CN112111445A - In-vitro monolayer culture and characterization method for constructing mouse intestinal epithelium by using Transwell - Google Patents
In-vitro monolayer culture and characterization method for constructing mouse intestinal epithelium by using Transwell Download PDFInfo
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Abstract
The invention discloses an in-vitro monolayer culture and characterization method for constructing mouse intestinal epithelium by using Transwell. The method comprises the following steps: (1) respectively inoculating intestinal crypt structures containing intestinal stem cells and intestinal epithelial layer sub-myofibroblasts on the upper and lower surfaces of a Transwell nested polycarbonate membrane, nesting the Transwell in a porous cell culture plate, adding an optimized culture medium into upper and lower chambers, and forming a single-layer intestinal epithelium after cocultivation; (2) regularly detecting the trans-epithelial resistance value of the single-layer intestinal epithelium every day in the culture process, representing the same cell composition and structural characteristics of the intestinal epithelium in vitro and in vivo by immunofluorescence and electron microscope results, and analyzing the permeability of the single-layer intestinal epithelium by using isothiocyanate labeled glucan as a tracing reagent. The invention co-cultures the intestinal crypts and intestinal epithelial and subcutaneous myofibroblasts for the first time to form an in-vitro intestinal epithelial monolayer culture model which has similar structural characteristics, cell composition and permeability with the intestinal epithelium in vivo.
Description
Technical Field
The invention relates to an in-vitro monolayer culture and characterization method for constructing mouse intestinal epithelium by using Transwell.
Background
The small intestine is the biggest nutrient absorption organ of human body, and the nutrient sensing, absorption and transportation at the intestinal layer are receiving increasing attention. At present, in vitro models for evaluating the functions of sensing, transporting and absorbing small intestine nutrients are mainly divided into two types: one is a model based on intestinal tissue, mainly based on the ews perfusion system; one is a model based on tumor cell lines, mainly uses Caco-2 or Caco-2 and HT29 combined culture and transmembrane analysis technology (Transwell) to generate polarized epithelial layer for in vitro epithelial cell response research. However, the in vitro intestinal function research models have certain defects, and the tumor cell line-based model cells are single in type, cannot truly reproduce the multi-cell composition characteristics of the intestinal tract, lack of interaction between cells and easily cause over-evaluation of absorption evaluation; the model based on the intestinal tissue has the problems of high technical requirement, large animal use amount, relatively poor experimental repeatability and stability and limited in-vitro test time. The discovery of intestinal stem cells and the establishment of an in-vitro 3D culture technology provide a new idea for solving the problems, but the application of the model in the aspect of nutrient absorption, transport and evaluation is greatly limited by the characteristics that the cell top end of the existing intestinal organoid cultured in vitro on the basis of the intestinal stem cells is located in a 3D structure.
Disclosure of Invention
In view of the above, the present invention aims to provide an in vitro monolayer culture and characterization method for constructing mouse intestinal epithelium by using Transwell. The method is different from the prior art, the intestinal crypts containing the intestinal stem cells and the trophoblast myofibroblasts are inoculated on different surfaces of a Transwell nest for co-culture to quickly form an in-vitro intestinal monolayer culture model, and further, the intestinal monolayer culture model obtained by the method has similar structural characteristics, cell composition and permeability of intestinal epithelium in vivo.
