assay was performed once per passage using AO / PI fluorescence double staining combined with flow cytometry, requiring cell viability ≥85%;

[0082] 2. Cell purity test: The cell purity was tested once every two passages using Giemsa staining combined with immunofluorescence markers. The cell purity was required to be ≥95%.

[0083] 3. Cell morphology and phenotype detection: Observe cell morphology under an inverted microscope daily to ensure that the cells are normal in shape, plump, and free from abnormal aggregation, lysis, degranulation, etc.

[0084] 4. Functional integrity test: The test was performed once every 3 passages using an endotoxin gradient stimulation assay. After incubating the cells with 0.01-10 EU / mL LPS, the cell degranulation response and coagulation protein release activity were detected. The EC50 response of the cells to endotoxin should deviate from that of freshly isolated cells by ≤15%.

[0085] 5. Sterility and exogenous factor detection: Detection is performed once a week, including bacterial, fungal, and mycoplasma detection, to ensure that the culture system is free from any exogenous contamination;

[0086] 6. Endotoxin residue testing: Each batch of culture medium and cell products must be tested, and the endotoxin residue must be ≤0.01EU / mL, which meets the requirements for pharmaceutical-grade raw materials.

[0087] Compared with the prior art, the present invention has the following outstanding substantive features and unexpected significant beneficial effects:

[0088] 1. High-purity and high-activity separation with extremely low cell damage. The self-developed composite anticoagulant protective solution has a triple effect of anticoagulation, inhibition of cell activation and protection of cell membrane. Combined with dual-mode gradient centrifugation and slow centrifugation parameters, the cell purity is ≥95%, recovery rate is ≥85% and viability is ≥92%, which is significantly better than existing technologies.

[0089] 2. Serum-free, fully chemically defined culture, long-term passage with functional fidelity, eliminating all animal-derived components, clearly defined culture medium composition, batch-stable, adapted to cell growth requirements, can achieve stable passage for more than 12 generations, culture cycle of more than 120 days, 120-day survival rate ≥65%, no significant attenuation of intracellular toxin response function, meeting GMP production requirements.

[0090] 3. Non-enzymatic hydrolysis and gentle passage, preserving the complete cell structure and function. Using the EDTA-mannitol non-enzymatic dissociation scheme to replace traditional trypsin digestion, it avoids irreversible damage to cell surface proteins. The cell adhesion rate after passage is ≥85%, and the genotype and function are highly stable after long-term passage.

[0091] 4. Standardized process throughout, adaptable to industrial-scale production, establishing standardized operating procedures throughout the entire process with high repeatability; equipped with a microcarrier-bioreactor culture system, enabling seamless scale-up from laboratory to 100 liters of mass production, with a single batch yielding 10 9 High-level functional cells to meet the needs of mass production in the industry.

[0092] 5. It is compatible with all species and has a wide range of applications. The process parameters can be flexibly adjusted, making it compatible with all existing horseshoe crab species in the world, such as the Chinese horseshoe crab and the American horseshoe crab, breaking through the limitations of single-species compatibility.

[0093] 6. GMP-level quality control, in compliance with pharmaceutical regulatory standards, has established a precise quality control system covering the entire process, including indicators such as cell viability, purity, function, sterility, and endotoxins. The products are safe, effective, and traceable, meeting the regulatory requirements of Chinese and foreign pharmacopoeias.

[0094] 7. Minimally invasive blood collection enables sustainable utilization of horseshoe crab resources. Low-damage blood collection combined with horseshoe crab resuscitation and reuse technology results in a near 100% survival rate of horseshoe crabs after blood collection. Blood can be collected repeatedly and combined with an in vitro culture system to reduce dependence on live blood collection from wild horseshoe crabs, thus balancing industry and ecological protection. Attached Figure Description

[0095] Figure 1 This is a flowchart illustrating the overall process framework of the method for in vitro isolation and culture of horseshoe crab blood amoeba-like cells according to the present invention.

