CN114854670A - Construction method of human cervical wart organ model and application of model in development of lead compound - Google Patents

Construction method of human cervical wart organ model and application of model in development of lead compound Download PDF

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CN114854670A
CN114854670A CN202210657877.2A CN202210657877A CN114854670A CN 114854670 A CN114854670 A CN 114854670A CN 202210657877 A CN202210657877 A CN 202210657877A CN 114854670 A CN114854670 A CN 114854670A
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wart
cervical
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organoid
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沈超
周利敏
王栋
杜欣
刘雪梅
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Hubei Maternal And Child Care Hospital
Wuhan University WHU
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Wuhan University WHU
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Abstract

The invention discloses a construction method of a human cervical wart organ model and application of the model in developing a lead compound, belonging to the technical field of biomedicine. The invention provides a method for establishing a human cervical wart tissue organoid model, which comprises the following steps: (1) digesting cervical wart tissues into single cells, mixing the single cells with matrix glue to form a dome sample, culturing, and adding a culture medium to form a gas-liquid exchange layer; the culture medium contains growth factors and extracellular matrix suitable for the growth of dry cells of cervical wart tissues; (2) and (2) mechanically treating the system obtained by culturing in the step (1) or digesting with collagenase to form cell masses or single cells, separating, and then resuspending with the culture medium to obtain the cervical wart tissue organoid model. The method can establish a specimen library of the cervical wart tissue organoid model, treat 106 cervical tissue samples together, and establish the library with the power of about 62.3 percent.

Description

Construction method of human cervical wart organ model and application of model in development of lead compound
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to a construction method of a human cervical wart organ model and application of the model in development of a lead compound.
Background
Cervical cancer, which is the fourth most common malignancy of women worldwide, especially in low-to-medium income countries, is often the second cancer affecting the health of women's life to breast cancer, mainly caused by persistent infection with Human Papillomavirus (HPV). In the course of the disease, HPV infects cervical epithelial cells, inducing epithelial cytopathy and gradually forming bulge, i.e. wart, the body activates immune clearance mechanism after HPV infection, and in many cases HPV is cleared by the immune system. However, when HPV forms persistent infections, especially with some high-risk types, warts gradually become cancerous. In 2018, 569847 new cases of cervical cancer are diagnosed globally, 311365 people die due to the malignant tumor, the related death rate is 54.6%, and the number of Chinese morbidity accounts for 18.6% of the world. Although the cervical cancer vaccine can effectively prevent the occurrence of cervical cancer, the cervical cancer vaccine can not treat patients infected with HPV and cervical cancer patients, and fundamentally, novel medicaments are needed to assist the human immune system to eliminate the infected HPV, namely inhibit the formation of warts or inhibit the continuous infection process of HPV, namely the cancer formation process. Therefore, there is an urgent need for a disease model that reproduces the characteristics of normal cervical tissue and can mimic the overall course of viral infection.
Over the past decade, organoid research and its applications have become standardized and practical. Organoids derived from common cancer tissues have been established and patient-derived organoids (PDOs) have been used to elucidate key scientific questions including the relationship between genetic/epigenetic changes and drug responses, cellular plasticity in disease progression, and resistance mechanisms. Organoids can summarize their organ-specific gene expression profiles, specific tissue spatial structure and function of the original tissue.
Organoid (organoid) culture is a leading-edge technology for three-dimensional culture of adult stem cell mass or tumor stem cells in vitro, a model simulating a human body internal tissue microenvironment is established, and stem cells form a tissue structure similar to an organ, so that the action conditions between cells and an extracellular matrix are well reserved. Therefore, compared with the traditional tumor cell line, the model has higher clinical relevance and individual diversity, and is more suitable for establishing an organoid specimen bank and evaluating the drug effect of the compound.
