CN115058384A - Full-molecular culture medium for culturing pig colorectal organoid - Google Patents

Full-molecular culture medium for culturing pig colorectal organoid Download PDF

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CN115058384A
CN115058384A CN202210829477.5A CN202210829477A CN115058384A CN 115058384 A CN115058384 A CN 115058384A CN 202210829477 A CN202210829477 A CN 202210829477A CN 115058384 A CN115058384 A CN 115058384A
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colorectal
pig
organoid
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CN115058384B (en
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崔立
李垚
赵婷婷
温智越
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Shanghai Jiaotong University
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Abstract

The invention discloses a full-molecular culture medium for culturing pig colorectal organoids, belonging to the technical field of organoid culture. The medium comprises the following components: HEPES, B27, Gastrin-I, N-acetyl-L-cysteine, nicotinamide, L-glutamine, N2, L-arginine, succinic acid, V D EGF, Noggin, R-spondin-1, Wnt3a, Y-27632, A-83-01, SB202190, prostaglandin E2, Tubastatin A and MS 275. The culture medium can improve the forming rate and activity of pig colorectal organoids to the maximum extent, has stable effect in the long-term organoid culture process, and the formed organoid stem cells have high activity and strong proliferation and differentiation capacity, thereby providing a powerful tool for pig intestinal development and pathological research, safety evaluation of intestinal disease-related drugs and clinical drug screening.

Description

Full-molecular culture medium for culturing pig colorectal organoids
Technical Field
The invention relates to the technical field of organoid culture, in particular to a full molecular culture medium for culturing pig colorectal organoids.
Background
Organoids are a novel model for in vitro studies, which are self-assembled from stem cells or tumor cells under three-dimensional culture conditions. The method can highly simulate the physiological structure and function of in-situ tissues, maintain the stability of genetic information after long-term passage, and has wide prospects in the aspects of constructing disease models, screening drugs, evaluating drug toxicity and safety and the like. In contrast to traditional two-dimensional cell culture, organoids have virtually all cell populations of the intestinal tract such as stem cells, Pangolin cells, goblet cells, secretory cells, and epithelial absorptive cells. The three-dimensionally cultured intestinal organoid has a structure similar to an intestinal cavity and a crypt, and can well simulate the high-level spatial structure of intestinal tissues in vitro, so that intestinal pathology and drug reaction can be better simulated.
The pig intestinal tract anatomy and physiology characteristics, blood biochemical indexes, the occurrence and development processes of diseases and the like are highly similar to those of a human body, but the pig intestinal tract anatomy and physiology characteristics, the blood biochemical indexes, the occurrence and development processes of the diseases and the like have the problems of complex operation, long modeling time and the like when being used as a large experimental animal. Colon organoid has been successfully applied in the drug evaluation research of human and mouse, but the application of pig intestine organoid in clinical drug screening and toxicology research is only reported. Since the pig intestine organoid with crypt source is reported for the first time in 2013, other researchers also report the establishment of the pig intestine organoid, but the pig intestine organoid cannot be subcultured for a long time. The pig colorectal organoid is a powerful model for simulating intestinal development, intestinal disease pathogenesis, drug safety and effect screening and pathogenic microorganism infection in vitro, and meanwhile, the pig colorectal organoid has important significance for reducing the use of experimental animals and improving the animal welfare.
At present, the formula of the pig colorectal organoid culture medium is disclosed by taking the reference of the formula of a mouse organoid and a human organoid, the concentration and the composition of various nutritional factors are different, and for example, the concentration difference of EGF in the mouse organoid culture and EGF in the human organ is 10 times. These concentration values are not very suitable for the culture of pig colon organoid, which results in the phenomena of low organoid forming efficiency, inability of long-term culture, easy apoptosis, serious vacuolation and the like. The invention discloses a full molecular culture medium for culturing pig colorectal organs, aiming at providing basis and reference for standardized culture and application in order to develop pig colorectal organ culture and provide a tool for clinical drug screening and pathological research.