[0096] Figure 2 This is a detailed flowchart illustrating the dual-mode in vitro isolation and purification process of horseshoe crab blood amoeboid cells according to the present invention.

[0097] Figure 3 This is a flowchart illustrating the primary culture and non-enzymatic passage culture of horseshoe crab blood amoeba-like cells according to the present invention.

[0098] Figure 4 This is a process flow diagram for the large-scale cultivation of microcarriers in a bioreactor according to the present invention. Detailed Implementation

[0099] The present invention will be further described in detail below with reference to specific embodiments. It should be understood that the specific embodiments described herein are only for explaining the present invention and are not intended to limit the present invention. 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.

[0100] The reagents and instruments used in the following examples are as follows: Reagents: L-15 basal culture medium powder (Gibco), iodixanol (OptiPrep™, Axis-Shield), sucrose, EDTA-Na2, caffeine, mannitol, heparin sodium, poly-L-lysine, type IV collagen, recombinant human bFGF, TGF-β, IGF-1 (PeproTech), insulin, transferrin, cholesterol-phospholipid complex, taurine, reduced glutathione, N-acetylcysteine, glutamine, HEPES, antibiotics, trypan blue, AO / PI fluorescence double staining kit, Giemsa staining solution. DMSO, trehalose, endotoxin standards (China National Institutes for Food and Drug Control), etc., are all commercially available analytical grade or GMP-grade biological reagents; Instruments: high-speed refrigerated centrifuge (Thermo Fisher Scientific), inverted fluorescence microscope (Nikon Scientific), flow cytometer (BD Scientific), constant temperature and humidity incubator (Thermo Fisher Scientific), 500mL bioreactor (Sartorius Scientific), sterile ultra-clean workbench (Sujing Group), water bath, electronic balance, pH meter, osmometer, 0.22μm sterile filter membrane, sterile syringe, centrifuge tubes, culture flasks, culture dishes, microcarriers, etc., are all standard laboratory equipment.

[0101] In the following examples, all horseshoe crabs used were healthy adult individuals, sourced from breeding bases approved by relevant departments, and the experimental procedures complied with the relevant regulations for Class II protected animals in China.

[0102] Example 1: Validation of the complete process for the standard isolation and culture of Chinese horseshoe crab blood amoeba-like cells

[0103] This embodiment provides a method for the in vitro isolation and culture of Chinese horseshoe crab blood amoeboid cells, the specific steps of which are as follows:

[0104] Step 1: Horseshoe crab pretreatment and minimally invasive blood collection

[0105] Ten healthy adult horseshoe crabs, weighing 450-750g (five males and five females), were selected and placed in an artificial rearing environment for 48 hours to acclimatize. The rearing conditions were: water temperature 25℃, salinity 28‰, pH 8.0, continuous aeration, and no feeding during the rearing period. After the rearing period, the blood collection sites were wiped with iodine solution, thoroughly disinfected with 75% medical alcohol, and rinsed three times with sterile saline.

[0106] Using an 18-gauge sterile, endotoxin-free syringe, the needle was inserted into the pericardial cavity through the joint space at the junction of the cephalothorax and plastron of the horseshoe crab. Blood was slowly drawn from the horseshoe crab at a rate of 0.8 mL / min, with the blood volume collected from each crab not exceeding 5% of its body weight, for a total of 280 mL. Immediately after collection, the blood was injected into a sterile anticoagulant tube pre-cooled to 4°C. The anticoagulant was pre-added to a modified caffeine-EDTA-mannitol composite anticoagulant solution at a volume ratio of 1:4.5 to the horseshoe crab blood, and the tube was gently inverted four times to mix.

