CN117363562A - Sheep small intestine organoid culture medium and culture method thereof - Google Patents
Sheep small intestine organoid culture medium and culture method thereof Download PDFInfo
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- HIJMSZGHKQPPJS-UHFFFAOYSA-N 3-(6-methylpyridin-2-yl)-n-phenyl-4-quinolin-4-ylpyrazole-1-carbothioamide Chemical compound CC1=CC=CC(C=2C(=CN(N=2)C(=S)NC=2C=CC=CC=2)C=2C3=CC=CC=C3N=CC=2)=N1 HIJMSZGHKQPPJS-UHFFFAOYSA-N 0.000 claims abstract description 6
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Abstract
The invention discloses a culture medium for a small intestine organoid of sheep, which is prepared by adding N2 supplement, B27 serum substitute, N-acetylcysteine, nicotinamide, A83-01, SB202190, Y27632, HEPES, glutaMax, a mixture of streptomycin, wnt3a, an R-spondin1 conditioned medium, noggin and EGF into an Advanced DMEM/F12 culture medium. Also, a method of culturing using the ovine small intestine organoid medium is disclosed. The culture medium can promote proliferation and differentiation of stem cells in the crypt of each intestinal segment of the sheep small intestine to form organoids in vitro; the method also has the advantages of less growth factors, low cost and high consistency.
Description
Technical Field
The invention relates to small intestine organoid culture, in particular to a culture medium for sheep small intestine organoids and a culture method thereof.
Background
The small intestine is the major organ for nutrient digestion and absorption in mammals. In monogastric animal studies such as human and mouse, cell lines and small intestine organoids can be used as models for in vitro studies, but there is a lack of corresponding models in ruminants, and thus it is difficult to develop corresponding in vitro studies.
Intestinal organoids offer an excellent in vitro model of research that contains almost all types of intestinal epithelial cells and has functions of nutrient transport and barrier. The application of the organoid to the research of intestinal epithelial cell function and intestinal disease can reduce the use of experimental animals, thereby greatly saving the experimental expenses.
Sheep are important ruminant livestock animals, and sheep and goats are among the most common breeds, and are widely used in ruminant research due to their moderate body forms. To date, culture media special for small intestine organoids of sheep animals have not been reported. Only one small intestine organoid of sheep reported the use ofA medium dedicated to human intestinal organoids of the company (reference number: PMC 8456012) showed that under such culture conditions, the intestinal organoids were only allowed to grow into spheroids and were not able to germinate to represent the villus structure of the small intestine. The conclusion that the human small intestine organoid culture medium grows slowly and has incomplete structure when the sheep small intestine organoid is cultured is also proved in the experiments of the inventor, and a model required by the research can not be established. There is therefore a real and urgent need to develop a culture medium that mimics the intestinal structure and cellular composition of the small intestine of an ovine animal.
Sheep and goats, on the other hand, are reproductive isolated and thus are two different species under sheep. As shown above (reference number: PMC 8456012), the same formulation is quite different in effect when culturing organoids of different species origin. Therefore, the research and development of the general small intestine organoid culture medium formula aiming at sheep and goats can greatly reduce the experimental cost and expand the application range.
Disclosure of Invention
The invention aims to: aiming at the defects and the shortcomings of the prior art, the invention provides a culture medium for small intestine organoids of sheep and a culture method thereof; the culture medium can promote proliferation and differentiation of stem cells in the crypt of each intestinal segment of the sheep small intestine to form organoids in vitro; the method also has the advantages of less growth factors, low cost and high consistency.
The technical scheme is as follows: the invention discloses a culture medium for a small intestine organoid of sheep, which is characterized in that: the culture medium is prepared by adding N2 supplement, B27 serum substitute, N-acetylcysteine, nicotinamide, A83-01, SB202190, Y27632, HEPES, glutaMax, a mixture of green streptomycin, wnt3a, R-spondin1 conditioned medium, noggin and EGF into an Advanced DMEM/F12 culture medium.
Wherein 1mL of N2 supplement, 2mL of B27 serum replacement, 1.25mM of N-acetylcysteine, 10mM of nicotinamide, 500nM of A83-01, 10nM of SB202190, 10 mu M Y27632, 1mL of HEPES, 1mL of GlutaMax, 1mL of a mixture of green streptomycin, 10 mu g of Wnt3a, 5mL of R-spondin1 conditioned medium, 10 mu g of Noggin, 5 mu g of EGF are added per 100mL of the Advanced DMEM/F12 medium.
