In-vitro culture method of giardia lamblia

Abstract

The invention relates to the technical field of animal parasitic pathology, and particularly discloses an in-vitro culture method of Giardia lanceolata, which comprises the following steps: S1, mixing an RPMI-1640 culture medium and a pre-sterilized bovine small intestine mucus index solution according to a volume ratio of 80: 20 to simulate a host small intestine mucous membrane surface environment to obtain a basic nutrition substrate; s2, obtaining an enhanced nitrogen source culture medium; s3, obtaining an initial attachment group; s4, obtaining a circulating proliferation population; s5, obtaining a purified trophozoite; s6, obtaining a stable passage strain with high activity and low pollution; by introducing the bovine small intestine mucus index liquid and finely regulating and controlling the osmotic pressure and the pH value, the method can highly reduce the host small intestine mucous membrane environment in chemical composition and physical properties, so that the affinity of flagellates and a matrix is remarkably improved, and attachment and directional growth of the flagellates are promoted; the calcium alginate microcarrier provides a bionic three-dimensional attachment structure, and the problems that cells are easy to gather and nutrients are not uniformly distributed in static culture are solved in cooperation with continuous circulating slight shearing force of the microfluid.

Description

Technical Field

[0001] The invention belongs to the technical field of animal parasitology, and particularly relates to an in vitro culture method of Giardia lamblia. Background Art

[0002] Giardia lamblia is an important intestinal protozoan that causes giardiasis in humans. It is prevalent worldwide and poses a serious threat to public health. In-depth research on Giardia lamblia, including drug screening, toxicological evaluation, and vaccine development, is crucial for the effective prevention and control of the disease. High-quality in vitro culture of Giardia lamblia is crucial for these studies.

[0003] However, existing in vitro culture technologies for Giardia lamblia have numerous shortcomings. Traditional static culture models have significant drawbacks. During static culture, cells are prone to aggregation, resulting in uneven nutrient distribution and localized nutrient excess or deficiency. This not only limits cell proliferation efficiency but can also lead to decreased cell health and affect the normal physiological functions of cells. Furthermore, static culture cannot simulate the effect of intestinal peristalsis on the exchange of nutrients and waste, and cannot provide Giardia lamblia with culture conditions closer to the in vivo physiological state, which is not conducive to its growth and reproduction.

[0004] To this end, the present application proposes an in vitro culture method for Giardia lamblia to solve the above problems. Summary of the Invention

[0005] The object of the present invention is to provide an in vitro culture method for Giardia lamblia to solve the problems raised in the above background technology.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] An in vitro culture method for Giardia lamblia comprises the following steps:

[0008] S1. Mix RPMI-1640 medium and pre-sterilized bovine small intestinal mucus index solution at a volume ratio of 80:20 to simulate the host small intestinal mucosal surface environment and obtain a basic nutrient substrate;

[0009] S2, 0.2M glycine and 2mM sodium bicarbonate are added to the basal nutrient medium for adjusting the osmolarity of the medium to 300mOsm / kg and the pH to 7.2, resulting in an optimized medium; 100U / mL penicillin, 100μg / mL streptomycin and 25μg / mL vancomycin are added to the optimized medium in a ratio of 100:100:25 for inhibiting bacterial and fungal contamination, resulting in a bacteriostatic medium; 5mM arginine and 2mM ornithine are additionally supplemented to the bacteriostatic medium for enhancing the nitrogen supply, resulting in an enhanced nitrogen source medium;

[0010] S3, the pre-coated collagen type I calcium alginate microcarriers are co-cultured with Giardia lamblia suspension containing 1x10^5 trophozoites / mL for providing a three-dimensional attachment surface, resulting in an initial attachment population;

[0011] S4, the culture solution containing the initial attachment population is placed in a microfluidic culture device with a flow rate of 0.1mL / min for continuous culture for 48h for simulating the peristalsis convection of small intestine and promoting proliferation, resulting in a circulating proliferation population;

[0012] S5, the circulating proliferation population is subjected to 30%-60% Percol l density gradient centrifugation for separating mature trophozoites from residual carriers, resulting in purified trophozoites;

[0013] S6, the purified trophozoites are re-inoculated into fresh enhanced nitrogen source medium for continuous three generations of subculture for obtaining a stable subculture strain with high activity and low contamination.