In order to achieve the purpose, the invention provides the following technical scheme:
an in vitro monolayer culture method for constructing mouse intestinal epithelium by using Transwell comprises the following steps:
step one, separating the intestinal crypt structure: collecting small intestine 7-8cm of ICR mouse with 8 weeks, and cleaning with 10ml injector containing phosphate buffer solution without calcium and magnesium ions precooled on ice until no residue is visible to naked eyes in the cleaning solution; cutting the intestinal tract along a longitudinal axis, putting the cut intestinal tract into a 100mm culture dish 1 which is placed on ice and contains phosphate buffer solution without calcium and magnesium ions, cutting the intestinal tract into intestinal sections of 0.5-1cm by using an ophthalmic scissors, transferring the cut intestinal sections into a 15ml centrifugal tube 1 containing 10ml of phosphate buffer solution without calcium and magnesium ions precooled on ice, turning the centrifugal tube 1 upside down for 10-20 times, then standing until the intestinal sections are settled at the bottom of the centrifugal tube 1, removing supernatant, and repeatedly cleaning for 3 times according to the method to remove the supernatant; transferring the washed tissue to a 50ml centrifuge tube 2 containing 20ml of 5mM EDTA solution; placing a 50ml centrifuge tube 2 in a 37 ℃ gas bath shaker at the rotating speed of 250rpm and oscillating for 10 minutes for digestion; after digestion, removing the EDTA solution in the centrifuge tube 2, adding the phosphate buffer solution without calcium and magnesium ions precooled on ice to wash the intestinal tissues for one time, removing the solution, adding 10ml of new phosphate buffer solution into the centrifuge tube 2, manually oscillating the centrifuge tube 2 up and down, standing until the intestinal section is settled at the bottom of the centrifuge tube 2, removing the supernatant in the centrifuge tube 2, adding 20ml of phosphate buffer solution without calcium and magnesium ions precooled on ice into the centrifuge tube 2, manually and forcefully oscillating the centrifuge tube for 240-50 times, and after standing still, transferring the supernatant into a new 50ml centrifuge tube 3 through a 100-micron-aperture cell sieve; centrifuging the centrifuge tube 3 at the rotation speed of 20g at 4 ℃ for 10 minutes, removing supernatant in the centrifuge tube 3 after centrifugation is finished, adding 1ml of culture medium 1 into the centrifuge tube 3, and then placing on ice for later use;
step two, composite gel treatment Transwell nesting: diluting the composite gel and the cold phosphate buffer solution without calcium and magnesium ions into a 1.5ml centrifuge tube 4 according to the volume ratio of 1:30, and placing on ice for later use; adding a diluent in a 100-microliter centrifuge tube 4 to the upper surface of a nest in a 24-culture plate 1 containing a Transwell, then placing the culture plate 1 in a carbon dioxide incubator at 37 ℃ for standing and incubating for 2h, and removing the solution on the upper surface of the nest of the culture plate 1 for later use after the incubation is finished;
step three, myofibroblast cell inoculation: the complex gel-treated Transwell nest was transferred from plate 1 to 6-well plate 2, inverted with the lower surface of the nest facing up, and then 100. mu.l was added at a concentration of 10ml5Inoculating the cell suspension of each cell on the lower surface of the nest in the culture plate 2, and placing the culture plate 2 in a carbon dioxide incubator at 37 ℃ for standing and incubating for 2 h; after the incubation is finished, the Transwell nest is transferred to a new 24-hole culture plate 3 in a re-upright mode, and then 500 ml of culture medium 2 is added into the nest lower chamber for later use;
step four, intestinal epithelium single culture in vitro: taking 200 microliters of the solution in the centrifuge tube 3 containing the intestinal crypt obtained in the step one, inoculating the solution in the nested upper chamber in the culture plate 3 inoculated with the myofibroblasts in the step three, then placing the culture plate 3 in a carbon dioxide incubator at 37 ℃ for culture, and replacing the culture media 1 and 2 in the upper chamber and the lower chamber of the Transwell nest once a day during the culture process.
Wherein, the culture medium 1 in the first step and the fourth step consists of Wint3a conditioned medium with the volume ratio of 50 percent, R-spondin conditioned medium with the volume ratio of 5 percent, DMEM/F12 medium, 4-hydroxyethyl piperazine ethanesulfonic acid, L-alanyl-L-glutamine dipeptide, epidermal growth factor, neuronal cell culture supplements N2 and B27, nicotinamide, Rho related protein forming serine/threonine kinase coiled coil inhibitor Y27632.
And in the second step, the composite gel consists of adhesive protein and collagen I.
And step three and step four, the culture medium 2 consists of a DMEM high-sugar culture medium, an unnecessary amino acid solution, penicillin and streptomycin.
