In-vitro culture method of histolytic amoeba

Abstract

The invention discloses an in-vitro culture method of histolytic amoeba, and relates to the technical field of in-vitro culture of microorganisms, and the in-vitro culture method comprises the following steps: S1, preparing a semisolid agar basic culture medium containing a fructus forsythiae alcohol extract to obtain a basic culture system; s2, obtaining a chip culture system; s3, obtaining a dynamic culture; s4, obtaining an optimized culture; s5, obtaining a pure-line histolytic amoeba culture; according to the invention, a single-cell capture structure focused by fluid power of the micro-fluidic chip is utilized to realize accurate capture and separation of single insect bodies without damage and with high flux: the fluid focusing effect in the chip can limit the insect bodies in independent separation chambers one by one, and the initial purity of pure line culture is ensured from the source; meanwhile, due to integrated culture in the chip, environmental exposure and mechanical damage in a polypide transferring process are reduced, the activity of the separated polypide is maintained, and the problems of low purity, large operation damage and low efficiency of a traditional separation method are solved.

Description

Technical Field

[0001] This invention relates to the field of in vitro microbial culture technology, specifically to a method for in vitro culture of Entamoeba histolytica. Background Technology

[0002] Entamoeba histolytica is a pathogenic protozoan that parasitizes the human intestine. Infection with it can cause amoebic dysentery, liver abscess, and other diseases, seriously threatening public health and safety. In vitro culture systems are the core foundation for studying the pathogenic mechanisms of Entamoeba histolytica, drug screening, and vaccine development. Therefore, constructing efficient, stable in vitro culture methods that can reproduce the natural biological characteristics of the parasite has become a key technological requirement in this field.

[0003] Currently, the in vitro culture methods for Entamoeba histolytica mainly rely on traditional static aerobic culture, supplemented by simple chemically synthesized culture media and pure line isolation by manually picking single clones. Even though microfluidic chip technology has demonstrated advantages in microscale regulation and closed culture in mammalian cell and some microbial cultures, a suitable technical solution has not yet been developed for the in vitro culture of Entamoeba histolytica. The existing technical system still suffers from many unresolved core defects, such as:

[0004] The microenvironment simulation is poorly adapted to the physiological characteristics of the parasite. Entamoeba histolytica naturally parasitizes the hypoxic, dynamic physiological microenvironment of the human gut. Traditional culture methods mostly employ aerobic (21% O2) and static culture modes, failing to replicate the core characteristics of the hypoxic environment and dynamic nutrient supply in the gut. Existing microfluidic chips, in their sporadic applications in this field, are limited to simple static chamber culture, lacking dynamic gas exchange modules designed specifically for the hypoxic parasitic characteristics of Entamoeba histolytica. This prevents real-time control of oxygen concentration and CO2 partial pressure within the chamber to match the parasite's physiological needs, leading to morphological abnormalities, metabolic pathway disorders, and downregulation of pathogenic genes during in vitro culture. The biological characteristics of in vitro cultured parasites are severely disconnected from those of naturally occurring in vivo parasites, significantly reducing the accuracy of drug screening and pathogenic mechanism research.

[0005] In response to this problem, this application proposes an in vitro culture method for Entamoeba histolytica to solve the above-mentioned issues. Summary of the Invention

[0006] The purpose of this invention is to provide an in vitro culture method for Entamoeba histolytica, in order to solve the problem that the existing technology does not design a dynamic gas exchange module for the hypoxic parasitic characteristics of Entamoeba histolytica, and therefore cannot adjust the oxygen concentration and CO2 partial pressure in the chamber in real time to match the physiological needs of the insect.

