In-vitro cultured calculus bovis and preparation method thereof

Abstract

The invention belongs to the technical field of in-vitro cultured calculus bovis, and provides in-vitro cultured calculus bovis and a preparation method thereof. The preparation method comprises the following steps: (1) separating cells; (2) primary culture; (3) cell inoculation and cultivation: adding the serum-free culture medium into the carrier matrix, putting the carrier matrix into a reactor, and performing cell inoculation on the carrier matrix after passage; the inoculated culture comprises a proliferation period; a synthesis period; a crystallization period; and (4) collecting and purifying the calculus bovis, namely stopping culturing after 45 days, and sequentially collecting, cleaning, degreasing, drying and crushing the calculus bovis. The preparation method aims at solving the problems of long culture period, unstable content of effective components, high production cost, large batch difference and the like in the existing in-vitro calculus bovis culture technology by optimizing a culture medium formula, regulating and controlling a culture process and a purification process.

Description

Technical Field

[0001] This invention belongs to the field of in vitro cultured bezoar technology, specifically, it relates to an in vitro cultured bezoar and its preparation method. Background Technology

[0002] Bezoar, a traditional and precious Chinese medicinal material, possesses the effects of clearing heat and detoxifying, calming the mind and soothing the nerves, and promoting bile secretion and opening the orifices. It is a core ingredient in classic Chinese patent medicines such as Angong Niuhuang Wan and Niuhuang Jiedu Pian. Natural bezoar comes from gallstones in the gallbladder, bile duct, or hepatic duct of cattle, but its formation rate is extremely low and limited by the cattle breeding cycle and resource distribution, resulting in a long-term shortage in market supply and high prices, which seriously restricts the development of related pharmaceutical industries.

[0003] To address the scarcity of natural bezoar resources, artificial cultivation techniques have emerged, mainly divided into two categories: in vivo cultivation and in vitro cultivation. In vivo cultivation involves surgically implanting a carrier into the gallbladder or bile duct of a cow to induce bezoar formation. While this method can produce a product with components similar to natural bezoar, it has the following drawbacks: it requires the sacrifice of healthy cattle, which contradicts animal welfare principles; the cultivation cycle is long, lasting 6-12 months, and is greatly affected by individual differences in physical condition, resulting in poor batch stability; the surgery and post-operative care costs are high, making large-scale production difficult.

[0004] In vitro culture, as a more promising alternative technology, simulates the in vivo bile secretion environment and uses cell culture and bioreactors to achieve the in vitro synthesis of bezoar, thus avoiding harm to animals.

[0005] However, existing in vitro culture techniques generally use culture media containing fetal bovine serum, which is not only costly but also introduces exogenous contaminants due to the complex composition of the serum, increasing the difficulty of quality control. Furthermore, the precise control of the culture medium during cell culture is not always sufficient. Therefore, existing in vitro culture techniques generally have long culture cycles, insufficient bilirubin content, relatively high production costs, and poor batch stability.

[0006] Therefore, developing a better method for preparing bezoar through in vitro culture is key to solving the shortage of bezoar resources and promoting the upgrading of the pharmaceutical industry. Summary of the Invention

[0007] To address the aforementioned shortcomings of existing technologies, the present invention aims to provide an in vitro cultured bezoar and its preparation method. This preparation method, through optimization of the culture medium formulation, cultivation process control, and purification process, aims to solve the problems of long cultivation cycles, unstable content of effective components, high production costs, and large batch-to-batch variations in existing in vitro cultured bezoar technologies.