The invention also provides an in vitro single-layer characterization method for constructing the mouse intestinal epithelium by using the Transwell, which comprises the following steps:
step one, measuring the transmembrane resistance of the intestinal epithelium in vitro monolayer culture: the resistance value of the transmembrane resistance between the upper and lower chambers of the Transwell insert in the plate 3 of step 4 of claim 1 was recorded by a daily timing using a resistance meter, and the calculation formula of the transmembrane resistance value was:
transmembrane resistance (intestinal monolayer culture-no intestinal monolayer culture control resistance) x membrane effective area;
step two, intestinal epithelium in vitro monolayer culture immunohistochemistry: the method of claim 1, step four, wherein the intestinal epithelium is cultured on the Transwell nest in plate 3 until day 4, the nested polycarbonate membrane is separated from the Transwell nest with a scalpel, and marked in the upper left corner of the membrane to distinguish the upper and lower surfaces of the polycarbonate membrane, and then the membrane is quickly placed in a 15ml centrifuge tube 5 containing 2ml of 4% paraformaldehyde solution for 15 minutes; removing the paraformaldehyde solution in the centrifuge tube 5, and then washing the membrane for three times with a phosphate buffer solution special for immunohistochemistry, wherein each time lasts for 5 minutes; after the cleaning is finished, adding 1ml of sealing liquid containing the primary antibody to be detected into the centrifugal tube 5, and standing for 12 hours in a refrigerator at 4 ℃; then, repeatedly washing for 3 times by using a phosphate buffer solution, wherein each time lasts for 5 minutes, adding a secondary antibody corresponding to the primary antibody source after washing is finished, and standing and incubating for 30 minutes at room temperature and 24 ℃; after incubation, the phosphate buffer solution is repeatedly washed for 4 times, each time for 5 minutes; after cleaning, adding 2- (4-amidinophenyl) -6-indoleamidine dihydrochloride solution, standing for 5 minutes at room temperature, and repeatedly cleaning twice with ultrapure water for 5 minutes each time; finally, transferring the nested polycarbonate membrane onto a glass slide with the upper surface of the membrane on the upper surface, adding a mounting solution, and then, performing cover slip mounting to obtain intestinal epithelium single-layer culture non-mounting sheets; observing the mounting plate by using a laser confocal microscope to obtain an immunohistochemical image;
step three, analyzing intestinal epithelium in vitro monolayer culture by electron microscopy: the Transwell nested polycarbonate membrane of claim 1, which contains monolayer culture of intestinal epithelium cultured to day 4-5 in culture plate 3, was separated from the Transwell nest with a scalpel, and was divided into two equal parts, and the upper left corner of the membrane was marked to distinguish the upper and lower surfaces; placing the separated nested membranes in 1.5ml centrifuge tubes 6 and 7 containing 1ml of glutaraldehyde respectively, and standing overnight at 4 ℃; removing the fixing solution, washing the sample with 0.1M (pH7.0) phosphate buffer solution for 15min for 3 times, and fixing the sample with 1% osmic acid solution for 1-2h after the washing; cleaning the sample for 3 times, dehydrating the sample by using ethanol solutions with six concentrations of 30%, 50%, 70%, 80%, 90% and 95%, treating each concentration for 15min, treating the sample by using 100% ethanol for one time for 20min, finally replacing the sample by using new 100% ethanol, and placing the sample in the 100% ethanol for later use; then, drying and coating the sample in the centrifuge tube 6, and observing the surface characteristics of the intestinal monolayer culture by using a scanning electron microscope; after a sample in the centrifuge tube 7 is treated overnight at 70 ℃ by a Spurr embedding medium, acetone mixed solution and pure embedding medium, a single-layer section of intestinal epithelium with the thickness of 70-90 nanometers is obtained by using a slicing machine, the section is respectively dyed by 5-10 parts by lead citrate solution and 50% ethanol saturated solution of uranyl acetate, and an image is obtained by transmission electron microscope observation;
step four, analyzing the permeability of the intestinal epithelium in vitro monolayer culture: detecting the permeability of intestinal monolayer culture by using isothiocyanate-labeled dextran with the molecular weight of 4.4kDa as a tracer, and washing Transwell nested polycarbonate membranes containing intestinal epithelial monolayer culture cultured to the day 4-5 in the four-layer culture plate 3 in the step of claim 1 by using a pink-free DMEM medium; then transferred to a new multi-well plate 4, 200 microliters of 5 milligrams per milliliter dextran solution at a concentration of 5 milligrams per milliliter was added to the nested upper chamber, 600 microliters of pink-free DMEM solution was added to the lower chamber, the entire process of the plate 4 was placed at 37 ℃, and 100 microliters of analytical sample was collected from the lower chamber into a new centrifuge tube 8 using a pipette gun at fixed time points, respectively, and the solution was replenished with the corresponding volume of phenol red-free DMEM medium; fluorescence in the sample of centrifuge tube 8 was then measured using a microplate reader at 495nm excitation and 520nm emission wavelengths and quantitatively calculated from a pre-established calibration curve of known concentration, using the formula:
the transmembrane transport of dextran equals the amount of dextran in the lower nested chamber/the initial amount of dextran in the upper nested chamber x 100%.