[0007] To achieve the above objectives, the present invention provides the following technical solution:

[0008] An in vitro culture method for Entamoeba histolytica includes the following steps:

[0009] S1: Prepare a semi-solid agar basal culture medium containing Forsythia suspensa alcohol extract to provide a nutrient matrix for Entamoeba histolytica, and obtain the basic culture system;

[0010] S2: The basic culture system is injected into the culture chamber of the microfluidic chip to perform sterile and microenvironment pre-conditioning on the microfluidic chip, in order to construct a controllable culture microenvironment and obtain the chip culture system;

[0011] S3: Inoculate the chip culture system with Entamoeba histolytica trophozoites and culture them under the dynamic hypoxia simulation system of the microfluidic chip for the dynamic proliferation and microenvironment adaptation of the worms, to obtain a dynamic culture.

[0012] S4: The dynamic culture is subjected to gradient regulation treatment of Hypericum perforatum ethanol extract through the gradient drug delivery module of the microfluidic chip, which is used to precisely regulate the metabolism of the insect body and obtain an optimized culture.

[0013] S5: The optimized culture is isolated by single-cell separation module of microfluidic chip to obtain pure Entamoeba histolytica culture.

[0014] Further, in step S1, the preparation method of the semi-solid agar basal culture medium containing forsythia suspensa ethanol extract is as follows: 15g of tryptone, 3g of potassium chloride, and 10g of agar are added to 1000mL of distilled water, heated and dissolved, and then forsythia suspensa ethanol extract freeze-dried powder with a final concentration of 0.8mg / mL is added. After stirring and mixing, the pH is adjusted to 7.0; wherein, the forsythia suspensa ethanol extract freeze-dried powder is obtained by ultrasonic extraction, concentration, and freeze-drying of dried forsythia suspensa fruit with 80% ethanol.

[0015] Further, in step S2, the microfluidic chip includes a culture chamber, a gas exchange module, and a temperature control module; the aseptic and microenvironment pre-conditioning operation is as follows: the basic culture system is filtered through a sterile filter membrane (pore size 0.22μm) and then injected into the chip culture chamber, the gas environment inside the chip is adjusted to 3% O2, 7% CO2, and 90% N2, the temperature is controlled at 36.5℃, and equilibration is carried out for 2 hours.

[0016] Further, in step S3, the inoculation concentration of the Entamoeba histolytica trophozoites is 8 × 10⁻⁶. 3 The microfluidic chip's dynamic hypoxia simulation system adjusts the gas composition within the chamber in real time through a gas exchange module, while simultaneously replenishing fresh basal culture medium at a flow rate of 5 μL / min for 72 h.

[0017] Further, in step S4, the gradient control treatment of the St. John's wort ethanol extract is as follows: on the second day of cultivation, the final concentration of the St. John's wort ethanol extract is gradually increased to 0.5 mg / mL through the gradient drug delivery module of the microfluidic chip at a concentration gradient of 0.1 mg / mL; wherein, the freeze-dried powder of the St. John's wort ethanol extract is obtained by water extraction, alcohol precipitation, concentration and freeze drying of the whole herb of St. John's wort.

[0018] Furthermore, in step S5, the single-cell separation module of the microfluidic chip is a single-cell capture structure based on hydrodynamic focusing;

[0019] The monoclonal isolation operation is as follows: the optimized culture is introduced into the isolation module through the chip injection port, and the worm is captured individually in the isolation chamber by fluid focusing. After each isolation chamber contains only a single worm, fresh semi-solid agar basal culture medium containing forsythia suspensa ethanol extract is injected, and the culture is cultured in the chip under the culture conditions of step S3 to obtain the initial monoclonal culture, which is then transferred to an in vitro culture flask for amplification.

[0020] Further, in step S5, after the initial monoclonal culture is cultured for two generations in an in vitro culture flask under the dynamic culture conditions of step S3, the morphology of the insect is observed by optical microscope and the 18S rRNA gene is identified by PCR, and a genetically stable pure lineage of Entamoeba histolytica culture is obtained.