[0008] To achieve the above objectives, the solution adopted by the present invention is as follows:

[0009] A method for preparing in vitro cultured bezoar includes: (1) cell separation: taking fresh gallbladders from healthy cattle, rinsing them with sterile physiological saline, cutting off the gallbladder mucosa layer, digesting it, filtering and collecting the cell suspension; centrifuging the cell suspension, discarding the supernatant, resuspending the cells in serum-free culture medium, and adjusting the concentration of the cell suspension to 1×10⁻⁶. 6 Cells / mL; Serum-free culture medium includes: 1L of DMEM / F12 medium, with 5μg / mL recombinant human insulin, 10μg / mL transferrin, 5ng / mL sodium selenite, 90μmol / L plant-derived cholesterol, and 50μmol / L synthetic taurine added; (2) Primary culture: The cell suspension is inoculated into a culture flask for primary culture, and passage is performed when the cell confluence reaches 80%-90%; (3) Cell inoculation and cultivation: The serum-free culture medium is added to the carrier matrix, placed in the reactor, and the passaged cells are inoculated into the carrier matrix and then cultured; The cultivation includes: Proliferation phase: 20ng / mL hepatocyte growth factor is added to the serum-free culture medium; The reactor temperature is set to 37℃ and the pH value is 7.2-7.4. Dissolved oxygen content is 5%-8%, and stirring rate is 50r / min; Synthesis period: 120μmol / L cholesterol and 5U / L recombinant glucuronyl transferase are added to serum-free culture medium after 5 days; The reactor temperature is set at 37℃, pH value is 7.2-7.4, dissolved oxygen content is 5%-8%, and stirring rate is 80r / min; Crystallization period: After 12 days, the calcium ion concentration in serum-free culture medium is increased to 1.5mmol / L, and 0.1% calcium taurocholate is added; The reactor temperature is set at 37℃, pH value is 7.2-7.4, dissolved oxygen content is 5%-8%, and stirring rate is 30r / min; (4) Bezoar harvesting and purification treatment: After 30 days, the culture is stopped, and bezoar is harvested, washed, defatted, dried and crushed in sequence.

[0010] Furthermore, in a preferred embodiment of the present invention, in step (2), the passaged cells are pretreated in a solution containing 10 μmol / L chenodeoxycholic acid for 24 h. This treatment can help activate bile synthesis-related genes within the cells.

[0011] Furthermore, in a preferred embodiment of the present invention, step (3) includes vaccination: on day 1, at 5 × 10 4 cells / cm 2 High-density vaccination, followed by a booster dose of 5×10⁻⁶ mmol / L 24 hours later. 4 cells / cm 2 This treatment can significantly improve the cell coverage on the carrier surface. The gradient density inoculation method described above, combined with the staged culture method during the culture period, can significantly shorten the in vitro culture cycle and significantly improve efficiency.

[0012] Furthermore, in a preferred embodiment of the present invention, step (3) further includes adding 10 μg / mL laminin to the inoculated reactor. This treatment significantly improves the adhesion rate between cells and the carrier matrix.

[0013] Furthermore, in a preferred embodiment of the present invention, step (4) includes harvesting: removing the carrier matrix and peeling off the particles on the surface with sterile tweezers to obtain bezoar particles that are brownish-yellow or reddish-brown.

[0014] Furthermore, in a preferred embodiment of the present invention, step (4) includes cleaning the bezoar granules with 0.01 mol / L PBS buffer at 37°C using 180W ultrasonic cleaning for 10 min.

[0015] Furthermore, in a preferred embodiment of the present invention, in step (4), defatting includes: using supercritical CO2 extraction at 40°C and 20MPa to defatt the bezoar granules for 2 hours.

[0016] Furthermore, in a preferred embodiment of the present invention, in step (4), drying includes: placing the defatted bezoar granules into a vacuum drying oven at 45°C and drying them until the moisture content is ≤3%.

[0017] An in vitro cultured bezoar prepared using the above-described method.

[0018] The beneficial effects of the in vitro cultured bezoar and its preparation method provided by this invention are:

[0019] (1) The method for preparing in vitro cultured bezoar provided by the present invention uses the serum-free culture medium provided in this application, which not only significantly reduces the formulation cost compared with the serum-containing culture medium, but also avoids the influence of serum batch differences on cells.