Disclosure of Invention
One of the purposes of the invention is to provide a method for constructing a human cervical wart organ model, which comprises the following steps:
1. human cervical wart tissue cell separation
Human cervical wart specimens were obtained from the external cervical os of donors by trained surgeons and should be collected and processed as soon as possible to at least 64mm 3 The tissue of (1). After surgical resection, the tissue should be kept at a low temperature of 4 ℃ in 1x DPBS until treatment.
(1) After surgical resection, the tissue should be kept at low temperature (4 ℃) in 1x DPBS until treatment (the tissue can be preserved in a basic culture medium at 4 ℃ for up to 48 hours), and the sample is preferably treated as soon as possible within 4 hours after the tissue is isolated, so as to maintain the maximum activity and improve the success rate of organoid culture;
(2) placing the tissue into a 10cm culture dish under the aseptic condition, and photographing and recording the tissue form before cleaning;
(3) rapidly cleaning the tissue for 3-4 times by using DPBS to remove blood stains and bacteria on the surface of the tissue;
(4) transferring the tissue to a new 10cm culture dish, and photographing and recording the shape of the cleaned tissue;
(5) shearing the tissue for organoid culture, transferring the sheared tissue into a 50ml centrifuge tube, adding pre-cooled DPBS (double-stranded phosphate buffer) to fix the volume to 40ml, slightly blowing the tissue fragments up and down by using a 10ml pipette, precipitating the tissue blocks, removing floating fragments in supernatant and the like;
(6) filtering the tissue and the supernatant by a 70um filter screen, and purging the tissue on the filter screen by using DPBS;
(7) transferring the tissues on the filter screen to a new 15ml centrifuge tube, and measuring the volume of the tissues by touch;
(8) adding IV Collagenase 4-5ml with final concentration of 2.5mg/ml and DNase 0.00001% with final concentration per ml tissue, and subjecting the mixture to shake digestion at 37 deg.C for about 2 h; or selecting 0.5mg/ml IV Collagenase for overnight digestion;
(9) after the tissues are digested in a shaking table for 1.5h, taking out the centrifuge tube to observe the digestion condition of the tissues, if the digestion is complete, carrying out the next step, if a large amount of parenchymal tissues still exist, adopting overnight digestion, wherein the digestion time of a small amount of tissues can be prolonged to 2.5h, and the adhered tissues cannot be digested. The goal of digestion is to obtain enough single cells (excluding red blood cells) to forego tissue re-digestion;
(10) violently pipetting up and down digestive juice by using a 10ml pipette to blow and beat tissues or shake a centrifuge tube, checking whether a large number of single cells exist in the solution, judging the tissues to be dead tissues if no single cells exist, and not performing subsequent operation;
(11) after digestion is finished, adding pre-cooled DPBS to a constant volume of 10ml, filtering digested substances into a 50ml centrifuge tube through a 70-100um filter, cleaning the centrifuge tube of 15ml, standing the filtered suspension at a low temperature, and adding pre-cooled DPBS to a constant volume of 40 ml;
(12) centrifuging for 5min at RT 450g to precipitate cells, discarding the supernatant, adding DPBS to a volume of 10ml, and transferring the resuspended cells to a 15ml centrifuge tube;
(13) centrifuging at RT 350g for 5min to precipitate cells, discarding supernatant, and observing precipitation amount to determine P 0 The number of culture wells (1-2 wells) was determined.
2. Cell plating
(1) The cells were pelleted by centrifugation at 350g for 5min and resuspended in the corresponding amount of Matrigel (40 ul Matrigel per well in a 24-well plate);
(2) add one drop of Matrigel cell suspension to the center of each well to inoculate cells, avoiding drippingTo the edge of the well, the plate was inverted and placed at 37 ℃ in 5% CO 2 Culturing in an incubator for 30min, or curing the Matrigel;
(3) 500ul of pre-heated complete medium was slowly added along the wall, and the plates were placed at 37 ℃ in 5% CO 2 Culturing in an incubator;
(4) changing the preheated complete culture medium every 3-4 days;
(5) the primary culture of the cervical organoid is carried out for 14 days at the maximum, and if the cervical organoid still does not grow out, the culture is abandoned.
