High-activity intestinal crypt as well as separation method and application thereof

Abstract

The invention relates to the technical field of tissue isolation, and particularly discloses a high-activity intestinal crypt and an isolation method and application thereof. The separation method comprises the following steps: S1, taking fresh intestinal tracts, cleaning contents, and scraping mucous membrane layer tissues to obtain intestinal mucous membrane tissues; s2, shearing intestinal mucosa layer tissues, mixing the intestinal mucosa layer tissues with the separation liquid after cleaning, and digesting and dissociating to obtain intestinal mucosa tissue digestive juice; s3, centrifuging the intestinal mucosa tissue digestive juice, removing supernate, mixing the supernate with the protective liquid to obtain intestinal crypt mixed liquid, filtering, centrifuging, and taking precipitate to obtain high-activity intestinal crypts; wherein the separation liquid comprises collagenase I, collagenase II, collagenase IV, elastase, DNA enzyme I and a ROCK inhibitor; the protection liquid comprises an RPMI1640 culture medium, antibiotics, fetal calf serum, bovine serum albumin and a ROCK inhibitor. The technical scheme provided by the invention aims to shorten the intestinal tract crypt separation time and improve the cell viability.

Description

Technical Field

[0001] The invention relates to the field of cell technology, and in particular to a highly active intestinal crypt and an isolation method and application thereof. Background Art

[0002] The intestine is the main site of nutrient absorption in mammals and the largest immune organ. The intestinal epithelium is renewed every 3 to 5 days on average, and is the fastest self-renewing tissue in adult mammals. The small intestinal mucosal epithelial tissue is mainly composed of intestinal villi-crypts. Villus epithelial cells can be mainly divided into absorptive enterocytes, secretory enterocytes, goblet cells and tuft cells according to their functions; crypt cells can be mainly divided into intestinal stem cells, Pan cells, progenitor cells and transitional amplification cells according to their functions and degree of differentiation. The traditional model used for intestinal physiology and pathology research is composed of a single type of cell, such as Caco-2 and IPEC-J2 cell lines. However, the traditional single cell model cannot truly simulate the structure, morphology, function, etc. of the intestine, which seriously limits the research on intestinal nutrition, disease and genetic regulation. In recent years, the development of intestinal 3D organoids has provided a new model for intestinal health research. Organoids are derived from stem cells or organ progenitor cells, and are cell clusters formed after in vitro 3D culture using the characteristics of stem cell proliferation and differentiation. Organoids are closely related to their source organs, have the three-dimensional cell structure of their source organs, can reproduce some of the functions and spatial structure of their source organs, and can truly reflect the physiological and pathological processes in the intestine. Currently, the main method for culturing primary intestinal organoids is to isolate the host intestinal crypts for culture.

[0003] Due to the similarity of intestinal structure between different species, researchers have explored the extraction methods of intestinal crypts of various species. It is worth noting that the methods used by researchers to extract and separate intestinal organoids are different, and the key lies in the different use of tissue digestion fluids. The current mainstream separation methods are mainly based on the traditional EDTA method and collagenase method. The basic principle of EDTA method to dissociate cells is to remove metal ions on the cell surface by chelation, thereby destroying the connection between cells and enabling the cells to be separated. This method is too lethal to cells, the dissociation time is extremely difficult to control, and the structural integrity, purity and activity of the extracted crypts are poor. The collagenase method can effectively hydrolyze proteins, polysaccharides and lipids in the extracellular matrix of connective tissue and epithelial tissue, so that the intestinal tissue is dissociated. However, the method of using collagenase to dissociate small intestinal tissue to extract organoids has a strict operation process, is time-consuming, and has a great risk of contamination by fibroblasts. Summary of the invention

[0004] The main purpose of the present invention is to provide a highly active intestinal crypt and an isolation method and application thereof, aiming to improve the viability of intestinal crypt cells.

[0005] To achieve the above object, the present invention provides a method for isolating highly active intestinal crypts, comprising the following steps:

[0006] S1. Take fresh intestines, wash the contents and scrape the mucosal layer to obtain intestinal mucosal tissue;

[0007] S2. chopping the intestinal mucosal tissue, washing it, mixing it with a separation solution, and digesting and dissociating it to obtain an intestinal mucosal tissue digestion solution;

[0008] S3. After the intestinal mucosal tissue digestion fluid is centrifuged, the supernatant is removed and mixed with the protective solution to obtain an intestinal crypt mixture, which is filtered and centrifuged to obtain a precipitate to obtain a highly active intestinal crypt;

[0009] Wherein, the separation solution comprises collagenase I, collagenase II, collagenase IV, elastase, DNase I and ROCK inhibitor;

[0010] The protective solution includes RPMI1640 culture medium, antibiotics, fetal bovine serum, bovine serum albumin and ROCK inhibitor.