[0107] The anticoagulant protective solution is formulated as follows: NaCl 8.0g, KCl 0.2g, Na2HPO4・12H2O 2.9g, KH2PO4 0.2g, caffeine 0.15g, EDTA-Na2 0.8g, mannitol 0.15mol / L, heparin sodium 3U / mL, add deionized water to make up to 1000mL, adjust the pH to 7.5, osmotic pressure 1050mOsm / kg, filter and sterilize through a 0.22μm filter membrane, and store at 4℃ for later use.

[0108] After blood collection, the blood collection site is rinsed with saline solution, sterile petroleum jelly is applied to seal the wound, and the horseshoe crab is placed back into the temporary holding environment for 8 days of culture. After recovery, it continues to be temporarily held for future use.

[0109] Step 2: In vitro cell isolation and purification

[0110] The collected anticoagulated horseshoe crab blood was allowed to stand at 4°C for 45 minutes. After initial stratification, it was separated and purified using a sucrose density gradient model. The specific steps are as follows:

[0111] 1. Pretreatment: A small amount of plasma from the upper layer was aspirated to remove impurities. The remaining blood was diluted 1.5 times with modified isotonic PBS buffer and gently blown to mix, resulting in 390 mL of diluted horseshoe crab blood.

[0112] 2. Density gradient preparation: Take 10 sterile centrifuge tubes of 50 mL each, and add 30%, 45%, and 60% sucrose gradient solutions to each tube in sequence, 4 mL per layer, adding slowly to avoid layering and mixing. Pre-cool at 4℃ for 12 min.

[0113] 3. Gradient centrifugation: Add 39 mL of diluted horseshoe crab blood to each centrifuge tube, with a volume ratio of diluted blood to gradient liquid of 1:2.5. Centrifuge at 4℃ and 1300 r / min (350×g) for 18 min, with both the acceleration and deceleration times being 5 min.

[0114] 4. Cell collection: After centrifugation, aspirate the milky white amoeboid cell layer between the 30% and 45% sucrose layers, collect a total of 85 mL of cell suspension, add 3 times the volume of modified isotonic PBS buffer and mix well.

[0115] 5. Washing and purification: Centrifuge at 4℃ and 900r / min for 6min, discard the supernatant, and repeat the washing 3 times until the supernatant is clear to obtain purified cell precipitate.

[0116] 6. Quality control testing: Trypan blue staining showed an initial cell viability of 92.3%; AO / PI fluorescence double staining combined with flow cytometry showed a cell viability of 91.8%; Giemsa staining showed a cell purity of 96.7%, meeting the quality control requirements.

[0117] Step 3: Primary Culture

[0118] 1. Culture medium preparation: Prepare a fully chemically defined serum-free culture medium based on a modified L-15 medium, adding the following components at final concentrations: recombinant human bFGF 8 ng / mL, TGF-β 3 ng / mL, IGF-1 8 ng / mL, insulin 8 μg / mL, transferrin 15 μg / mL, glutamine 3 mmol / L, non-essential amino acids 50 mg / L, sodium pyruvate 0.15 g / L, glucose 5.0 mmol / L, vitamin C 10 mg / L, biotin 0.5 mg / L, and nicotinamide 1.5 mg / L. The following ingredients were added: mg / L cholesterol-phospholipid complex 12 mg / L taurine 1.5 mmol / L, reduced glutathione 0.8 mmol / L, N-acetylcysteine ​​0.2 mmol / L, HEPES 12 mmol / L, penicillin 100 U / mL, streptomycin 100 μg / mL, gentamicin 50 μg / mL, mannitol 0.12 mol / L, and NaCl 6.2 g / L. The pH was adjusted to 7.5, the osmotic pressure was 1055 mOsm / kg, and the mixture was sterilized by filtration through a 0.22 μm filter membrane and stored at 4°C for later use.

[0119] 2. Coating of culture containers: T25 culture flasks were incubated with a mixture of poly-L-lysine and type IV collagen at 37°C for 2 hours, and then washed 3 times with PBS before use.