The invention has the following advantages and beneficial effects:
compared with the prior art methods, the method provided by the invention has the following remarkable advantages and progresses:
1. the technical method of the invention improves the existing intestinal crypt separation method, shortens the exposure time of the intestinal crypt digestive solution, is beneficial to improving the activity of intestinal stem cells, and can ensure that the monolayer culture of the small intestine in vitro is successfully completed.
2. The technical method of the invention co-cultures intestinal crypts containing intestinal stem cells and myofibroblasts, and overcomes the problems of slow formation speed of intestinal epithelial monolayers and low transmembrane resistance of intestinal epithelia in the prior art through the interaction of the myofibroblasts and the intestinal epithelial cells.
3. The technical method of the invention also optimizes the in-vitro identification method of the intestinal tract in-vitro monolayer culture model, and can determine the reliability of the in-vitro application of the intestinal tract in-vitro monolayer culture model.
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FIG. 1 shows the transmembrane resistance (A), permeability (B), immunohistochemistry (C) and electron microscopy identification of intestinal monolayer culture in vitro (D and E).
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The invention discloses a method for constructing mouse intestinal epithelium in vitro monolayer culture and characterization by using Transwell, and a person skilled in the art can realize the culture by using the contents in the text and properly improving parameters. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the invention without departing from the spirit and scope of the invention.
In order to better illustrate the invention, the following examples are given.
Example 1:
step one, separating the intestinal crypt structure: collecting small intestine 7-8cm of ICR mouse with 8 weeks, and cleaning with 10ml injector containing phosphate buffer solution without calcium and magnesium ions precooled on ice until no residue is visible to naked eyes in the cleaning solution; cutting the intestinal tract along a longitudinal axis, putting the cut intestinal tract into a 100mm culture dish 1 which is placed on ice and contains phosphate buffer solution without calcium and magnesium ions, cutting the intestinal tract into intestinal sections of 0.5-1cm by using an ophthalmic scissors, transferring the cut intestinal sections into a 15ml centrifugal tube 1 containing 10ml of phosphate buffer solution without calcium and magnesium ions precooled on ice, turning the centrifugal tube 1 upside down for 10-20 times, then standing until the intestinal sections are settled at the bottom of the centrifugal tube 1, removing supernatant, and repeatedly cleaning for 3 times according to the method to remove the supernatant; transferring the washed tissue to a 50ml centrifuge tube 2 containing 20ml of 5mM EDTA solution; placing a 50ml centrifuge tube 2 in a 37 ℃ gas bath shaker at the rotating speed of 250rpm and oscillating for 10 minutes for digestion; after digestion, removing the EDTA solution in the centrifuge tube 2, adding the phosphate buffer solution without calcium and magnesium ions precooled on ice to wash the intestinal tissues for one time, removing the solution, adding 10ml of new phosphate buffer solution into the centrifuge tube 2, manually oscillating the centrifuge tube 2 up and down, standing until the intestinal section is settled at the bottom of the centrifuge tube 2, removing the supernatant in the centrifuge tube 2, adding 20ml of phosphate buffer solution without calcium and magnesium ions precooled on ice into the centrifuge tube 2, manually and forcefully oscillating the centrifuge tube for 240-50 times, and after standing still, transferring the supernatant into a new 50ml centrifuge tube 3 through a 100-micron-aperture cell sieve; centrifuge tube 3 was centrifuged at 4 ℃ for 10 minutes at 20g, the supernatant in centrifuge tube 3 was removed after centrifugation was completed, and 1ml of medium 1 was added to centrifuge tube 3, followed by placing on ice for future use.
Step two, composite gel treatment Transwell nesting: diluting the composite gel and the cold phosphate buffer solution without calcium and magnesium ions into a 1.5ml centrifuge tube 4 according to the volume ratio of 1:30, and placing on ice for later use; adding a diluent in a 100 microliter centrifuge tube 4 to the upper surface of the nest in the 24 culture plate 1 containing the Transwell, then placing the culture plate 1 in a carbon dioxide incubator at 37 ℃ for standing incubation for 2h, and removing the solution on the upper surface of the nest of the culture plate 1 for standby after the incubation is finished.
Step three, myofibroblast cell inoculation: transferring the Transwell nest treated by the composite gel from the culture plate 1 to a 6-hole culture plate 2, turning and inverting the Transwell nest with the lower surface of the nest upward, then inoculating 100 microliters of cell suspension with a concentration of 105 cells per milliliter to the lower surface of the nest in the culture plate 2, and placing the culture plate 2 in a carbon dioxide incubator at 37 ℃ for standing and incubating for 2 hours; after incubation was complete the Transwell nest was re-transferred upright to a new 24 well plate 3 and 500 ml of medium 2 was added to the nest lower chamber for future use.