Disclosure of Invention
The invention aims to provide a full-molecular culture medium for culturing pig colorectal organoids, which can solve the problems in the prior art, can improve the formation rate and activity of the pig colorectal organoids to the maximum extent, has stable effect in the long-term organoid culture process, forms organoid stem cells with high activity and strong proliferation and differentiation capacity, and provides a powerful tool for interaction research of pathogenic microorganisms and intestinal tracts, development research of the intestinal tracts, safety evaluation of medicines related to intestinal diseases, clinical medicine screening and pathological research.
In order to achieve the purpose, the invention provides the following scheme:
the invention provides a full molecular culture medium for culturing pig colorectal organoids, which comprises the following components in percentage by weight: 4-hydroxyethylpiperazine ethanesulfonic acid (HEPES)10-15mM, B272-2.5 v%, Gastrin-I12-18 nM, N-acetyl-L-cysteine (N-acetyl-L-cysteine)1-2mM, Nicotinamide (Nicotinamide)10-15mM, L-Glutamine (L-Glutamine)1.5-2.5nM, N22-2.5 v%, L-Arginine (L-Arginine) 10-15. mu.M, Succinic acid (Succinic acid) 8-12. mu. M, V D 10-20nM, EGF 80-120ng/mL, Noggin 100-150ng/mL, R-spondin-1500-800 ng/mL, Wnt3a 100-150ng/mL, Y-276328-12 μ M/mL, A-83-010.5-1.0 μ M/mL, SB 2021908-12 μ M/mL, prostaglandin E21-2 v%, Tubastatin A5-10 μ M and MS 2750.5-1 μ M.
Further, the components and contents are as follows: HEPES 15mM, B272 v%, Gastrin-I15 nM, N-acetyl-L-cysteine 1.5mM, nicotinamide 12mM, L-glutamine 2nM, N22 v%, L-arginine 10. mu.M, succinic acid 10. mu. M, VD 15nM, EGF 100ng/mL, Noggin 130ng/mL, R-spondin-1600 ng/mL, Wnt3a 120ng/mL, Y-2763210. mu.m/mL, A-83-010.8. mu.m/mL, SB 20219010. mu.m/mL, prostaglandin E21 v%, Tubastatin A8. mu.M and MS 2750.6. mu.M.
Further, the culture medium also comprises basal medium Advanced DMEM/F12.
The invention also provides a method for culturing the pig colorectal organs, which comprises the steps of pretreating pig colorectal tissues to obtain crypt cell precipitates, adding the full-molecular culture medium and matrigel into the cell precipitates, solidifying, adding the full-molecular culture medium into the cell precipitates, and culturing at constant temperature to obtain the pig colorectal organs.
Further, the constant temperature culture was a culture in an incubator at 37 ℃ and the whole molecule medium was changed every 3 days before passage.
Further, passaging was performed at a passaging ratio of 1: 4.
The invention also provides the pig colorectal organoid obtained by the culture method.
The invention also provides a full molecular culture medium for pig colorectal organoid differentiation, Wnt3a is removed from the full molecular culture medium, and the concentration of the R-spondin component is increased to 900ng/mL, so that the full molecular culture medium for pig colorectal organoid differentiation is obtained.
The invention also provides a regulation and control intervention method for the culture and differentiation of pig colorectal organs, which comprises the following steps:
adding the full-molecular culture medium into the pig colorectal crypt cell sediment for proliferation culture to obtain a pig colorectal organoid sphere; and adjusting the whole molecular culture medium to the whole molecular culture medium for pig colorectal organoid differentiation, and promoting the pig colorectal organoid sphere to be differentiated into a mature pig colorectal organoid.
The invention also provides the application of the whole molecular culture medium or the pig colorectal organoid or the whole molecular culture medium for pig colorectal organoid differentiation in drug screening and/or drug analysis.