[0020] (2) The method for preparing in vitro cultured bezoar provided by this invention includes the following steps: During the proliferation phase, a serum-free culture medium with a specific amount of hepatocyte growth factor is added to promote a doubling of cell number; during the synthesis phase, a specific amount of cholesterol and glucuronyl transferase is added to activate the bilirubin synthesis pathway; during the crystallization phase, the calcium ion concentration is increased, and a specific "crystallization promoter," calcium taurocholate, is added to accelerate particle aggregation; simultaneously, the stirring rate during the synthesis phase is increased from 50 r / min during the proliferation phase to 80 r / min to increase nutrient exchange efficiency; and during the crystallization phase, the stirring rate is reduced to 30 r / min to prevent the crystallized particles from falling off. The synergistic cooperation between the above process steps can significantly shorten the cultivation cycle and increase the content of key components in bezoar.

[0021] (3) The method for preparing in vitro cultured bezoar provided by the present invention adopts a gradient density inoculation method combined with a staged culture method during the culture period, which can significantly shorten the in vitro culture cycle and significantly improve efficiency.

[0022] This application, through improvements to the culture medium, inoculation and cultivation methods, and optimization of other process conditions, and the synergistic coordination between each step, produces in vitro cultured bezoar under specific technical conditions. This preparation method provides a feasible solution for the standardized and large-scale production of high-quality bezoar, and has significant medicinal and economic value. Detailed Implementation

[0023] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0024] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0025] Example 1

[0026] This embodiment provides a method for preparing in vitro cultured bezoar, including:

[0027] (1) Cell isolation: Fresh gallbladders from healthy cattle were rinsed three times with sterile saline; gallbladder mucosa (0.5cm × 0.5cm pieces) were cut off and digested with 0.25% trypsin at 37℃ for 30 min. After digestion was terminated, the cells were filtered (through a 200-mesh sieve) to collect the cell suspension. The suspension was centrifuged (1000 r / min, 5 min), the supernatant was discarded, and the cells were resuspended in culture medium. The concentration of the cell suspension was adjusted to 1 × 10⁻⁶. 6 Cells / mL; Serum-free culture medium includes: 1L of DMEM / F12 medium, with 5μg / mL recombinant human insulin, 10μg / mL transferrin, 5ng / mL sodium selenite, 90μmol / L plant-derived cholesterol, and 50μmol / L synthetic taurine added.

[0028] (2) Primary culture: Inoculate the cell suspension into a culture flask and place it in a CO2 incubator (37℃, 5% CO2) for culture. Change the culture medium after 24 hours, and change the medium every 2-3 hours thereafter. When the cell confluence reaches 80%-90%, passage the cells.

[0029] (3) Cell seeding and culture: Add serum-free culture medium to the carrier matrix, place the reactor, and seed the passaged cells into the carrier matrix at a rate of 1×10⁶. 5 cells / cm 2Inoculate at high density and culture statically for 48 hours; environmental parameters set as follows: temperature 37℃, pH 7.2-7.4, dissolved oxygen 5%-8%;

[0030] Next, cultivation, including:

[0031] Proliferation phase: Add 20 ng / mL hepatocyte growth factor to serum-free culture medium; set the reactor temperature to 37℃, pH to 7.2-7.4, dissolved oxygen to 5%-8%, and stirring rate to 50 r / min;

[0032] Synthesis period: 120 μmol / L cholesterol and 5 U / L recombinant glucuronyl transferase were added to the serum-free culture medium after 5 days; the reactor temperature was set at 37℃, pH at 7.2-7.4, dissolved oxygen at 5%-8%, and stirring rate at 80 r / min.

[0033] Crystallization period: After 12 days, the calcium ion concentration in the serum-free culture medium was increased to 1.5 mmol / L, and 0.1% calcium taurocholate was added; the reactor temperature was set at 37℃, the pH value at 7.2-7.4, the dissolved oxygen content at 5%-8%, and the stirring rate at 30 r / min.

[0034] (4) Harvesting and purification of bezoar: After 30 days of cultivation, the carrier matrix was removed and the particles on the surface were peeled off with sterile tweezers to obtain bezoar particles that are brownish-yellow or reddish-brown. The bezoar particles were ultrasonically cleaned with 0.01 mol / L PBS buffer at 37°C for 10 min at 180 W. The bezoar particles were degreased by supercritical CO2 extraction at 40°C and 20 MPa for 2 h. The degreased bezoar particles were placed in a vacuum drying oven at 45°C and dried until the moisture content was ≤3%. The particles were then pulverized through a 100-mesh sieve to obtain bezoar powder.

