CN115449504A - Method for establishing 2D culture model of porcine small intestine organs - Google Patents
Method for establishing 2D culture model of porcine small intestine organs Download PDFInfo
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Abstract
The application discloses a method for establishing a stable 2D culture model of a porcine small intestine organoid, which comprises the steps of firstly obtaining a passable porcine small intestine organoid, preparing a porcine small intestine organoid tissue suspension, and then culturing to form a porcine small intestine organoid 2D growth model. The application changes the method for obtaining the porcine small intestine organoid monolayer, changes the traditional mechanical dissociation into the enzymatic dissociation and is beneficial to keeping the integrity of cells; 2D two-dimensional culture of cells is utilized to help reduce the influence of interaction between injected substances and apical membranes due to fragments accumulated in an inner cavity in the 3D culture process; the 2D intestinal organoid model enables functional research to better accord with real physiological conditions, can provide simpler and more convenient operation for high-flux nutrient screening and bioactive substance function identification, and solves the problem that the intestinal nutrition and health related research feasibility is poor due to the fact that the intestinal luminal surface of the 3D intestinal organoid cannot be in direct contact with a culture environment.
Description
Technical Field
The invention belongs to the technical field of animal histology. In particular to a method for establishing a 2D culture model of a porcine small intestine organoid.
Background
In the last decade organoid technology has been applied systematically in multiple research disciplines in biology, medicine, etc. as an integrated technology area, the harvesting of stem cells with the ability to induce the formation of multifunctional organs, based on their self-organizing ability, allows the formation of 3D structures in vitro that are structurally and functionally similar to their in vivo counterparts (Juan He, et al. Organoid culture techniques are widely used in intestinal studies. Under proper in vitro culture conditions, intestinal stem cells initially form a saccular vacuolar structure with a single central lumen, the stem cells further divide and proliferate to generate transitional expansion cells, the saccular cavity is expanded outwards to form bud-shaped bulges, the transitional expansion cells generate a plurality of terminally differentiated cells such as intestinal epithelial cells, intestinal secretory cells, goblet cells and Pangolian cells after 4-5 divisions, so that the saccular structure presents bud characteristics, and a typical organoid structure comprising all intestinal epithelial cell types and an intact crypt-villus structure is formed after 5-7 days (Akkerman N, et al. Methods have been developed to optimize Organoid in vitro culture, demonstrating the possibility of long term culture in vitro, which can be continued for at least 1 and a half years with stable genetic properties (Sugimoto S, et al. Establishment of 3d endogenous organic Cultures from endogenous animal cells, methods in Molecular biology.2017, 1612. Intestinal organoids are not only highly similar in cell lineage composition to Intestinal epithelial tissue, but also have functional properties that are highly consistent with the source tissue (Foulke-AbelJ, et al. Human enterprises as a Model of Upper Small Intestinal intracellular Transport Physiology and Pathophysiology. Gastroenterology.2016, 150 (3): 638-649). Therefore, the introduction of a brand new technology of small intestine organs will deepen the understanding of the regulation and control of the intestinal mucosa barrier function by the bioactive substances, provide a new thought for researching the intestinal health improvement function of nutrient substances, and lay a theoretical and practical foundation for individual accurate nutrition and metabolism research.
In related studies in the field of nutrition impact on intestinal health, organoid models are used in cross-applications with multiple disciplines. The most remarkable characteristics of organoids are their strong self-renewal capacity and high similarity to the microenvironment in vivo (gaoyun et al. Gut organoids research and application. International J. Digestive tract.2017, 37 (2): 87-91). The advantages of the characteristics are that the organoid in vitro culture technology develops rapidly. Although considerable progress has been made in the development of enteral nutrition biology based on animal models such as rodents and dogs, the use of these models has been limited by the existence of clear differences in anatomical and physiological properties between animals and humans (beer J, et al. Regulation and plasticity of intestinal cells and regeneration. Development.2016 (20): 3639-3649). For example, mouse models lack the key clinical symptoms and pathological changes that represent human gastrointestinal disease, thereby limiting the use of mice as models for clinical nutritional studies; dogs are also widely restricted in their use in related research due to social ethical issues (Sara Rahmani, et al. Intelligent oligonucleotides: A new paradigm for engineering the internal epitope in vitro. Biomaterials.2019, 194). In comparison, swine is a relatively reliable animal model with the advantages of similarity of physiological features and anatomical features of the porcine and human gut (Koopmans, sietase Jan, schuurman, teun. Considerations on pig models for apetite, metabolic syndrome and organism type 2diabetes [ from food intake to metabolic disease j ]. European Journal of pharmacology.2015, 759. Therefore, the pig intestinal organoids combine the advantages of the in vivo and in vitro research model. At present, a relatively complete pig intestinal organoid culture method is researched and developed, but organoids formed by the culture method still have certain defects in some applications for simulating real intestinal environments.