[0014] Preferably, the bovine small intestine mucosa indicator solution used in the basal nutrient medium is derived from healthy human small intestine mucosa swab solution, which is sterilized by 0.22μm filter membrane and used.

[0015] Preferably, in step S2, the addition amount of glycine and sodium bicarbonate is in the range of 0.15-0.25M and 1.5-2.5mM, respectively, so as to maintain the osmolarity of the medium at 280-320mOsm / kg and the pH at 7.0-7.4.

[0016] Preferably, 0-50μg / mL chloramphenicol is further added to the bacteriostatic medium for further inhibiting gram-negative bacterial contamination.

[0017] Preferably, the addition amount of arginine and ornithine in step S2 is in the range of 3-7mM and 1-3mM, respectively, so as to increase the proliferation rate of Giardia lamblia.

[0018] Preferably, the microcarriers are calcium alginate beads with a diameter of 200-500μm, and 1mg / mL collagen type I is pre-coated on the surface of the microcarriers for enhancing the adhesion efficiency of flagellate.

[0019] Preferably, the microfluidic culture device is a closed loop system containing a one-way peristaltic pump and a 37℃ constant temperature tank, the flow rate is 0.05-0.2 mL / min, and the culture time is 24-72 h to adapt to different culture scale requirements.

[0020] Compared with the prior art, the present application has the following beneficial effects:

[0021] (1) The present application can significantly improve the affinity of flagellates and matrix, promote their attachment and directional growth by introducing bovine small intestine mucus index liquid and fine control of osmotic pressure and pH, thereby highly reducing the chemical composition and physical properties of the host small intestine mucosa environment; the calcium alginate microcarrier provides a biomimetic three-dimensional attachment framework, which, combined with the slight shear force of the microfluidic continuous circulation, not only avoids the problems of easy aggregation of cells and uneven distribution of nutrients in static culture, but also simulates the promoting effect of intestinal peristalsis on the exchange of nutrients and waste, thereby improving the proliferation efficiency and cell health degree.

[0022] (2) The present application comprehensively uses various antibacterial agents, and cooperates with the design of a closed circulation system to greatly inhibit the invasion and colonization of bacteria and fungi, reduce the risk of repeated liquid change and cross contamination of culture instruments, and ensure the long-term sterile stability of the culture system; combined with Percol density gradient separation and continuous fluid culture, the mature trophozoites can be quickly separated and the carrier fragments can be effectively removed, so that the acquisition of purified high-activity population is realized; continuous dynamic passage of multiple generations further ensures the activity consistency and genetic stability of the strain, and provides a reliable material basis for subsequent drug screening, toxicological evaluation and vaccine research and development. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 A flow chart of the in vitro culture method of Giardia lamblia according to the present application. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0025] Embodiment one:

[0026] Please refer to Figure 1 Fig. 1 shows an in vitro culture method of Giardia lamblia, which comprises:

[0027] 1. Materials and reagents:

[0028] Giardia lamblia stock: derived from human clinical isolate, type GL-2025, 1 x 10^6 trophozoites / mL stored in liquid nitrogen.

[0029] RPMI-1640 medium (without phenol red, Gibco): supplemented with 10% (v / v) sterilized fetal bovine serum (FBS), 100 U / mL penicillin, 100 μg / mL streptomycin before use.

[0030] Bovine small intestine mucus index fluid: fresh healthy volunteer jejunal mucosa swab fluid (with ethical approval), 0.22 μm filter sterilized and stored at -20 °C.

[0031] Amino acids and salts: glycine (Sigma, analytical pure), sodium bicarbonate (analytical pure), arginine, ornithine (all purchased from Sigma).

[0032] Antibacterial agents: vancomycin (25 μg / mL), chloramphenicol (50 μg / mL).

[0033] Microcarriers: calcium alginate microspheres (diameter 250 μm), surface pre-coated with 1 mg / mL collagen type I (Sigma).

[0034] Percol l density gradient reagent: Sigma, 30% - 60% fractionally configured.

[0035] Reagent consumables: sterile centrifuge tubes, sterile pipettes, 0.22 μm filters, etc.

[0036] 2. Instruments and equipment:

[0037] Microfluidic culture device: containing a unidirectional peristaltic pump (flow rate adjustable 0.05 - 0.2 mL / min), 37 °C thermostat, closed loop circulation pipeline.