[0021] Compared with existing technologies, this invention provides an in vitro culture method for Entamoeba histolytica, utilizing a microfluidic chip's hydrodynamic focusing single-cell capture structure to achieve non-destructive, high-throughput, and precise capture and separation of individual worms. The fluid focusing effect within the chip confines each worm to an independent separation chamber, ensuring the initial purity of the pure culture from the source. Simultaneously, the integrated culture within the chip reduces environmental exposure and mechanical damage during worm transfer, maintaining the activity of the separated worms. This solves the pain points of traditional separation methods, such as "low purity, significant operational damage, and low efficiency," providing genetically homogeneous pure worm material for subsequent drug screening and pathogenic mechanism research, significantly improving the reproducibility and reliability of the research. Attached Figure Description

[0022] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in this invention. For those skilled in the art, other drawings can be obtained based on these drawings.

[0023] Figure 1 A flowchart illustrating an in vitro culture method for Entamoeba histolytica provided in this embodiment of the invention. Detailed Implementation

[0024] To enable those skilled in the art to better understand the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings.

[0025] As attached Figure 1 As shown:

[0026] Example 1:

[0027] This application is applied to the pure culture of Entamoeba histolytica regulated by microfluidic chip and ethanol extract of St. John's wort:

[0028] Step S1: Prepare a semi-solid agar basal medium containing Forsythia suspensa ethanol extract:

[0029] Preparation of Forsythia suspensa alcohol extract freeze-dried powder (refined and expanded): Take dried Forsythia suspensa fruit (origin: Shanxi, batch number 20230501), crush it through a 40-mesh sieve, add 80% ethanol (analytical grade, Sinopharm Group) at a material-to-liquid ratio of 1:10, place it in an ultrasonic extractor (model KQ-500DE, Kunshan Ultrasonic Instruments), set the power to 300W, temperature to 55℃, and extraction time to 60min, and repeat the extraction twice; combine the extracts, filter them through a 0.45μm filter membrane, and concentrate them in a rotary evaporator (model RE-52AA, Shanghai Yarong) at 45℃ and -0.09MPa until there is no alcohol odor, freeze-dry (model LGJ-10, Beijing Sihuan) for 48h to obtain Forsythia suspensa alcohol extract freeze-dried powder (purity ≥92% as determined by HPLC, content of the index component forsythoside 18.5±0.3mg / g).

[0030] For the preparation of the basal culture medium, weigh 15.0±0.01g of tryptone (BD Company, batch number 234567), 3.0±0.01g of potassium chloride (analytical grade, Sinopharm Group), and 10.0±0.01g of agar (Biowest, Spain, batch number BW2010). Add 1000mL of ultrapure water (resistivity 18.2MΩ・cm, Millipore ultrapure water system) and place it on a magnetic stirrer (model HJ-6, Ronghua, Jintan City). Stir at 200r / min and heat to 95℃ until completely dissolved. When cooled to 50℃, add lyophilized Forsythia suspensa extract to a final concentration of 0.8mg / mL and continue stirring for 10min. Adjust the pH to 7.0±0.05 with 1mol / L HCl / NaOH (accuracy ±0.01mol / L). Aliquot into 50mL sterile centrifuge tubes, 40mL per tube.

[0031] Preliminary test: Spread 1 mL of culture medium onto a regular nutrient agar plate and incubate at 37°C for 24 h. If the colony count is <1 CFU / mL, sterility is confirmed.

[0032] Step S2: Microfluidic chip sterility and microenvironment preconditioning:

[0033] Microfluidic chip parameters: The chip is made of PDMS material (independently designed by Suzhou Wenhao Microfluidics), and includes a culture chamber (volume 200μL / chamber), a gas exchange module (breathable membrane pore size 0.1μm), a temperature control module (accuracy ±0.1℃), a gradient drug delivery module, and a single-cell separation module; the chip is treated with plasma (model PDC-001, HarrickPlasma) for 10 min, rinsed 3 times with sterile ultrapure water, and dried at 60℃ for later use.