The intestinal organoid 3D culture model is an emerging in-vitro research system for researching the growth and differentiation of tissue somatic stem cells and organ formation, and is a milestone type research result in the field of intestinal biology research. However, the study on the nutritional health of the intestine is hindered because the intestinal luminal surface of the 3D cultured intestinal organoid is wrapped inside the cavity of the spherical structure, and in addition, the inner cavity of the 3D cultured organoid accumulates debris from cell renewal, which can bind or block the interaction of injected substances with the apical membrane, thereby affecting the culture of the whole organoid. Therefore, it is of practical significance to optimize the culture procedure and to form an in vitro organoid application model that is more convenient to operate, and it is the technical problem that is solved herein.
Disclosure of Invention
The invention aims to establish a stable establishment method of a 2D culture model of a porcine small intestine organoid, which comprises the steps of firstly obtaining a passable porcine small intestine organoid, preparing a porcine small intestine organoid tissue suspension, and then culturing to form a 2D growth model of the porcine small intestine organoid.
The method is realized by the following technical measures: a method for establishing a pig small intestine organoid 2D culture model comprises the following steps: obtaining passable porcine small intestine organoids: aseptically collecting 5-10 cm from the middle section of the pig jejunum, and clearing the pig jejunum twice until the supernatant is clear; sucking supernatant, transferring the intestinal segment into a solution containing 30mL of 8mM EDTA-PBS, flatly placing the intestinal segment on ice, shaking for 40min, removing the supernatant, adding a new 30mL of 8mM EDTA-PBS, shaking for 40min on ice again, sucking the EDTA solution, shaking and cleaning with 30mL of PBS, placing the intestinal segment on ice to allow intestinal tissue blocks to settle by gravity, removing the supernatant, and repeating the process for 1-2 times; the pellet was transferred to a 15mL centrifuge tube at 5Resuspend the pellet in mL DMEM/F12 (containing 10% FBS,1% triple antibiotics) medium, count crypts by microscopy 3-5 μ L; centrifuging at 4 deg.C for 5min at 500g, discarding supernatant, mixing the precipitate with Matrigel matrix, and spotting in 48-well plate at 20 μ L/well; transferring 48-well cell culture plate into carbon dioxide incubator, 37 deg.C, 5% 2 Incubating for 15min under the condition until the Matrigel is solidified; adding preheated complete culture medium with 200 mu L/hole after the Matrigel is solidified; placing the mixture in an incubator at 37 ℃ for continuous culture, and replacing a fresh culture medium every 2-3 days; carrying out passage according to growth conditions every 5-7 days;
preparing a porcine small intestine organoid tissue suspension: when the cultured pig organs grow to be passable, absorbing the culture medium, adding 500 mu L of precooled PBS into each hole, and incubating for 1min at room temperature; the pipette sucks PBS to uniformly mix the intestinal organs, and transfers the mixture into a 15mL centrifuge tube; adding 1mL of PBS to each hole for washing, and adding the PBS to the same 15mL centrifuge tube; centrifuging at 4 deg.C for 5min at 200g, and removing supernatant; adding proper amount of TrypL E according to the cell amount, and carrying out water bath at 37 ℃ for 5min, wherein the TrypL E is shaken every 2 min; addition of an appropriate amount of DMEM/F12 (containing 10% FBS) medium to terminate digestion; centrifuging at room temperature of 500g for 5min, and discarding the supernatant; adding a proper amount of culture medium, and beating by blowing to obtain a porcine small intestine organoid tissue suspension;
establishing a 2D model of the pig small intestine: and (3) connecting the prepared porcine small intestine organoid tissue suspension to the surface of the cell culture plate paved with the glue or adding the cell culture plate into the cell culture plate to form a porcine small intestine organoid 2D monolayer.
