CN109750065B - Mouse intestinal epithelial crypt cell line and construction and culture method thereof - Google Patents
Mouse intestinal epithelial crypt cell line and construction and culture method thereof Download PDFInfo
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Abstract
The invention belongs to the technical field of molecular and cell biology, and particularly relates to a method for constructing a mouse small intestine epithelial crypt cell line. The cell line well maintains the morphology of the intestinal epithelial primary cells and has vigorous proliferation capacity. It can form characteristic organoid structure under three-dimensional culture condition, and specific staining indicates that the cells composing the structure comprise various intestinal epithelial differentiated filial generations. The crypt cells after being established can only grow under the primary culture condition, can be applied to establishing a mouse small intestine epithelial in vitro multidimensional research model, is used for researching the physiological functions of the intestinal tract and the pathological mechanisms including infection, cancer occurrence and the like, and provides a tool and a platform for the treatment target of intestinal diseases including intestinal cancer and the screening and evaluation of drugs.
Description
Technical Field
The invention belongs to the technical field of molecular and cellular biology, and particularly relates to a mouse intestinal epithelial crypt cell line and a construction and culture method thereof.
Background
Intestinal epithelial cells are one of the cell types with the fastest in vivo renewal speed, crypt cells provide a continuous cell supply for the renewal, cells leaving crypts along a basement membrane are gradually differentiated into different types of mature epithelial cells, such as absorptive epithelial cells, goblet cells, Pan cells, M cells and the like, and the daughter cells play different functions to jointly maintain the physiological function of an intestinal epithelial system. The intestinal epithelial system is an elegant model for studying stem cell biology and cell lineage development, and is the simplest model for studying mammalian tissue renewal, pluripotent stem cells, binary lineage determination, and programmed cell death. Therefore, a large number of intestinal epithelial crypt cell lines are required as research tools during the course of the study.
Most of primary cells for commercialization or research have the problem of limited passage times, and the cells of different passage times have great characteristic difference, so that the repeatability of the research result based on the cells is poor. To solve this problem, it is necessary to immortalize a certain cell type and construct a corresponding immortalized cell line.
Disclosure of Invention
In order to solve the problems, the inventor tries to construct an immortalized cell line of the crypt cell, firstly tries to culture the crypt cell in vitro, and because the intestinal epithelial structure comprises a plurality of daughter cells such as epithelial cells, goblet cells, Pan cells and M cells, and the crypt cell is positioned at the bottom of the crypt, the complete, pollution-free and considerable number of crypt structures are often difficult to obtain when the crypt cells are separated by the traditional separation method. Even if a certain amount of crypt cells are obtained through a large amount of separation work, primary cells can not grow adherent to the wall and grow slowly or can not grow when the traditional culture medium is adopted for in vitro culture.
Through the long-term research on the crypt structure of the inventor, two buffer solutions without enzyme are selected to separate the crypt cells, the pollution of interstitial cells and other filial cells is avoided, a large number of crypt structures without pollution are obtained, and when the pollution-free crypt cells are cultured by adopting a traditional culture medium, the crypt cells grow slowly or cannot grow. The inventors further studied the medium, and finally prepared a serum-free medium (ENR medium) mainly characterized by the combination of EGF (heel) WNT (heel) BMP (↓) molecular signals, and cultured primary crypt cells, in which the primary epithelial crypt cells could grow adherent to the wall, the growth rate was high, and the crypt cell phenotype was maintained.
After the in vitro culture of the crypt cells is completed, the inventor tries the immortalization of primary cells, after a plurality of immortalization systems are tried, the immortalization system in CN105002217A is selected to transfect the crypt cells to obtain the immortalized crypt cells, then the immortalized crypt cells are subcultured by adopting the serum-free culture medium, the original characteristics of the crypt cells can still be kept after the number of passages reaches 20, meanwhile, the dependence on the signal combination is also kept in the cells after the establishment of the lineages, the multidirectional differentiation potential of the crypt cells is kept, and the conditional immortalization of the crypt cells is realized.
