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CN114717182B - Molecular culture medium for 3D culture of goat colon organoids - Google Patents

Molecular culture medium for 3D culture of goat colon organoids Download PDF

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CN114717182B
CN114717182B CN202210565637.XA CN202210565637A CN114717182B CN 114717182 B CN114717182 B CN 114717182B CN 202210565637 A CN202210565637 A CN 202210565637A CN 114717182 B CN114717182 B CN 114717182B
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goat
inhibitor
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CN114717182A (en
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刘勇
燕爱飞
梁开阳
汤少勋
周传社
谭支良
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Institute of Subtropical Agriculture of CAS
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Abstract

The invention discloses a molecular culture medium for 3D culture of goat colon organoids, which is prepared by adding an N2 additive, a B27 serum substitute, N-acetylcysteine, niacinamide, noggin, R-spondin-1, epidermal growth factor EGF, wnt3a, a ROCK kinase inhibitor, a P38 MAPK inhibitor, a TGF-beta I receptor inhibitor, a gastrin I, HEPES buffer, glutamine I and penicillin streptomycin mixed solution into a ADVANCED DMEM/F12 buffer basal medium; the ADVANCED DMEM/F12 buffer basic culture medium is prepared by adding glutamine I and HEPE buffer into ADVANCED DMEM/F12 culture medium. The culture medium is suitable for colon crypt separation and 3D culture of colon organoids of newborn lambs, lactating lambs, weaning lambs and fattening lambs, and is used for long-term subculture of goat colon organoids, intestinal nutrition physiology research, bionics research and disease modeling.

Description

Molecular culture medium for 3D culture of goat colon organoids
Technical Field
The invention belongs to the technical field of biological medicine, and particularly relates to a molecular culture medium for 3D culture of goat colon organoids.
Background
Intestinal organoids (INTESTINAL ORGANOIDS) are three-dimensional (3D) in vitro tissue models that have many physiologically relevant characteristics of in vivo intestinal tissue, including polarized epithelial layers surrounding functional lumens, and all types of intestinal epithelial cells are arranged in the proportions and spaces of the in vivo intestinal tract.
The intestinal organoids are capable of maintaining genetic background and apparent stability, which is a considerable advantage for long-term subculturing of intestinal organoids and research of intestinal biological functions. The intestinal organoids are able to establish symbiotic systems with other specific cell types introduced into the culture system for studying interactions between different cell types. The introduction of immune cells into the extracellular matrix of organoid cultures can be used to study the expected interactions between immune cells and the basal side of epithelial cells. Likewise, co-culture of intestinal microorganisms with intestinal organoids is achieved by microinjection to mimic interactions between somatic intestinal epithelial cells and intestinal microorganisms.
The intestinal organoid culture technique is gradually maturing for different species, with the development of human and murine organoids being most rapid. In recent years, livestock-derived organoid studies have also attracted extensive interest to researchers, such as those reported in swine-and bovine-derived organoids. However, the micro-habitat required for the growth and development of goat intestinal stem cells is different from the intestinal organoids of murine or other animal species due to species differences, and there is a difference in the requirement for the concentration of key regulatory factors for 3D in vitro biomimetic culture. In addition, the primary structure and function of the protein of the key regulatory factor between different animal species are different, for example, the amino acid sequence of the goat Wnt3a protein is different from the amino acid sequence of the protein of other animal species. Based on the early preliminary work of the invention, the mature organoid culture medium comprising the murine, porcine and bovine sources is used for separating and culturing the colonoscopy of the goat, which has the phenomena of slow organoid growth, low balling rate, low germination rate, easy apoptosis and the like, which indicates that the structure, function and concentration of the key regulatory factors are insufficient to support the proliferation and differentiation of the colon stem cells of the goat. Aiming at the lack of related research reports, which are not mature in the technical proposal of separating and culturing the goat colonoscopy organs at home and abroad at present, the invention aims to provide a separating and culturing method, a molecular culture medium and a preparation method of the goat colonoscopy organs.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a molecular culture medium for 3D culture of goat colon organoids.