Preferably, the method for establishing the 2D model of the pig small intestine by adopting a Matrigel glue spreading method comprises the following steps:
(1) Diluting Matrigel and ice bath PBS according to the proportion of 1: 100 and then using the diluted Matrigel and ice bath PBS to coat a 48-hole cell culture plate;
(2) Adding 100 mu L of matrigel into each hole for coating the hole plate;
(3) Dropping 100 μ LPBS, and pressing on the Matrigel upper layer;
(4) Standing in a carbon dioxide cell incubator at 37 ℃ for 2h, and sucking out PBS after matrigel is solidified;
(5) Adding PBS, standing in a carbon dioxide incubator at 37 deg.C for 5min, and repeating for three times;
(6) Adding 100 mu LPBS into the hole, and standing in a carbon dioxide incubator at 37 ℃ for later use;
(7) Taking appropriate amount of prepared porcine small intestine organoid tissue suspension according to the ratio of about 10 4 And (4) attaching the cells to the surface of a 48-hole cell culture plate paved with the glue at the density of one cell/mL, standing and culturing in a cell culture box at 37 ℃, and finally converging to form 2D monolayer cells.
Preferably, the ratio of the Matrigel to ice-bath PBS is 1: 20.
Preferably, the 2D model of the small intestine of the pig is established by adopting a Transwell method, which specifically comprises the following steps: (1) The prepared porcine small intestine organoid tissue suspension is processed according to the 2 x 10 4 Pieces/well were added to nested Transwell nested 24-well cell culture plates; (2) Culturing in a cell carbon dioxide incubator for about 3 days, measuring resistance every 24h, changing the liquid, and observing the resistance change condition; and (3) culturing until 2D monolayer culture of the porcine small intestine organoid is formed.
Preferably, the method for establishing the 2D culture model of the pig small intestine by using the Transwell method and observing the cell structure by using a transmission electron microscope specifically comprises the following steps: a. washing the Transwell membrane inoculated with the porcine small intestine organoid and cultured for 3 days twice by PBS (phosphate buffer solution), cutting the Transwell membrane by a blade, placing the membrane in a glutaraldehyde solution (2.5 percent), and fixing the membrane at 4 ℃ overnight; b. removing the fixative, adding 0.1M phosphate buffer (pH7.0) to rinse the membrane, repeating for three times, each time for 15min; c. adding 1% osmate solution to fix the sample for 1-2h; d. aspirating osmate waste solution, adding sufficient 0.1M phosphate buffer (pH7.0) to rinse the membrane three times for 15min each time; e. sequentially immersing samples for 15min by preparing ethanol solutions with the concentrations of 30%,50%,70% and 80% respectively to dehydrate the samples, and then sequentially immersing the samples into 90% and 95% acetone solutions for 15min respectively; finally, the sample is placed in pure acetone for 20min, and the steps are repeated once; in the waiting process, in order to completely dehydrate the sample, the sample needs to be properly oscillated; f. soaking the sample in a mixed solution of Spurr embedding agent and acetone = 1: 1 (volume ratio) for 1h; g. soaking the sample in a mixed solution of Spurr embedding medium and acetone = 3: 1 (volume ratio) for 3 hours; h. placing the sample in a pure embedding medium, and standing overnight at room temperature; i. embedding, slicing and staining observation: embedding the sample subjected to the permeation treatment, heating at 70 ℃ overnight to obtain an embedded sample, slicing the sample in an LEICA EM UC7 type ultrathin slicer to obtain slices of 70-90nm, dyeing the slices for 5-10min by using lead citrate solution and 50% ethanol saturated solution of uranyl acetate respectively, and observing in a Hitachi H-7650 type transmission electron microscope.
Preferably, the method for obtaining a passable porcine small intestine organoid and/or the method for preparing a porcine small intestine organoid tissue suspension is applied to the research related to porcine intestinal organoids.
Preferably, the method for establishing the pig small intestine 2D culture model by adopting the Matrigel glue spreading method and/or the method for establishing the pig small intestine 2D culture model by adopting the Transwell method are applied to the related research of pig intestinal tract organoids.
The beneficial effect of this application: the application changes the method for obtaining the pig intestinal tract organoid monolayer, changes the traditional mechanical dissociation into the enzymatic dissociation and is beneficial to keeping the integrity of cells; 2D (two-dimensional) culture of cells is utilized to help reduce the influence of the interaction between injected substances and the apical membrane due to the obstruction of debris accumulated in the inner cavity during the 3D culture process; the 2D small intestine organoid model is utilized to enable the functional research of bioactive substances to better accord with the real physiological condition, provide simpler and more convenient operation for high-flux nutrient screening and bioactive substance function identification, and solve the problem that the intestinal cavity surface of a 3D intestinal organoid can not be in direct contact with a culture environment, so that the feasibility of the related research on the intestinal nutrition and the health is poor.