The technical scheme of the invention is as follows:
a method for constructing a mouse intestinal epithelial crypt cell line (iMICs) comprises sequentially separating and removing non-crypt epithelium with buffer solution I and buffer solution II to obtain crypt cells; transfecting the crypt cells by using a plasmid vector containing an SV40TAg expression element and a piggyBac transposase shearing site, and infecting by using adenovirus or plasmid vector expressing piggyBac transposase to obtain immortalized crypt cells; culturing the immortalized crypt cells by using an ENR culture medium, screening and passaging to obtain an intestinal epithelial crypt cell line; the buffer solution I is phosphate buffer salt solution of EDTA, and the buffer solution II is phosphate buffer salt solution of D-sorbitol and sucrose.
Preferably, the concentration of EDTA in the buffer solution I is 0.5-4 mmol/L, the concentration of D-sorbitol in the buffer solution II is 40-70mmol/L, and the concentration of sucrose is 30-60mmol/L, more preferably, the concentration of EDTA in the buffer solution I is 2mmol/L, the concentration of D-sorbitol in the buffer solution II is 54.9 m mmol/L, and the concentration of sucrose is 43.4 mmol/L.
Preferably, the ENR culture medium is a serum-free culture medium based on Advanced DMEM/F12, and comprises epidermal growth factors EGF, Noggin and R-Spondin 2.
Preferably, in the ENR culture medium, the concentration of the epidermal growth factor EGF is 30-80ng/mL, the concentration of the Noggin protein is 80-120ng/mL, and the volume ratio of the conditioned medium of R-Spondin2 to the Advanced DMEM/F12 culture medium is 08-1.2: 8-12.
More preferably, the construction method comprises the following specific steps:
(1) isolation of intestinal epithelial crypt cells: cleaning the middle-lower part of jejunum and the middle-upper part of ileum of a mouse, shearing intestinal canal tissues, transferring the intestinal canal tissues into a separation buffer solution I, turning and shaking the intestinal canal tissues, sucking the buffer solution I out, adding a buffer solution II, violently shaking the intestinal canal tissues, filtering the intestinal canal tissues, removing non-crypt epithelium, centrifugally collecting crypt cells from filtrate, digesting the crypt cells by pancreatin to obtain a single cell suspension, terminating digestion, and centrifugally collecting the crypt cells;
(2) construction of intestinal epithelial crypt cell line iMICs: and (2) resuspending the single-cell suspension finally collected in the step (1) by using an ENR culture medium, inoculating, transfecting a plasmid vector containing an SV40TAg expression element and a piggyBac transposase shearing site, infecting by using an adenovirus expressing the piggyBac transposase within 24 hours, replacing a new ENR culture medium, and carrying out drug screening and continuous passage to obtain the intestinal epithelial crypt cell line.
In the construction method, the crypt cells are effectively separated by adopting a specific buffer solution, and an immortalized intestinal epithelial crypt cell line is established by combining an immortalized system based on the piggyBac principle and a specific ENR culture medium, wherein the cell line has multidirectional differentiation potential, specifically, a single iMIC cell can form a unique Organoid (Organoid) structure under a three-dimensional culture condition and consists of a plurality of differentiated intestinal epithelial filial generations; the cells after the establishment of the system also keep the dependence on an ENR culture medium, and can be completely treated according to the conventional cell culture method, so that the operation of any gene level can be conveniently carried out on the cells, including plasmid transfection, virus infection and the like, and the cells are certainly a very practical tool in the field of intestinal epithelium; in addition, the same ENR culture medium is also adopted by the three-dimensional culture condition of a single iMIC cell, so that the switching between two-dimensional culture and three-dimensional culture is very convenient, and a researcher can observe multi-dimensional information.
Therefore, the intestinal epithelial crypt cell line constructed by the construction method also belongs to the protection scope of the invention.
The invention also provides a culture method of the intestinal epithelial crypt cell line, which comprises the steps of digesting the intestinal epithelial crypt cell line by pancreatin, carrying out passage, and culturing by using an ENR culture medium, wherein the passage is carried out once every 2-3 days.
The specific structures of the plasmid vector containing the SV40TAg expression element and the piggyBac transposase splicing site, the adenovirus or plasmid vector for expressing the piggyBac transposase, the immortalized plasmid pMPH86 and the adenovirus for expressing the piggyBac transposase are disclosed in the invention patent CN 105002217A.