[0011] In one embodiment, in the separation solution, the ROCK inhibitor is ROCK inhibitor Y27632;

[0012] The mass concentration of collagenase I is 0.3-0.7 mg / mL;

[0013] The mass concentration of collagenase II is 0.3-0.7 mg / mL;

[0014] The mass concentration of the collagenase IV is 0.8-1.2 mg / mL;

[0015] The mass concentration of the elastase is 0.3-0.7 mg / mL;

[0016] The mass concentration of the DNase I is 0.01-0.2 mg / mL;

[0017] The molar concentration of the ROCK inhibitor is 8-12 μM.

[0018] In one embodiment, in the protective solution, the antibiotics include penicillin and / or streptomycin, and the ROCK inhibitor is ROCK inhibitor Y27632;

[0019] The mass percentage of the antibiotic is 0.5% to 1.5%;

[0020] The mass proportion of the fetal bovine serum is 8% to 12%;

[0021] The mass proportion of the bovine serum albumin is 0.5% to 1.5%;

[0022] The molar concentration of the ROCK inhibitor is 8-12 μM.

[0023] In one embodiment, in step S2, when the intestinal mucosal layer tissue is cut into pieces with a diameter of 1 to 3 mm; and / or,

[0024] During digestion and dissociation, the digestion and dissociation are carried out while oscillating, the oscillation speed is 150-200 rpm, the digestion and dissociation temperature is 36-38° C., and the digestion and dissociation time is 10-15 min.

[0025] In one embodiment, in step S3, when the intestinal mucosal tissue digestion fluid is centrifuged, the centrifugal temperature is 2-8° C., the centrifugal force is 200 g, and the centrifugal time is 2-4 min; and / or,

[0026] Before filtering, the intestinal crypt mixture is blown 15 to 20 times.

[0027] In one embodiment, in step S3, filtering includes the following steps:

[0028] The intestinal crypt mixture was filtered through a 70 μm cell strainer, and the filtrate was recorded as the first fraction;

[0029] The first fraction was filtered using a 40 μm cell strainer, the filtrate was recorded as the second fraction, and the retentate was recorded as the third fraction;

[0030] The cell filter is backwashed with the protective solution and the third fraction is collected and centrifuged to obtain the precipitate to obtain the highly active intestinal crypts.

[0031] The present invention also provides a highly active intestinal crypt, including the highly active intestinal crypt isolated by the aforementioned method for isolating highly active intestinal crypts.

[0032] The present invention also proposes a use of intestinal crypts in the culture of intestinal organoids, wherein the intestinal crypts include the highly active intestinal crypts as described above.

[0033] The present invention also provides an application of intestinal crypts in flow cytometry separation of intestinal mucosal epithelial cells, wherein the intestinal crypts include the highly active intestinal crypts as described above.

[0034] The present invention also proposes an application of intestinal crypts in single-cell transcriptomic sequencing of intestinal mucosal epithelial cells, wherein the intestinal crypts include the highly active intestinal crypts as described above.

[0035] In the technical scheme of the present invention, collagenase I, collagenase II, collagenase IV, elastase, DNase I and ROCK inhibitor are used as separation liquids, wherein collagenase I, collagenase II, collagenase IV and elastase can decompose the skeleton structure between cells, thereby separating the mucosal tissue into single cells or cell clumps; ROCK inhibitor mainly inhibits cell apoptosis by inhibiting the kinase activity of ROCK, promotes cell survival, and significantly reduces dissociation-induced cell apoptosis; RPMI1640 culture medium, antibiotics, fetal bovine serum, bovine serum albumin and ROCK inhibitor are used as protective liquids, wherein antibiotics are used to prevent or control the growth of microorganisms, thereby avoiding contamination of cell culture, fetal bovine serum and bovine serum albumin are used as protein stabilizers, which can bind and neutralize free radicals and other potentially harmful substances in the solution, thereby protecting cells from damage, and fetal bovine serum contains rich nutrients, including amino acids, vitamins, hormones, minerals and lipids, etc., which can maintain the growth environment of cells, and ROCK inhibitor significantly reduces dissociation-induced cell apoptosis by inhibiting the kinase activity of ROCK. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0037] Figure 1 This is a graph showing the cell counting results of intestinal crypts provided in Example 1, Comparative Example 1 and Comparative Example 2 of the present invention;