In order to achieve the above purpose, the technical scheme provided by the invention is as follows:
the molecular culture medium for 3D culture of the goat colon organoids is prepared by preparing N2 additive (N2 supplement), B27 serum substitute (B27 supplement), N-acetylcysteine (N-Acetyl-L-cysteine), niacinamide (niacinamide), noggin, R-spondin-1, epidermal growth factor EGF, wnt3a, ROCK kinase inhibitor (Y27632), P38 MAPK inhibitor (SB 202190), TGF-beta I receptor inhibitor (A83-01), gastrin I (Gastrin I), HEPES buffer, glutamine I (GlutataMAX-I) and penicillin streptomycin mixed solution in ADVANCED DMEM/F12 buffer basal medium; the ADVANCED DMEM/F12 buffer basic culture medium is prepared by adding glutamine I and HEPE buffer into ADVANCED DMEM/F12 culture medium; wherein the concentration or volume percent concentration of the glutamine I and HEPE buffer in ADVANCED DMEM/F12 medium is: 1-5%, 10-25mM; the concentration or volume percentage concentration of the N2 additive, the B27 serum substitute, the N-acetylcysteine, the niacinamide, the Noggin, the R-spondin-1, the EGF, the Wnt3a, the ROCK kinase inhibitor, the P38 MAPK inhibitor, the TGF-beta I type receptor inhibitor, the gastrin I, HEPES buffer solution, the glutamine I and the penicillin streptomycin mixed solution in the ADVANCED DMEM/F12 culture medium is respectively ：1-2％、1-2％、1-2mM、10-15mM、100-200ng/mL、500-1000ng/mL、100-150ng/mL、100-200ng/mL、10-20μM、10-50μM、500-100nM、10-15nM、10-15mM、1-2％、1-2％.
Preferably, the concentration or volume percentage concentration of the N2 additive, the B27 serum replacement, the N-acetylcysteine, the niacinamide, the Noggin, the R-spondin-1, the epidermal growth factor EGF, the Wnt3a, the ROCK kinase inhibitor, the P38 MAPK inhibitor, the TGF-beta type I receptor inhibitor, the gastrin I, HEPES buffer, the glutamine I and the penicillin streptomycin mixed solution in the ADVANCED DMEM/F12 culture medium are respectively: 1%, 1mM, 10mM, 100ng/mL, 1000ng/mL, 100ng/mL, 10. Mu.M, 500nM, 10nM, 15mM, 1%.
Preferably, the concentration or volume percentage concentration of the N2 additive, the B27 serum replacement, the N-acetylcysteine, the niacinamide, the Noggin, the R-spondin-1, the epidermal growth factor EGF, the Wnt3a, the ROCK kinase inhibitor, the P38 MAPK inhibitor, the TGF-beta type I receptor inhibitor, the gastrin I, HEPES buffer, the glutamine I and the penicillin streptomycin mixed solution in the ADVANCED DMEM/F12 culture medium are respectively: 1%, 1mM, 10mM, 100ng/mL, 500ng/mL, 100ng/mL, 10. Mu.M, 500nM, 10nM, 15mM, 1%.
Preferably, the concentration or volume percentage concentration of the N2 additive, the B27 serum replacement, the N-acetylcysteine, the niacinamide, the Noggin, the R-spondin-1, the epidermal growth factor EGF, the Wnt3a, the ROCK kinase inhibitor, the P38 MAPK inhibitor, the TGF-beta type I receptor inhibitor, the gastrin I, HEPES buffer, the glutamine I and the penicillin streptomycin mixed solution in the ADVANCED DMEM/F12 culture medium are respectively: 1%, 1mM, 10mM, 200ng/mL, 1000ng/mL, 150ng/mL, 100ng/mL, 10. Mu.M, 500nM, 10nM, 15mM, 1%.
Preferably, the concentration or volume percentage concentration of the N2 additive, the B27 serum replacement, the N-acetylcysteine, the niacinamide, the Noggin, the R-spondin-1, the epidermal growth factor EGF, the Wnt3a, the ROCK kinase inhibitor, the P38 MAPK inhibitor, the TGF-beta type I receptor inhibitor, the gastrin I, HEPES buffer, the glutamine I and the penicillin streptomycin mixed solution in the ADVANCED DMEM/F12 culture medium are respectively: 1%, 1mM, 10mM, 150ng/mL, 500ng/mL, 150ng/mL, 100ng/mL, 10. Mu.M, 500nM, 10nM, 15mM, 1%.
The molecular culture medium and matrigel are mixed according to the volume ratio of 1:1 to prepare the inoculation culture medium for the 3D culture of the goat colon organoids.
Details of the components in the molecular medium for the 3D culture of the goat colonography organ are shown in Table 1.