The invention has the beneficial effects that:
1. the intestinal epithelial crypt cell line iMICs constructed by the construction method provided by the invention realizes condition immortalization, can be stably passaged for more than 20 generations, well keeps the form of intestinal epithelial primary cells, has vigorous proliferation capacity, and expresses specific molecular markers Villin, E-Cadherin, ZO-1, PCNA and the like; therefore, the iMICs can be applied to an in vitro multidimensional research model, is used for researching the physiological functions of the intestinal tract and pathological mechanisms including infection, cancer occurrence and the like, and provides a high-efficiency and convenient platform for carrying out treatment targets and drug screening and evaluation on intestinal diseases including intestinal cancer.
2. The intestinal epithelial crypt cells constructed by the construction method provided by the invention can form a characteristic Organoid structure (Organoid) under a three-dimensional condition, one cell forms an Organoid under a certain culture condition, the Organoid is an important reference for embodying the pluripotency of the cell, and the Organoid is a functional standard for verifying the stem/progenitor cell characteristics of the cell under an in vitro condition, and the cell line established by the invention not only has the Organoid forming capability, but also contains a plurality of intestinal epithelial differentiation filial generations in the formed Organoid (figure 7).
3. The construction method of the invention adopts the separation buffer solutions I and II to realize the separation of the intestinal epithelial cell crypt cells, obtains a large amount of high-purity primary epithelial crypt structures through the collagenase-free buffer solution and mechanical vibration, and avoids the pollution of differentiated crypt epithelium and interstitial cells.
4. The construction method of the invention adopts the ENR culture medium to culture the intestinal epithelial crypt cells, avoids the adoption of animal serum used in other culture media, has complex serum components and contains various growth factors, and the influence of the serum components on the growth, proliferation and differentiation of the cells is difficult to control.
5. The operations of cell digestion, passage and cryopreservation of iMIC in the intestinal epithelial crypt cell line are similar to those of primary cells, so that the operation and the editing aiming at the iMIC cell gene level are very convenient, and the iMIC cell line becomes a convenient platform for the gene function research or the cell modification of the intestinal epithelial stem cell.
Drawings
FIG. 1 is a flow chart of the isolation and construction of intestinal crypt cells into immortalized cell lines.
FIG. 2 is a schematic representation of freshly isolated mouse intestinal crypt cells, wherein, A, the intestinal epithelial villus-crypt anatomy; b, mouse intestinal crypts separated in large quantity under a low power microscope; c, single complete Crypt under the high power mirror (Crypt, left side is the Crypt bottom, right side is the Crypt top), scale: b is 100 mu m, and C is 500 mu m.
Fig. 3 is primary and different generation crypt cell microscopic morphology showing that cells can be serially passaged and maintain the basic epithelial cell morphology, in which P0: primary cells, P3, 5, 9, 20 represent the corresponding passage number of the under-the-lens morphology, scale: 100 μm.
FIG. 4 shows the growth of crypt cells in different media after successful establishment of the line, wherein A, iMIC is observed under a low power microscope; b, observing the growth condition of iMIC under a high power lens; c, comparing the number of living cells of each group after plating for 48 hours at the same time by crystal violet staining, and measuring the number of the living cells by a scale: 100 mu m for A and 50 mu m for B.
FIG. 5 is HE staining and immunofluorescence staining of crypt cells.
FIG. 6 is a three-dimensional culture system of organoids of individual intestinal epithelial crypt cells, wherein A is a three-dimensional culture pattern. The centrifugally collected iMIC single-cell suspension and Matrigel are mixed and seeded in a 24-well plate, the plate is placed in a 37 ℃ incubator, and after complete solidification, ENR culture medium is added. B, iMIC-oligonucleotides (low power mirror). C, iMIC-oligonucleotides (high power mirror). A scale: b:100 μm, C: 25 μm.
FIG. 7 is an iMIC-Organoid specific stain formed under three-dimensional culture conditions, wherein ALP-alkaline phosphatase stain, PAS-periodate Schiff's stain, pharmacin Blue-Aryland stain, scale: 50μm.
Detailed Description
The present invention will be further described with reference to the following examples and the accompanying drawings.