Wherein the Advanced DMEM/F12 culture medium is Gibco TM Model 12634010 medium from company. (PMC 3969856)
Wherein, the R-Spondin1 conditioned medium comprises 99 percent by volume of Advanced DMEM/F12 medium and 1 percent by volume of glutaMax culture HA-R-Spondin1-Fc 293T cell supernatant. The preparation method of the R-Spondin1 conditioned medium is described in the specification of Cultrex HA-R-Spondin1-Fc 293T cells (R & D, catalog number: 3710-001-01).
The invention relates to a method for culturing a small intestine organoid of sheep, which is characterized by comprising the following steps: comprising the following steps:
1) Isolating crypt from the sheep small intestine;
2) Culturing small intestine crypt of sheep to obtain organoids;
3) Digestion and passage of the intestinal organoids of sheep;
4) Freezing and preserving intestinal organoids of sheep;
5) Resuscitation of the intestinal organoids of sheep.
Wherein, the step 1) comprises the following steps:
1) Obtaining samples of each section of sheep small intestine, wherein the length of the samples is 4.5cm-5.5cm, cutting off the sections of the small intestine, washing the content of the small intestine by using PBS containing the streptomycin, and scraping mucus and fluff in the inner wall of the small intestine;
2) Cutting the cleaned intestinal segment, washing with precooled PBS containing green streptomycin, adding 10mL of 2mM EDTA, and putting on ice for digestion for 45min;
3) Standing to naturally settle intestinal fragments, discarding supernatant, adding pre-cooled PBS, shaking to remove crypt, passing the whole crypt suspension through 70 μm filter screen, centrifuging at 900rpm for 3min, and collecting crypt.
Wherein, in the step 1), PBS containing 3% of the green streptomycin is used for three times of washing; in said step 2), pre-chilled PBS containing 1% of penicillin is used for washing until there is no foam.
Wherein, the step 2) comprises the following steps:
1) Mixing sheep small intestine organoid culture medium with growth factor-free matrigel 1:1, inoculating 500 crypts/50 μl onto 24-well plate;
2) Placing the culture dish in a 37 ℃ incubator for 20min to solidify the matrigel, and then adding 600 mu L of the ovine small intestine organoid culture medium;
3) After the culture medium is updated once for 72 hours, a large number of globus-shaped sheep intestinal organoids can be seen after 9-11 days.
Wherein, the step 3) comprises the following steps:
1) Discarding the supernatant, and adding precooled PBS to blow away matrigel;
2) Centrifuging at 900rpm for 3min, discarding supernatant, digesting at 37deg.C for 3min with TrypLE, and adding equal volume of culture medium for small intestine organoids of Caprae Seu Ovis to terminate digestion;
3) The 24-well plate was seeded with 1000 single cells per well;
4) Repeating the step 2) to culture the small intestine crypt of the sheep to become organoid, and completing digestion and passage.
Wherein, during the freezing and preserving of the step 4), the adopted freezing and preserving liquid comprises 45% volume fraction intestinal organoid culture medium, 45% volume fraction FBS and 10% volume fraction DMSO.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: the invention creates a whole intestinal organoid culture solution suitable for various sheep animals through a great deal of creative labor of the inventor, and discloses a scheme for establishing organoids. By adopting the method for separating the small intestine organoids of sheep, provided by the invention, a large number of crypts with better activity can be obtained. The sheep small intestine organoid culture medium provided by the invention is suitable for each intestinal section of sheep small intestine, namely duodenum, ileum and jejunum, has good universality and multi-species adaptability. The sheep small intestine organoid culture medium provided by the invention has simple composition, and the R-spondin1 conditioned medium is used for replacing the R-spondin1 growth factor, so that the test cost is reduced.
Drawings
FIG. 1 shows the use of the duodenum, ileum and jejunum of a sheepComparing the company human small intestine organoid culture medium with the sheep small intestine organoid culture medium of the patent for optical observation;
FIG. 2 is a photomicrograph of the duodenal, ileal and jejunal crypts of a sheep;
FIG. 3 is a composition diagram of the culture of sheep jejunum crypt in different media;
FIG. 4 is a graph showing the results of culturing jejunum crypts in different media;
FIG. 5 is a graph showing the survival rate and the germination area of jejunum organoids cultured in different media;
FIG. 6 is a light-microscopic view showing the gradual growth of jejunum organoids and a graph showing the detection results of different cell marker genes;
FIG. 7 is a photomicrograph of the progressive growth of a single duodenal, ileal, jejunal organoid of a sheep;
FIG. 8 is an observation of H & E sections of the duodenum, ileum, jejunum organoids of sheep;
FIG. 9 is a photomicrograph of various passage degrees of the duodenum, ileum, jejunum organoids of a sheep;
FIG. 10 is a photomicrograph of a re-cultured chorionic organoid after recovery of the duodenum, ileum, jejunum organoids of sheep.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings and the specific embodiments.