[0035] Example 2

[0036] This embodiment provides a method for preparing in vitro cultured bezoar. The difference from Embodiment 1 is that step (2) further includes: pretreating the passaged cells in a solution containing 10 μmol / L chenodeoxycholic acid for 24 h.

[0037] Example 3

[0038] This embodiment provides a method for preparing in vitro cultured bezoar. The difference from embodiment 2 is that step (3) further includes adding 10 μg / mL laminin to the inoculated reactor.

[0039] Example 4

[0040] This embodiment provides a method for preparing in vitro cultured bezoar. The difference from Embodiment 3 is that, in step (3), the seeding of cells after passage into the carrier matrix includes: on day 1, at 5 × 10⁻⁶ cells / day. 4 cells / cm 2 High-density vaccination, followed by a booster dose of 5×10⁻⁶ mmol / L 24 hours later. 4 cells / cm 2 .

[0041] Comparative Example 1

[0042] This comparative example provides a method for preparing in vitro cultured bezoar, including:

[0043] (1) Cell isolation: Fresh gallbladders from healthy cattle were rinsed three times with sterile saline; gallbladder mucosa (0.5cm × 0.5cm pieces) were cut off and digested with 0.25% trypsin at 37℃ for 30 min. After digestion was terminated, the cells were filtered (through a 200-mesh sieve) to collect the cell suspension. The suspension was centrifuged (1000 r / min, 5 min), the supernatant was discarded, and the cells were resuspended in culture medium. The concentration of the cell suspension was adjusted to 1 × 10⁻⁶. 6 cells / mL;

[0044] The culture medium consisted of 1L of DMEM / F12 medium supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin antibiotics, 50μmol / L cholesterol, 20μmol / L chenodeoxycholic acid, 50μg / mL vitamin C, and 10μg / mL insulin.

[0045] (2) Primary culture: Inoculate the cell suspension into a culture flask and place it in a CO2 incubator (37℃, 5% CO2) for culture. Change the culture medium after 24 hours, and change the medium every 2-3 hours thereafter. When the cell confluence reaches 80%-90%, passage the cells.

[0046] (3) Cell seeding and culture: Add the culture medium to the carrier matrix, place it in the reactor, and seed the passaged cells into the carrier matrix at a rate of 1×10⁻⁶. 5 cells / cm 2 Inoculate at high density and culture statically for 48 hours; environmental parameters set as follows: temperature 37℃, pH 7.2-7.4, dissolved oxygen 5%-8%;

[0047] The subsequent cultivation process included: adding 90 μmol / L cholesterol, 10 U / L glucuronyl transferase, 1.2 mmol / L calcium ions and 50 μmol / L taurcholic acid to the culture medium; cultivating for 8 weeks; setting the reactor temperature to 37℃, pH to 7.2-7.4, dissolved oxygen to 5%-8%, and stirring speed to 70 r / min.

[0048] (4) Harvesting and purification of bezoar: After 8 weeks of cultivation, the carrier matrix was removed, and the particles on the surface were peeled off with sterile tweezers to obtain bezoar particles that are brownish-yellow or reddish-brown. The particles were rinsed with deionized water 3 times, soaked in ether for 2 hours, washed with distilled water to remove the ether, and dried in a vacuum drying oven at 45℃ until the moisture content was ≤5%. The particles were then pulverized through a 100-mesh sieve to obtain bezoar powder.

[0049] Comparative Example 2

[0050] This comparative example provides a method for preparing in vitro cultured bezoar. The difference from Example 1 is that in steps (2) and (3), serum-free culture medium was not used. The culture medium used included: 1L of DMEM / F12 medium with 10% fetal bovine serum, 1% penicillin-streptomycin antibiotics, 50μmol / L cholesterol, 20μmol / L chenodeoxycholic acid, 50μg / mL vitamin C, and 10μg / mL insulin.