[0038] Figure 2 This is a comparison chart of the cell activities of the intestinal crypts provided in Example 1, Comparative Example 1 and Comparative Example 2 of the present invention;

[0039] Figure 3 A morphological comparison diagram of intestinal crypts provided in Example 1, Comparative Example 1 and Comparative Example 2 of the present invention;

[0040] Figure 4 This is a morphological diagram of primary organoid culture of intestinal crypts provided in Examples 1 to 3 of the present invention;

[0041] Figure 5 This is a diagram showing the application of flow cytometry sorting of immune cells in the intestinal mucosa provided in Example 4 of the present invention;

[0042] Figure 6 This is a diagram of the application of single-cell transcriptome sequencing of epithelial cells in the intestinal mucosa provided in Example 5 of the present invention.

[0043] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0044] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0045] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0046] At present, the mainstream separation methods are mainly based on the traditional EDTA method and collagenase method. The basic principle of the EDTA method to dissociate cells is to remove metal ions on the cell surface through chelation, thereby destroying the connection between cells and allowing the cells to be separated. This method is too lethal to cells, the dissociation time is extremely difficult to control, and the structural integrity, purity and activity of the extracted crypts are poor. The collagenase method can effectively hydrolyze proteins, polysaccharides and lipids in the extracellular matrix of connective tissue and epithelial tissue, thereby dissociating intestinal tissue. However, the method of using collagenase to dissociate small intestinal tissue to extract organoids has a strict operation process, is time-consuming, and has a great risk of contamination by fibroblasts.

[0047] For example, the EDTA method described in CN118165915A is used to separate mouse small intestinal crypts; the type IV collagenase method described in CN117821371A is used to separate rhesus monkey intestinal crypts; the EDTA method described in CN117844739A is used to separate sheep small intestinal crypts; and the type I collagenase method described in CN117586941A is used to separate chicken intestinal crypts. The feeding habits, intestinal mucosal barrier physiology, intestinal microbial composition and major intestinal diseases of pigs are similar to those of humans, and can be used as a good research model. The prior art has been studied on the separation and purification of intestinal crypts, such as the use of the EDTA method to separate intestinal crypts as described in CN104745525B, but the structural integrity, purity and activity of the crypts obtained by this method are poor. Although the above methods can successfully separate intestinal crypts, there is a huge room for improvement in the operation time of the entire process, the efficiency of crypt extraction, the activity of crypt stem cells, the number of crypts and the cost.

[0048] In view of this, the present invention proposes a method for isolating highly active intestinal crypts, comprising the following steps:

[0049] S1. Take fresh intestines, wash the contents and scrape the mucosal layer to obtain intestinal mucosal tissue;

[0050] S2. chopping the intestinal mucosal tissue, washing it, mixing it with a separation solution, and digesting and dissociating it to obtain an intestinal mucosal tissue digestion solution;

[0051] S3. After the intestinal mucosal tissue digestion fluid is centrifuged, the supernatant is removed and mixed with the protective solution to obtain an intestinal crypt mixture, which is filtered and centrifuged to obtain a precipitate to obtain a highly active intestinal crypt;

[0052] Wherein, the separation solution comprises collagenase I, collagenase II, collagenase IV, elastase, DNase I and ROCK inhibitor;

[0053] The protective solution includes RPMI1640 culture medium, antibiotics, fetal bovine serum, bovine serum albumin and ROCK inhibitor.

[0054] In the technical scheme of the present invention, collagenase I, collagenase II, collagenase IV, elastase, DNase I and ROCK inhibitor are used as separation liquids, wherein collagenase I, collagenase II, collagenase IV and elastase can decompose the skeleton structure between cells, thereby separating the mucosal tissue into single cells or cell clumps, and ROCK inhibitor mainly inhibits cell apoptosis by inhibiting the kinase activity of ROCK, promotes cell survival, and significantly reduces dissociation-induced cell apoptosis. The reagent combination involved in the present invention maximizes the physiological and biochemical functions of enzyme preparations and inhibitors, can stably and efficiently separate intestinal crypt cells, and protect cell activity to the greatest extent while shortening the operation time;

[0055] RPMI1640 culture medium, antibiotics, fetal bovine serum, bovine serum albumin and ROCK inhibitor are used as protective solutions, wherein antibiotics are used to prevent or control the growth of microorganisms, thereby avoiding contamination of cell cultures; fetal bovine serum and bovine serum albumin, as protein stabilizers, can bind to and neutralize free radicals and other potentially harmful substances in the solution, thereby protecting cells from damage; and fetal bovine serum contains rich nutrients, including amino acids, vitamins, hormones, minerals and lipids, which can maintain the growth environment of cells; and ROCK inhibitors significantly reduce dissociation-induced cell apoptosis by inhibiting ROCK kinase activity.