[0120] 3. Cell seeding: Resuspend purified cells in culture medium and adjust the concentration to 3 × 10⁻⁶. 5 Inoculate 5 mL of the solution into 10 T25 culture flasks at a density of 3 × 10⁶ cells / mL, with each flask containing 5 mL of the solution. 4 Cells / cm². 3.4 Culture conditions: 22℃ constant temperature incubator, 90% relative humidity, normal air environment, culture in the dark. Do not change the medium for the first 24 hours, change half of the medium after 24 hours, and then change half of the medium every 60 hours thereafter.

[0121] 4. Observation during culture: 24 hours after inoculation, cells begin to adhere to the wall and extend pseudopodia; after 7 days of culture, the cell density reaches 85%, forming a monolayer of adherent cells with uniform morphology, full cytoplasmic granules, and no abnormal lysis, and can be passaged.

[0122] Step 4: Subculture

[0123] 1. Non-enzymatic dissociation: When the cell confluence reaches 85%, discard the old culture medium, wash 3 times with modified PBS, add 2 mL / bottle of EDTA-mannitol non-enzymatic dissociation solution, incubate at 22℃ for 5 min, and when the cells are observed to become round and detach, immediately add 4 mL of culture medium to stop dissociation, and gently pipette to form a uniform cell suspension.

[0124] 2. Seeding and passage: Centrifuge the cell suspension at 900 rpm for 6 min, discard the supernatant, resuspend in culture medium, count the cells, and adjust the concentration to 3 × 10⁻⁶. 5 Inoculate at a ratio of 1:3 into new coated T25 culture flasks.

[0125] 3. Culture Maintenance: The culture conditions for passage were the same as those for the primary culture, with half the medium changed every 60 hours. In this example, the cells were continuously and stably passaged for 12 generations, with cell viability maintained above 88% in each generation, normal morphology, and stable proliferation capacity.

[0126] Step 5: Cryopreservation and Thawing

[0127] 1. Cryopreservation: Cells in the 5th generation logarithmic growth phase were non-enzymatically digested and centrifuged to collect the precipitate. The precipitate was then resuspended in serum-free, chemically defined cryopreservation buffer (70% culture medium, 0.25 mol / L trehalose, 8 mg / L recombinant human serum albumin substitute peptide, 10% DMSO), and the concentration was adjusted to 3 × 10⁻⁶. 6 The samples were dispensed at a rate of 1 / mL into cryovials, cooled using a programmed gradient, and then transferred to a liquid nitrogen tank for storage.

[0128] 2. Thawing: After one month of cryopreservation, the cryovials were removed and thawed rapidly in a 38°C water bath. Culture medium was added for dilution, followed by centrifugation. The supernatant was discarded, and the cells were resuspended and inoculated into culture flasks. The medium was changed after 24 hours. Results showed that the cells retained 88.2% viability after thawing, adhered well, and exhibited normal morphology, allowing for normal subculturing.

[0129] Step 6: Overall Quality Control Results

[0130] 1. Cell viability: The viability was maintained at 90%-93% during primary culture, and remained at 88.2% at the 12th passage. The viability was 88.2% after revival, all meeting the requirement of ≥85%.

[0131] 2. Cell purity: The purity was 96.7% after primary isolation and remained at 95.8% after passage to the 12th generation, with no impurities contaminating the cells.

[0132] 3. Morphological observation: The cells were uniform in morphology throughout the process, with full granules, clear pseudopodia, and no abnormal degranulation or lysis.

[0133] 4. Functional integrity: Cells from passages 3, 6, 9, and 12, stimulated with a gradient of 0.01-10 EU / mL endotoxin, all showed dose-dependent degranulation and coagulation protein release. The EC50 of the LPS response deviated from that of freshly isolated cells by ≤10%, indicating good functional integrity.

[0134] 5. Sterility test: The entire culture system is free from bacterial, fungal, and mycoplasma contamination, and the endotoxin residue is ≤0.01EU / mL, which meets pharmaceutical grade requirements.