3. Organoid library construction
Observing the growth state and the size quantity of the wart organs under a microscope, sucking out the culture medium, adding 1ml of cold 1 XDPBS, collecting a certain quantity of the organs into a 15ml centrifuge tube, placing on ice for 15 minutes, centrifuging 350g for 5 minutes to remove Matrigel, adding the cryopreservation liquid into 200/500 ul tubes, subpackaging into cryopreservation tubes, placing in a programmed cooling box, slowly cooling at-80 ℃, and transferring into liquid nitrogen for long-term storage the next day.
In the construction method of the human cervical wart tissue organoid model, the complete culture medium contains 1XGlutamax TM 、10mM HEPES、10mM Nicotinamide、10nM N 2 supplement, 1mM N-acetyl-L-cysteine (NAC), 2. mu. MA 83-01, 10. mu. MY27632, 1. mu.M Jagged-1, 100ng/ml Human Noggin, 25ng/ml Human EGF, 10. mu.M Forskolin, 0.5ug/ml Hydrocortisone, 1. mu.M SB202190, 1XPenicillin-Streptomycin Advanced DEME/F12.
In the method for constructing the human cervical wart tissue organoid model, the frozen stock solution is Advanced DMEM/F12 containing 20% FBS and 10% DMSO.
4. Identification of molecular characteristics of wart-like organs
And verifying whether the molecular characteristics of the organoid model are consistent with the tissues or not by an immunofluorescence technique.
The invention also aims to provide the application of the cervical wart organ model in the development of a lead compound, which comprises the following specific steps:
organoids were collected from 24-well plates, digested with collagenase, resuspended in screening medium, and then 15u per wellThe seeds are placed in a 96-well plate, the organoid density is about 2000-3000/well, after being inverted and solidified for 10min, 150ul of culture medium is added for 1 day; selecting brefeldin A as a positive control, and selecting a DMSO group as a negative control; the concentration of the drug to be screened is diluted in a gradient manner, 6 concentrations are selected, and three times of dilution are carried out; adding 20ul of diluted drug (DMSO final concentration is 0.1%), culturing for 3-4 days, and adding Cell
Figure BDA0003689063280000031
2.0 luminescennt Cell viatilityassay (Promega) the chemiluminescence of ATP in surviving organoids was measured to assess the effectiveness of the drug.
Compared with the prior art, the invention has the following beneficial effects:
(1) the method can be used for quickly and effectively establishing the cervical wart tissue organoid model which can be cultured in vitro, and molecular biology and genetic research show that the cervical wart tissue organoid model greatly reserves the genetic information and biological characteristics of the actual cervical wart tissue of a patient, and the cervical wart tissue organoid model can be used for further research and study as the in vitro model of the wart tissue.
(2) The cervical wart tissue organoid model established by the method can be subjected to in vitro passage and cryopreservation to establish a library, and the sensitivity of the lead compound can be rapidly and effectively screened in vitro to evaluate the drug effect of the compound.
(3) The method can be used for establishing a specimen bank containing a large number of cervical wart tissue organoid models, greatly covers the heterogeneity and diversity of cervical wart tissues, and becomes a high-efficiency platform for researching the generation and development mechanism of the cervical wart tissues.
Drawings
FIGS. 1 to 3 are graphs showing the growth state of organoids in culture in example 1.
FIG. 4 is a graph of HE staining of tissues and corresponding organoids of example 1.
FIG. 5 is an immunofluorescence plot of specific biomarkers (MUC5AC, PAX8, Ki67, TP63) in the tissues and corresponding organoids of example 1.
FIG. 6 is an immunofluorescence plot of specific biomarkers (CK14, CK13, CK17, EpCAM) in tissues and corresponding organoids of example 1.
FIG. 7 is a graph showing the results of organoid drug sensitivity screening in example 2.
FIG. 8 is a graph showing the results of the effects of different growth factors on organoid growth in comparative example 1.