As shown in figure 1, experiments prove that the human small intestine organoid culture medium has the phenomena of slow growth and incomplete structure when the sheep small intestine organoid is cultured, and a model required by research cannot be built.
Organoids can be induced from adult stem cells or pluripotent stem cells, and organoid culture is critical to the isolation of adult stem cells and the construction of organoid media for organoids induced from adult stem cells. The growth factors in the organoid culture medium are key to organoid maturation, the inventor tries more than ten culture medium formulas in the early stage, and finally determines the optimal formula taking the components as the sheep intestinal organoid culture medium.
1mL of N2 supplement, 2mL of B27 serum replacement, 1.25mM of N-acetylcysteine, 10mM of nicotinamide, 500nM of A83-01, 10nM of B202190, 10 mu M Y27632, 1mL of HEPES, 1mL of GlutaMax, 1mL of a mixture of green streptomycin, 10 mu g of Wnt3a, 5mL of R-spondin1 conditioned medium, 10 mu g of Noggin and 5 mu g of EGF are added to 100mL of Advanced DMEM/F12 medium.
Examples:
1. isolation of crypts from sheep small intestine:
1. tissue acquisition: the carotid artery of the newborn lamb is exsanguinated and killed, and the tissue of each intestinal section of the small intestine is rapidly dissected and obtained, and each tissue is about 5cm;
2. small intestine tissue was placed in a sterile petri dish and surface mucus was gently scraped using a glass slide;
3. soaking in 30mL 3 times of double-resistant precooled PBS, and shaking sufficiently to enable chyme and microorganisms to fall off;
4. repeating the steps for 3 times to thoroughly remove microorganisms;
5. placing the washed small intestine tissue into a 50mL centrifuge tube, shearing the tissue by using clean scissors, adding 30mL PBS containing double pre-cooling, reversing upside down, standing on ice for 1min, and carefully discarding the supernatant;
6. repeating the steps until no obvious turbidity exists after the steps are evenly mixed upside down;
7. discarding the supernatant, adding 10mL of 2mM EDTA, and performing low-temperature digestion on a rocker shaking table for 45min;
8. centrifuging at low speed briefly, removing supernatant, adding PBS, and lightly washing;
9. centrifuging at low speed for a short time, removing supernatant, adding PBS with volume of 2 times, and shaking forcefully to remove crypt;
10. filtering with 70 μm filter screen, collecting filtrate, and centrifuging at 900rpm for 3min;
11. the supernatant was discarded, and the washing was repeated twice with PBS.
2. Culturing small intestine crypt of sheep as organoid:
1. measuring the density of the crypt suspension by sucking 50 μl of the suspension, observing the crypt separation condition by sucking an appropriate amount of the suspension as shown in fig. 1, and adjusting the density to 10 crypts/. Mu.L;
2. sucking the culture solution with the required volume, adding the matrix glue with the same volume, blowing and uniformly mixing, and then placing on ice for 1min;
3. 50 mu L of each hole of the 24-hole plate is added, and the mixture is left to stand at 37 ℃ for 20min for solidification;
4. adding 600 mu L of intestinal organoid culture medium into each hole, and placing into a cell incubator for culture;
5. every 72h of medium replacement, crypt grew rapidly, a distinct cell mass was observed after 3d, and cells were significantly increased after 8d, as shown in fig. 5;
6. the intestinal crypts can grow into the chorionic organoids with single-layer columnar epithelium at about 10d every 72h by changing the culture medium.
3. Digestion passaging of ovine intestinal organoids:
1. the culture medium is discarded, 1mL of ice-cold PBS is added to blow off matrigel, and the matrigel is collected into a 1.5mL centrifuge tube;
2. centrifuging at 900rpm for 3min, discarding supernatant, adding 500 μl of TrypLE (GIBCO, 12604013), and standing at 37deg.C for 3min;
3. adding 500 mu L of intestinal organoid culture medium to stop digestion, and blowing by using a liquid-transferring gun until no obvious cell mass exists;
4. centrifuging at 1000rpm for 3min, discarding supernatant, and repeating the second step.
4. Cryopreservation of ovine intestinal organoids:
1. dispersing matrigel by ice-cold PBS, and collecting into a 1.5mL centrifuge tube;
2. centrifuging at 600rpm for 3min;
3. the supernatant was discarded, and a prepared frozen stock solution (45% volume fraction intestinal organoid medium+45% volume fraction FBS and 10% volume fraction DMSO) was added;
4. placed in a freezer, transferred to a-80 ℃ refrigerator overnight, transferred to liquid nitrogen for storage the next day.