TABLE 1
Three necessary growth factors R-spondin-1, EGF and Noggin exist in the molecular culture medium for 3D culture of goat colonocytes. Wherein R-spondin-1 is a Wnt agonist and is also a ligand of Lgr5, and can regulate proliferation of crypt stem cells. EGF factor promotes mainly proliferation of small intestine epithelial cells; noggin is an inhibitor of BMP pathway and can increase the number of crypts in organoids. For colonic organoids, wnt3a is also required to maintain self-renewal of stem cells and their ability to differentiate. The invention mainly relates to concentration exploration of key regulatory factors such as R-spondin-1, EGF, noggin and the like required by goat colon organoid culture.
The invention establishes a molecular culture medium and a culture system for culturing the goat colon organoid by utilizing the bionic culture technology of the goat gastrointestinal cells/organoids. The molecular culture medium and Matrigel (Matrigel) are mixed according to the proportion of 1:1 to form colon crypt seed-grafting mixed solution, meanwhile, by a counting method, crypts are inoculated according to the inoculation concentration of 500 crypts inoculated in 50 mu l of the crypt seed-grafting mixed solution, and 3D three-dimensional culture of the colon crypts can be carried out.
When the molecular culture medium for 3D culture of the goat colon organoid is used for organoid culture, the colon distal end of a newborn lamb is taken, and the colon intestinal gland (crypt) is obtained by sequentially carrying out washing, shearing, washing, digestion and filtering treatment; the goat colon gland (crypt) is resuspended, diluted and embedded in matrigel, inoculated and cultured in a growth medium to obtain the goat colon organoid. Preferably, the goat colon gland can be derived from newborn lambs, lactating lambs, weaned lambs and fattening lambs.
Specifically, the invention relates to a separation and purification process of a colon crypt and processes of 3D culture, passage, freezing storage and the like of the colon of a goat, wherein the separation and culture of the colon crypt of the 3D culture of the colon of the goat is carried out based on the molecular culture medium for the 3D culture of the colon of the goat.
The colonic crypt separation process involves intestinal tract cleaning, shredding, crypt separation and crypt inoculation. Preferably, the precooled PBS buffer solution without calcium and magnesium ions washes the colon for more than 30 times to remove impurities such as intestinal contents; the colon nubs are obtained by scraping intestinal mucosa of the colon and then cutting into 2-3mm small pieces.
The intestinal crypt separation and purification process. Preferably, an intestinal crypt suspension is obtained by incubating for 1 hour with 20-30ml of a 2.5-5mM EDTA (ethylenediamine tetraacetic acid) solution on a shaker at a shaking frequency of 30 rpm. Preferably, the crypt suspension is screened through a 100 μm cell strainer, centrifuged at 300g for 5min at 4℃and the supernatant discarded, and the pellet resuspended in 10ml of 0.1% bovine serum albumin solution (BSA solution) and centrifuged again with the same centrifugation parameters, and repeated 3 times.
The intestinal crypt inoculation is carried out by utilizing mixed solution of colonic crypt inoculation to resuspend the separated and purified intestinal crypt, and the inoculation is carried out. Preferably, the intestinal crypt heavy suspension is prepared at an inoculation concentration of 10-15 crypts in 1 μl of the crypt inoculation mixture, and 20-40 μl of the intestinal crypt heavy suspension is inoculated in a 24-well or 48-well plate. The experimental process involves the use process of matrigel to ensure that the operation is finished on ice, after inoculation is finished, the inoculated 24-hole or 48-hole plate is placed into 37 ℃ for 10min of thermal incubation, so that matrigel solidification is finished, and 500 μl of molecular culture medium is added into the culture holes for 3D culture of organoids.
In the 3D culture process of the organoids, preferably, after inoculation, the first molecular culture medium is added with 10 mu M of ROCK kinase inhibitor besides all the functional components in the previous step, and the molecular culture medium without the ROCK kinase inhibitor is added when the liquid is changed for 24 hours.
And (3) subculturing the intestinal organoids, and carrying out organoid subculture after the primary organoids balling and budding (about 5-7 days). Preferably, based on the characteristics of high temperature coagulation and low temperature softening of matrigel, 1ml of precooled PBS buffer solution at 4 ℃ is utilized, ice is placed, centrifugation is carried out for 5min to 10min at 300 Xg, supernatant is discarded, organoids are collected, and inoculated culture is carried out by utilizing the inoculation liquid.