[0135] Example 2: Comparison of the effects of different anticoagulant systems on separation performance

[0136] This embodiment compares the separation effect of the composite anticoagulant protective liquid of the present invention with that of a traditional anticoagulant. Other operating steps are the same as in Example 1, and the experimental groups are as follows:

[0137] The control group used a 1:3% sodium chloride solution as an anticoagulant, with the anticoagulant to horseshoe crab blood volume ratio being 1:4.5.

[0138] Control group 2: 0.1 mol / L sodium citrate anticoagulant was used as the anticoagulant, and the volume ratio of anticoagulant to horseshoe crab blood was 1:4.5;

[0139] Control group 3: 20 mmol / L theophylline-EDTA anticoagulant was used as the anticoagulant, and the ratio of anticoagulant to horseshoe crab blood volume was 1:4.5;

[0140] Experimental group: The modified caffeine-EDTA-mannitol composite anticoagulant protective solution of this invention has an anticoagulant to horseshoe crab blood volume ratio of 1:4.5.

[0141] Each group was set up with 3 replicates. The detection indicators were cell purity, cell recovery rate, and cell viability. The experimental results are shown in the table below:

[0142] sheet

[0143] Control group 1 78.3±2.5 65.7±3.2 76.2±2.8 Some cells aggregate and degranulate. Control group 2 82.5±2.1 69.8±2.9 80.5±2.3 A small number of cells aggregated, with abnormal morphology Control group 3 89.6±1.8 76.2±2.5 86.3±1.9 Slight de-granulation experimental group 96.5±1.2 85.3±2.7 92.1±1.5 Normal shape, no aggregation or degranulation

[0144] The results show that the composite anticoagulant protective solution of the present invention has significantly better anticoagulant and cell protection effects than traditional anticoagulants and existing theophylline anticoagulant systems. It can effectively inhibit blood coagulation, cell aggregation and degranulation, and greatly improve separation purity, recovery rate and cell viability, laying a core foundation for subsequent culture.

[0145] Example 3: Comparative verification of fully chemically defined culture medium and traditional serum culture medium

[0146] This embodiment compares the culture effects of the fully chemically defined serum-free culture medium of the present invention with those of existing traditional serum culture media. Other operational steps are the same as in Example 1, and the experimental groups are as follows:

[0147] Control group: conventional serum culture medium (2×L-15 medium + 15% fetal bovine serum + 1% penicillin antibiotics);

[0148] Experimental group: The present invention uses a chemically defined, serum-free culture medium.

[0149] Each group was set up with 3 replicates. The detection indicators were the maximum number of passages, cell viability at different culture times, and functional integrity. The experimental results are shown in the table below:

[0150] sheet

[0151] Maximum number of generations 3rd generation 12 generations and above Cell viability (%) after 10 days of culture 82.3±2.4 92.5±1.4 Cell viability (%) after 30 days of culture 65.7±3.1 89.7±1.8 Cell viability (%) after 60 days of culture 5.2±1.3 82.1±2.2 Cell viability (%) after 120 days of culture All cells lysed 65.7±2.8 EC50 bias in LPS response of third-generation cells (%) 38.2±4.5 9.2±1.5 Inter-batch variation CV (%) >25% <8%

[0152] The results show that the fully chemically defined serum-free culture medium of the present invention completely breaks through the technical bottleneck of traditional serum culture medium. It not only achieves long-term stable cell passage and greatly extends the culture cycle, but also precisely maintains the functional activity of cells, with extremely strong batch consistency, fully meeting GMP production requirements. The technical effect is significantly better than the existing technology.

[0153] Example 4: Comparison of the effects of non-enzymatic passaging and trypsin passaging

[0154] This embodiment compares the effects of the non-enzymatic mild passage system of the present invention with that of traditional trypsin digestion and passage. Other operational steps are the same as in Example 1, and the experimental groups are as follows:

[0155] Control group: 0.25% trypsin-EDTA digestion and passage, digestion time 2.5 min;

[0156] Experimental group: The EDTA-mannitol non-enzymatic dissociation solution of this invention was passaged and incubated for 5 minutes.