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The accompanying drawings are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application and not to limit the application. In the drawings:
FIG. 1 is a light microscope picture of a pig small intestine organoid plate for three days;
FIG. 2 shows the microscopic morphology of 2D monolayers of porcine small intestine organoids cultured for three days under observation by a transmission electron microscope.
Detailed Description
Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.
The invention relates to a method for obtaining passable porcine small intestine organoids, which comprises the following steps:
(1) Aseptically collecting the pig jejunum middle section about 5-10 cm, placing the pig jejunum middle section in precooled PBS (containing 1% triple antibiotics) and placing the pig jejunum middle section on ice to serve as an experimental intestine section I for standby application;
(2) Transferring the experimental intestinal section to a plate containing 10mL of PBS (containing 1% of triple antibiotics), removing connective tissues attached to the outer surface of the intestinal section, cleaning the inner cavity of the intestinal section by using a 10mL syringe, longitudinally peeling the intestinal section, scraping contents in the intestinal section by using a sterile glass slide, washing by using the PBS (containing 1% of triple antibiotics), and transferring to a clean bench;
(3) Transferring the intestinal section into a sterile centrifuge tube filled with 30mL PBS, reversely washing the intestinal section, sucking a supernatant after the intestinal tissue naturally settles, adding 30mL PBS again, and repeating the steps until the supernatant is clear;
(4) Transferring the intestinal section to a sterile culture dish containing 10mL PBS, slightly scraping the surface of the intestinal section by using a sterile glass slide to remove residual contents, and shearing the intestinal section to about 2mm as an experimental intestinal section II;
(5) Transferring the experimental intestinal section II into a sterile centrifuge tube filled with 30mL of PBS, and reversely washing the intestinal section until supernatant is clarified;
(6) Sucking the supernatant, transferring the second intestine section to 30mL of PBS solution containing 8mM EDTA, flatly placing on ice, shaking for 40min, removing the supernatant, adding a new 30mL of PBS solution containing 8mM EDTA, shaking again on ice for 40min, sucking the EDTA solution, shaking and cleaning with 30mL of PBS, placing on ice to settle the intestinal tissue blocks by gravity, removing the supernatant, and repeating the process for 1-2 times;
(7) Transferring the precipitated intestinal tissue mass to a new sterile 15mL centrifuge tube, resuspending in 5mL DMEM/F12 (containing 10% FBS,1% triple antibiotic) medium, counting the number of individual crypts by microscopic examination of 3-5. Mu.L;
(8) Centrifuging at 4 deg.C for 5min at 500g, discarding supernatant, mixing precipitate with Matrigel, and spotting into 48-well cell culture plate at 20 μ L/well;
(9) Transferring 48-well cell culture plate into carbon dioxide incubator, 37 deg.C, 5% 2 Incubating for 15min under the condition until Matrigel is solidified;
(10) After the Matrigel is solidified, adding preheated 200 muL/well complete culture medium according to the amount of 200 muL/well; placing the culture medium in an incubator at 37 ℃ for continuous culture, and replacing the fresh culture medium every 2-3 days; the generation was carried out every 5 to 7 days according to the growth.
A preparation method of a porcine small intestine organoid tissue suspension comprises the following steps:
(1) When the cultured porcine small intestine organoids grow to a passable state, removing the culture medium by suction, adding 500 mu L of precooled PBS into each hole, and incubating for 1min at room temperature;
(2) The liquid transfer gun sucks PBS to uniformly mix the small intestine organs, and the small intestine organs are transferred to a 15mL centrifuge tube;
(3) Adding 1mL of PBS to each hole for washing, and adding the PBS to the same 15mL centrifuge tube;
(4) Centrifuging at 4 deg.C for 5min at 200g, and removing supernatant;
(5) Adding proper amount of TrypL E according to the cell amount, and carrying out water bath at 37 ℃ for 5min, wherein the TrypL E is shaken every 2 min;
(6) Terminating the digestion by adding an appropriate amount of DMEM/F12 (containing 10% FBS) medium;
(7) Centrifuging at room temperature of 500g for 5min, and removing supernatant;
(8) Adding a proper amount of culture medium, and beating by blowing to obtain the porcine small intestine organoid tissue suspension.