[0056] Collagenase type I, II and IV, each of these three collagenases has its own preference in application: Collagenase I contains relatively uniform enzyme activities and is often used for the preparation of epithelial cells, liver, lung, fat and adrenal tissue cells; Collagenase II contains higher clostripain activity and is usually used for the preparation of tissue-derived cells such as heart, bone, muscle, thymus and cartilage; Collagenase IV contains at least 7 protease components and is usually used in cell preparation experiments that need to maintain receptor integrity;

[0057] Elastase has esterase and acyl gelatinase activities, and has the function of hydrolyzing a large number of protein substrates and natural elastin (a protein that cannot be hydrolyzed by pancreatin, chymotrypsin or pepsin);

[0058] DNase I can enter necrotic cells and gather near the nucleus, efficiently hydrolyzing the chromatin DNA of necrotic cells, and DNase I has the function of inhibiting cell aggregation. When type I, type II, type IV collagenase and elastase over-dissociate some cells, DNase I can play its physiological function. On the one hand, DNase I can promote apoptosis of cells with low activity, and on the other hand, it can promote cell separation, play an auxiliary role in separation in the separation solution, and improve separation efficiency and cell activity.

[0059] It should be noted that the fresh intestine can be the intestine of an artiodactyl, a rodent, or a primate, and is not limited here, as long as there are crypts in the intestine. Specifically, in one embodiment of the present invention, fresh pig intestine is used as the fresh intestine.

[0060] In one embodiment, in the separation solution, the ROCK inhibitor is ROCK inhibitor Y27632; the mass concentration of collagenase I is 0.3-0.7 mg / mL; the mass concentration of collagenase II is 0.3-0.7 mg / mL; the mass concentration of collagenase IV is 0.8-1.2 mg / mL; the mass concentration of elastase is 0.3-0.7 mg / mL; the mass concentration of DNase I is 0.01-0.2 mg / mL; and the molar concentration of the ROCK inhibitor is 8-12 μM.

[0061] In the technical solution of the present invention, by using ROCK inhibitor Y27632, ROCK inhibitor Y27632 is a small molecule inhibitor of Rho-related protein kinase p160ROCK, which has the function of inhibiting the "anoestrus" effect of intestinal crypt stem cells, preventing stem cell apoptosis, and improving the survival rate of stem cells; by adjusting the mass concentration of various enzymes and the molar concentration of ROCK inhibitor, it can be adjusted to the optimal concentration to ensure the separation efficiency of crypt cells and avoid damage to the crypt cells by the separation fluid.

[0062] In one embodiment, in the protective solution, the antibiotics include penicillin and / or streptomycin, and the ROCK inhibitor is ROCK inhibitor Y27632; the mass proportion of the antibiotics is 0.5% to 1.5%; the mass proportion of the fetal bovine serum is 8% to 12%; the mass proportion of the bovine serum albumin is 0.5% to 1.5%; and the molar concentration of the ROCK inhibitor is 8 to 12 μM.

[0063] In the technical solution of the present invention, by adopting penicillin and / or streptomycin, the growth of microorganisms can be prevented or controlled, thereby avoiding contamination of cell culture; by adding ROCK inhibitor Y27632 to the protective solution, dissociation-induced cell apoptosis can be significantly reduced; by adjusting the content of each component in the protective solution, the nutrients required for the growth and development of crypt cells can be provided, a certain degree of buffering environment can be provided, the stability of the cell culture environment can be maintained, and the toxic effects on cells can be reduced.

[0064] In one embodiment, in step S2, when the intestinal mucosal tissue is sheared, the intestinal mucosal tissue is sheared into fragments with a diameter of 1 to 3 mm; and / or, during digestion and dissociation, the digestion and dissociation are performed while oscillating, the oscillation speed is 150 to 200 rpm, the digestion and dissociation temperature is 36 to 38°C, and the digestion and dissociation time is 10 to 15 minutes.

[0065] In the technical scheme of the present invention, the intestinal mucosal layer tissue is cut into fragments with a diameter of 1 to 3 mm to increase the specific surface area of ​​the tissue, thereby increasing the contact area between the tissue and the separation liquid and accelerating the digestion and dissociation speed; the digestion and dissociation are performed by oscillating, so that the tissue and the separation liquid are fully mixed to increase the degree of digestion and dissociation, and the digestion and dissociation temperature is controlled to 36 to 38°C so that the enzyme in the separation liquid is within the optimal working temperature range. At the same time, a suitable temperature is provided for the intestinal mucosal layer tissue to ensure the viability of the intestinal mucosal layer cells; by adjusting the digestion and dissociation time to 10 to 15 minutes, the mucosal tissue is separated into single cells or cell clusters, maintaining appropriate connections between cells, while not completely preventing necessary dissociation, thereby reducing cell apoptosis and ensuring cell viability.