FIG. 9 is a graph showing the results of Forskolin effect on organoid growth in comparative example 2.
FIG. 10 is a graph showing the effect of Hydrocortisone on organoid growth in comparative example 3.
FIG. 11 is a graph of organoid growth over time in the same field of view in culture in basal medium for two different patient-derived organoids of comparative example 4.
Fig. 12 is a graph of statistical results in example 2.
Detailed Description
Penicilin-Streptomyces used in the examples of the present invention was purchased from Life Technologies, cat # 15140-122.
The advanced DEME/F12 used in the examples of the present invention was purchased from Life Technologies, cat # 12634-010.
GlutaMax adopted by embodiment of the invention TM Purchased from Life Technologies under catalog number 35050-.
10mM HEPES used in the examples of the present invention was purchased from Life Technologies, cat 15630-056.
Nicotinamide used in the examples of the present invention was purchased from Sigma-Aldrich under the reference number N0636.
N adopted by the embodiment of the invention 2 supplement was purchased from Invitrogen, cat # 17502-.
N-acetyl-L-cysteine (NAC) adopted in the embodiment of the invention is purchased from Sigma-Aldrich and has the code number of A9165.
A83-01 used in the examples of the present invention was purchased from Tocris Bioscience under a product number of 2939.
Y27632 used in the examples of the present invention is purchased from MCE, and the product number is HY-10071.
Jagged-1 adopted in the embodiment of the invention is purchased from MCE and has a cargo number HY-P70399.
The Human Noggin adopted in the embodiment of the invention is purchased from MCE, and the commodity number is HY-P70558.
The Human EGF adopted in the embodiment of the invention is purchased from MCE, and the product number is HY-P7017.
Forskolin used in the examples of the present invention was purchased from MCE under the reference HY-15371.
Hydrocortisone adopted in the embodiment of the invention is purchased from MCE and has the product number of HY-N0583.
SB202190, available from MCE under the trade designation HY-10295, was used in the examples of this invention.
DPBS, available from Gibco under the trade designation 21300058, was used in the examples of the present invention.
IV Collagenase used in the examples of the present invention was purchased from Gibco under the cat # 17104019.
Matrigel used in the examples of the present invention was purchased from corning under the cat # 356231.
FBS (Total bone serum) adopted in the embodiment of the invention is purchased from the green-year-round of FBS, and the product number is 11011-.
DMSO used in the examples of the invention was purchased from Sigma under the designation D8418.
Adopted by the embodiment of the invention
Figure BDA0003689063280000051
3D was purchased from Promega under the accession number G9683.
Example 1
The embodiment provides a method for constructing a human cervical wart organ model, which comprises the following steps:
(1) study object
Specimen source patient inclusion criteria: 1) can be used for obtaining a cervical wart tissue specimen by operation. 2) The patient voluntarily took the trial and signed an informed consent.
(2) Step of Collection
1) Collecting a tissue sample: after surgery, wart tissue was soaked in PBS for a short period of time and transferred from hospital to laboratory.
2) Tissue sample washing: and (3) clamping the tissues by using forceps, putting the tissues into PBS cleaning solution, slightly washing and swinging for 4-5 times, and taking out the tissues by using the forceps.
3) Tissue sample preservation: placing into tissue specimen preserving fluid, labeling sample information (case number, name), and sealing the tube cover.
4) Information acquisition: collecting clinical information such as pathological data of patients.
(3) Organoid culture
1) Obtaining single cells from tissue
After sterilization treatment and high-activity transfer, the tissue is cleaned for 2 times by phosphate buffer solution in a laboratory, the tissue is cut into pieces by scissors, the tissue is cleaned for one time by PBS and collected by filtering by using a filter screen, a proper amount of enzymolysis compound solution (2.5mg/ml IV collagen + 0.00001% DNase) is added, enzymolysis is carried out for 2 hours in a shaker at 37 ℃ and 200rpm, the cell passes through a cell sieve to obtain single cell suspension, and the cell suspension is centrifuged twice by 300g and then is re-suspended by a basic culture medium for counting.
