CN104818238B - Chicken intestine epithelial cell separation culture method - Google Patents
Chicken intestine epithelial cell separation culture method Download PDFInfo
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Abstract
The invention relates to a chicken intestinal epithelial cell separation culture method, which comprises the following steps: step 1: digesting the chicken embryo intestinal tissue block by using protease to obtain a digestion product; step 2: filtering the digestion product by using a cell sieve, and collecting cell blocks with the size of 100-500 meshes; and step 3: and centrifuging the cell mass at 800-1500r/min for 5-15min, and collecting cell sediment. The invention also provides the chicken intestinal epithelial cells obtained by the method. The method provided by the invention can greatly shorten the test time, save reagents and improve the purity of the separated intestinal epithelial cells.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a chicken intestinal epithelial cell separation culture method.
Background
The intestinal epithelial cells are the medium of the internal and external environment of the intestinal tract, are also an important component of the immune barrier of the organism, and have important physiological functions of digestion, absorption, secretion, immunity and the like. The intestinal epithelial cells can be isolated and cultured in vitro under certain conditions. The in vitro separation and culture of the intestinal epithelial cells provide a simple, rapid and accurate means for researching the biological functions of the intestinal tract, the absorption mechanism and regulation of nutrient substances, and the proliferation, differentiation and apoptosis of the intestinal epithelial cells, and provide an ideal in vitro model for researching the action of nutrients and other external factors on the intestinal epithelium. However, the separation and culture difficulty of the intestinal epithelial cells is high, and reports that a monoclonal intestinal epithelial cell line can be successfully established are few. For a long time, intestinal cancer cell lines are mostly used as experimental models in the research of the biological functions of intestinal epithelial cells, the regulation factors of cell proliferation and differentiation, substance absorption and metabolism and the like. However, there is a certain difference between the intestinal cancer epithelial cells and the normal epithelial cells in terms of basic cellular structure, metabolism, and functional characteristics, and there is a great limitation in explaining normal behaviors with the research results of cancer cell lines.
At present, no patent literature about a method for separating or culturing primary chicken intestinal epithelial cells in vitro is disclosed, and although the conventional cell culture technology is adopted in the conventional chicken intestinal epithelial cell culture method, the aim of culture can be achieved to a certain extent, the problems of complex operation, difficult adherence of cells, slow growth, difficult separation of other miscellaneous cells such as fibroblasts and the like exist, and the requirements of cell experiments cannot be met.
The inventor of the invention finds out through long-term experimental research that the intestinal epithelial cell mixed with fibroblast usually cannot separate the intestinal epithelial cell from the fibroblast when separating the cell, and some documents separate the fibroblast component by adopting a differential adherence or low-speed centrifugation mode, however, the process is complicated and time-consuming, generally requires 5-7 hours and has low cell purity.
Therefore, it is necessary to develop a method for separating and culturing chicken intestinal epithelial cells in vitro, which has the advantages of simple operation process, good cell adherence effect, rapid growth and high cell purity.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and develop the chicken intestinal epithelial cell in-vitro separation culture method which is simple and convenient in operation process, good in cell adherence effect, rapid in growth, good in state and high in purity.
The inventor of the invention deeply researches and compares the method for culturing and separating the intestinal epithelial cells at home and abroad, improves the in vitro culture method of the chicken intestinal epithelial cells through a large amount of experiments, and provides an experimental model for the subsequent biological action research of the intestinal epithelial cells.
The invention provides a chicken intestinal epithelial cell separation culture method, which comprises the following steps:
step 1: digesting the chicken embryo intestinal tissue block by using protease to obtain a digestion product;
step 2: filtering the digestion product by using a cell sieve, and collecting cell blocks with the size of 100-500 meshes;
and step 3: and centrifuging the cell mass at 800-1500r/min for 5-15min, and collecting cell sediment.
Optionally, the protease is type i collagen protease and/or hyaluronidase.
Optionally, when the protease is type I collagenase and hyaluronidase, the ratio of the type I collagenase to the hyaluronidase is 1-2: 1.
optionally, in the digestion process of step 1, the concentration of the type I collagen protease in the digestion system is 0.8-1.6 mg/mL; the concentration of hyaluronidase is 0.4-1.6 mg/mL.
Optionally, the method comprises soaking the chicken embryo intestinal tissue block in DMEM/F12 medium before digestion.
Optionally, the digestion condition is shaking digestion at 37 ℃ for 25-35 min.
Optionally, the method further comprises subjecting the cell pellet to a primary culture after step 3, the conditions of the primary culture comprising: adding DMEM/F12 containing 20% fetal calf serum into the cell precipitate, performing rotary blowing to obtain cell suspension, inoculating the cell suspension into a rat tail collagen-coated culture plate, and placing at 37 deg.C and 5% CO2Culturing in an incubator for 24-48 hours, and replacing DMEM/F12 culture medium with fetal calf serum concentration of 10% to continue culturing after adherence.
Optionally, the size of the chicken embryo intestinal tissue block is 0.5-1mm3。
Optionally, the chick embryo intestinal tissue mass is taken from a 15 day old chick embryo.