[0066] In one embodiment, in step S3, when the intestinal mucosal tissue digestion fluid is centrifuged, the centrifugal temperature is 2 to 8°C, the centrifugal force is 200g, and the centrifugal time is 2 to 4 minutes; and / or, before filtering, the intestinal crypt mixture is blown 15 to 20 times.

[0067] In the technical solution of the present invention, the centrifugal temperature is adjusted to 2-8°C to avoid further dissociation of cells by the separation fluid, thereby reducing the possibility of cell death; the centrifugal force and centrifugation time are adjusted to facilitate the separation of intestinal mucosal tissue from the separation fluid; the protective fluid is added to completely stop the digestive effect of the separation fluid on the cells, thereby avoiding excessive cell digestion and apoptosis; and the intestinal crypts can be blown away by blowing 15-20 times to fully release the crypts and clean the crypt cells at the same time.

[0068] In one embodiment, in step S3, filtering includes the following steps:

[0069] The intestinal crypt mixture was filtered through a 70 μm cell strainer, and the filtrate was recorded as the first fraction;

[0070] The first fraction was filtered using a 40 μm cell strainer, the filtrate was recorded as the second fraction, and the retentate was recorded as the third fraction;

[0071] The cell filter is backwashed with the protective solution and the third fraction is collected and centrifuged to obtain the precipitate to obtain the highly active intestinal crypts.

[0072] In the technical solution of the present invention, by filtering with a 70 μm cell filter, large tissue fragments can be filtered out, thereby improving the purity of crypt cells in the first fraction; by filtering with a 40 μm cell filter, broken cells and small cells (such as red blood cells) can be screened out, thereby improving the purity of crypt cells in the third fraction.

[0073] The present invention also provides a highly active intestinal crypt, including the highly active intestinal crypt isolated by the aforementioned method for isolating highly active intestinal crypts.

[0074] Since the intestinal crypt adopts all the technical solutions of all the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be described one by one here.

[0075] The present invention also proposes a use of intestinal crypts in the culture of intestinal organoids, wherein the intestinal crypts include the highly active intestinal crypts as described above.

[0076] In one embodiment, the culturing of intestinal organoids comprises the following steps:

[0077] After resuspending the intestinal crypts in culture medium, they were mixed with Matrigel;

[0078] After the matrix gel solidifies, add the culture medium and start culturing the intestinal organoids.

[0079] The present invention also provides an application of intestinal crypts in flow cytometry separation of intestinal mucosal epithelial cells, wherein the intestinal crypts include the highly active intestinal crypts as described above.

[0080] In one embodiment, the flow cytometry separation of intestinal mucosal epithelial cells comprises the following steps:

[0081] After combining the second fraction with the intestinal crypts, centrifuge at 4°C, 300g for 3 minutes, add 0.25% trypsin-EDTA, digest at 37°C for 5-10 minutes, and then add the protective solution to terminate the digestion;

[0082] Centrifuge at 4°C, 500g for 3 min, discard the supernatant, resuspend the precipitate and blow it evenly to obtain a single-cell suspension, which can be combined with specific antibodies or fluorescent labeling reagents for flow cytometry sorting.

[0083] The present invention also proposes an application of intestinal crypts in single-cell transcriptomic sequencing of intestinal mucosal epithelial cells, wherein the intestinal crypts include the highly active intestinal crypts as described above.

[0084] In one embodiment, the single-cell transcriptomic sequencing of intestinal mucosal epithelial cells comprises the following steps:

[0085] After the second fraction was combined with the intestinal crypts, they were centrifuged at 4°C, 300 g for 3 min, 0.25% trypsin-EDTA was added, and digested at 37°C for 5-10 min, and then the crypt protection solution was added to terminate the digestion;

[0086] Centrifuge at 4°C, 500g for 3 min, discard the supernatant, resuspend the precipitate and blow it evenly to obtain a single-cell suspension. After selectively combining red blood cell lysis and dead cell removal, single-cell oil-in-water sequencing can be performed.

[0087] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0088] Experimental Materials

[0089] Unless otherwise specified, the reagents and materials used in the examples of the present invention are commercially available.