[0157] Each group was set up with 3 replicates. The detection indicators were cell adhesion rate after passage, cell viability, and functional integrity after continuous passage. The experimental results are shown in the table below:

[0158] sheet

[0159] Adhesion rate 24 hours after subculturing (%) 72.5±3.2 88.3±1.8 Cell viability after passage (%) 81.2±2.5 90.5±1.4 EC50 bias in LPS response of 6th generation cells (%) 42.6±5.3 10.5±1.7 Maximum stable generation count 5th generation 12 generations and above

[0160] The results show that the non-enzymatic passage system of the present invention can effectively avoid irreversible damage to cells by trypsin, significantly improve the cell adhesion rate and viability after passage, and better maintain the functional integrity of cells during long-term passage, providing key support for long-term stable cell culture.

[0161] Example 5: Applicability verification for multiple horseshoe crab species

[0162] This embodiment verifies the applicability of the method of the present invention to different horseshoe crab species. Chinese horseshoe crab, American horseshoe crab, and roundtail horseshoe crab were used as experimental subjects, and the same operating procedure as in Example 1 was adopted. Three replicates were set up for each species. The experimental results are shown in the table below:

[0163] sheet

[0164] Chinese horseshoe crab 96.7±1.1 92.3±1.4 90.2±1.6 12 generations and above horseshoe crab 95.8±1.3 91.5±1.6 88.7±1.9 11 generations and above Horseshoe crab 94.2±1.5 89.7±1.8 85.3±2.2 More than 10 generations

[0165] The results show that the method of the present invention has excellent adaptability to Chinese horseshoe crabs, American horseshoe crabs, and roundtail horseshoe crabs. The isolation and culture effects of different horseshoe crab species are significantly better than those of existing technologies, and it has broad species applicability, which can cover the mainstream raw horseshoe crab species in the global horseshoe crab reagent industry.

[0166] Example 6: Validation of large-scale microcarrier-bioreactor culture

[0167] This embodiment verifies the feasibility of industrial-scale production of the method of the present invention. A 500mL bioreactor combined with a microcarrier culture system is used, and the operation steps are as follows:

[0168] 1. Microcarrier pretreatment: After autoclaving, Cytodex3 microcarriers were incubated with a mixed coating solution at 37°C with shaking for 2 hours, and then washed 3 times with PBS before use.

[0169] 2. Inoculation and Culture: The purified *Hydroxella asiatica*-like cells from Example 1 were inoculated at a rate of 1.5 × 10⁻⁶ cells / year. 5 Cells / mL were seeded into a bioreactor containing 200 mL of chemically defined culture medium, and pretreated microcarriers were added. The stirring speed was set to 15 rpm, the culture temperature to 22 °C, and the dissolved oxygen was maintained at 60% air saturation.

[0170] 3. Medium replacement and passage: 48 hours after inoculation, 50 mL of fresh culture medium was added for the first time, and then 20% of the volume was added every 72 hours thereafter. On the 8th day of culture, when the cell coverage on the surface of the microcarrier reached 80%, the cells were detached using a non-enzymatic dissociation solution and re-inoculated into a new microcarrier system at a ratio of 1:3.

[0171] Experimental results: After five consecutive batches (40 days), cell viability remained between 88% and 92%, LPS response function was stable, and the total number of cells harvested per batch reached 3.2 × 10⁻⁶. 9 One sample can meet the raw material extraction requirements for 100 liters of horseshoe crab reagent, proving that the method of the present invention has excellent feasibility for industrial scale-up.

[0172] The above embodiments are merely preferred embodiments of the present invention and are not intended to limit the present invention. Those skilled in the art can make appropriate adjustments to the process parameters and component concentrations without departing from the spirit and scope of the present invention, all of which fall within the protection scope of the present invention.