The invention also provides the chicken intestinal epithelial cells obtained by the method.
The invention utilizes the difference of the intestinal epithelial cell mass and the fibroblast in physical size, and adopts the cell screen with a certain aperture to filter the digestion product so as to separate and take out most of the components of the fibroblast.
Drawings
FIG. 1 is a histological image of intestinal epithelial cell growth, and the direct staining and the result of negative identification of staining were observed under a microscope at a magnification of 200 times after 24 hours of culture (control group, no staining).
FIG. 2 is a histological morphology of intestinal epithelial cell growth, which is a result of positive staining when direct staining is observed by microscope magnification of 200 times after 24 hours of culture.
FIG. 3 is a result of identification of negative staining after 24 hours of incubation and trypsinization into single cells under observation at a microscope magnification of 200 times (control group, no staining).
FIG. 4 shows the results of positive staining identification after incubation for 24 hours under a microscope at 200-fold magnification and after trypsinization into single cells.
Detailed Description
The present invention will be described in detail below by way of specific embodiments. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.
The invention provides a chicken intestinal epithelial cell separation culture method, which comprises the following steps:
step 1: digesting the chicken embryo intestinal tissue block by using protease to obtain a digestion product;
step 2: filtering the digestion product by using a cell sieve, and collecting cell blocks with the size of 100-500 meshes;
and step 3: and centrifuging the cell mass at 800-1500r/min for 5-15min, and collecting cell sediment.
The chicken intestine tissue block can be derived from 15-day-old chicken embryo intestine tissue, and the mesentery of the intestine tissue is removed and cleaned. The washing method may be performed by washing with PBS solution containing antibiotics at multiple times, and the antibiotics may include penicillin at a concentration of 100U/L and streptomycin at a concentration of 100. mu.g/L.
The method provided by the invention comprises the steps of soaking the chicken embryo intestinal tissue block in a DMEM/F12 culture medium and then digesting, wherein in the digestion process, the concentration of type I collagen protease in a digestion system is 0.8-1.6 mg/mL; the concentration of hyaluronidase is 0.4-1.6 mg/mL.
In one embodiment of the invention, the mesenteric membrane-removed and washed intestinal tissue can be soaked in DMEM/F12 medium for shearing to obtain chicken embryo intestinal tissue blocks with the size of 0.5-1mm3。
In the methods provided herein, the protease is a type i collagen protease and/or a hyaluronidase. Preferably, the protease is a type i collagen protease and a hyaluronidase.
When the protease is type I collagenase and hyaluronidase, the dosage ratio of the type I collagenase to the hyaluronidase is 1-2: 1.
in the invention, the type I collagenase is dissolved into an enzyme solution with the concentration of 1-2mg/mL and then is contacted with the tissue block to be digested; dissolving hyaluronidase into enzyme solution with concentration of 0.5-2mg/mL, and contacting with tissue block to be digested; or preparing the type I collagenase and the hyaluronidase into a mixed enzyme solution, and then contacting the mixed enzyme solution with the tissue block to be digested, wherein the concentration of the type I collagenase in the mixed enzyme solution is 1-2mg/mL, and the concentration of the hyaluronidase is 0.5-2 mg/mL.
Optionally, the digestion condition is shake digestion at 37 deg.C for 25-35min, preferably digestion for 30 min.
Optionally, the method further comprises subjecting the cell pellet to a primary culture after step 3, the conditions of the primary culture comprising: adding DMEM/F12 containing 20% fetal calf serum into the cell precipitate, performing rotary blowing to obtain cell suspension, inoculating the cell suspension into a rat tail collagen-coated culture plate, and placing at 37 deg.C and 5% CO2Culturing in an incubator for 24-48 hours, and replacing DMEM/F12 culture medium with fetal calf serum concentration of 10% to continue culturing after adherence.
Wherein the medium for primary culture and continuous culture may further contain penicillin at a concentration of 100U/L and streptomycin at a concentration of 100. mu.g/L.
In one embodiment of the present invention, the method provided by the present invention comprises the steps of:
the digestion product was first passed through a 100 mesh cell sieve, and mixture 1, which was able to pass through the 100 mesh cell sieve, and fraction 1, which was not able to pass through the 100 mesh cell sieve, were collected separately.
Wherein, the mixture 1 which can pass through the 100-mesh cell sieve is centrifuged at 1000r/min for 10 minutes, and then the supernatant 1 and the precipitate 1 are collected. Wherein, the main components in the supernatant 1 are single cell, collagenase type I and/or hyaluronidase liquid. This supernatant 1 can be used for the subsequent digestion step.
Wherein the main components of the sediment 1 are intestinal epithelial crypt units and partial single cells, DMEM/F12 culture medium can be added into the collected sediment 1 for basic suspension to prevent the cells from dehydration and death, and the resuspension 1 is obtained.