2) Organoid primary culture procedure
The separated cells were added to a three-dimensional culture medium (Matrigel), seeded in 24-well plates or 48-well plates at low temperature, the plates were inverted and incubated in an incubator at 37 ℃ for 15 minutes, and after the Matrigel solidified, complete medium was added.
3) Organoid library construction procedure
Observing the growth state and the size quantity of the wart organs under a microscope, sucking out the culture medium, adding 1ml of cold 1x DPBS, collecting the organs into a 15ml centrifuge tube, placing on ice for 15 minutes, centrifuging 300g for 5 minutes to remove matrigel, adding the cryopreservation liquid into 200/500 ul tubes, subpackaging into cryopreservation tubes, cooling at-80 ℃ by using a programmed cooling box, and transferring into liquid nitrogen for long-term storage the next day.
The growth status of organoids during culture is shown in fig. 1-3, where the numbers in fig. 2-3 are different sample numbers.
The matrix material used in this example was Matrigel, thawed on ice overnight at 4 ℃;
the basal medium used in this example was 1 XGlutaMax-containing TM 、10mM HEPES、10mM Nicotinamide、10nM N 2 Advanced DEME/F12 of supplement.
The complete medium used in this example was 1XGlutaMax containing TM 、10mM HEPES、10mM Nicotinamide、10nM N 2 supplement, 1mM N-acetyl-L-cysteine (NAC), 2. mu. MA 83-01, 10. mu. MY27632, 1. mu.M Jagged-1, 100ng/ml Human Noggin, 25ng/ml Human EGF, 10. mu.M Forskolin, 0.5ug/ml Hydrocortisone, 1. mu.M SB202190, 1X Penicillin-Streptomyces AdvancedDEME/F12.
4) Molecular characterization of wart organs
By immunofluorescence technique, it is proved whether the molecular characteristics of organoid model are consistent with the tissues.
The cervix connects the uterine cavity and the vagina, and includes an endocervix consisting of a monolayer of columnar cells secreting mucus and an exocervix consisting of stratified squamous epithelium, and a squamous epithelial junction (SCJ) formed by the transition of columnar epithelium to squamous epithelium. Squamous epithelia specifically express biomarkers of KRT5, KRT14, KRT17, p63, CDH1, etc., while columnar lineages show characteristic expression of KRT7, KRT8, KRT18, KRT19, etc. After embedding and cryosectioning the tissue and corresponding organoid samples, immunofluorescence analysis of the expression of specific biomarkers, including the expression of the corresponding biomarkers in the tissue and organoids and the similarity of the marker locations to verify consistency, see fig. 4-6 for results.
To determine whether human cervical organoids can reproduce the structure and function of cervical tissue, we explored the expression and distribution of specific biomarkers in tissues and corresponding organoids. The immunofluorescence plots shown in fig. 5 and 6 show that the biochemical composition of organoids is similar to cervical tissue. Furthermore, we found that basal layer cells of exocervical epithelium expressing TP63, CK14 and proliferating cells expressing Ki67 were localized to the outer side of organoids.