Step four, intestinal epithelium single culture in vitro: taking 200 microliters of the solution in the centrifuge tube 3 containing the intestinal crypts obtained in the step one, and inoculating the solution in the nested upper chamber in the culture plate 3 inoculated with the myofibroblasts in the step three; the plate 3 was then incubated in a 37 ℃ carbon dioxide incubator. The Transwell nested upper and lower chamber media 1 and 2 were changed once a day during the culture.
Step five, measuring the transmembrane resistance of the intestinal epithelium in vitro monolayer culture: the resistance value of the transmembrane between the upper and lower chambers of the Transwell insert in the plate 3 of claim 4 was recorded periodically on a daily basis using a resistance meter. The transmembrane resistance value is calculated by the formula:
transmembrane resistance value (number of intestinal monolayer containing cultures-no intestinal monolayer cultures control number) x membrane effective area.
FIG. 1A is a cross-membrane resistance measurement result during in vitro co-culture based on jejunal crypt and subcutaneous myofibroblast, from which it can be seen that with the increase of culture time, the in vitro monolayer culture transmembrane resistance of intestinal epithelium gradually increases, the transmembrane resistance of intestinal epithelium in the third day of culture basically reaches the in vivo mouse intestinal epithelium transmembrane resistance range of 40-100 Ω cm2, and then the resistance continuously rises to enter the resistance stabilization phase in the fourth day of culture, and the result proves that stable tight connection is formed between cells constituting the in vitro culture of intestinal tract after the fourth day of culture, and the functional maturation can be analyzed by subsequent functions.
Step six, culturing the intestinal epithelium on the Transwell nest in the culture plate 3 in the step four of claim 1 to the 4 th day, separating the nested polycarbonate membrane from the Transwell nest by using a scalpel, marking the upper left corner of the membrane for distinguishing the upper surface and the lower surface of the polycarbonate membrane, and then quickly placing the membrane into a 15ml centrifuge tube 5 containing 2ml of paraformaldehyde solution with the concentration of 4% for fixing for 15 minutes; removing the paraformaldehyde solution in the centrifuge tube 5, and then washing the membrane for three times with a phosphate buffer solution special for immunohistochemistry, wherein each time lasts for 5 minutes; after the cleaning is finished, adding 1ml of sealing liquid containing the primary antibody to be detected into the centrifugal tube 5, and standing for 12 hours in a refrigerator at 4 ℃; then, repeatedly washing for 3 times by using a phosphate buffer solution, wherein each time lasts for 5 minutes, adding a secondary antibody corresponding to the primary antibody source after washing is finished, and standing and incubating for 30 minutes at room temperature and 24 ℃; after incubation, the phosphate buffer solution is repeatedly washed for 4 times, each time for 5 minutes; after cleaning, adding 2- (4-amidinophenyl) -6-indoleamidine dihydrochloride solution, standing for 5 minutes at room temperature, and repeatedly cleaning twice with ultrapure water for 5 minutes each time; finally, transferring the nested polycarbonate membrane onto a glass slide with the upper surface of the membrane on the upper surface, adding a mounting solution, and then, performing cover slip mounting to obtain intestinal epithelium single-layer culture non-mounting sheets; the mounting was observed using a confocal laser microscope to obtain immunohistochemical images.
FIG. 1C shows immunohistochemistry results of intestinal epithelial monolayer cultures, using ZO-1 as a tight junction marker protein formed between intestinal epithelial cells, where white bright linear structures (as indicated by the various cuts in the figure) between cells are positive signals for the tight junction protein ZO-1, indicating that four days of in vitro intestinal monolayer culture formed a structural basis for maintaining the mechanical barrier and permeability of intestinal mucosal epithelium.