The invention discloses the following technical effects:
the culture medium for culturing the pig colorectal organoids is developed based on physiological and biochemical characteristics of pig intestines, has clear components, is all full molecules, is specially used for culturing the pig colorectal organoids, has good repeatability, can improve the formation rate and the activity of the pig colorectal organoids to the maximum extent, has stable effect in the long-term organoid culture process, forms organoid stem cells with high activity and strong proliferation and differentiation capacity, and provides a powerful tool for interaction research of pathogenic microorganisms and the intestines, research of pig intestinal development and pathological process, safety evaluation of medicines related to intestinal diseases and clinical medicine screening.
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In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings can be obtained by those skilled in the art according to the published technology without creative efforts.
FIG. 1 shows colorectal epithelial cells and crypts of suckling pigs; a: an intestinal epithelial layer; b: the colonic epithelial crypt; c: rectal epithelial crypts;
FIG. 2 shows the differentiation results of the pig colorectal organoid culture in example 3; the left image is proliferation medium and proliferation microscope image; the right picture is a structure picture of a differentiation medium and a differentiation bud;
FIG. 3 is an electron microscope image of a colon organoid of a pig; a: pig colon organoid culture 1d, B: culturing the colon organoid of the pig for 7 d;
FIG. 4 is a statistical chart of different media for culturing pig colorectal organoids; a: organoid formation rate; b: the official rate of budding organs; c: a surface area;
FIG. 5 is an electron micrograph of porcine colorectal organoids passed to passage 5; the upper graph is as follows: pig colon, lower panel: pig rectum;
FIG. 6 is an electron micrograph of porcine colorectal organoids passed to the 10 th generation; the upper diagram: pig colon, lower panel: pig rectum;
FIG. 7 is an electron microscope result chart of pig colorectal organoids cultured in different culture media; a: culturing the 2 nd molecular culture medium; b: comparative example 1 culture medium 2 d; c: culturing 12d with the full molecular culture medium; d: comparative example 1 culture medium 12 d.
Detailed Description
Reference will now be made in detail to various exemplary embodiments of the invention, the detailed description should not be construed as limiting the invention but as a more detailed description of certain aspects, features and embodiments of the invention.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. In addition, for numerical ranges in the present disclosure, it is understood that each intervening value, to the upper and lower limit of that range, is also specifically disclosed. Every smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in a stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the documents are cited. In case of conflict with any incorporated document, the present specification will control.
It will be apparent to those skilled in the art that various modifications and variations can be made in the specific embodiments of the present disclosure without departing from the scope or spirit of the disclosure. Other embodiments will be apparent to those skilled in the art from consideration of the specification. The description and examples are intended to be illustrative only.
As used herein, the terms "comprising," "including," "having," "containing," and the like are open-ended terms that mean including, but not limited to.
The materials, instruments and reagents used in the present invention are commercially available unless otherwise specified; the experimental methods used are all routine experimental methods in the field unless otherwise specified.
Example 1A full molecular culture Medium for the culture of porcine colorectal organoids
The invention adopts 30 piglets (three-element hybrid white pigs with 4-5 weeks old) to respectively collect small intestines and large intestines. Extracting each intestinal crypt tissue. Key growth factors EGF, Noggin, R-spondin and Wnt3a concentrations were then determined by ELISA for guidance in organoid medium formulation, since this step could not be omitted because of organoid species specificity. The values (average values) obtained were as follows:
TABLE 1
Figure BDA0003747634440000061
And the optimal concentration range of various components of the pig colorectal organoid is obtained by combining large-scale colorectal organoid concentration screening, and meanwhile, a brand-new pig colorectal organoid culture medium formula with definite nutrient components is determined by combining physiological and biochemical characteristics and nutritional requirements of pig intestines. The main components of a full molecular culture medium for culturing pig colorectal organoids are shown in the following table:
TABLE 2
Figure BDA0003747634440000071
Example 2 culture of porcine Colon organoids
(1) Collecting colon segments of 3-day-old newborn piglets, longitudinally splitting, and washing with 4 deg.C precooled DPBS. The villi were scraped off with a glass slide, rinsed three times with 4 ℃ pre-cooled DPBS containing 5% Primocin, and then mucosal tissue containing crypt structures were scraped off and collected in a 50mL centrifuge tube.