[0038] Inverted microscope (Nikon Ti-E) with 100x phase contrast objective lens.

[0039] Blood cell counting chamber and trypan blue staining reagent (Sigma).

[0040] Tabletop high-speed centrifuge (Eppendorf 5810R).

[0041] pH / osmolarity detector: pH meter (Mettler Toledo), osmometer (OsmoTECH).

[0042] 3. Culture method steps:

[0043] 3.1 Preparation of basic nutrient medium:

[0044] Take RPMI-1640 medium 1L, add 250mL of sterilized bovine small intestine mucosa indicator liquid;

[0045] In 4℃ ice bath, gently invert and mix for 15min, stand for 5min, blow off the bubbles, and mark as the basic nutrient medium.

[0046] 3.2 Optimization of medium preparation:

[0047] Add glycine 200mmol (20mL 10M stock solution) and sodium bicarbonate 2mmol (4mL 0.5M stock solution) to the basic nutrient medium;

[0048] Correct to 7.20±0.02 using a pH meter, and to 300±5mOsm / kg using an osmometer, and mark as the optimized medium.

[0049] 3.3 Preparation of bacteriostatic medium:

[0050] Add penicillin 100U / mL, streptomycin 100μg / mL, and vancomycin 25μg / mL to the optimized medium;

[0051] Optionally, add chloramphenicol 50μg / mL to inhibit gram-negative bacteria, and obtain the bacteriostatic medium.

[0052] 3.4 Preparation of enhanced nitrogen source medium:

[0053] Add arginine 5mmol (5mL 1M stock solution) and ornithine 2mmol (2mL 1M stock solution) to the bacteriostatic medium;

[0054] 37℃ water bath equilibration for 30min, and mark as the enhanced nitrogen source medium.

[0055] 3.5 Construction of initial attachment population:

[0056] Add 5mL of collagen type I pre-coated calcium alginate microspheres (about 1×10^7) to the bacterial suspension (1×10^5 trophozoites / mL, final volume 50mL);

[0057] Incubate at 37℃, 5% CO2 for 2h, and three-dimensional attached trichomonads can be seen on the surface of the microspheres, and mark as the initial attachment population.

[0058] 3.6 Microfluidic circulation proliferation:

[0059] Connect the culture solution containing the initial attachment population to the microfluidic device, set the flow rate to 0.1mL / min, and circulate at 37℃ for 48h;

[0060] Take 1mL every 12h, count the viable cells with trypan blue, and inspect for contamination;

[0061] 48h after microspheres surface coverage reached more than 85%, the number of living organisms was 1x10^5>8x10^6 trophozoites / mL, marked as the circulating proliferation population.

[0062] 3.7 Gradient separation purification:

[0063] The circulating proliferation population was collected in a 50mL centrifuge tube, and centrifuged at 800g for 10min with a 30%-60% Percol l gradient.

[0064] The middle layer (enriched mature trophozoites) was separated, and the supernatant and bottom residual carrier were discarded.

[0065] 3.8 Stable passage acquisition:

[0066] The enriched mature trophozoites were re-inoculated into 50mL fresh culture medium, and the microfluidic circulation was repeated for 48h, marked as the first generation.

[0067] After 3 consecutive passages, the living proliferation curve tended to be stable, the contamination rate was <1%, and a stable strain with high activity and low contamination was obtained.

[0068] As can be seen from the above, the small intestine mucosa environment is simulated: the introduction of bovine small intestine mucus index liquid significantly improves the flagellate attachment rate, thereby increasing the amplification speed by 2.5 times;

[0069] Physicochemical coupling optimization: fine control of osmotic pressure and pH with glycine / sodium bicarbonate, normal rate of flagellate morphology >95%;

[0070] Triple antibacterial strategy: penicillin + streptomycin + vancomycin (optionally chloramphenicol), bacterial and fungal contamination rate <1%, significantly better than traditional single antibiotic;

[0071] Nitrogen source enhancement: arginine / ornithine synergistic energy supply, proliferation rate 40% higher than that without nitrogen source medium;

[0072] Three-dimensional microcarriers + microfluidics: calcium alginate microspheres provide a three-dimensional attachment framework, and microfluidics simulate small intestine peristalsis, greatly improving the proliferation efficiency, with a proliferation of about 80 times within 48h, while reducing the aggregation death easily produced in static culture;

[0073] Gradient purification + stable passage: Percol l density separation combined with continuous microfluidic passage ensures intergenerational stability, with Viability maintained at >90% for three consecutive generations, with controllable contamination, meeting the long-term high-quality in vitro maintenance needs.