[0090] Collagenase I (Gibco, catalog number: 17100017);

[0091] Collagenase II (Yisun, catalog number: 40508ES76);

[0092] Collagenase IV (Gibco, catalog number: 17104019);

[0093] Elastase (Maokang, catalog number: MX1010-200MG);

[0094] DNase I (Thermo Scientific, Catalog No.: 90083);

[0095] ROCK inhibitor Y27632 (Biyuntian, catalog number: SC0326-5mg);

[0096] RPMI1640 culture medium (Gibco, catalog number 72400047);

[0097] Penicillin / streptomycin (Gibco, Cat. No. 15070063);

[0098] Fetal bovine serum FBS (Gibco, cat. no. 10091148);

[0099] Bovine serum albumin (BSA) (Sigma Aldrich, Catalog No. A1933);

[0100] 0.25% Trypsin-EDTA (Gibco, Cat. No. 25200114);

[0101] DPBS (Gibco, Cat. No. 14190144);

[0102] AO / PI fluorescent dye (Logos Biosystems, catalog number: F23001).

[0103] Example 1

[0104] This example provides a method for isolating porcine jejunal crypts, and the specific steps are as follows:

[0105] 1) Take 3-5 cm of fresh pig jejunum, cut it open longitudinally, and repeatedly rinse the contents and chyme with saline;

[0106] 2) Use a glass slide to scrape the intestinal mucosal tissue, discard the intestinal muscle tissue, use ophthalmic scissors to cut the tissue into 1-2 mm fragments, and collect them into a 50 mL centrifuge tube filled with pre-cooled DPBS;

[0107] 3) Let stand for 30 seconds, use a 10 mL pipette to aspirate the upper layer of liquid as much as possible, add 40 mL of pre-cooled DPBS, and use a 10 mL pipette to pipette repeatedly for 5 times;

[0108] 4) Repeat step 3) 15 times, centrifuge at 4°C, 200g, for 3 min, and remove the supernatant;

[0109] 5) Add 20 mL of the highly active porcine intestinal crypt isolation solution prepared in advance (0.5 mg / mL collagenase I; 0.5 mg / mL collagenase II; 1.0 mg / mL collagenase IV; 0.5 mg / mL elastase; 0.1 mg / mL DNase I; 10 μM ROCK inhibitor Y27632; solvent DPBS), 37°C, 200 rpm, shake and dissociate for 12 min;

[0110] 6) Centrifuge at 4°C, 200g for 3 min and remove the supernatant;

[0111] 7) Add 20 mL of pre-prepared and pre-cooled high-activity porcine intestinal crypt protection solution (RPMI1640 medium + 1% penicillin / streptomycin + 10% FBS + 1% BSA + 10 μM ROCK inhibitor Y27632), and use a 10 mL pipette to repeatedly pipette 20 times to fully release the crypts;

[0112] 8) Filter using a 70 μm cell strainer, and the filtrate obtained after filtration is marked as fraction 1;

[0113] 9) Filter fraction 1 using a 40 μm cell strainer. The filtrate obtained after filtration is labeled as fraction 2, and the material retained on the filter is labeled as fraction 3;

[0114] 10) Use crypt protection solution to reversely flush and collect fraction 3, centrifuge at 4°C, 300g for 5 minutes, and mark the precipitate as fraction 4, which is the crypt with high concentration, high purity and high activity;

[0115] After fraction 2 and fraction 4 were combined, centrifuged at 4°C, 300g for 3 min. 1 mL of 0.25% trypsin-EDTA was added, and digested at 37°C for 5 min. The crypt protection solution was added to terminate the digestion. Centrifuged at 4°C, 500g for 3 min, and the supernatant was discarded.

[0116] This example also proposes a primary organoid culture method, the specific steps are as follows:

[0117] Fraction 4 was resuspended in a small amount of porcine intestinal organoid culture medium, mixed with matrix gel in a certain proportion, inoculated into a cell culture plate, placed in a cell culture incubator, and porcine intestinal organoid culture medium was added after the matrix gel solidified.

[0118] Example 2

[0119] This example proposes a method for isolating porcine ileal crypts and culturing primary organoids, and the specific steps are similar to those in Example 1.

[0120] Example 3

[0121] This example proposes a method for isolating porcine colonic crypts and culturing primary organoids, and the specific steps are similar to those in Example 1.