(2) In a clean bench, DPBS supernatant was pipetted off and incubated for 2 minutes with DMEM-F12 medium containing 10% serum to preserve crypt tissue activity.
(3) Discarding the supernatant of the DMEM-F12 culture medium, adding about 20mL of DPBS precooled at 4 ℃, slowly blowing and beating for 3 times up and down by using a pipette, adding new DPBS precooled at 4 ℃ again after tissue fragments are settled, and repeating the step for 20 times until no obvious suspended fragments exist in the supernatant part.
(4) Discarding the supernatant of DPBS, adding 15mL of mild dissociation agent (GCDR, STEMCELL), shaking at 37 deg.C for 60min, incubating at 200rpm, and allowing the centrifuge tube to be horizontal and the crypt fragments to shake back and forth to promote crypt separation.
(5) After dissociation was complete, centrifugation was carried out at 300rpm for 5min at 4 ℃ and 20mL of DMEM-F12 medium containing 20% serum was added immediately to resuspend the crypts.
(6) The suspension was filtered through a 100 μm sieve, and the collected suspension was centrifuged at 800rpm at 4 ℃ for 5min to obtain crypt precipitates.
(7) Adding 1mL of whole organoid medium (whole molecule medium in example 1), adding equal amount of matrigel, mixing well the crypt pellet, inoculating 50 μ L per well onto 24-well plate, and placing at 37 deg.C with 5% CO 2 The incubator (2) was allowed to solidify for 20 min.
(8) And taking out the solidified culture plate, adding 5mL of complete organoid culture medium into each hole, taking a picture by using a microscope to record the crypt shape, and then putting the crypt shape into an incubator for continuous culture.
(9) Organoid formation and status were observed daily, medium was changed every 3 days, and passage was initiated on day 7.
(10) And (5) carrying out organoid passage according to the ratio of 1:4 to obtain the pig colorectal organoids.
EXAMPLE 3 cultivation of porcine Colon organoids
1. Tissue washing: wiping the intestinal mucosa with sterile gauze, scraping and massaging for 5 times with forceps elbow to promote mucus layer and aged intestinal epithelium to fall off; transfer the mucosa to a 15mL tube, add 10mL of tissue wash (provided by ogono biotechnology, hangzhou), shake vigorously up and down with the hand and discard the supernatant at least 3 times until the wash is clear and free of scum.
2. Epithelial layer peeling (total time about 10 minutes): the basolateral aspect is unfolded upwards and the stromal cells on the basolateral aspect are removed as much as possible with scissors or cutting forceps, leaving only a thin layer (i.e. epithelial layer) on the surface of the intestinal tract. The clean epithelial layer is peeled off by scraping with a blunt tip of forceps or scissors to promote dissociation and peel thoroughly until the epithelial layer begins to become soft and stringy from a soft, slippery tough, and no longer springs back significantly (see fig. 1A: piglet intestine epithelial layer).
3. Shearing: transfer to a 2mL tube, replenish the level with chelation buffer (supplied by aogono biotechnology, hangzhou) to a level of about 1cm, and shear the epithelial layer with an ophthalmic shear, which was found to swell soft and shear easily (indicating incomplete chelation if tough and easily bounced off, return to step 4).
4. Chelating: in most cases, if the epithelial layer is still tough, the layer can be kept on ice for 15-30min by adding a chelating solution, and observed 1 time every 10 min.
5. Blowing and sucking: transferring the mixture into a 15mL tube by using a 1mL tip (low adsorption + pre-rinse + shearing width), supplementing the chelating buffer solution to 1.5mL, and violently blowing (the range is adjusted to about 700 mu L) epithelial fragments (generally about 10-15 min) by using a 1mL tip (low adsorption + pre-rinse + shearing width). The gun head can be cut by scissors in a gradient way according to the size of the fragments so as to obtain the gun head with the smallest caliber but not blocked. Until the formation of the suspension was observed, particles (i.e., crypts) were visible to the naked eye through the light.