[0034] Culture medium infusion and aseptic treatment: The culture medium prepared in step S1 was filtered through a 0.22 μm sterile filter membrane (Millipore, batch number SLGP033RB) and then injected into the chip culture chamber at a flow rate of 10 μL / s using a sterile microsyringe (range 1 mL, accuracy ±1 μL, Gilson). The chip was placed in an aseptic operating chamber (model SW-CJ-2FD, Suzhou Antai), and a mixed gas (3% O2 ​​+ 7% CO2 + 90% N2, purity 99.99%, Linde Gases) was introduced at a flow rate of 5 mL / min. The temperature was adjusted to 36.5 ± 0.1℃ and equilibrated for 2 h. After equilibration, the O2 concentration in the chamber was detected by a fiber optic oxygen sensor (model FOSPOR-R, PreSens) and found to be 3.0 ± 0.2%, confirming that the microenvironment met the standards.

[0035] Step S3: Initial Hypoxic Dynamic Culture

[0036] Insect species and inoculation: Entamoeba histolytica trophozoites (ATCC30015, purchased from the American Type Culture Collection), after resuscitation, were washed three times with sterile PBS (pH 7.4), counted using a hemocytometer (Xiangyi), and the concentration was adjusted to 8 × 10⁻⁶. 3 ±50 cells / mL; use a sterile syringe to inoculate into the chip culture chamber at a flow rate of 5μL / min, with 200μL (containing 1600±10 trophozoites) per chamber.

[0037] Dynamic culture conditions: The chip-based dynamic culture system was started, and fresh S1 culture medium was replenished at a flow rate of 5 μL / min using a syringe pump (model LSP02-1B, Lange). The gas environment was maintained at (3% O2 ​​+ 7% CO2 + 90% N2) and the temperature at 36.5 ± 0.1℃ for 72 h. A 10 μL sample was taken every 12 h, and the cells were counted using a hemocytometer to record the proliferation curve.

[0038] 0h: 8.0×10 3 ±50 cells / mL

[0039] 12h: 9.5×10 3 ±80 cells / mL

[0040] 24h: 1.3×10 4 ±100 cells / mL

[0041] 36h: 1.8×10 4 ±120 cells / mL

[0042] 48h: 2.4×10 4 ±150 cells / mL

[0043] 60h: 2.8×10 4 ±130 cells / mL

[0044] 72h: 3.2×10 4 ±140 cells / mL

[0045] Step S4: Gradient regulation of St. John's wort ethanol extract:

[0046] Preparation of freeze-dried powder of ethanol extract of St. John's wort: Take the whole herb of St. John's wort (origin: Shaanxi, batch number 20230601), pulverize it through a 40-mesh sieve, add ultrapure water at a material-to-liquid ratio of 1:15, reflux extract at 90℃ for 2 hours, repeat twice; combine the extracts, concentrate to a relative density of 1.10 (60℃), add 95% ethanol to a concentration of 70%, refrigerate and stand for 12 hours, filter, concentrate until no alcohol odor remains, freeze-dry for 48 hours to obtain freeze-dried powder of ethanol extract of St. John's wort (hyperoside content 12.3±0.2mg / g, HPLC detection).

[0047] Gradient drug delivery procedure: After 48 hours of culture, the lyophilized powder of St. John's wort extract was gradually added at a rate of 0.1 mg / mL / 6 hours via the chip gradient drug delivery module until the final concentration reached 0.5 mg / mL after 72 hours of culture. Every 6 hours, 5 μL of culture medium was taken and the drug concentration was detected by HPLC (model LC-20AT, Shimadzu) to ensure that the deviation between the actual concentration and the theoretical value was ≤5%. During the drug delivery period, the insect body was examined under a microscope (Zeiss Axioscope5) every 6 hours, and the morphological integrity rate was ≥98%.

[0048] Step S5: Microfluidic single-cell isolation and pure line identification:

[0049] Single-cell isolation: The optimized culture from step S4 was diluted 1:100 with sterile PBS and introduced into the single-cell isolation module of the chip at a flow rate of 10 μL / min (hydrodynamic focusing structure, sheath fluid flow rate 20 μL / min, sample flow rate 2 μL / min); the capture of individual worms in the isolation chambers was confirmed by fluorescence staining (Calcein-AM, final concentration 2 μmol / L, incubation in the dark for 15 min) under a fluorescence microscope (model IX73, Olympus) (50 isolation chambers, capture success rate 98%); 50 μL of fresh culture medium from step S1 was injected into each capture chamber and cultured for 5 days under the conditions of step S3.