The materials and reagents used in the present invention can be commercially available without specific reference.
Example 1
1. Isolation of intestinal crypt cells.
Preparing a reagent:
basic culture medium:
Advanced DMEM/F12
L-Glutamine (2mM)
Penicillin streptomycin Penn/Strep (100 units/ml)
Hepes (10 mM)
1:100 N2 supplement
1:50 B27 supplement
ENR medium: when the culture medium is prepared, 4 factors are added on the basis of a basic culture medium:
epidermal growth factor EGF (final concentration 50ng/ml)
Noggin protein (final concentration 100 ng/ml or conditioned medium added at volume ratio of 1: 10)
R-spondin (conditioned Medium added at a volume ratio of 1: 10)
Y27632 (ROCK inhibitor, final concentration 10µM)
Separation buffer I: 2mM EDTA, PBS, filtered and stored at 4 ℃.
Separation buffer II: 54.9 mM D-sorbitol, 43.4 mM sucrose, PBS, filtered and stored at 4 ℃.
1) Euthanizing 2-week-old CD1 mice; the middle lower part of the jejunum and the middle upper part of the ileum are dissected out, transferred to a culture dish containing cold PBS, and the intestinal canal is cut open longitudinally and rinsed repeatedly to remove the intestinal content.
2) The intestinal wall was cut transversely into small pieces of about 1cm x 1 cm.
3) The tissue pieces were transferred to 15 ml centrifuge tubes containing 5ml ice-cold PBS (containing cyan/streptomycin).
4) Shaking at 4 ℃ for 5 minutes with tumbling.
5) The PBS was aspirated as far as possible with a pipette and 5ml of cold intestinal epithelium isolation buffer I was added.
6) Shaking at 4 ℃ for 30 minutes with tumbling.
7) Buffer I was pipetted out and then replaced with 5ml of cold intestinal epithelial separation buffer II.
8) After shaking vigorously for 1-2 min, the solution becomes turbid and the transparency of the intestinal canal increases.
9) A small amount of liquid can be sucked under a mirror to see whether free crypts exist, and if no or few, the step 8 can be repeated.
10) A100 μm sterile cell filter was fitted over a 50 ml conical tube and placed on ice (operating in a biosafety cabinet).
11) All contents of buffer II were filtered through a filter.
12) Carefully rinsed with buffer II to ensure that there was sufficient separation of crypts to pass through the filter (gut and large)
The piece of differentiated intestinal epithelium will be removed at this step).
13) 20 microliter of the filtered solution was observed under a microscope. May be roughly counted.
14) The filtrate was dispensed into 1ml centrifuge tubes and centrifuged at 150G and 4 ℃ for 5 minutes.
15) The centrifugally collected intestinal crypts were digested with 0.25% pancreatic enzyme until a majority of the crypts formed a single cell suspension.
16) Digestion was stopped with DMEM (10% FBS) and collected by centrifugation.
The isolated crypt cells are observed by using a high power microscope, and the result is shown in figure 2, so that a large number of crypt cells can be obtained after the isolation method is filtered, and the complete crypt structure is ensured.
2. Establishment of intestinal epithelial crypt cell immortalized cell line
1) The crypt cells collected in the previous step were resuspended in Organoid medium and plated in 6-well plates.
2) An immortalized plasmid pMPH86 (expressing SV40-T-Antigen flanked by piggyBac transposase recognition regions) was transfected within 12 hours using a liposome method.
3) After 4 hours the medium was changed, the cells were infected with adenovirus Ad-pBase (green fluorescent) expressing piggyBac transposase, and after 6 hours the medium was changed with fresh Organoid.
4) When the cells grow to 80% in the pores, the cells are digested with 0.1% pancreatin and passaged.
5) Freezing and storing a part of the seeds when the seeds are transferred to the 3 rd generation, and preserving the seeds every two generations.
6) Hygromycin (Hygromycin) drug was added for 2 rounds of screening.
7) The passage is continued, once every 3 days or so, until more than 20 generations.
By observing the morphology of the primary and different generations of crypt cells, it can be seen from FIG. 3 that the cells can be passaged continuously and all maintain the basic epithelial cell morphology.