[0051] Comparative Example 3

[0052] This comparative example provides a method for preparing in vitro cultured bezoar, which differs from Example 1 in that (3) cell seeding and culture: serum-free culture medium is added to the carrier matrix, placed in a reactor, and passaged cells are seeded into the carrier matrix at a ratio of 1×10 5 cells / cm 2 Inoculate at high density and culture statically for 48 hours; environmental parameters set as follows: temperature 37℃, pH 7.2-7.4, dissolved oxygen 5%-8%;

[0053] The subsequent incubation process included: adding 90 μmol / L cholesterol, 10 U / L glucuronyl transferase, 1.2 mmol / L calcium ions and 50 μmol / L taurcholic acid to the serum-free medium; incubating for 8 weeks; setting the reactor temperature to 37℃, pH to 7.2-7.4, dissolved oxygen to 5%-8%, and stirring speed to 70 r / min.

[0054] Comparative Example 4

[0055] This comparative example provides a method for preparing in vitro cultured bezoar, which differs from Example 1 in that (3) cell seeding and culture: serum-free culture medium is added to the carrier matrix, placed in a reactor, and passaged cells are seeded into the carrier matrix at a ratio of 1×10 5 cells / cm 2 Inoculate at high density and culture statically for 48 hours; environmental parameters set as follows: temperature 37℃, pH 7.2-7.4, dissolved oxygen 5%-8%;

[0056] Next, cultivation, including:

[0057] Proliferation phase: Add 20 ng / mL hepatocyte growth factor to serum-free culture medium; set the reactor temperature to 37℃, pH to 7.2-7.4, dissolved oxygen to 5%-8%, and stirring rate to 70 r / min;

[0058] Synthesis period: 120 μmol / L cholesterol and 5 U / L recombinant glucuronyl transferase were added to the serum-free culture medium after 7 days; the reactor temperature was set at 37℃, pH at 7.2-7.4, dissolved oxygen at 5%-8%, and stirring rate at 70 r / min.

[0059] Crystallization period: After 21 days, the calcium ion concentration in the serum-free culture medium was increased to 1.5 mmol / L, and 0.1% calcium taurocholate was added; the reactor temperature was set at 37℃, the pH value at 7.2-7.4, the dissolved oxygen content at 5%-8%, and the stirring rate at 70 r / min.

[0060] Comparative Example 5

[0061] This comparative example provides a method for preparing in vitro cultured bezoar, which differs from Example 1 in that: (3) Cell inoculation and culture: serum-free culture medium is added to the carrier matrix, placed in a reactor, and passaged cells are inoculated into the carrier matrix at a rate of 1×10 5 cells / cm 2 Inoculate at high density and culture statically for 48 hours; environmental parameters set as follows: temperature 37℃, pH 7.2-7.4, dissolved oxygen 5%-8%;

[0062] Next, cultivation, including:

[0063] Proliferation phase: Add 90 μmol / L cholesterol, 10 U / L glucuronyl transferase, 1.2 mmol / L calcium ions and 50 μmol / L taurcholic acid to serum-free culture medium; set the reactor temperature to 37℃, pH to 7.2-7.4, dissolved oxygen to 5%-8%, and stirring rate to 50 r / min.

[0064] Synthesis period: After 7 days, set the reactor temperature to 37℃, pH value to 7.2-7.4, dissolved oxygen content to 5%-8%, and stirring rate to 80r / min;

[0065] Crystallization period: After 21 days, set the reactor temperature to 37℃, pH value to 7.2-7.4, dissolved oxygen content to 5%-8%, and stirring rate to 30r / min.

[0066] Experimental Example 1

[0067] Three batches of bezoar powder were prepared according to the preparation methods of in vitro cultured bezoar provided in Examples 1-4 and Comparative Examples 1-5, respectively.

[0068] (1) Detect the bilirubin, bile acid and cholesterol in the prepared bezoar powder.