The invention discloses two establishing methods of pig small intestine organoid 2D culture models, which respectively comprise the following steps:
matrigel spreading method:
(1) Diluting Matrigel and ice bath PBS according to the proportion of 1: 100 and then using the diluted Matrigel and ice bath PBS to coat a 48-hole cell culture plate;
(2) Adding 100 mu L of matrigel into each hole for coating the hole plate;
(3) Dropping 100 μ L PBS, pressing it on the Matrigel upper layer;
(4) Standing in a carbon dioxide cell incubator at 37 ℃ for 2h, and sucking out PBS after matrigel is solidified;
(5) Adding PBS, standing in a carbon dioxide incubator at 37 deg.C for 5min, and repeating for three times;
(6) Adding 100 μ L PBS into the well, and standing in a carbon dioxide incubator at 37 deg.C for use;
(7) Taking appropriate amount of prepared porcine small intestine organoid tissue suspension according to the ratio of about 10 4 The cells/mL are attached to the surface of a 48-well cell culture plate with the glue spread, and are statically cultured in a cell culture box at 37 ℃, and the cells can be converged into a 2D monolayer usually for about 72 hours.
Wherein the ratio of Matrigel to cold PBS is determined: the single cell suspension after passage of the porcine small intestine organoid is connected with three different Matrigel glue spreading ratios, which are respectively 1: 10, 1: 15 and 1: 20.
As can be seen from FIG. 1D, three days after the intestinal organoid tissue suspension was attached, the experimental group with glue spreading ratio of 1: 10 did not form a monolayer structure, but appeared a 3D organoid structure, indicating that the glue spreading ratio was too thick at 1: 10, resulting in cells tending to organoid 3D growth; the thickness of the glue spreading is proper in the experimental groups with the glue spreading ratio of 1: 15 and 1: 20, and after three days of culture, a monolayer epithelial-like structure can be formed, and as can be seen from fig. 1E and 1F, the monolayer confluence degree is higher than that of the glue spreading ratio of 1: 20 when the glue spreading ratio is 1: 15. Therefore, studies were performed using a 2D monolayer culture of porcine small intestine organoids confluent after three days of continuous culture using Matrigel to PBS in a ratio of 1: 20 for gel spreading.
Transwell method:
(1) The prepared porcine small intestine organoid tissue suspension is processed according to the 2 x 10 4 One/well was added to a Transwell nested 24-well cell culture plate;
(2) Placing the mixture in a cell culture box for culturing for about 3d, measuring the resistance once every 24h, changing the solution, and observing the change condition of the resistance value;
(3) And after the 2D monolayer culture of the porcine small intestine organoid is formed, subsequent identification and analysis can be carried out.
Adopting a transmission electron microscope to observe the structure of the 2D monolayer culture:
(1) washing the Transwell membrane inoculated with the porcine small intestine organoid and cultured for 3 days twice with PBS, cutting the Transwell membrane with a blade, placing the membrane in a glutaraldehyde solution (V/V = 2.5%), and fixing the membrane overnight at 4 ℃;
(2) removing the stationary liquid, adding 0.1M phosphate buffer (pH7.0) to rinse the membrane, repeating for three times, each time for 15min;
(3) samples were fixed for 1-2h by adding osmic acid solution (V/V = 1%);
(4) aspirating the osmic acid waste solution, adding sufficient 0.1M phosphate buffer (pH7.0) to rinse the sample three times, each time for 15min;
(5) the samples were dehydrated by sequentially immersing the samples for 15min in ethanol solutions having concentrations of 30%,50%,70%, and 80%, respectively. Then, sequentially immersing the sample into 90% and 95% acetone solutions, and respectively soaking for 15min; finally, the sample is placed in pure acetone for 20min, and the steps are repeated once; in the waiting process, in order to completely dehydrate the sample, the sample needs to be properly oscillated;
(6) soaking the sample in a mixed solution of Spurr embedding agent and acetone = 1: 1 (volume ratio) for 1h;
(7) then, placing the sample in a mixed solution of Spurr embedding medium and acetone = 3: 1 (volume ratio) for soaking for 3 hours;
(8) placing the sample in pure embedding medium, and standing overnight at room temperature;
(9) embedding, slicing and staining observation: embedding the sample subjected to the permeation treatment by using an embedding medium, and standing overnight at 70 ℃ to obtain the embedded sample.