Seventhly, analyzing the intestinal epithelium in-vitro monolayer culture by an electron microscope: the culture plate 3 in the fourth step contains a Transwell nested polycarbonate membrane which is cultured to intestinal epithelial monolayer culture on days 4-5, the Transwell nested polycarbonate membrane is separated from the Transwell nest by a scalpel and then evenly divided into two parts, and marks are used for distinguishing the upper surface and the lower surface of the membrane at the upper left corner; placing the separated nested membranes in 1.5ml centrifuge tubes 6 and 7 containing 1ml of glutaraldehyde respectively, and standing overnight at 4 ℃; removing the fixing solution, washing the sample with 0.1M (pH7.0) phosphate buffer solution for 15min for 3 times, and fixing the sample with 1% osmic acid solution for 1-2h after the washing; cleaning the sample for 3 times, dehydrating the sample by using ethanol solutions with six concentrations of 30%, 50%, 70%, 80%, 90% and 95%, treating each concentration for 15min, treating the sample by using 100% ethanol for one time for 20min, finally replacing the sample by using new 100% ethanol, and placing the sample in the 100% ethanol for later use; then, drying and coating the sample in the centrifuge tube 6, and observing the surface characteristics of the intestinal monolayer culture by using a scanning electron microscope; after a sample in the centrifuge tube 7 is treated overnight at 70 ℃ by a Spurr embedding medium, acetone mixed solution and pure embedding medium, a single-layer section of intestinal epithelium with the thickness of 70-90 nanometers is obtained by using a slicing machine, the section is respectively dyed by 5-10 parts by lead citrate solution and 50% ethanol saturated solution of uranyl acetate, and an image is obtained by transmission electron microscope observation.
Fig. 1D and E are results of a scanning electron microscope and a transmission electron microscope for monolayer culture of intestinal epithelium, respectively, and the results confirm that the intestinal epithelium in vitro culture surface forms a microvilli structure identical to that of the intestinal epithelium in vivo, forms polarized epithelial cells, and can be used as a substitute for the intestinal epithelium in vivo for functional analysis.
Step eight, analyzing the permeability of the intestinal epithelium in vitro monolayer culture: detecting the permeability of intestinal monolayer culture by using isothiocyanate-labeled dextran with the molecular weight of 4.4kDa as a tracer, and washing Transwell nested polycarbonate membranes containing intestinal epithelial monolayer culture cultured to the day 4-5 in the four-layer culture plate 3 in the step of claim 1 by using a pink-free DMEM medium; then transferred to a new multi-well plate 4, 200 microliters of 5 milligrams per milliliter dextran solution at a concentration of 5 milligrams per milliliter was added to the nested upper chamber, 600 microliters of pink-free DMEM solution was added to the lower chamber, the entire process of the plate 4 was placed at 37 ℃, and 100 microliters of analytical sample was collected from the lower chamber into a new centrifuge tube 8 using a pipette gun at fixed time points, respectively, and the solution was replenished with the corresponding volume of phenol red-free DMEM medium; fluorescence in the sample of centrifuge tube 8 was then measured using a microplate reader at 495nm excitation and 520nm emission wavelengths and quantitatively calculated from a pre-established calibration curve of known concentration, using the formula:
the transmembrane transport of dextran equals the amount of dextran in the lower nested chamber/the initial amount of dextran in the upper nested chamber x 100%.
Fig. 1B is a result of intestinal epithelium in vitro monolayer culture permeability analysis, and compared with the control group without cell inoculation and with only several myofibroblasts, intestinal in vitro monolayer culture has significantly lower permeability, indicating that intestinal in vitro monolayer forms complete barrier function and permeability after 4-5 days of culture.
Therefore, the steps and the results show that the method for constructing the mouse intestinal epithelium in vitro monolayer culture and characterization by using the Transwell is stable, reliable and effective.
The foregoing is directed to embodiments of the present invention and it is understood that various modifications and enhancements may be made by those skilled in the art without departing from the principles of the invention, which should be considered as within the scope of the invention.