Claims

1. A method for in vitro isolation and culture of horseshoe crab blood amoeboid cells, characterized in that: Includes the following steps: (1) Pretreatment of horseshoe crabs and minimally invasive blood collection: After temporary rearing and sterilization of healthy adult horseshoe crabs, blood was collected using a minimally invasive method. The horseshoe crab blood was mixed with a modified caffeine-EDTA-mannitol composite anticoagulant protective solution. (2) Cell isolation and purification: After the anticoagulated horseshoe crab blood was allowed to stand, it was separated by dual-mode gradient centrifugation and washed to obtain purified horseshoe crab blood amoeboid cells; (3) Primary culture: The purified cells were seeded into the coated culture container and cultured in the dark using a chemically defined serum-free culture medium. (4) Subculture: After the cells reach the target confluence, they are subcultured using a non-enzymatic mild dissociation method.

2. The method for in vitro isolation and culture of horseshoe crab blood amoeboid cells according to claim 1, characterized in that: The modified caffeine-EDTA-mannitol composite anticoagulant protective solution described in step (1) contains NaCl, KCl, Na2HPO2・12H2O, KH2PO4, caffeine, EDTA-Na2, mannitol, and sodium heparin, with a pH of 7.4-7.6 and an osmotic pressure of 1020-1080 mOsm / kg.

3. The method for in vitro isolation and culture of horseshoe crab blood amoeboid cells according to claim 1, characterized in that: The dual-mode gradient centrifugation in step (2) is either sucrose density gradient centrifugation or iodixanol density gradient centrifugation. The centrifugation temperature is 4℃, the rotation speed is 1200-1500r / min, and the centrifugation time is 15-20min.

4. The method for in vitro isolation and culture of horseshoe crab blood amoeboid cells according to claim 1, characterized in that: The fully chemically defined serum-free culture medium described in step (3) is based on the modified L-15 culture medium, with added growth regulators, basic nutrients, antioxidants and anti-apoptotic components. The system has an osmotic pressure of 1045-1065 mOsm / kg and a pH of 7.4-7.

6.

5. The method for in vitro isolation and culture of horseshoe crab blood amoeboid cells according to claim 1, characterized in that: In step (3), the culture vessel is coated with a mixture of poly-L-lysine and type IV collagen, and the inoculation density is 1×10⁻⁶. 4 -5×10 4 Units / cm² or 1×10 6 -5×10 6 Cells / mL, culture temperature 20-24℃.

6. The method for in vitro isolation and culture of horseshoe crab blood amoeboid cells according to claim 1, characterized in that: The non-enzymatic hydrolysis and mild dissociation described in step (4) uses EDTA-mannitol non-enzymatic dissociation solution, incubated at 20-24℃ for 3-8 min, with a subculture ratio of 1:2-1:

4.

7. The method for in vitro isolation and culture of horseshoe crab blood amoeboid cells according to claim 1, characterized in that: It also includes a large-scale cultivation step: using microcarrier-bioreactor suspension culture, stirring speed of 10-20 rpm, and maintaining dissolved oxygen at 60% air saturation.

8. The method for in vitro isolation and culture of horseshoe crab blood amoeboid cells according to claim 1, characterized in that: Cells were cryopreserved using serum-free, chemically defined cryopreservation solution via programmed gradient cooling, and the cell viability was ≥85% after thawing.

9. The method for in vitro isolation and culture of horseshoe crab blood amoeboid cells according to claim 1, characterized in that: It also includes full-process GMP-level quality control steps, and the test indicators include cell viability, purity, functional integrity, sterility, and endotoxin residue.

10. The method according to any one of claims 1-9, characterized in that, The horseshoe crab is any one of the following: Chinese horseshoe crab, Indian horseshoe crab, American horseshoe crab, and roundtail horseshoe crab.

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