[0074] From the above, by introducing bovine small intestine mucus index liquid and fine control of osmotic pressure and pH, the method can highly reduce the host small intestine mucosa environment in chemical composition and physical properties, thereby significantly improving the affinity of flagellate and matrix, promoting its attachment and directional growth.

[0075] Calcium alginate microcarriers provide a biomimetic three-dimensional attachment framework, combined with the slight shear force of microfluidic continuous circulation, not only avoiding the problem of easy aggregation of cells and uneven distribution of nutrients in static culture, but also simulating the promoting effect of intestinal peristalsis on the exchange of nutrients and waste, thereby improving the proliferation efficiency and cell health degree.

[0076] Example two:

[0077] 1. Proliferation curve and doubling time determination:

[0078] Inoculation and sampling:

[0079] Take the stable strain suspension and inoculate it in 50 mL enhanced nitrogen source medium at an initial concentration of 1 × 10^5 trophozoites / mL.

[0080] Continue the culture under the conditions of the microfluidic device (flow rate 0.1 mL / min, 37°C).

[0081] Take 1 mL of culture medium every 6 h for cell counting and trypan blue staining, respectively.

[0082] Cell counting:

[0083] The trypan blue method is used to distinguish live / dead cells, and the number of live cells is counted using a hemocytometer.

[0084] Record the live cell concentration at each time point and draw a logarithmic growth curve.

[0085] Doubling time calculation:

[0086] Fit a straight line with the data in the logarithmic linear growth phase, and calculate the doubling time (Td) according to the following formula:

[0087]

[0088] Where slope refers to the rate constant of the natural logarithm value of cell number changing with time in the logarithmic growth phase, i.e., the growth rate;

[0089] Compare the Td under the conditions of traditional RPMI-1640 static culture (same initial inoculation concentration, same batch of FBS and antibiotics) with the Td.

[0090] 2. Morphology and adhesion evaluation:

[0091] Phase contrast microscopic observation:

[0092] At the end of 48h culture, microcarrier microspheres samples were taken and random 5 fields were photographed using inverted phase contrast microscope (100x).

[0093] The attachment coverage (number of attached cells per total visible area of microsphere surface) and morphological integrity (flagella integrity, nucleus structure) were evaluated.

[0094] Scanning electron microscopy (SEM) analysis:

[0095] Attached and circulating populations were fixed (2.5% glutaraldehyde, 4°C, 2h), dehydrated in graded ethanol, critical point dried and metallized.

[0096] The attachment structure and flagella arrangement were observed to verify the promoting effect of three-dimensional microcarriers on attachment.

[0097] 3. Viability and survival rate detection:

[0098] Flow cytometry viability staining:

[0099] SYTO 9 / PI double staining was used to identify live and dead cells in circulating proliferating population and control group cultured in traditional way.

[0100] 10,000 events were counted to calculate the percentage of live cells.

[0101] MTS metabolic activity assay:

[0102] 1 x 10^5 cells were taken at each time point (24h, 48h), MTS reagent was added and incubated at 37°C for 2h, and the absorbance at 490nm was measured.

[0103] The absorbance is related to the number of viable cells and is used to evaluate metabolic activity.

[0104] 4. Contamination detection and antibacterial effect evaluation:

[0105] Bacterial / fungal growth plate detection:

[0106] After the end of culture (48h), 100μL of culture medium was taken and spread on LB plates and Sabouraud plates, which were incubated at 37°C for 24h.

[0107] The colonies were observed and counted to evaluate the contamination rate.

[0108] 16S / 18S rRNA PCR detection:

[0109] Total DNA was extracted from samples and PCR amplification was performed using universal 16S (bacteria) and 18S (fungi) primers.

[0110] No significant microbial contamination was determined in the absence of amplification product.

[0111] 5. Passage stability verification:

[0112] Continuous passage monitoring:

[0113] Stable strain was continuously passaged for 5 generations, and sampling was performed after 48h for each generation.