Slicing the sample by an LEICA EM UC7 type ultrathin slicer to obtain 70-90nm slices, staining the slices with a lead citrate solution and a uranyl acetate 50% ethanol saturated solution for 5-10min respectively, and observing 2D monolayer cells formed by organoids in a Hitachi H-7650 type transmission electron microscope.
And (4) observing the results: microscopic morphology of the 2D monolayer cultures of porcine small intestine organoids after approximately three days of culture was observed using a transmission electron microscope and the results are shown in fig. 2. It can be clearly observed from the figure that the porcine small intestine organoid 2D monolayer has differentiated the intestinal villi and tight junctions, the part indicated by the arrows in the figure is the intercellular tight junctions, and the circled border is the intestinal villi structure.
It will be apparent to those skilled in the art that various changes and modifications may be made in the present application without departing from the spirit and scope of the application. Thus, if such modifications and variations of the present application fall within the scope of the claims of the present application and their equivalents, the present application is intended to include such modifications and variations as well.
Claims (8)
1. A method for establishing a pig small intestine organoid 2D culture model is characterized by comprising the following steps:
obtaining passable porcine small intestine organoids: aseptically collecting the pig jejunum from the middle section of the pig jejunum by about 5-10 cm, and clearing the pig jejunum twice until the supernatant is clear; sucking supernatant, transferring the intestinal segment into a solution containing 30mL 8mM EDTA-PBS, flatly placing on ice, shaking for 40min, removing the supernatant, adding a new 30mL 8mM EDTA-PBS solution, shaking again on ice for 40min, sucking out the EDTA solution, shaking and cleaning with 30mL PBS, placing on ice to allow the intestinal tissue blocks to settle by gravity, removing supernatant, and repeating the process for 1-2 times; transferring the pellet to a 15mL centrifuge tube, resuspending the pellet in 5mL DMEM/F12 (containing 10% FBS,1% Anti-Anti) medium, and counting the number of crypts by microscopic examination, taking 3-5. Mu.L; centrifuging at 4 deg.C for 5min at 500g, discarding supernatant, mixing the precipitate with Matrigel matrix, and spotting in 48-well plate at 20 μ L/well; transferring 48-well cell culture plates to a carbon dioxide incubator at 37 ℃ and 5% CO 2 Incubating for 15min under the condition until the Matrigel is solidified; adding preheated complete culture medium with 200 mu L/hole after the Matrigel is solidified; placing the culture medium in an incubator at 37 ℃ for continuous culture, and replacing the fresh culture medium every 2-3 days; carrying out passage according to growth conditions every 5-7 days;
preparing a porcine small intestine organoid tissue suspension: when the cultured pig organs grow to be passable, absorbing the culture medium, adding 500 mu L of precooled PBS into each hole, and incubating for 1min at room temperature; the pipette sucks PBS to uniformly mix the intestinal organs, and transfers the mixture into a 15mL centrifuge tube; adding 1mL of PBS to each hole for washing, and adding into the same 15mL centrifuge tube; centrifuging at 4 deg.C for 5min at 200g, and removing supernatant; adding proper amount of TrypL E according to the cell amount, and carrying out water bath at 37 ℃ for 5min, wherein the TrypL E is shaken every 2 min; terminating the digestion by adding an appropriate amount of DMEM/F12 (containing 10% FBS) medium; centrifuging at room temperature of 500g for 5min, and removing supernatant; adding a proper amount of culture medium, and beating by blowing to obtain a porcine small intestine organoid tissue suspension;
establishing a 2D model of the pig small intestine: and (3) connecting the prepared porcine small intestine organoid tissue suspension to the surface of the cell culture plate paved with the glue or adding the cell culture plate into the cell culture plate to form a porcine small intestine organoid 2D monolayer.
2. The method for establishing the 2D culture model of the porcine small intestine-like organ according to claim 1, wherein the establishment of the porcine small intestine 2D model by a Matrigel glue spreading method specifically comprises:
(1) Diluting Matrigel and ice bath PBS according to the proportion of 1: 100 and then using the diluted Matrigel and ice bath PBS to coat a 48-hole cell culture plate;
(2) Adding 100 mu L of matrigel into each hole for coating the hole plate;
(3) Dropping 100. Mu.L PBS, and pressing the PBS on the Matrigel upper layer;
(4) Standing in a carbon dioxide cell incubator at 37 ℃ for 2h, and sucking out PBS after matrigel is solidified;
(5) Adding PBS, standing in a carbon dioxide incubator at 37 deg.C for 5min, and repeating for three times;
(6) Adding 100 μ L PBS into the well, and standing in a carbon dioxide incubator at 37 deg.C for use;
(7) Taking appropriate amount of prepared porcine small intestine organoid tissue suspension according to the ratio of about 10 4 And (3) attaching the cells/mL to the surface of a 48-hole cell culture plate paved with the glue, standing and culturing the cells in a cell culture box at 37 ℃, and finally converging the cells into a 2D monolayer.