Claims (5)
1. An in vitro monolayer culture method for constructing mouse intestinal epithelium by using Transwell is characterized by comprising the following steps:
step one, separating the intestinal crypt structure: collecting small intestine 7-8cm of ICR mouse with 8 weeks, and cleaning with 10ml injector containing phosphate buffer solution without calcium and magnesium ions precooled on ice until no residue is visible to naked eyes in the cleaning solution; cutting the intestinal tract along a longitudinal axis, putting the cut intestinal tract into a 100mm culture dish 1 which is placed on ice and contains phosphate buffer solution without calcium and magnesium ions, cutting the intestinal tract into intestinal sections of 0.5-1cm by using an ophthalmic scissors, transferring the cut intestinal sections into a 15ml centrifugal tube 1 containing 10ml of phosphate buffer solution without calcium and magnesium ions precooled on ice, turning the centrifugal tube 1 upside down for 10-20 times, then standing until the intestinal sections are settled at the bottom of the centrifugal tube 1, removing supernatant, and repeatedly cleaning for 3 times according to the method to remove the supernatant; transferring the washed tissue to a 50ml centrifuge tube 2 containing 20ml of 5mM EDTA solution; placing a 50ml centrifuge tube 2 in a 37 ℃ gas bath shaker at the rotating speed of 250rpm and oscillating for 10 minutes for digestion; after digestion, removing the EDTA solution in the centrifuge tube 2, adding the phosphate buffer solution without calcium and magnesium ions precooled on ice to wash the intestinal tissues for one time, removing the solution, adding 10ml of new phosphate buffer solution into the centrifuge tube 2, manually oscillating the centrifuge tube 2 up and down, standing until the intestinal section is settled at the bottom of the centrifuge tube 2, removing the supernatant in the centrifuge tube 2, adding 20ml of phosphate buffer solution without calcium and magnesium ions precooled on ice into the centrifuge tube 2, manually and forcefully oscillating the centrifuge tube for 240-50 times, and after standing still, transferring the supernatant into a new 50ml centrifuge tube 3 through a 100-micron-aperture cell sieve; centrifuging the centrifuge tube 3 at the rotation speed of 20g at 4 ℃ for 10 minutes, removing supernatant in the centrifuge tube 3 after centrifugation is finished, adding 1ml of culture medium 1 into the centrifuge tube 3, and then placing on ice for later use;
step two, composite gel treatment Transwell nesting: diluting the composite gel and the cold phosphate buffer solution without calcium and magnesium ions into a 1.5ml centrifuge tube 4 according to the volume ratio of 1:30, and placing on ice for later use; adding a diluent in a 100-microliter centrifuge tube 4 to the upper surface of a nest in a 24-culture plate 1 containing a Transwell, then placing the culture plate 1 in a carbon dioxide incubator at 37 ℃ for standing and incubating for 2h, and removing the solution on the upper surface of the nest of the culture plate 1 for later use after the incubation is finished;
step three, myofibroblast cell inoculation: the complex gel-treated Transwell nest was transferred from plate 1 to 6-well plate 2, inverted with the lower surface of the nest facing up, and then 100. mu.l was added at a concentration of 10ml5Inoculating the cell suspension of each cell on the lower surface of the nest in the culture plate 2, and placing the culture plate 2 in a carbon dioxide incubator at 37 ℃ for standing and incubating for 2 h; after the incubation is finished, the Transwell nest is transferred to a new 24-hole culture plate 3 in a re-upright mode, and then 500 ml of culture medium 2 is added into the nest lower chamber for later use;
step four, intestinal epithelium single culture in vitro: taking 200 microliters of the solution in the centrifuge tube 3 containing the intestinal crypt obtained in the step one, inoculating the solution in the nested upper chamber in the culture plate 3 inoculated with the myofibroblasts in the step three, then placing the culture plate 3 in a carbon dioxide incubator at 37 ℃ for culture, and replacing the culture media 1 and 2 in the upper chamber and the lower chamber of the Transwell nest once a day during the culture process.
2. The method according to claim 1, wherein the medium 1 in the first and fourth steps consists of Wint3a conditioned medium at a volume ratio of 50%, R-spondin conditioned medium at a volume ratio of 5%, DMEM/F12 medium, 4-hydroxyethylpiperazine ethanesulfonic acid, L-alanyl-L-glutamine dipeptide, epidermal growth factor, neuronal cell culture supplements N2 and B27, nicotinamide, Rho-associated protein-forming serine/threonine kinase coiled-coil inhibitor Y27632.
3. The method according to claim 1, wherein the complex gel in step two is composed of fibronectin and collagen I.
4. The method as set forth in claim 1, wherein said medium 2 in the third and fourth steps consists of DMEM high sugar medium, non-essential amino acid solution, penicillin, streptomycin.