During the frozen preservation of the intestinal organoids, the organoids are frozen by using the organoid frozen liquid. Preferably, the organoid frozen stock solution is DMSO: FBS: ADVANCED DMEM/f12=1:2:7, and after long-term freezing in liquid nitrogen, the activity of organoid cells and the continuous passage performance are ensured.
In summary, the culture medium of the invention contains various cytokines, signal channel regulatory factors, amino acids, serum-free additives and the like which are required for the culture of the goat colon organoids, and the various cytokines and the regulatory factors are directly and closely affected with each other and are coordinated, so that the goat colon organoids can maintain high cell and tissue activity in the culture process.
The culture medium is suitable for colon crypt separation and 3D culture of colon organoids of newborn lambs, lactating lambs, weaning lambs and fattening lambs, and is used for long-term subculture of the colon organoids of goats, intestinal nutrition physiology research, bionics research and disease modeling.
Drawings
FIG. 1 shows organoid growth and sprouting in example 1 of the present invention when R-spondin-1 (1000 ng/ml), wnt3a (100 ng/ml), EGF (100 ng/ml), noggin (100 ng/ml);
FIG. 2 shows organoid growth and sprouting when R-spondin-1 (500 ng/ml), wnt3a (100 ng/ml), EGF (100 ng/ml), noggin (100 ng/ml) are used in example 2 of the present invention;
FIG. 3 shows organoid growth and sprouting in example 3 of the present invention when R-spondin-1 (1000 ng/ml), wnt3a (100 ng/ml), EGF (150 ng/ml), noggin (200 ng/ml);
FIG. 4 shows organoid growth and sprouting in example 4 of the present invention when R-spondin-1 (500 ng/ml), wnt3a (100 ng/ml), EGF (150 ng/ml), noggin (150 ng/ml).
Detailed Description
Example 1
This example details the isolation culture, medium and configuration method of goat colonoscopy.
The growth medium used for the goat intestinal organoid culture in this example consisted of the following components: b27, N2, EGF, gastrin I, N-acetylcysteine, R-spondin-1, niacinamide, noggin, wnt3a, ROCK kinase inhibitor, glutamine I, penicillin streptomycin cocktail, HEPES buffer, P38 MAPK inhibitor, TGF-beta type I receptor inhibitor, ADVANCED DMEM/F-12 medium.
The preparation method of the culture medium for the goat colon organoids comprises the following steps:
The growth medium for goat intestinal organoid culture of this example was formulated with the following concentrations of each functional ingredient per 50ml of medium: b27,1%; n2,1%; EGF,100ng/ml; gastrin I,10nM; n-acetylcysteine, 1mM; r-spondin-1, 1000ng/ml; niacinamide, 10mM; noggin,100ng/ml; wnt3a,100ng/ml; ROCK kinase inhibitors, 10 μm; glutamine I,1%; penicillin streptomycin mixed solution, 1%; HEPES buffer, 15mM; p38 MAPK inhibitor, 10 μm; TGF-beta type I receptor inhibitors, 500nM. ADVANCED DMEM/F-12 was formulated as a 50ml system.
And uniformly mixing the raw materials to obtain the culture solution for colorectal cancer organoid culture. After the preparation of the culture broth, it was sterilized and filtered with a 0.22 μm filter and stored at 4℃for less than 2 weeks.
The method for separating and culturing the goat intestinal organoids in the example comprises the following steps.
The experimental procedure of the method for obtaining goat colon mucosa segments (2-3 mm), extracting goat colon crypt and culturing was as follows.
And uniformly mixing the raw materials to obtain the culture solution for colorectal cancer organoid culture. After the preparation of the culture solution, the culture solution was sterilized and filtered by a 0.22 μm filter, and stored at 4℃for not more than 2 weeks.
Sample cleaning: the colon was washed several times with 1% diabody in PBS, and obvious adipose tissue was removed.
Sample shearing and cleaning: the tissue was cut into small pieces of about 2-3mm, and the colon pieces were washed 30 times to clarify the supernatant.
Colon crypt digestion: the sheared colonic mucosa pieces were placed in 2.5mM EDTA solution, on ice, and digested at 30rpm/min for 1h.
Enrichment: the digestate from step 3 was removed, resuspended in 10ml of 0.1% BSA solution and the 100 μm cell strainer was filtered into a new 50ml centrifuge tube and repeated several times. Selecting a centrifuge tube with high crypt purity, centrifuging at 300 Xg and 4 ℃ for 5min, and enriching intestinal crypts.