Example 2
This example provides a method for screening organoid drugs, comprising the steps of:
collecting organoids from a 24-well plate, carrying out trypsin digestion, then suspending the organoids in a complete culture medium, then placing 15ul of the organoids in a 96-well plate according to the density of the organoids in each well, wherein the organoids are about 2000-3000 per well, inverting and solidifying for 10min, then adding 150ul of the culture medium, and culturing for 1 day; brefeldin A (5uM) was selected as a positive control, and DMSO group was negativeControl (organoid with 0.1% DMSO), blank control contained no organoid, 0.1% DMSO; carrying out gradient dilution on the concentration of the screening medicine of Obatoclax (CAS is 803712-79-0), selecting 6 concentrations (respectively 25uM, 7.813uM, 2.441uM, 0.763uM, 0.238uM and 0.075uM), and carrying out three repetitions; adding 20ul diluted drug (DMSO final concentration is 0.1%), culturing for 3-4 days, and using Cell
Figure BDA0003689063280000061
2.0Luminescent Cell viatilityassay (Promega) to measure the amount of chemiluminescence of ATP in the surviving organoids, and to evaluate the effectiveness of the drug, the results are shown in fig. 7, where fig. 7 shows sensitivity screening of the same drug using two organoids, testing of 6 different concentrations using semilogarithmic dilution, adding the drug to organoids after culturing for one day in a 96-well plate, growth differences of organoids at different concentrations after culturing for 5 days, measuring Cell activity using celltiter-glo, calculating the rate of inhibition of growth of organoids by the drug, and calculating the semiinhibitory concentration using prism software, IC 50. As can be seen, the organoids established by the tissues of the HPV infected patients are more sensitive to drugs than the organoids established by the uninfected HPV patients, and the organoids from different sample sources have different drug sensitivities to the same drug, so that the cervical organoids from the ectocervical epithelial lesion tissues can be used as a drug screening model.
Criteria for evaluating the effectiveness of a drug: the measured chemiluminescence value is converted into the inhibition rate of the drug on the growth of the organoid, the IC50 of the drug is calculated, the HPV infected organoid is more sensitive to the drug, the uninfected organoid is not sensitive, and the lesion can be inhibited by using the drug with lower concentration.
Inhibition ═ 1- [ (experimental group-blank) - (positive control-blank) ]/[ (negative control-blank) - (positive control-blank) ].
Half inhibitory concentrations of the drug, IC50, were calculated using the formula log (inhibitor) vs. response- -Variable slope (four parameters).
The following comparative examples explore the optimal composition of cervical organoid medium. The basic culture medium is added with different combinations of cytokines for culture, the growth state of the organoids, including growth rate, size and quantity, is observed, so as to evaluate the promoting or inhibiting effect of a certain growth factor on the growth of the cervical organoids, and the culture medium which is most suitable for culturing the cervical organoids is prepared.
Comparative example 1
The basal medium used in this comparative example contained 1XGlutaMax TM 、10mM HEPES、10mM Nicotinamide、10nM N 2 Advanced DEME/F12 of supplement.
The test procedure of this comparative example was the same as example 1 except that:
100ng/ml of Human Noggin, 1uM of SB202190 and 2 mu of MA 83-01 are respectively added into a basal culture medium to explore the promoting effect of the growth factor on organoid growth.
As shown in FIG. 8, it is clear from FIG. 8 that Noggin, A83-01 and SB202190 had a significant effect on organoid growth, and that R-linking-1 at 250ng/mL had no significant effect on organoid growth, and might be involved in maintaining the organoid dryness.
Comparative example 2
The basal medium used in this comparative example contained 1XGlutaMax TM 、10mM HEPES、10mM Nicotinamide、10nM N 2 Advanced DEME/F12 of supplement.
The test procedure of this comparative example was the same as example 1 except that:
the basic culture medium is added with 10uM Forskolin to facilitate the research of the promotion effect of the Forskolin on the organoid growth, and the result is shown in FIG. 9, and the Forskolin has an obvious promotion effect on the organoid growth as shown in FIG. 9.
Comparative example 3
The test procedure of this comparative example was the same as example 1 except that:
the Hydrocortisone is added into the basal culture medium by 0.5ug/ml so as to facilitate the research of the promoting effect of the Hydrocortisone on the growth of the organoids, and the result is shown in figure 10, and as can be seen from figure 10, the Hydrocortisone has obvious promoting effect on the growth of the organoids.