Meanwhile, the crypt cells are cultured in different culture media, and the culture results are shown in figure 4, so that the immortalization in the line establishing process is conditional immortalization, and the cells of the crypt cell line are reacted from one side and keep the original characteristics.
And 3, three-dimensional culture of iMIC cells.
1) The iMIC of the two-dimensional culture was collected by conventional digestion and centrifugation. The Matrigel is added into a centrifuge tube (the Matrigel is a temperature-sensitive material, is in a liquid state at 4 ℃, and is polymerized to be converted into a solid state at 37 ℃, and the conversion can be interchanged along with the temperature change).
2) The mixture was blown by a 200. mu.L pipette and dropped into the center of a well of an empty 24-well plate without the culture medium, 50. mu.L per well. The well plate was placed in a 37 ℃ incubator.
3) After 15 minutes the Matrigel was allowed to polymerize into a solid state, removed and ENR medium was carefully added to the liquid level above the top of the Matrigel (without floating the Matrigel pellet).
4) The solution was changed every 2 days, and a photograph was taken when organoids with a distinct morphology had grown.
The above picture is shown in fig. 6, wherein most of the cells form organoids, so that single intestinal epithelial crypt cells can form Organoid structures, and the structure has multiple functions.
4. iMIC cellular immunohistochemistry.
1) After removing the culture medium from the two-dimensional cultured cells, rinsing the cells once by using PBS, and immediately adding 4% paraformaldehyde or glacial methanol for fixing for 10 minutes;
2) the fixative was aspirated off, rinsed 3 times with PBS, 3 minutes each;
3) adding 1% NP40, and treating for 10 minutes at room temperature;
4) PBS washing 3 times;
5) adding goat serum (same species serum as the secondary antibody), and incubating at room temperature for 10-20 min;
6) serum was aspirated, no wash, and primary antibody freshly prepared with PBS was added;
7) rinsing with PBS 3 times;
8) adding a fluorescent labeled secondary antibody aiming at the primary antibody (operation in a dark place after the step), and incubating for 30 minutes at 37 ℃;
9) rinsing with PBS 3 times, and counterstaining with DAPI;
10) after rinsing with PBS, pictures were taken under a fluorescent microscope.
Results of staining referring to fig. 5, intestinal crypt cells were HE (hematoxylin-eosin stained) stained for cell-free eosin staining, indicating that the iMICs cell line was not contaminated with mesenchymal cells.
5. Ascidin Blue Staining
Preparing an Ascidin staining solution:
Alcian Blue GX 1g
0.1M HCl 100ml
the dyeing method comprises the following steps:
1) cells were fixed with 0.1% glutaraldehyde for 10 minutes.
2) PBS was rinsed 3 times.
3) Staining with the prepared Ascidin solution for 5-30 min, and observing under a mirror, and stopping staining when no/low background specific staining appears.
4) Rinse 1 time, 3 minutes with 0.1% HCl.
5) PBS was rinsed twice.
6) Fresh PBS was added and the image was taken. The positive coloration was blue-green.
6. Alkaline phosphatase (ALP) staining
Citric acid working solution: 2ml of citric acid concentrate (Sigma-Aldrich) was diluted into 100 ml of deionized water.
Fixing liquid: 2 volumes of citric acid working solution were mixed with 3 volumes of acetone at room temperature.
Diazonium salt solution: a small amount of Fast Blue R.R Salt was taken from a 10. mu.l pipette tip and dissolved completely in 48 ml of distilled water at room temperature.
1) Preparing a basic dye mixed solution: 2ml of Naphthol AS-MX phosphate alkaline solution is added to the diazonium salt solution.
2) Cells were fixed with fixative for 30-60 seconds at room temperature and rinsed 2 times with PBS.
3) Cells were incubated with alkaline-dye mixtures for 10-30 minutes at room temperature (monitoring staining under a microscope) and the samples were protected from direct light with aluminum foil paper.
4) The basic dye mixture is removed after dyeing is complete. Rinse thoroughly with PBS 2 x 3 min (note: do not let the sample dry, counterstain if necessary).
5) Fresh PBS was added and the image was taken. The positive coloration is purple.
7. Periodic Acid Schiff's (PAS) staining
Cells were fixed in 10% neutral formalin and staining procedure was performed according to kit instructions. Positive cells will typically develop a magenta coloration.