[0069] Bilirubin, bile acids, and cholesterol were determined by high-performance liquid chromatography (HPLC) according to the Chinese Pharmacopoeia (2020 edition). The test results are shown in Table 1.

[0070] Table 1

[0071] Group Bilirubin (%) cholic acid(%) cholesterol(%) Example 1 38.2±0.8 14.6±0.6 1.8±0.3 Example 2 38.8±0.7 15.0±0.6 1.7±0.4 Example 3 39.2±0.8 15.5±0.7 1.6±0.4 Example 4 39.5±0.6 15.8±0.8 1.5±0.3 Pharmacopoeia Standard ≥35.0 ≥12.0 ≤3.0 Comparative Example 1 34.5±1.2 11.8±0.9 2.7±0.4 Comparative Example 2 35.4±1.3 12.2±0.8 2.4±0.3 Comparative Example 3 35.2±1.1 12.5±0.8 2.5±0.3 Comparative Example 4 36.1±1.2 13.2±0.9 2.1±0.3 Comparative Example 5 35.8±1.1 12.0±0.8 2.3±0.4

[0072] The results in Table 1 show that the bilirubin and bile acid content of the bezoar powder prepared in Examples 1-4 were significantly higher than those in Comparative Examples 1-5 (P<0.05), and the cholesterol content was significantly lower than that in Comparative Examples 1-5, all of which met the pharmacopoeia standards.

[0073] (2) Detect the content of heavy metals and harmful elements in the prepared bezoar powder.

[0074] Heavy metals and harmful elements: The contents of lead (Pb) and arsenic (As) were determined by inductively coupled plasma mass spectrometry (ICP-MS). The test results are shown in Table 2.

[0075] Table 2

[0076] Group Lead (mg / kg) Arsenic (mg / kg) Example 1 2.3±0.2 0.8±0.2 Example 2 2.1±0.2 0.7±0.3 Example 3 2.0±0.3 0.6±0.2 Example 4 1.9±0.2 0.5±0.2 Pharmacopoeia Standard ≤5 ≤2 Comparative Example 1 3.8±0.3 1.5±0.3 Comparative Example 2 3.3±0.3 1.2±0.4 Comparative Example 3 3.4±0.4 1.3±0.3 Comparative Example 4 2.8±0.3 1.0±0.4 Comparative Example 5 3.4±0.4 1.2±0.4

[0077] The results in Table 2 show that the heavy metal content of the bezoar powder prepared in Examples 1-4 is significantly lower than that in Comparative Examples 1-5, and far below the pharmacopoeia limit, indicating better safety.

[0078] (3) Detect the microbial limit in the prepared bezoar powder.

[0079] Microbial limits: The total number of aerobic bacteria, molds, and yeasts were determined according to the pharmacopoeia microbial counting method, and Escherichia coli was detected using the chromogenic medium method. The test results are shown in Table 3.

[0080] Table 3

[0081]

[0082]

[0083] The results in Table 3 show that the bezoar powder prepared in Examples 1-4 had a lower level of microbial contamination and no Escherichia coli was detected, meeting the stringent requirements for injectable preparations; Comparative Example 1 had a poorer microbial control effect.

[0084] (4) The relative standard deviations (RSDs) of bilirubin, bile acid and heavy metal indices in the three batches of samples provided in Example 1 and Comparative Example 1 were calculated. The results are shown in Table 4.

[0085] Table 4

[0086]

[0087] The results in Table 4 show that the batch-to-batch RSD of the bezoar powder prepared in Example 1 is less than 3%, and its stability is much better than that of Comparative 1 (RSD ≥ 8%).

[0088] (5) The contents of bilirubin, bile acid and heavy metals in the samples of Example 1 and Comparative Example 1 were retested after being sealed and stored at 4°C for 6 months. The results are shown in Table 5.

[0089] Table 5

[0090]

[0091] The results in Table 5 show that after 6 months of storage, the loss rate of key components of the bezoar powder prepared in Example 1 was significantly lower than that in Comparative Example 1, indicating excellent stability.