[0114] The proliferation fold, percentage of viable cells and contamination rate were compared among generations, and the intergenerational stability graph was drawn.

[0115] Genetic diversity detection:

[0116] Genomic DNA was extracted from the 1st and 5th generation cells, and polymorphism analysis was performed using Giardia-specific SSR markers.

[0117] The SSR profiles were compared to confirm the genetic stability.

[0118] Comparative example:

[0119] Traditional method implementation:

[0120] 1. Materials and reagents:

[0121] Giardia lamblia stock: GL-2025, a clinical isolate, 1x10^6 trophozoites / mL, stored in liquid nitrogen.

[0122] RPMI-1640 medium (containing phenol red, Gibco): 1L, supplemented with 10% (v / v) sterilized fetal bovine serum (FBS), 100 U / mL penicillin, and 100 μg / mL streptomycin before use.

[0123] Antibacterial agent (optional): vancomycin 25 μg / mL (if stronger antibacterial effect is needed, it can be added).

[0124] PBS buffer: pH 7.2, used for cell washing and suspension.

[0125] Trypan blue staining solution: Sigma, used for live and dead cell differentiation.

[0126] 2. Instruments and equipment:

[0127] 37°C CO2 incubator: humidity about 90%, CO2 concentration 5%.

[0128] 75cm 2 Cell culture flask (with filter port) or 25cm 2 flat-bottomed culture tube.

[0129] Inverted phase contrast microscope (Nikon Ti-E, 100x objective).

[0130] Hemocytometer and pipette.

[0131] Eppendorf 5810R.

[0132] 3. Culture method steps:

[0133] 3.1. Thawing and primary culture:

[0134] Take the cryo tube out of the liquid nitrogen tank and thaw quickly in a 37°C water bath for about 1 min;

[0135] Transfer the thawed suspension into a 10 mL pre-warmed to 37°C RPMI-1640 supplemented with FBS and penicillin-streptomycin, and mix gently;

[0136] Centrifuge at 100 x g for 3 min, discard the supernatant, and resuspend the cells in 10 mL pre-warmed RPMI-1640;

[0137] Inoculate the cell suspension into a 75 cm 2 Incubate at 37°C, 5% CO2.

[0138] 3.2. Daily maintenance and subculture:

[0139] Daily observation: observe the trichomonad morphology and density under an inverted microscope every 24 h;

[0140] Medium change: when the density of both suspended and adherent trichomonads reaches about 1 x 10^6 trophozoites / mL, discard the old medium gently and add 10 mL fresh pre-warmed RPMI-1640;

[0141] Subculture: perform subculture every 3-4 days:

[0142] Take all the culture medium into a 15 mL centrifuge tube and centrifuge at 100 x g for 3 min;

[0143] Discard the supernatant and resuspend to 1 x 10^5 trophozoites / mL with pre-warmed medium;

[0144] Transfer to a new flask and make up to 10 mL, and continue incubation under the same conditions.

[0145] 3.3. Cell counting and viability detection:

[0146] Take 100 μL of the suspension and mix with 100 μL trypan blue before each subculture;

[0147] Count the number of live (no blue staining) and dead (blue staining) cells in a hemocytometer, and calculate the live cell rate;

[0148] And determine the inoculation density for the next subculture according to the live cell concentration.

[0149] 3.4. Contamination detection:

[0150] Each time the liquid is taken 1 mL of culture, spread on LB plate, 37°C culture 24h;

[0151] Observation of bacterial colony growth, if there are colonies, it is determined that there is bacterial contamination;

[0152] If there are colonies for two consecutive times, consider supplementing vancomycin or chloramphenicol, or directly discarding the contaminated bottle.

[0153] Although this "traditional static RPMI-1640 culture method" is a commonly used online solution, compared with the three-dimensional microcarrier + microfluidic dynamic circulation culture of the present application, there are obvious deficiencies in proliferation efficiency, viability maintenance and contamination inhibition.

[0154] The results obtained in Example 2 and the comparative examples are compared as shown in the following Table 1:

[0155] Table 1

[0156]

[0157] As can be seen from the above, supplementing arginine and ornithine rich in energy and synthetic precursors in the basic medium can effectively activate the core metabolic pathway of Giardia lamblia, enhance the cell division ability and biosynthesis level, and provide sufficient support for high-speed proliferation.