3. The method for establishing the 2D culture model of the porcine small intestine-like organ according to claim 2, wherein the ratio of the Matrigel to ice-bath PBS is 1: 20.
4. The method for establishing the 2D pig small intestine organoid culture model according to claim 1, wherein the establishment of the 2D pig small intestine model by the Transwell method specifically comprises:
(1) The prepared porcine small intestine organoid tissue suspension is processed according to the 2 x 10 4 Add one/well to a nested Transwell nested 24-well cell culture plate;
(2) Culturing in a cell carbon dioxide incubator for about 3 days, measuring the resistance every 24h, changing the liquid, and observing the change condition of the resistance value;
(3) Culturing until 2D monolayer culture of porcine small intestine organoid is formed.
5. The method for establishing the 2D pig small intestine organoid culture model according to claim 4, wherein the establishment of the 2D pig small intestine model by the Transwell method and observation of cell structure by a transmission electron microscope specifically comprises:
a. washing the Transwell membrane inoculated with the porcine small intestine organoid and cultured for 3 days twice by PBS (phosphate buffer solution), cutting the Transwell membrane by a blade, placing the membrane in a glutaraldehyde solution (2.5 percent), and fixing the membrane at 4 ℃ overnight;
b. removing the fixative, adding 0.1M phosphate buffer (pH7.0) to rinse the membrane, repeating for three times, each time for 15min;
c. adding 1% osmate solution to fix the sample for 1-2h;
d. aspirating osmate waste solution, adding sufficient 0.1M phosphate buffer (pH7.0) to rinse the membrane three times for 15min each time;
e. gradient eluting with 30%,50%,70%,80% ethanol solution for 15min, sequentially transferring to 90% and 95% acetone solutions, and soaking for 15min; finally, immersing the sample in pure acetone for 20min twice; in the waiting process, in order to completely dehydrate the sample, the sample needs to be oscillated;
f. soaking the sample in a mixed solution of Spurr embedding agent and acetone = 1: 1 (volume ratio) for 1h;
g. soaking the sample in a mixed solution of Spurr embedding agent and acetone = 3: 1 (volume ratio) for 3h;
h. placing the sample in a pure embedding medium, and standing overnight at room temperature;
i. embedding, slicing and staining observation: embedding the sample subjected to the permeation treatment, heating at 70 ℃ overnight to obtain an embedded sample, slicing the sample in an LEICA EM UC7 type ultrathin slicer to obtain slices of 70-90nm, dyeing the slices for 5-10min by using lead citrate solution and 50% ethanol saturated solution of uranyl acetate respectively, and observing in a Hitachi H-7650 type transmission electron microscope.
6. The method of claim 1, wherein the method of obtaining a passable porcine small intestine organoid and/or the method of preparing a porcine small intestine organoid tissue suspension is applied to studies relating to porcine small intestine organoids.
7. The method for establishing the 2D pig small intestine organoid culture model according to claim 2, wherein the method for establishing the 2D pig small intestine culture model by using the Matrigel spreading method is applied to the research on pig small intestine organoids.
8. The method for establishing the 2D pig small intestine organoid culture model according to claim 4 or 5, wherein the method for establishing the 2D pig small intestine organoid culture model by the Transwell method is applied to research on pig small intestine organoids.
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| CN116590235A (en) * | 2023-07-14 | 2023-08-15 | 北京嘉士腾医学检验实验室有限公司 | Efficient digestive tract tumor organoid culture method |
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| CN116590235A (en) * | 2023-07-14 | 2023-08-15 | 北京嘉士腾医学检验实验室有限公司 | Efficient digestive tract tumor organoid culture method |
| CN116590235B (en) * | 2023-07-14 | 2023-11-21 | 北京嘉士腾医学检验实验室有限公司 | Efficient digestive tract tumor organoid culture method |
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