The target tube was placed in a centrifuge, centrifuged at 300 Xg for 5min at 4℃and the supernatant discarded, the pellet resuspended in 10ml of 0.1% BSA solution, centrifuged at 200g for 3min and 10. Mu.l of suspension counted after resuspension and the total number of crypts in the centrifuge tube calculated. Preferably, the inoculum is mixed at a total of 12 crypt numbers of 1 μl matrigel, and the intestinal crypt is resuspended, and the procedure is performed on ice.
Sucking 50 μl of the suspension with a pipette, injecting into a 24-well plate incubated in an incubator, incubating the 24-well plate in the incubator for 10min after inoculation until matrigel is completely polymerized and solidified, adding 500 μl of organoid growth medium along the hole wall after matrigel is solidified, culturing the 24-well plate in a 5% CO 2 incubator at 37deg.C, and replacing molecular medium every 2-3 d. Preferably, 50. Mu.l of the resuspended inoculum/well is inoculated and allowed to solidify in an incubator for 10min; ROCK kinase inhibitor was added to the first-added medium after inoculation, and the medium without ROCK kinase inhibitor was replaced for 24 hours.
The culture was carried out at 37℃and 5% CO 2 concentration for 5-7 days with medium changes at 1d intervals. Organoid morphological structures under an optical microscope are shown in fig. 1.
Example 2
The growth medium used for the goat intestinal organoid culture in this example consisted of the following components: b27, N2, EGF, gastrin I, N-acetylcysteine, R-spondin-1, niacinamide, noggin, wnt3a, ROCK kinase inhibitor, glutamine I, penicillin streptomycin cocktail, HEPES, P38 MAPK inhibitor, TGF-beta type I receptor inhibitor, ADVANCED DMEM/F-12 medium.
The preparation method of the culture medium for the goat colon organoids comprises the following steps:
The following functional ingredient concentrations were formulated per 50ml of ADVANCED DMEM/F-12 medium for the growth medium for goat intestinal organoids of this example and referenced below: b27,1%; n2,1%; gastrin I,10nM; EGF,100ng/ml; n-acetylcysteine, 1mM; r-spondin-1, 500ng/ml; niacinamide, 10mM; noggin,100ng/ml; wnt3a,100ng/ml; ROCK kinase inhibitors, 10 μm; glutamine I,1%; penicillin streptomycin mixed solution, 1%; HEPES,15mM; p38 MAPK inhibitor, 10 μm; TGF-beta type I receptor inhibitors, 500nM.
And uniformly mixing the raw materials to obtain the culture solution for colorectal cancer organoid culture. After the preparation of the culture broth, it was sterilized and filtered with a 0.22 μm filter and stored at 4℃for less than 2 weeks.
The method for separating and culturing the goat intestinal organoids in the example comprises the following steps.
The experimental procedure of the method for obtaining goat colon mucosa segments (2-3 mm), extracting goat colon crypt and culturing was as follows.
Sample cleaning: the colon was washed several times with 1% diabody in PBS, and obvious adipose tissue was removed.
Sample shearing and cleaning: the tissue was cut into small pieces of about 2-3mm, and the colon pieces were washed 30 times to clarify the supernatant.
Colon crypt digestion: the sheared colonic mucosa pieces were placed in 2.5mM EDTA solution, on ice, and digested at 30rpm/min for 1h.
Enrichment: the digestate from step 3 was removed, resuspended in 10ml of 0.1% BSA solution, and the 100 μm cell strainer was filtered into a fresh 50ml centrifuge tube and repeated several times. Selecting a centrifuge tube with high crypt purity, and centrifuging at a temperature of 300 Xg for 5min at a temperature of 4 ℃.
The target tube was placed in a centrifuge, centrifuged at 300 Xg for 5min at 4℃and the supernatant discarded, the pellet resuspended in 10ml of 0.1% BSA solution, centrifuged at 200 Xg for 3min and after the last 1 resuspension 10. Mu.l of suspension was counted and the total number of recesses in the centrifuge tube calculated. Preferably, the matrigel volume to be added is calculated as 15 crypts of 1 μl matrigel, and the intestinal crypts are resuspended in a centrifuge tube with a pipette, step operated on ice.