[0158] This scheme comprehensively uses a variety of antibacterial agents, and cooperates with the closed circulation system design, greatly inhibits the invasion and colonization of bacteria and fungi, reduces the risk of repeated liquid change and cross contamination of culture instruments, and ensures the long-term sterile stability of the culture system.

[0159] Combined with Percoll density gradient separation and continuous fluid culture, mature trophozoites can be quickly separated and carrier fragments can be effectively removed, realizing the acquisition of high-activity purified population; continuous dynamic passage of multiple generations further ensures the activity consistency and genetic stability of the strain, providing a reliable material basis for subsequent drug screening, toxicological evaluation and vaccine research and development applications.

[0160] In the description of the specification, the description of the terms "one embodiment", "some embodiments", "example", "specific example" or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present application. In the specification, the illustrative description of the above terms is not necessarily for the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, the skilled in the art can combine and combine the different embodiments or examples described in the specification and the features of the different embodiments or examples without contradiction.

[0161] In the drawings of the embodiments of the present application, only the structures related to the embodiments of the present application are involved, and other structures can be referred to the general design. In the case of no conflict, the same embodiments and different embodiments of the present application can be combined with each other.

[0162] Although the embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, alternatives, and variations can be made thereto without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.

Claims

1. A method for culturing Giardia lamblia in vitro, characterized in that: The following steps are involved: S1. Mix RPMI-1640 medium and pre-sterilized bovine small intestinal mucus index solution in a volume ratio of 80:20 to simulate the host small intestinal mucosal surface environment and obtain a basic nutrient substrate; S2. Adding glycine and sodium bicarbonate to the basal nutrient substrate to adjust the osmotic pressure of the culture medium to 280–320 mOsm / kg and the pH to 7.0–7.4 to obtain an optimized culture medium; adding 100 U / mL penicillin, 100 μg / mL streptomycin, and 25 μg / mL vancomycin to the optimized culture medium to obtain an antibacterial culture medium; and additionally supplementing the antibacterial culture medium with arginine and ornithine to obtain an enhanced nitrogen source culture medium; S3, co-culturing calcium alginate microcarriers pre-coated with collagen type I with a Giardialamblia suspension containing 1×10^5 trophozoites / mL to provide a three-dimensional attachment surface and obtain an initial attachment population; S4, placing the culture solution containing the initial attached population in a microfluidic culture device at a flow rate of 0.1 mL / min and continuously culturing for 48 hours to obtain a circulating proliferating population; S5. Perform 30%–60% Percol density gradient centrifugation on the circulating proliferating population to separate mature trophozoites from residual vectors to obtain purified trophozoites. S6. Re-inoculating the purified trophozoites into fresh enhanced nitrogen source medium for three consecutive passages to obtain stable passage strains.

2. The in vitro culture method of Giardia lamblia according to claim 1, wherein: The bovine small intestinal mucus indicator liquid used in the basic nutrient substrate is derived from healthy human small intestinal mucosal swab fluid and is sterilized through a 0.22 μm filter membrane before use.

3. The in vitro culture method of Giardia lamblia according to claim 1, wherein In step S2, the amounts of glycine and sodium bicarbonate added are in the range of 0.15–0.25 M and 1.5–2.5 mM, respectively, so as to maintain the osmotic pressure of the culture medium at 280–320 mOsm / kg and the pH at 7.0–7.

4.

4. The in vitro culture method of Giardia lamblia according to claim 1, wherein 0-50 μg / mL chloramphenicol is also added to the antibacterial culture medium.

5. The in vitro culture method of Giardia lamblia according to claim 1, characterized in that: The amounts of arginine and ornithine added in step S2 ranged from 3–7 mM and 1–3 mM, respectively.

6. The in vitro culture method of Giardia lamblia according to claim 1, characterized in that: The microcarriers are calcium alginate spheres with a diameter of 200-500 μm, and are pre-coated with 1 mg / mL collagen type I on their surfaces.

7. The in vitro culture method of Giardia lamblia according to claim 1, characterized in that: The microfluidic culture device is a closed-loop system containing a unidirectional peristaltic pump and a 37°C constant temperature bath, with a flow rate of 0.05-0.2 mL / min and a culture time of 24-72 h.

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