6. Gravity settling: let stand and let the sample pieces settle under gravity for 1 minute. The supernatant was removed and examined by inverted microscopy for crypts in the supernatant and the crypt-containing supernatant was collected into 15mL low binding centrifuge tubes (pre-rinse). If a floating residual mass of tissue is found and the tissue is sufficient, it can be screened with a 100 μm or 70 μm cell sieve to filter out the tissue residue.
7. Discarding the single cells: the crypt suspension was centrifuged once at 175g for 3min at 4 ℃ and the supernatant (containing single cells in the supernatant) was discarded to obtain crypt pellets (FIG. 1B: pig colon crypt; C: pig rectal crypt). The single cells and debris are discarded as clean as possible to ensure that the crypts are pure. If necessary, the single cells can be removed by sieving with a cell sieve (< 100. mu.m, > 40 μm) and then washing in reverse.
8. Adding 1mL of whole organoid medium (whole molecule medium in example 1), adding equal amount of matrigel, mixing well the crypt pellet, inoculating 50 μ L per well onto 24-well plate, and placing at 37 deg.C with 5% CO 2 The incubator (2) was allowed to solidify for 20 min.
9. And taking out the solidified culture plate, adding 5mL of complete organoid culture medium into each hole, taking a picture by using a microscope to record the shape of the crypt, and then putting the crypt into an incubator to continue culturing.
10. Organoid formation and status were observed every day, medium was changed every 3 days, and passage was started on day 7 (FIG. 3 is electron micrograph of porcine colon organoid, A: porcine colon organoid culture 1d, B: culture 7 d).
11. And (3) carrying out multiple passages on the organoids according to the ratio of 1:4 to obtain the porcine colorectal organoids (see figure 5: an electron microscope picture of the 5 th generation porcine colorectal organoids, an upper picture: a porcine colon, a lower picture: a porcine rectum; and figure 6: an electron microscope picture of the 10 th generation porcine colorectal organoids, an upper picture: a porcine colon, a lower picture: a porcine rectum).
Example 4 Regulation and control intervention method for pig colorectal organoid culture differentiation
The proliferation medium was the whole molecular medium of example 1; the differentiation medium is based on the proliferation medium, Wnt3a is removed, the concentration of R-spondin is increased to 900ng/mL, and BMP factor can be additionally added to the medium until the concentration is 100 ng/mL.
Pig colorectal organoid spheres were cultured by proliferation according to the method of example 2 (fig. 2, left panel), and the proliferation medium was adjusted to a differentiation medium to promote the organoid spheres (FO) to begin to differentiate into mature organoids (AO), which forms the budding structures as shown in fig. 2, right panel. Lays a foundation for initially establishing a regulation and control intervention method for the culture and differentiation of the colorectal organs of the pigs.
Comparative example 1
The difference from example 2 is that the whole molecular culture medium (PG) of the invention was replaced with a commercial brand human-derived basal medium: merck Michibo MD164 Medium (ST).
Effect verification
The porcine colon organoids were cultured by proliferation according to the technical protocols of example 2 and comparative example 1, respectively.
The cell type specific passage frequency is defined by a comprehensive morphological index. The frequency of passage of intestinal organoids depends on maturation markers such as intraluminal sprouting and debris accumulation. The real-time living cell imaging analyzer can be used for dynamically quantifying the indicators of the eccentricity (budding) or darkness (fragment accumulation in the lumen) of the organoids. Organoid surface area was photographed by microscope and calculated by image J software to give a value.