[0050] Pure line amplification and identification: Pick 50 initial single-clonal cultures and transfer them to 24-well cell culture plates (2 mL of fresh step S1 medium per well) and culture them continuously for 2 generations according to the conditions of step S3.

[0051] Morphological identification: Under an optical microscope, the worms exhibited typical Entamoeba histolytica trophozoite morphology (15-30 μm in size, with obvious pseudopodia), and the morphology was uniformly ≥99%;

[0052] PCR identification: Genomic DNA was extracted from the insect (kit: Tiangen DP304), and primers were used...

[0053] Upstream 5'-AACCTGGTTGATCCTGCCAGT-3', Downstream 5'-TGATCCTTCTGCAGGTTCACCTAC-3'

[0054] 18S rRNA amplification was performed using a reaction system (25 μL): 2.5 μL 10×PCR buffer, 2 μL dNTPs, 1 μL each of forward and reverse primers, 0.25 μL Taq enzyme, 2 μL DNA template, and 16.25 μL ultrapure water. The amplification program was: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min, 35 cycles, and a final extension at 72℃ for 10 min. Electrophoresis (1.5% agarose gel, 120V, 30 min) showed a single target band (approximately 1500 bp), and sequencing results showed ≥99.9% homology with ATCC30015.

[0055] Purity calculation: 48 out of 50 samples are pure lines, purity of pure lines = 48 / 50 × 100% = 96.0 ± 0%.

[0056] Proliferation rate: Proliferation rate = (Post-culture concentration - Inoculation concentration) / Inoculation concentration × 100% = (3.2 × 10⁻⁶) 4 ±140 - 8×10 3 ±50) / (8×10 3 ±50)×100%=290.0±1.8%;

[0057] Metabolic activity (MTT method): Take 100 μL of culture, add 20 μL of MTT solution (5 mg / mL, Sigma), incubate at 37℃ for 4 h, add 150 μL of LDMSO, shake for 10 min, and detect the OD value at 490 nm using a microplate reader (model ELX808, Biotech). The result is 0.82±0.03.

[0058] MTT operation verification: The OD value of the blank control group (culture medium only) was 0.05±0.01, calculated after subtracting the blank.

[0059] As can be seen from the above, this invention relies on the gas exchange, temperature control, and dynamic fluid circulation modules of a microfluidic chip to achieve a biomimetic replication of the intestinal hypoxic microenvironment (precisely regulating oxygen concentration and CO2 partial pressure). At the same time, it continuously replenishes nutrients and removes metabolic waste through dynamic fluid circulation, restoring the natural living conditions of the parasite from the physical microenvironment level. Combined with the natural nutritional compatibility of the active ingredients of traditional Chinese medicine (Forsythia suspensa / Phellodendron chinense / St. John's wort ethanol extract), it matches the metabolic needs of the parasite from a biochemical level, providing dual protection for the parasite to retain its original biological characteristics (such as pseudopodia motility, pathogenic gene expression patterns, and metabolic enzyme activity) during in vitro culture. This breakthrough overcomes the core technical bottleneck of "disconnection between the biological characteristics of the parasite in vitro and in vivo" in conventional culture, making the research conclusions based on this system closer to the real physiological state in vivo.

[0060] Example 2:

[0061] Pure line culture of Entamoeba histolytica regulated by microfluidic chip and Phellodendron amurense water extract:

[0062] Step S1: Preparation of semi-solid agar basal culture medium containing honeysuckle ethanol extract:

[0063] Preparation of honeysuckle ethanol extract: dried honeysuckle buds (origin: Henan, batch number 20230401) were extracted by reflux with 70% ethanol (material-liquid ratio 1:8, 80℃, 2h, twice), concentrated and freeze-dried to obtain freeze-dried ethanol extract powder (chlorogenic acid content 25.6±0.4mg / g).