[0092] In summary, the in vitro cultured bezoar and its preparation method provided by this invention address the problems of long culture cycles, unstable content of effective components, high production costs, and large batch-to-batch variations in existing in vitro cultured bezoar technologies by optimizing the culture medium formulation, controlling the culture process, and purifying the process.

[0093] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A method for preparing in vitro cultured bezoar, characterized in that: include: (1) Cell isolation: Fresh gallbladders from healthy cattle were rinsed with sterile saline, and the gallbladder mucosa was excised, digested, filtered, and the cell suspension was collected. The cell suspension was centrifuged, the supernatant was discarded, and the cells were resuspended in serum-free culture medium. The concentration of the cell suspension was adjusted to 1×10⁻⁶. 6 cells / mL; The serum-free culture medium comprises: 1L of DMEM / F12 medium, supplemented with 5μg / mL recombinant human insulin, 10μg / mL transferrin, 5ng / mL sodium selenite, 90μmol / L plant-derived cholesterol, and 50μmol / L synthetic taurine; (2) Primary culture: The cell suspension is seeded into a culture flask for primary culture, and passage is performed when the cell confluence reaches 80%-90%. (3) Cell seeding and culture: The serum-free culture medium is added to the carrier matrix, placed in a reactor, and the cells after passage are seeded and cultured in the carrier matrix. The cultivation includes: Proliferation phase: 20 ng / mL hepatocyte growth factor was added to the serum-free culture medium; the reactor temperature was set to 37℃, pH to 7.2-7.4, dissolved oxygen to 5%-8%, and stirring rate to 50 r / min. Synthesis period: After 5 days, 120 μmol / L cholesterol and 5 U / L recombinant glucuronyl transferase were added to the serum-free culture medium; the reactor temperature was set at 37℃, pH at 7.2-7.4, dissolved oxygen at 5%-8%, and stirring rate at 80 r / min. Crystallization period: After 12 days, the calcium ion concentration in the serum-free culture medium was increased to 1.5 mmol / L, and 0.1% calcium taurocholate was added; the reactor temperature was set to 37℃, the pH value to 7.2-7.4, the dissolved oxygen content to 5%-8%, and the stirring rate to 30 r / min. (4) Harvesting and purification of bezoar: After 30 days, stop the culture and carry out bezoar harvesting, washing, defatting, drying and pulverizing in sequence.

2. The method for preparing in vitro cultured bezoar according to claim 1, characterized in that: In step (2), the passaged cells are pretreated in a solution containing 10 μmol / L chenodeoxycholic acid for 24 h.

3. The method for preparing in vitro cultured bezoar according to claim 1, characterized in that: In step (3), the vaccination includes: on day 1, 5 × 10 4 cells / cm 2 High-density vaccination, followed by a booster dose of 5×10⁻⁶ mmol / L 24 hours later. 4 cells / cm 2 .

4. The method for preparing in vitro cultured bezoar according to claim 3, characterized in that: Step (3) also includes adding 10 μg / mL laminin to the inoculated reactor.

5. The method for preparing in vitro cultured bezoar according to claim 1, characterized in that: In step (4), the harvesting includes: taking out the carrier matrix, peeling off the particles on the surface with sterile tweezers, and obtaining bezoar particles that are brownish-yellow or reddish-brown.

6. The method for preparing in vitro cultured bezoar according to claim 5, characterized in that: In step (4), the cleaning process includes: ultrasonically cleaning the bezoar granules at 180W for 10 min with 0.01 mol / L PBS buffer at 37°C.

7. The method for preparing in vitro cultured bezoar according to claim 5, characterized in that: In step (4), defatting includes: using supercritical CO2 extraction at 40°C and 20MPa to defatt the bezoar granules for 2 hours.

8. The method for preparing in vitro cultured bezoar according to claim 5, characterized in that: In step (4), drying includes: placing the defatted bezoar granules into a vacuum drying oven at 45°C and drying them until the moisture content is ≤3%.

9. An in vitro cultured bezoar prepared using the method for preparing in vitro cultured bezoar according to any one of claims 1-8.

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