5. Resuscitation of ovine intestinal organoids:
1. after being taken out of the liquid nitrogen, the mixture is quickly thawed in a water bath at 37 ℃;
2. transferring to a centrifuge tube, and resuspending the organoids with 3 volumes of complete medium;
3. centrifuging at 500rpm for 3min;
4. the supernatant was discarded and inoculated directly into 24-well plates.
Claims (10)
1. A sheep small intestine organoid culture medium, characterized in that: the culture medium is prepared by adding N2 supplement, B27 serum substitute, N-acetylcysteine, nicotinamide, A83-01, SB202190, Y27632, HEPES, glutaMax, a mixture of green streptomycin, wnt3a, R-spondin1 conditioned medium, noggin and EGF into an Advanced DMEM/F12 culture medium.
2. The ovine small intestine organoid medium of claim 1, wherein: 1mL of N2 supplement, 2mL of B27 serum replacement, 1.25mM of N-acetylcysteine, 10mM of nicotinamide, 500nM of A83-01, 10nM of SB202190, 10 mu M Y27632, 1mL of HEPES, 1mL of GlutaMax, 1mL of a mixture of penicillin, 10 mu g of Wnt3a, 5mL of R-spondin1 conditioned medium, 10 mu g of Noggin, 5 mu g of EGF were added per 100mL of the Advanced DMEM/F12 medium.
3. According to claim 1 or 2The sheep small intestine organoid culture medium is characterized in that: the Advanced DMEM/F12 culture medium is Gibco TM Model 12634010 medium from company.
4. The ovine small intestine organoid medium according to claim 1 or 2, wherein: the R-Spondin1 conditioned medium comprises 99% by volume of Advanced DMEM/F12 medium and 1% by volume of glutaMax culture HA-R-Spondin1-Fc 293T cell supernatant.
5. The method for culturing an ovine small intestine organoid according to claim 1, wherein: comprising the following steps:
1) Isolating crypt from the sheep small intestine;
2) Culturing small intestine crypt of sheep to obtain organoids;
3) Digestion and passage of the intestinal organoids of sheep;
4) Freezing and preserving intestinal organoids of sheep;
5) Resuscitation of the intestinal organoids of sheep.
6. The method for culturing an ovine small intestine organoid according to claim 5, wherein: said step 1) comprises the steps of:
1) Obtaining samples of each section of sheep small intestine, wherein the length of the samples is 4.5cm-5.5cm, cutting off the sections of the small intestine, washing the content of the small intestine by using PBS containing the streptomycin, and scraping mucus and fluff in the inner wall of the small intestine;
2) Cutting the cleaned intestinal segment, washing with precooled PBS containing green streptomycin, adding 10mL of 2mM EDTA, and putting on ice for digestion for 45min;
3) Standing to naturally settle intestinal fragments, discarding supernatant, adding pre-cooled PBS, shaking to remove crypt, passing the whole crypt suspension through 70 μm filter screen, centrifuging at 900rpm for 3min, and collecting crypt.
7. The method for culturing an ovine small intestine organoid according to claim 6, wherein: in the step 1), PBS containing 3% of the green streptomycin is used for three times of washing; in said step 2), pre-chilled PBS containing 1% of penicillin is used for washing until there is no foam.
8. The method for culturing an ovine small intestine organoid according to claim 5, wherein: said step 2) comprises the steps of:
1) Mixing sheep small intestine organoid culture medium with growth factor-free matrigel 1:1, inoculating 500 crypts/50 μl onto 24-well plate;
2) Placing the culture dish in a 37 ℃ incubator for 20min to solidify the matrigel, and then adding 600 mu L of the ovine small intestine organoid culture medium;
3) After the culture medium is updated once for 72 hours, a large number of globus-shaped sheep intestinal organoids can be seen after 9-11 days.
9. The method for culturing an ovine small intestine organoid according to claim 5, wherein: said step 3) comprises the steps of:
1) Discarding the supernatant, and adding precooled PBS to blow away matrigel;
2) Centrifuging at 900rpm for 3min, discarding supernatant, digesting at 37deg.C for 3min with TrypLE, and adding equal volume of culture medium for small intestine organoids of Caprae Seu Ovis to terminate digestion;
3) The 24-well plate was seeded with 1000 single cells per well;
4) Repeating the step 2) to culture the small intestine crypt of the sheep to become organoid, and completing digestion and passage.
10. The method for culturing an ovine small intestine organoid according to claim 5, wherein: in the step 4), the adopted frozen stock solution comprises 45% volume fraction of intestinal organoid culture medium, 45% volume fraction of FBS and 10% volume fraction of DMSO.
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