Sucking 50 μl of the suspension with a pipette, injecting into a 24-well plate incubated in an incubator, placing the 24-well plate in the incubator after the plates are incubated for 10min until the matrigel is completely polymerized and cured, adding 500 μl of organoid growth medium along the hole wall after the matrigel is completely cured, placing the 24-well plate in a 37 ℃ and 5% CO 2 incubator for culturing, and replacing the culture solution every 2-3 d. Preferably, the mixture is inoculated in a 50. Mu.l volume/well and polymerized in an incubator for 10min; ROCK kinase inhibitor was added to the first-added medium after inoculation, and the medium without ROCK kinase inhibitor was replaced for 24 hours.
The culture was carried out at 37℃and 5% CO 2 concentration for 5-7 days with medium changes at 1d intervals. Organoid morphological structures under an optical microscope are shown in fig. 2.
Example 3
The growth medium used for the goat intestinal organoid culture in this example consisted of the following components: b27, N2, EGF, gastrin I, N-acetylcysteine, R-spondin-1, niacinamide, noggin, wnt3a, ROCK kinase inhibitor, glutamine I, penicillin streptomycin cocktail, HEPES buffer, P38 MAPK inhibitor, TGF-beta type I receptor inhibitor, ADVANCED DMEM/F-12 medium.
The preparation method of the culture medium for the goat colon organoids comprises the following steps:
The following functional ingredient concentrations were formulated per 50ml of ADVANCED DMEM/F-12 medium for the growth medium for goat intestinal organoids of this example and referenced below: b27,1%; n2,1%; gastrin I,10nM; EGF,150ng/ml; n-acetylcysteine, 1mM; r-spondin-1, 1000ng/ml; niacinamide, 10mM; noggin,200ng/ml; wnt3a,100ng/ml; ROCK kinase inhibitors, 10 μm; glutamine I,1×; penicillin streptomycin mixed solution, 1%; HEPES,15mM; p38 MAPK inhibitor, 10 μm; TGF-beta type I receptor inhibitors, 500nM.
And uniformly mixing the raw materials to obtain the culture solution for colorectal cancer organoid culture. After the preparation of the culture broth, it was sterilized and filtered with a 0.22 μm filter and stored at 4℃for less than 2 weeks.
The method for separating and culturing the goat intestinal organoids in the example comprises the following steps.
Sample cleaning: the colon was washed several times with 1% diabody in PBS, and obvious adipose tissue was removed.
Sample shearing and cleaning: the tissue is cut into small pieces of about 2-3mm, and the colon pieces are washed for 20-30 times to clarify the supernatant.
Colon crypt digestion: the sheared colonic mucosa pieces were placed in 2.5Mm EDTA solution and digested for 1h at 30rpm/min on ice.
Enrichment: the digestate from step 3 was removed, resuspended in 10ml of 0.1% BSA solution and the 100 μm cell strainer was filtered into a new 50ml centrifuge tube and repeated several times. Selecting a tube with high crypt purity, centrifuging for 5min at 300g and 4 ℃.
The target tube was placed in a centrifuge, centrifuged at 4℃for 5min at 300g, the supernatant discarded, the pellet resuspended in 10ml of 0.1% BSA solution, centrifuged at 200g for 3min, and after 1 final resuspension 10. Mu.l of suspension was counted and the total number of recesses in the centrifuge tube calculated. Preferably, the matrigel volume to be added is calculated as 1 μl matrigel 12.5 crypts, and the intestinal crypts are resuspended in a centrifuge tube with a pipette, step operated on ice.
Sucking 50 μl of the suspension with a pipette, adding into 24 well plates incubated in an incubator, incubating the 24 well plates in the incubator for 10min after the plates are placed in the incubator until matrigel is completely polymerized, adding 500 μl of organoid growth medium along the wall of the well after matrigel is completely polymerized, culturing the 24 well plates in a incubator with 5% CO 2 at 37deg.C, and replacing the culture medium every 2-3d (medium yellowing). Preferably, the mixture is inoculated in a 50. Mu.l volume/well and polymerized in an incubator for 10min; ROCK kinase inhibitor was added to the first-added medium after inoculation, and after 24 hours the medium without ROCK kinase inhibitor was replaced.
The culture was carried out at 37℃and 5% CO 2 concentration for 5-7 days with medium changes at 1d intervals. Organoid morphological structures under an optical microscope are shown in fig. 3.