In the above 5, 6 and 7, iMIC-Organoid specific staining was formed under three-dimensional culture conditions, and three specific chemical staining were performed on progenies of the differentiation lineage of intestinal epithelium, namely alkaline phosphatase staining (ALP stain, expressed on intestinal epithelial absorptive cells for alkaline phosphatase), Ascinum staining (Alcian Blue stain, mainly from intestinal epithelial secretory cells for secreted mucin), and Periodic Acid Schiff staining (Periodic Acid-Schiff stain, PAS, glycogen component, specific to goblet cells). Referring to FIG. 7, specific positive staining can be detected under all three staining conditions, wherein the staining of Ascidin is more dispersed, and the positive staining of alkaline phosphate and periodic acid is more limited in scope but higher in specificity.
Claims (7)
1. A method for constructing a mouse intestinal epithelial crypt cell line is characterized by comprising the following specific steps:
(1) isolation of intestinal epithelial crypt cells: cleaning the middle-lower part of jejunum and the middle-upper part of ileum of a mouse, shearing intestinal canal tissues, transferring the intestinal canal tissues into a separation buffer solution I, turning and shaking the intestinal canal tissues, sucking the buffer solution I out, adding a buffer solution II, violently shaking the intestinal canal tissues and the non-crypt epithelium, removing the intestinal canal and the non-crypt epithelium from the intestinal canal tissues, centrifugally collecting crypt cells from filtrate, digesting the crypt cells by pancreatin to obtain a single cell suspension; the buffer solution I is phosphate buffer salt solution of EDTA, and the buffer solution II is phosphate buffer salt solution of D-sorbitol and sucrose;
(2) construction of intestinal epithelial crypt cell line: resuspending the crypt cells finally collected in the step (1) by using an ENR culture medium, inoculating, transfecting a plasmid vector containing an SV40TAg expression element and a piggyBac transposase shearing site, infecting by using adenovirus expressing the piggyBac transposase within 24 hours, replacing a new ENR culture medium, and carrying out drug screening and continuous passage to obtain an intestinal epithelial crypt cell line; the obtained intestinal epithelial crypt cell line has the characteristics of intestinal epithelial progenitor/stem cells, and single cells can form characteristic organoid under the condition of three-dimensional culture.
2. The method according to claim 1, wherein the concentration of EDTA in the buffer solution I is 0.5 to 4 mmol/L, the concentration of D-sorbitol in the buffer solution II is 40 to 70mmol/L, and the concentration of sucrose is 30 to 60 mmol/L.
3. The method according to claim 2, wherein the concentration of EDTA in the buffer I is 2mmol/L, the concentration of D-sorbitol in the buffer II is 54.9 mmol/L, and the concentration of sucrose is 43.4 mmol/L.
4. The method of claim 1, wherein the ENR medium is a serum-free medium based on Advanced DMEM/F12, comprising EGF, Noggin protein and R-Spondin 2.
5. The method for constructing an Epidermal Growth Factor (EGF) in an ENR medium, wherein the concentration of the EGF is 30-80ng/mL, the concentration of the Noggin protein is 80-120ng/mL, and the volume ratio of the conditioned medium of R-Spondin2 to the Advanced DMEM/F12 medium is 08-1.2: 8-12.
6. The intestinal epithelial crypt cell line constructed by the construction method according to any one of claims 1 to 5, wherein the obtained intestinal epithelial crypt cell line has intestinal epithelial progenitor/stem cell characteristics, and a single cell can form a characteristic organoid under three-dimensional culture conditions.
7. The method for culturing the intestinal epithelial crypt cell line according to claim 6, wherein the intestinal epithelial crypt cell line is digested with trypsin, passaged, and cultured in ENR medium, and the passaging is performed every 2 to 3 days.
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| CN102212504B (en) * | 2011-05-05 | 2012-12-19 | 吉林农业大学 | Rabbit intestinal epithelial cell line and preparation method thereof |
| CN105002217B (en) * | 2014-05-20 | 2020-03-31 | 中国人民解放军第三军医大学第一附属医院 | Transposon vector for preparing immortalized cell, system and use method thereof |
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