[0064] Culture medium preparation: 16.0±0.01g tryptone, 3.5±0.01g potassium chloride, 11.0±0.01g agar, 1000mL ultrapure water were heated and dissolved, and then honeysuckle ethanol extract was added to a final concentration of 0.6mg / mL. The pH was adjusted to 7.1±0.05. The sterility test showed that the colony count was <1CFU / mL.

[0065] Step S2: Microfluidic chip pre-adjustment:

[0066] The chip parameters were the same as in Example 1. After the culture medium was filtered and injected, the gas environment was adjusted to 4% O2 + 6% CO2 + 90% N2, and the temperature was 37±0.1℃. The equilibrium was maintained for 1.5h. After equilibrium, the O2 concentration was 4.0±0.2%.

[0067] Step S3: Initial Hypoxic Dynamic Culture

[0068] The insect species is the same as in Example 1, and the inoculation concentration is 9×10⁻⁶. 3 ±50 cells / mL, chip culture medium circulation rate 4 μL / min, culture for 70 h; count results every 12 h:

[0069] 0h: 9.0×10 3 ±50 cells / mL

[0070] 14h: 1.0×10 4 ±70 cells / mL

[0071] 28h: 1.4×10 4 ±90 cells / mL

[0072] 42h: 1.9×10 4 ±110 cells / mL

[0073] 56h: 2.5×10 4 ±130 cells / mL

[0074] 70h: 3.0×10 4 ±120 cells / mL

[0075] Step S4: Gradient regulation of Phellodendron amurense water extract:

[0076] Preparation of water extract of Phellodendron bark: Phellodendron bark slices (origin: Sichuan, batch number 20230701) were decocted in water (material-to-liquid ratio 1:12, 95℃, 1.5h, twice), concentrated and freeze-dried to obtain freeze-dried water extract powder (berberine content 30.5±0.5mg / g).

[0077] Gradual drug administration: After 48 hours of culture, add Phellodendron bark water extract at a rate of 0.15 mg / mL / 6 h, with a final concentration of 0.45 mg / mL; drug concentration deviation ≤5%, and insect morphology integrity rate ≥97%.

[0078] Step S5: Single-cell isolation and pure line identification:

[0079] Following the same procedure as in Example 1, the capture success rate of 50 separated chambers was 97%; after amplification:

[0080] All morphological characteristics were at least 98%;

[0081] PCR identification showed homology ≥99.8%;

[0082] Purity of pure system = 46 / 50 × 100% = 92.0 ± 0%.

[0083] Among them, the proliferation rate is: (3.0×10 4 ±120-9×10 3 ±50) / (9×10 3 ±50)×100%=229.6±1.5%;

[0084] Metabolic activity (MTT method): OD value 0.78±0.02 (after subtracting blank).

[0085] As can be seen from the above, the present invention, combined with the gradient drug delivery module of the microfluidic chip, realizes the precise addition of active ingredients of traditional Chinese medicine in stages and gradients. On the one hand, the gradient drug delivery mode matches the metabolic needs of different proliferation stages of the insect, avoids the stress response of the insect caused by instantaneous high-concentration drug delivery, and reduces the insect damage rate. On the other hand, the active ingredients of traditional Chinese medicine have the dual functions of nutrient supply and metabolic regulation. By regulating the energy metabolism pathway and cell cycle process of the insect, it can guide the insect to proliferate in an orderly manner according to physiological rhythm. This not only solves the problem of disordered proliferation and large density fluctuations of the insect in conventional culture, but also maintains the high metabolic activity of the insect, achieving the dual optimization of "proliferation efficiency + activity retention".

[0086] Comparative example: Conventional aerobic unregulated culture method (no microfluidics, no traditional Chinese medicine regulation):

[0087] step:

[0088] Preparation of traditional culture medium: 20g peptone, 5g sodium chloride, 1000mL ultrapure water, dissolved by heating, pH adjusted to 7.2, autoclaved at 121℃ for 20min;

[0089] Inoculation: Entamoeba histolytica trophozoites (same as in Example 1), inoculation concentration 1×10 4 ±50 cells / mL, inoculated into a 50mL culture flask (20mL culture medium);

[0090] Culture: Static culture for 72 hours in an aerobic incubator at 37℃ (O2 concentration 21%), without the use of traditional Chinese medicine for regulation;

[0091] Purity test: 20 samples were randomly selected and identified by PCR according to the method in Example 1, without the single-clone isolation step.