Example 4
The growth medium used for the goat intestinal organoid culture in this example consisted of the following components: b2, N2, EGF, gastrin I, N-acetylcysteine, R-spondin-1, niacinamide, noggin, wnt3a, ROCK kinase inhibitor, glutamine I, penicillin streptomycin cocktail, HEPES, P38 MAPK inhibitor, TGF-beta type I receptor inhibitor, ADVANCED DMEM/F-12 medium.
The preparation method of the culture medium for the goat colon organoids comprises the following steps:
The following functional ingredient concentrations were formulated per 50ml of ADVANCED DMEM/F-12 medium for the growth medium for goat intestinal organoids of this example and referenced below: b27,1%; n2,1%; gastrin I,10nM; EGF,150ng/ml; n-acetylcysteine, 1mM; r-spondin-1, 500ng/ml; niacinamide, 10mM; noggin,150ng/ml; wnt3a,100ng/ml; ROCK kinase inhibitors, 10 μm; glutamine, 1%; penicillin streptomycin mixed solution, 1%; HEPES,15mM; p38 MAPK inhibitor, 10 μm; TGF-beta type I receptor inhibitors, 500nM.
The method for separating and culturing the goat intestinal organoids in the example comprises the following steps.
The experimental procedure of the method for obtaining goat colon mucosa segments (2-3 mm), extracting goat colon crypt and culturing was as follows.
And uniformly mixing the raw materials to obtain the culture solution for colorectal cancer organoid culture. After the preparation of the culture solution, the culture solution was sterilized and filtered by a 0.22 μm filter, and stored at 4℃for not more than 2 weeks.
Sample cleaning: the colon was washed several times with 1% diabody in PBS, and obvious adipose tissue was removed.
Sample shearing and cleaning: the tissue was cut into small pieces of about 2-3mm, and the colon pieces were washed 30 times to clarify the supernatant.
Colon crypt digestion: the sheared colonic mucosa pieces were placed in 2.5mM EDTA solution, on ice, and digested at 30rpm/min for 1h.
Enrichment: the digestate from step 3 was removed, resuspended in 10ml of 0.1% BSA solution and the 100 μm cell strainer was filtered into a new 50ml centrifuge tube and repeated several times. Selecting a centrifuge tube with high crypt purity, centrifuging at 300 Xg and 4 ℃ for 5min, and enriching intestinal crypts.
The target tube was placed in a centrifuge, centrifuged at 300 Xg for 5min at 4℃and the supernatant discarded, the pellet resuspended in 10ml of 0.1% BSA solution, centrifuged at 200g for 3min and 10. Mu.l of suspension counted after resuspension and the total number of crypts in the centrifuge tube calculated. Preferably, the inoculum is mixed at a total of 12 crypt numbers of 1 μl matrigel, and the intestinal crypt is resuspended, and the procedure is performed on ice.
Sucking 50 μl of the suspension with a pipette, injecting into a 24-well plate incubated in an incubator, incubating the 24-well plate in the incubator for 10min after inoculation until matrigel is completely polymerized and solidified, adding 500 μl of organoid growth medium along the hole wall after matrigel is solidified, culturing the 24-well plate in a 5% CO 2 incubator at 37deg.C, and replacing molecular medium every 2-3 d. Preferably, 50. Mu.l of the resuspended inoculum/well is inoculated and allowed to solidify in an incubator for 10min; ROCK kinase inhibitor was added to the first-added medium after inoculation, and the medium without ROCK kinase inhibitor was replaced for 24 hours.
The culture was carried out at 37℃and 5% CO 2 concentration for 5-7 days with medium changes at 1d intervals. Organoid morphological structures under an optical microscope are shown in fig. 1.
The concentration changes of R-spondin-1, wnt3a, EGF and Noggin referred to in the above examples are shown in Table 2.