FIG. 7 is an electron microscope result chart of pig colorectal organoids cultured in different culture media; a: culturing the 2 nd molecular culture medium; b: comparative example 1 medium culture 2 d; c: culturing 12d with the full molecular culture medium; d: comparative example 1 culture medium 12 d. As can be seen from the micrographs of FIGS. 7A-D, the use of the complete molecular culture medium of the invention enables the culture to produce more morphologically stable and larger porcine colorectal organoids. As can be seen from FIGS. 4A-C, the organoid formation rate and surface area of the present invention were significantly increased as compared to comparative example 1, demonstrating that the whole-molecule culture medium of the present invention is more suitable for the culture of porcine intestinal organoids. Compared with comparative example 1, the invention has improved colorectal organoid formation rate, long-term culture stability and activity. Therefore, the formula combination of the full molecular culture medium can improve the forming rate and activity of the pig colorectal organoids to the maximum extent, the effect is stable in the long-term organoid culture process, the formed organoid stem cells have high activity and strong proliferation and differentiation capacity, and a powerful tool is provided for clinical drug screening and pathological research of colorectal cancer.
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 full molecular culture medium for culturing pig colorectal organoids is characterized by comprising the following components in percentage by weight: HEPES 10-15mM, B272-2.5 v%, Gastrin-I12-18 nM, N-acetyl-L-cysteine 1-2mM, nicotinamide 10-15mM, L-glutamine 1.5-2.5nM, N22-2.5 v%, L-arginine 10-15. mu.M, succinic acid 8-12. mu. M, V D 10-20nM, EGF 80-120ng/mL, Noggin 100-150ng/mL, R-spondin-1500-800 ng/mL, Wnt3a 100-150ng/mL, Y-276328-12 μ M/mL, A-83-010.5-1.0 μ M/mL, SB 2021908-12 μ M/mL, prostaglandin E21-2 v%, Tubastatin A5-10 μ M and MS 2750.5-1 μ M.
2. The method of claim 1The full-molecular culture medium is characterized by comprising the following components in percentage by weight: HEPES 15mM, B272 v%, Gastrin-I15 nM, N-acetyl-L-cysteine 1.5mM, nicotinamide 12mM, L-glutamine 2nM, N22 v%, L-arginine 10. mu.M, succinic acid 10. mu. M, V D 15nM, EGF 100ng/mL, Noggin 130ng/mL, R-spondin-1600 ng/mL, Wnt3a 120ng/mL, Y-2763210 μ M/mL, A-83-010.8 μ M/mL, SB 20219010 μ M/mL, prostaglandin E21 v%, Tubastatin A8 μ M and MS 2750.6 μ M.
3. The complete molecular culture medium according to any one of claims 1 to 2, further comprising basal medium Advanced DMEM/F12.
4. A method for culturing pig colorectal organs, which is characterized in that pig colorectal tissues are pretreated to obtain cell precipitates, then the whole molecular culture medium and matrigel according to any one of claims 1 to 3 are added into the cell precipitates, after solidification, the whole molecular culture medium is added into the cell precipitates, and the pig colorectal organs are cultured at constant temperature.
5. The culture method according to claim 4, wherein the constant-temperature culture is a culture in an incubator at 37 ℃ and the whole-molecule medium is replaced every 3 days before passage.
6. The culture method according to claim 5, wherein the passaging is performed at a passaging ratio of 1: 4.
7. A porcine colorectal organoid obtainable by the culture method according to any one of claims 4 to 6.
8. A whole molecular culture medium for pig colorectal organoid differentiation, which is prepared by removing Wnt3a component from the whole molecular culture medium according to any one of claims 1 to 3, and increasing the concentration of R-spondin component to 900 ng/mL.
9. A regulation and control intervention method for pig colorectal organoid culture differentiation is characterized by comprising the following steps:
adding the whole molecular culture medium of any one of claims 1-3 into the pig colorectal crypt cell sediment for proliferation culture to obtain a pig colorectal organoid sphere; and adjusting the whole molecular culture medium to the whole molecular culture medium for the differentiation of the pig colorectal organoid according to claim 8, so as to promote the differentiation of the pig colorectal organoid sphere into a mature pig colorectal organoid.
10. Use of the whole molecule culture medium of any one of claims 1 to 3 or the porcine colorectal organoid of claim 7 or the whole molecule culture medium for differentiation of porcine colorectal organoids of claim 8 for drug screening and/or drug analysis.
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