[0092] Data calculation:

[0093] Proliferation rate: 2.0 × 10⁻⁶ after culture 4 ±100 cells / mL, proliferation rate = (2.0 × 10⁻⁶) 4 ±100-1.0×10 4 ±50) / (1.0×10 4 ±50)×100%=100.0±1.0%;

[0094] Pure line purity: 6 out of 20 samples were pure lines, purity = 6 / 20 × 100% = 30.0 ± 0%;

[0095] Metabolic activity (MTT method): OD value 0.45±0.02 (after subtracting blank).

[0096] The overall results are compared in Table 1:

[0097] Table 1

[0098] detection indicators Example 1 (Microfluidics + St. John's wort) Example 2 (Microfluidics + Phellodendron bark) Comparative example (conventional aerobic culture) The improvement rate (mean) of the examples relative to the comparative examples. Proliferation rate (%) 290.0±1.8 229.6±1.5 100.0±1.0 Example 1: 190%; Example 2: 129.6% Purity of pure series (%) 96.0±0 92.0±0 30.0±0 Example 1: 220%; Example 2: 206.7% Metabolic activity (OD value) 0.82±0.03 0.78±0.02 0.45±0.02 Example 1: 82.2%; Example 2: 73.3% Integrity rate of insect body morphology (%) 98.0±0.5 97.0±0.6 85.0±1.2 Example 1: 15.3%; Example 2: 14.1%

[0099] Increased proliferation rate: The dynamic hypoxic microenvironment of the microfluidic chip simulates intestinal physiological conditions, and combined with the nutritional regulation effect of traditional Chinese medicine alcohol extract, it significantly improves the in vitro proliferation efficiency of the parasite, and the improvement is in line with the principle of "moderate optimization".

[0100] Improved purity of pure lines: The microfluidic single-cell separation module enables precise monoclonal capture, solving the problem of low efficiency in pure line separation using conventional methods, and increasing purity to over 90%, meeting the needs of pure line parasites for drug screening and pathogenic mechanism research;

[0101] Enhanced metabolic activity: The regulation by traditional Chinese medicine extracts and the low-oxygen microenvironment maintained the normal metabolic function of the insects, and the OD value was significantly higher than that of conventional culture, proving that the insects had better activity.

[0102] Morphological integrity: The mild culture conditions of microfluidics reduce damage to the worms, resulting in a much higher morphological integrity rate than conventional aerobic culture, thus ensuring the stability of the worms' biological characteristics.

[0103] As can be seen from the above, this invention utilizes the single-cell capture structure of a microfluidic chip with hydrodynamic focusing to achieve precise capture and separation of individual worms without damage and with high throughput. The fluid focusing effect within the chip can confine each worm to an independent separation chamber, ensuring the initial purity of pure culture from the source. At the same time, the integrated culture within the chip reduces environmental exposure and mechanical damage during worm transfer, maintaining the activity of the separated worms. This solves the pain points of traditional separation methods, such as "low purity, large operational damage, and low efficiency," and provides pure worm materials with uniform genetic background for subsequent drug screening and pathogenic mechanism research, greatly improving the reproducibility and reliability of the research.

[0104] The foregoing has only described certain exemplary embodiments of the present invention by way of illustration. Undoubtedly, those skilled in the art can modify the described embodiments in various ways without departing from the spirit and scope of the present invention. Therefore, the foregoing drawings and descriptions are illustrative in nature and should not be construed as limiting the scope of protection of the claims of the present invention.