TABLE 2 summary of key regulatory factor concentration changes in goat colonoids

Claims (8)

1. The molecular culture medium for 3D culture of the goat colon organoid is characterized in that the molecular culture medium for 3D culture of the goat colon organoid is prepared by adding N2 additive, B27 serum substitute, N-acetylcysteine, niacinamide, noggin, R-spondin-1, epidermal growth factor EGF, wnt3a, ROCK kinase inhibitor, P38 MAPK inhibitor, TGF-beta I receptor inhibitor, gastrin I, HEPES buffer, glutamine I and penicillin streptomycin mixed solution into ADVANCED DMEM/F12 buffer basal medium; the ADVANCED DMEM/F12 buffer basic culture medium is prepared by adding glutamine I and HEPE buffer into ADVANCED DMEM/F12 culture medium; wherein the concentration or volume percent concentration of the glutamine I and HEPE buffer in ADVANCED DMEM/F12 medium is: 1-5%, 10-25mM; the concentration or volume percentage concentration of the N2 additive, the B27 serum substitute, the N-acetylcysteine, the niacinamide, the Noggin, the R-spondin-1, the EGF, the Wnt3a, the ROCK kinase inhibitor, the P38 MAPK inhibitor, the TGF-beta I type receptor inhibitor, the gastrin I, HEPES buffer solution, the glutamine I and the penicillin streptomycin mixed solution in the ADVANCED DMEM/F12 culture medium is respectively ：1-2%、1-2%、1-2 mM、10-15 mM、100-200 ng/mL、500-1000 ng/mL、100-150 ng/mL、100-200 ng/mL、10-20 μM、10-50 µM、500-100 nM、10-15 nM、10-15 mM、1-2%、1-2%.
2. The molecular medium for 3D culture of goat colonoscopy according to claim 1, wherein the concentrations or volume percent concentrations of N2 additive, B27 serum replacement, N-acetylcysteine, niacinamide, noggin, R-spondin-1, epidermal growth factor EGF, wnt3a, ROCK kinase inhibitor, P38 MAPK inhibitor, TGF- β type I receptor inhibitor, gastrin I, HEPES buffer, glutamine I, penicillin streptomycin cocktail in ADVANCED DMEM/F12 medium are respectively: 1%, 1mM, 10 mM, 100 ng/mL, 1000 ng/mL, 100 ng/mL, 100 ng/mL, 10 μM, 500 nM, 10nM, 15 mM, 1%.
3. The molecular medium for 3D culture of goat colonoscopy according to claim 1, wherein the concentrations or volume percent concentrations of N2 additive, B27 serum replacement, N-acetylcysteine, niacinamide, noggin, R-spondin-1, epidermal growth factor EGF, wnt3a, ROCK kinase inhibitor, P38 MAPK inhibitor, TGF- β type I receptor inhibitor, gastrin I, HEPES buffer, glutamine I, penicillin streptomycin cocktail in ADVANCED DMEM/F12 medium are respectively: 1%, 1mM, 10mM, 100 ng/mL, 500 ng/mL, 100 ng/mL, 100 ng/mL, 10 μM, 500 nM, 10nM, 15 mM, 1%.
4. The molecular medium for 3D culture of goat colonoscopy according to claim 1, wherein the concentrations or volume percent concentrations of N2 additive, B27 serum replacement, N-acetylcysteine, niacinamide, noggin, R-spondin-1, epidermal growth factor EGF, wnt3a, ROCK kinase inhibitor, P38 MAPK inhibitor, TGF- β type I receptor inhibitor, gastrin I, HEPES buffer, glutamine I, penicillin streptomycin cocktail in ADVANCED DMEM/F12 medium are respectively: 1%, 1 mM, 10 mM, 200 ng/mL, 1000 ng/mL, 150 ng/mL, 100 ng/mL, 10 μM, 500 nM, 10 nM, 15 mM, 1%.
5. The molecular medium for 3D culture of goat colonoscopy according to claim 1, wherein the concentrations or volume percent concentrations of N2 additive, B27 serum replacement, N-acetylcysteine, niacinamide, noggin, R-spondin-1, epidermal growth factor EGF, wnt3a, ROCK kinase inhibitor, P38 MAPK inhibitor, TGF- β type I receptor inhibitor, gastrin I, HEPES buffer, glutamine I, penicillin streptomycin cocktail in ADVANCED DMEM/F12 medium are respectively: 1%, 1mM, 10mM, 150 ng/mL, 500 ng/mL, 150 ng/mL, 100 ng/mL, 10 μM, 500 nM, 10nM, 15 mM, 1%.
6. An inoculation culture medium for 3D culture of a goat colonocyte, which is characterized in that the inoculation culture medium for 3D culture of the goat colonocyte is prepared by mixing the molecular culture medium in the volume ratio of 1:1 with matrigel.
7. Use of a molecular culture medium for 3D culture of a goat colonocytes according to any one of claims 1 to 5 for the preparation of goat intestinal organoids.
8. Use of an inoculation medium for 3D cultivation of a goat colonocyte as claimed in claim 6 for the preparation of a goat intestinal organoid.
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