[0122] Example 4

[0123] This embodiment proposes a method for isolating porcine ileal crypts and isolating and flow sorting ileal mucosal immune cells, wherein the method for isolating porcine ileal crypts is similar to the method for isolating porcine jejunal crypts in Example 1, with the difference that, after step 10), fraction 2 and fraction 4 are combined and centrifuged at 4°C, 300g for 3 min. Add 0.25% trypsin-EDTA, digest at 37°C for 5 min, and then add crypt protection solution to terminate digestion. Centrifuge at 4°C, 500g for 5-10 min, and discard the supernatant. Wash with PBS containing 2% BSA, resuspend with 8mL of 40% Percoll solution and transfer to a 15mL centrifuge tube; use a long needle syringe to slowly add 5mL of 70% Percoll solution to the bottom of the centrifuge tube, and spin at 2000r·min. -1 Centrifuge for 20 minutes; carefully aspirate the lymphocytes in the middle layer and transfer them to a 15 mL centrifuge tube. After washing with PBS containing 2% BSA, resuspend the cells with an appropriate amount of PBS to obtain preliminary separation of intestinal lymphocytes. After resuspending and blowing the precipitate, a single cell suspension of immune cells is obtained, which is used for flow cytometry sorting.

[0124] Example 5

[0125] This example proposes a single-cell transcriptome sequencing of porcine jejunal mucosal epithelial cells, comprising the following steps:

[0126] Take fraction 2 and fraction 4 in Example 1, combine fraction 2 and fraction 4, centrifuge at 4°C, 300g for 3min. Add 0.25% trypsin-EDTA, digest at 37°C for 5min, and then add crypt protection solution to terminate digestion. Centrifuge at 4°C, 500g for 3min, and discard the supernatant. Resuspend the precipitate and blow it evenly to obtain a single cell suspension. Combined with the red blood cell lysis operation, perform single cell oil-in-water machine detection.

[0127] Comparative Example 1

[0128] This comparative example proposes a method for isolating porcine jejunal crypts (EDTA method)

[0129] The experimental method is similar to that of Example 1, except that the dissociation solution used in step 5) is: 10.0 mM HEPES; 30.0 mM Na2EDTA-2H2O; 0.50 mM DTT; 0.25 mM BSA; 2.5 mM glucose; 2.5 mM glutamine; 0.50 mM sodium DLβ-hydroxybutyrate; 10 mM Y-27632; 2.7 mM KCl; 1.5 mM KH2PO4; 138 mM NaCl; 8 mM Na2HPO4 7H2O, and the dissociation conditions are 4°C, 100 rpm, and 20 min.

[0130] Comparative Example 2

[0131] This comparative example proposes a method for separating porcine jejunal crypts (traditional enzymatic hydrolysis)

[0132] The experimental method is similar to that of Example 1, except that the dissociation solution used in step 5) is formulated as follows: 1% penicillin / streptomycin+2 mg / mL collagenase IV+10 μM / mL Rock inhibitor Y27632.

[0133] Performance Testing

[0134] The porcine jejunal crypt cells provided in Example 1, Comparative Example 1 and Comparative Example 2 were resuspended and blown uniformly, and then mixed with AO / PI fluorescent dyes. The activity of the porcine intestinal crypt cells was measured using a Luna cell counter (Logos Biosystems). Figure 1 and Figure 2 As shown, the viable cell rate of Example 1 can reach more than 90%, and the viable cell rates of the comparative example and comparative example 2 are approximately 55% and 73%, respectively.

[0135] The porcine jejunal crypt cells provided in Example 1, Comparative Example 1 and Comparative Example 2 were placed under an optical microscope to observe the morphology of the porcine jejunal crypts. Figure 3 As shown, the crypts extracted in Example 1 are abundant in number, of appropriate size, with few broken cells and a clean background, while the crypts extracted in Comparative Examples 1 and 2 are moderate in number, of small size, with many broken cells and a messy background.

[0136] The crypts provided in Examples 1 to 3 were placed in an incubator for culture. After 72 hours of culture, the morphology of the porcine jejunal organoids, porcine ileal organoids, and porcine colonic organoids were observed under an optical microscope. Figure 4As shown; it can be seen that the pig jejunal organoids have normal morphology, are in a budding state, and are in good growth condition; the pig ileal organoids have normal morphology, are in a budding state, and are in good growth condition; the pig colon organoids have normal morphology, are vacuolar, and are in good growth condition.

[0137] The application results of flow sorting in Example 4 are as follows Figure 5 shown. Figure 5 The HC group used the immune cell suspension separated in Comparative Example 1 for flow sorting analysis, and the OSA group used the immune cell suspension separated in Example 1 for subsequent flow sorting analysis. The results showed that the proportion of innate lymphocytes ILC1 / 3 in the OSA group was significantly higher than that in the HC group, indicating that the immune cells extracted and separated by the method established in Example 1 of this scheme are easier to perform flow sorting analysis, even for the innate lymphocytes that account for a small proportion of immune cells, their analysis ratio can still be increased, thereby proving that the viability and purity of the extracted immune cells are high, and the results are more reliable and accurate.