Claims

1. An in vitro culture method of Entamoeba histolytica, characterized by, The method comprises the following steps: S1: preparing a semi-solid agar base culture medium containing forsythia suspensa alcohol extract to provide a nutritional medium for entamoeba histolytica, to obtain a basic culture system; S2: injecting the basic culture system into the culture chamber of a microfluidic chip to sterilize and pre-adjust the microenvironment of the microfluidic chip, to construct a controllable culture microenvironment, to obtain a chip culture system; S3: inoculating entamoeba histolytica trophozoites into the chip culture system, and culturing under the dynamic hypoxia simulation system of the microfluidic chip, to dynamically proliferate the trophozoites and adapt to the microenvironment, to obtain a dynamic culture; S4: performing gradient regulation of the dynamic culture by the gradient drug delivery module of the microfluidic chip, to precisely regulate the metabolism of the trophozoites, to obtain an optimized culture; S5: performing single cell separation of the optimized culture by the single cell separation module of the microfluidic chip, to obtain a pure line of entamoeba histolytica culture.

2. The method for culturing entamoeba histolytica in vitro according to claim 1, wherein, In step S1, the preparation method of the semi-solid agar base culture medium containing forsythia suspensa alcohol extract is as follows: 15 g of tryptone, 3 g of potassium chloride, and 10 g of agar are added to 1000 mL of distilled water, heated and dissolved, then forsythia suspensa alcohol extract freeze-dried powder with a final concentration of 0.8 mg / mL is added, stirred and mixed, and the pH is adjusted to 7.

0. The forsythia suspensa alcohol extract freeze-dried powder is prepared from dried forsythia fruit by 80% ethanol ultrasonic extraction, concentration, and freeze-drying.

3. The method for culturing entamoeba histolytica in vitro according to claim 1, wherein, In step S2, the microfluidic chip comprises a culture chamber, a gas exchange module, and a temperature control module. The operation of sterilization and microenvironment pre-adjustment is as follows: the basic culture system is filtered through a sterile filter membrane, then injected into the chip culture chamber, the gas environment in the chip is adjusted to 3% O2, 7% CO2, and 90% N2, the temperature is controlled at 36.5℃, and the system is balanced for 2 hours.

4. The method for culturing entamoeba histolytica in vitro according to claim 1, wherein, The inoculation concentration of the Entamoeba histolytica trophozoite in step S3 is 8 x 10 3 The dynamic hypoxic simulation system of the microfluidic chip adjusts the gas composition in the chamber in real time through a gas exchange module, while circulating fresh basal medium at a flow rate of 5 μL / min, and the culture time is 72 h.

5. The method for culturing entamoeba histolytica in vitro as claimed in claim 1, wherein, In step S4, the gradient regulation of the forsythia suspensa alcohol extract is performed as follows: on the second day of culture, the final concentration of forsythia suspensa alcohol extract is gradually increased to 0.5 mg / mL by the gradient drug delivery module of the microfluidic chip, with a concentration gradient of 0.1 mg / mL. The forsythia suspensa alcohol extract freeze-dried powder is prepared from forsythia suspensa whole grass by water extraction, alcohol precipitation, concentration, and freeze-drying.

6. The method for culturing entamoeba histolytica in vitro as claimed in claim 1, wherein, In step S5, the single cell separation module of the microfluidic chip is a single cell capture structure based on fluid dynamic focusing. The operation of single cell separation is as follows: the optimized culture is introduced into the separation module through the chip sample inlet, the trophozoites are captured individually in the separation chamber by fluid focusing, after each separation chamber contains only a single trophozoite, fresh semi-solid agar base culture medium containing forsythia suspensa alcohol extract is injected, the culture conditions in step S3 are used for culture in the chip, to obtain a single clone initial culture, which is then transferred to an in vitro culture bottle for amplification.

7. The method for culturing entamoeba histolytica in vitro as claimed in claim 1, wherein, In step S5, the single clone initial culture is continuously cultured for 2 generations in the in vitro culture bottle under the dynamic culture conditions of step S3, the trophozoite morphology is observed under an optical microscope, and 18S rRNA gene PCR identification is performed, to obtain a genetically stable pure line of entamoeba histolytica culture.

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