From comparative examples 1 to 3, suitable for cervical organoidsThe complete medium for growth (containing various growth factors) was: containing 1X GlutaMax TM 、10mM HEPES、10mM Nicotinamide、10nM N 2 supplement, 1mM N-acetyl-L-cysteine (NAC), 2. mu. M A83-01, 10. mu. M Y27632, 1. mu.M Jagged-1, 100ng/ml Human Noggin, 25ng/ml Human EGF, 10. mu.M Forskolin, 0.5ug/ml Hydrocortisone, 1. mu.M SB202190, 1 XPenicillin-Streptomyces AdvancedDEME/F12.
Comparative example 4
FIG. 11 in comparative example 4 shows the growth of organoids cultured with basal medium over time in example 1. Lines 1 and 2 are the growth of the organoids from two different patients over time in minimal medium, respectively.
Example 2
The experimental method adopted in this example is the same as example 1, except that the basic culture medium adopted is the optimal basic culture medium obtained in comparative examples 1-3, 106 experiments are performed, and the success rate is counted, and the result is shown in fig. 12, and it can be seen from fig. 12 that the success rate of constructing the cervical organoid by the method provided by the present invention is 62.3%.
The above-described embodiments are only intended to illustrate the preferred embodiments of the present invention, and not to limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims (10)

1. A complete medium, characterized in that the complete medium contains 1X GlutaMax TM 、10mM HEPES、10mM Nicotinamide、10nM N 2 supplement, 1mM N-acetyl-L-cysteine (NAC), 2. mu. M A83-01, 10. mu. MY27632, 1. mu.M Jagged-1, 100ng/ml Human Noggin, 25ng/ml Human EGF, 10. mu.M Forskolin, 0.5ug/ml Hydrocortisone, 1. mu.M SB202190, 1X Penicillin-Streptomycin Advanced DEME/F12.
2. A construction method of a human cervical wart organ model is characterized by comprising the following steps:
(1) pretreating a human cervical wart tissue cell line to obtain a single cell;
(2) plating single cells and culturing using the complete medium of claim 1;
(3) building a wart organ library.
3. The method of claim 2, further comprising the step of (4) identifying the molecular characteristics of the wart-like organ.
4. The construction method according to claim 3, wherein the specific operation steps of the step (2) are as follows:
(1) centrifuging at RT 350g for 5min to precipitate cells, and resuspending the cells with a corresponding amount of Matrigel;
(2) add a drop of Matrigel cell suspension to the center of each well to inoculate cells, avoid dropping to the edge of the well, place the plate upside down at 37 deg.C, 5% CO 2 Culturing in an incubator for 30min, or curing the Matrigel;
(3) 500ul of pre-heated complete medium was slowly added along the wall, and the plates were placed at 37 ℃ in 5% CO 2 Culturing in an incubator;
(4) changing the preheated complete culture medium every 3-4 days;
(5) the primary culture of the cervical organoid is carried out for 14 days at the maximum, and if the cervical organoid still does not grow out, the culture is abandoned.
5. The construction method according to claim 4, wherein the specific operation steps of the step (3) are as follows:
observing the growth state and the size quantity of the wart organs under a microscope, sucking out the culture medium, adding 1ml of cold 1 XDPBS, collecting a certain quantity of the organs into a 15ml centrifuge tube, placing on ice for 15 minutes, centrifuging 350g for 5 minutes to remove Matrigel, adding the cryopreservation liquid into 200/500 ul tubes, subpackaging into cryopreservation tubes, placing in a programmed cooling box, slowly cooling at-80 ℃, and transferring into liquid nitrogen for long-term storage the next day.
6. The method of claim 5, wherein the molecular characterization in step (4) comprises characterization of the biomarkers MUC5AC, PAX8, Ki67, TP63, CK14, CK13, CK17 and EpCAM.
7. The human cervix wart organoid model constructed by the construction method of any one of claims 2-6.
8. Use of the complete medium of claim 1 in the construction of a human cervical wart organoid model.
9. Use of the construction method of any one of claims 2 to 6 in the construction of a human cervical wart organoid model.
10. Use of the human cervical wart organomodel of claim 7 in the development of lead compounds.
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