[0138] The quality control results of single-cell transcriptome sequencing in Example 5 are as follows Figure 6 As shown, according to Figure 6 It can be seen that the quality control results of cell transcriptome sequencing are well reflected, and more than 10,000 cells can be easily captured. Various data such as the number of gene tests meet the qualified requirements.

[0139] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. A method for isolating highly active intestinal crypts, characterized in that: The following steps are involved: S1. Take fresh intestines, wash the contents and scrape the mucosal layer to obtain intestinal mucosal tissue; S2. chopping the intestinal mucosal tissue, washing it, mixing it with a separation solution, and digesting and dissociating it to obtain an intestinal mucosal tissue digestion solution; S3. After the intestinal mucosal tissue digestion fluid is centrifuged, the supernatant is removed and mixed with the protective solution to obtain an intestinal crypt mixture, which is filtered and centrifuged to obtain a precipitate to obtain a highly active intestinal crypt; Wherein, the separation solution comprises collagenase I, collagenase II, collagenase IV, elastase, DNase I and ROCK inhibitor; The protective solution includes RPMI1640 culture medium, antibiotics, fetal bovine serum, bovine serum albumin and ROCK inhibitor.

2. The method for isolating highly active intestinal crypts according to claim 1, characterized in that: In the separation solution, the ROCK inhibitor is ROCK inhibitor Y27632; The mass concentration of collagenase I is 0.3-0.7 mg / mL; The mass concentration of collagenase II is 0.3-0.7 mg / mL; The mass concentration of the collagenase IV is 0.8-1.2 mg / mL; The mass concentration of the elastase is 0.3-0.7 mg / mL; The mass concentration of the DNase I is 0.01-0.2 mg / mL; The molar concentration of the ROCK inhibitor is 8-12 μM.

3. The method for isolating highly active intestinal crypts according to claim 1, characterized in that: In the protective solution, the antibiotics include penicillin and / or streptomycin, and the ROCK inhibitor is ROCK inhibitor Y27632; The mass percentage of the antibiotic is 0.5% to 1.5%; The mass proportion of the fetal bovine serum is 8% to 12%; The mass proportion of the bovine serum albumin is 0.5% to 1.5%; The molar concentration of the ROCK inhibitor is 8-12 μM.

4. The method for isolating highly active intestinal crypts according to claim 1, characterized in that: In step S2, when the intestinal mucosal layer tissue is cut into pieces, the intestinal mucosal layer tissue is cut into pieces with a diameter of 1 to 3 mm; and / or, During digestion and dissociation, the digestion and dissociation are carried out while oscillating, the oscillation speed is 150-200 rpm, the digestion and dissociation temperature is 36-38° C., and the digestion and dissociation time is 10-15 min.

5. The method for isolating highly active intestinal crypts according to claim 1, characterized in that: In step S3, when the intestinal mucosal tissue digestion fluid is centrifuged, the centrifugal temperature is 2 to 8° C., the centrifugal force is 200 g, and the centrifugal time is 2 to 4 min; and / or, Before filtering, the intestinal crypt mixture is blown 15 to 20 times.

6. The method for isolating highly active intestinal crypts according to claim 1, characterized in that: In step S3, filtering includes the following steps: The intestinal crypt mixture was filtered through a 70 μm cell strainer, and the filtrate was recorded as the first fraction; The first fraction was filtered using a 40 μm cell strainer, the filtrate was recorded as the second fraction, and the retentate was recorded as the third fraction; The cell filter is backwashed with the protective solution and the third fraction is collected and centrifuged to obtain the precipitate to obtain the highly active intestinal crypts.

7. A highly active intestinal crypt, characterized in that It comprises the highly active intestinal crypts isolated by the method for isolating highly active intestinal crypts as described in any one of claims 1 to 6.

8. A use of intestinal crypts in the culture of intestinal organoids, characterized in that: The intestinal crypts include the highly active intestinal crypts as described in claim 7.

9. An application of intestinal crypts in flow cytometry separation of intestinal mucosal epithelial cells, characterized in that: The intestinal crypts include the highly active intestinal crypts as described in claim 7.

10. An application of intestinal crypts in single-cell transcriptomic sequencing of intestinal mucosal epithelial cells, characterized in that: The intestinal crypts include the highly active intestinal crypts as described in claim 7.

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