5. An in vitro monolayer characterization method for constructing mouse intestinal epithelium by using Transwell, which is characterized by comprising the following steps:
step one, measuring the transmembrane resistance of the intestinal epithelium in vitro monolayer culture: the resistance value of the transmembrane resistance between the upper and lower chambers of the Transwell insert in the plate 3 of step 4 of claim 1 was recorded by a daily timing using a resistance meter, and the calculation formula of the transmembrane resistance value was:
transmembrane resistance (intestinal monolayer culture-no intestinal monolayer culture control resistance) x membrane effective area;
step two, intestinal epithelium in vitro monolayer culture immunohistochemistry: the method of claim 1, step four, wherein the intestinal epithelium is cultured on the Transwell nest in plate 3 until day 4, the nested polycarbonate membrane is separated from the Transwell nest with a scalpel, and marked in the upper left corner of the membrane to distinguish the upper and lower surfaces of the polycarbonate membrane, and then the membrane is quickly placed in a 15ml centrifuge tube 5 containing 2ml of 4% paraformaldehyde solution for 15 minutes; removing the paraformaldehyde solution in the centrifuge tube 5, and then washing the membrane for three times with a phosphate buffer solution special for immunohistochemistry, wherein each time lasts for 5 minutes; after the cleaning is finished, adding 1ml of sealing liquid containing the primary antibody to be detected into the centrifugal tube 5, and standing for 12 hours in a refrigerator at 4 ℃; then, repeatedly washing for 3 times by using a phosphate buffer solution, wherein each time lasts for 5 minutes, adding a secondary antibody corresponding to the primary antibody source after washing is finished, and standing and incubating for 30 minutes at room temperature and 24 ℃; after incubation, the phosphate buffer solution is repeatedly washed for 4 times, each time for 5 minutes; after cleaning, adding 2- (4-amidinophenyl) -6-indoleamidine dihydrochloride solution, standing for 5 minutes at room temperature, and repeatedly cleaning twice with ultrapure water for 5 minutes each time; finally, transferring the nested polycarbonate membrane onto a glass slide with the upper surface of the membrane on the upper surface, adding a mounting solution, and then, performing cover slip mounting to obtain intestinal epithelium single-layer culture non-mounting sheets; observing the mounting plate by using a laser confocal microscope to obtain an immunohistochemical image;
step three, analyzing intestinal epithelium in vitro monolayer culture by electron microscopy: the Transwell nested polycarbonate membrane of claim 1, which contains monolayer culture of intestinal epithelium cultured to day 4-5 in culture plate 3, was separated from the Transwell nest with a scalpel, and was divided into two equal parts, and the upper left corner of the membrane was marked to distinguish the upper and lower surfaces; placing the separated nested membranes in 1.5ml centrifuge tubes 6 and 7 containing 1ml of glutaraldehyde respectively, and standing overnight at 4 ℃; removing the fixing solution, washing the sample with 0.1M (pH7.0) phosphate buffer solution for 15min for 3 times, and fixing the sample with 1% osmic acid solution for 1-2h after the washing; cleaning the sample for 3 times, dehydrating the sample by using ethanol solutions with six concentrations of 30%, 50%, 70%, 80%, 90% and 95%, treating each concentration for 15min, treating the sample by using 100% ethanol for one time for 20min, finally replacing the sample by using new 100% ethanol, and placing the sample in the 100% ethanol for later use; then, drying and coating the sample in the centrifuge tube 6, and observing the surface characteristics of the intestinal monolayer culture by using a scanning electron microscope; after a sample in the centrifuge tube 7 is treated overnight at 70 ℃ by a Spurr embedding medium, acetone mixed solution and pure embedding medium, a single-layer section of intestinal epithelium with the thickness of 70-90 nanometers is obtained by using a slicing machine, the section is respectively dyed by 5-10 parts by lead citrate solution and 50% ethanol saturated solution of uranyl acetate, and an image is obtained by transmission electron microscope observation;
step four, analyzing the permeability of the intestinal epithelium in vitro monolayer culture: detecting the permeability of intestinal monolayer culture by using isothiocyanate-labeled dextran with the molecular weight of 4.4kDa as a tracer, and washing Transwell nested polycarbonate membranes containing intestinal epithelial monolayer culture cultured to the day 4-5 in the four-layer culture plate 3 in the step of claim 1 by using a pink-free DMEM medium; then transferred to a new multi-well plate 4, 200 microliters of 5 milligrams per milliliter dextran solution at a concentration of 5 milligrams per milliliter was added to the nested upper chamber, 600 microliters of pink-free DMEM solution was added to the lower chamber, the entire process of the plate 4 was placed at 37 ℃, and 100 microliters of analytical sample was collected from the lower chamber into a new centrifuge tube 8 using a pipette gun at fixed time points, respectively, and the solution was replenished with the corresponding volume of phenol red-free DMEM medium; fluorescence in the sample of centrifuge tube 8 was then measured using a microplate reader at 495nm excitation and 520nm emission wavelengths and quantitatively calculated from a pre-established calibration curve of known concentration, using the formula:
the transmembrane transport of dextran equals the amount of dextran in the lower nested chamber/the initial amount of dextran in the upper nested chamber x 100%.
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