Wherein, the component 1 which can not pass through the 100-mesh cell sieve is recovered and added into DMEM/F12 culture medium for re-suspension, the re-suspended mixture is mixed with the supernatant 1 collected before and then is subjected to secondary digestion, the mixture 2 which can pass through the 100-mesh cell sieve is collected, the mixture 2 which can pass through the 100-mesh cell sieve is centrifuged at 1000r/min for 10 minutes, the precipitate 2 is collected, and the re-suspension 2 is obtained after the precipitate 2 is re-suspended by DMEM/F12 culture medium.
Mixing the heavy suspension 1 and the heavy suspension 2 to obtain a mixed suspension, sieving the mixed suspension through a 500-mesh cell sieve to remove single cells, collecting components which can not pass through the 500-mesh cell sieve, centrifuging the components at a low speed of 1000r/min, removing supernatant, collecting precipitate 3, and carrying out primary culture on the precipitate 3.
The invention also provides the chicken intestinal epithelial cells obtained by the method.
The present invention is illustrated in detail by the following examples:
in the following examples: test subjects: 15 day old chick embryos.
PBS (Hyclone), DMEM/F12 (Hyclone), fetal bovine serum (Gibico), collagenase type I (Sigma), hyaluronidase (Sigma); BCIP/NBT alkaline phosphatase color reagent (Biyunnan Co., Ltd.), rat tail collagen (Sigma Co., Ltd.), cell sieve (Solebao Co., Ltd.) CO2Incubator (Thermo Scientific), inverted microscope (Olympus).
Example 1
1. Primary culture: taking out 2 15-day-old chick embryo intestinal tissues under aseptic condition, carefully removing mesentery, and repeatedly washing with PBS containing antibiotic for 3-4 times. Collecting intestinal tissue without impurities in 20mL small beaker, adding 5mL DMEM/F12, and shearing into pieces smaller than 1mm by ophthalmic scissors3The tissue mass of (a) was transferred to a 50mL centrifuge tube and digested with 20mL of collagenase type i at a concentration of 2mg/mL and 20mL of hyaluronidase at a concentration of 1mg/mL in combination, stirred in a 37 water bath for 30min at 80r/min, and then the mixture of the digested products was filtered using a 100 mesh cell sieve, and after centrifugation through the mixture of the cell sieve for 10min at 1000r/min, the supernatant (mainly consisting of collagenase type i and hyaluronidase solution and single cells) was transferred to a new centrifuge tube and suspended to the pellet (mainly consisting of intestinal epithelial crypt unit and a portion of single cells) with a culture basis weight of 5mL DMEM/F12 to prevent cell dehydration death, and was used. The tissue mass which failed to pass through the 100 mesh cell sieve was recovered and the previous digestion step was repeated with the mixed enzyme digest recovered from the previous step, and crypt units were collected as described above, mixed with the previous crypt units, and re-spun and trimmed to 50mL with DMEM/F12. Then, the 500-mesh fine mesh is adoptedCell-sieve the crypt mixture was filtered to remove individual cells, intestinal crypt units that failed to pass through the 500 mesh cell sieve were recovered in DMEM/F12, the supernatant was discarded after centrifugation at low speed of 1000r/min, and the pellet was resuspended in 20mL of DMEM/F12 medium containing 20% FBS, 100U/L penicillin and 100. mu.g/L streptomycin and plated in rat tail collagen-coated 6-well cell plates at 3mL per well. Finally placing the mixture at 37 ℃ and 5% CO2After 24 hours of culture in an incubator, the culture was continued after the adherence in DMEM/F12 medium containing 10% FBS, 100U/L penicillin and 100. mu.g/L streptomycin, and the medium was changed every 48 hours thereafter. And observing the adherent growth of the cells under an inverted microscope every day.
2. Identification of intestinal epithelial cells: the intestinal mucosa epithelial cells can secrete mucus, and the alkaline phosphatase is a marker enzyme on the microvilli of the intestinal epithelial cells. After the cells are fully paved on the culture plate, the cells are digested into single cells by pancreatin, cell suspension is prepared, the cells are dyed and identified by a BCIP/NBT alkaline phosphatase developing kit, the dyeing condition is observed under a microscope, positive epithelial cells are shown to be black according to the appearing result, negative controls are not developed, and counting and photographing are carried out under the microscope.
3. As a result: and (3) primary culture results: chicken intestinal epithelial cell crypts adhere generally at 24 hours in an "island" shape. The culture medium liquid is changed and then is continuously cultured for 24 hours, so that the radiation growth from the island to the periphery is observed, and the materials are gradually converged into a sheet in a paving stone shape after 48-60 hours.
Identification of intestinal epithelial cells: after the cells are fully paved on the culture plate, the cells are dyed and identified by using a BCIP/NBT alkaline phosphatase color development kit, the positive cells are dyed into blue black, the positive rate reaches more than 80 percent, and the vast majority of cells are intestinal epithelial cells.
Referring to fig. 1 to 4: FIG. 1 is a histological image of intestinal epithelial cell growth, and the direct staining and the result of negative identification of staining were observed under a microscope at a magnification of 200 times after 24 hours of culture (control group, no staining). FIG. 2 is a histological morphology of intestinal epithelial cell growth, which is a result of positive staining when direct staining is observed by microscope magnification of 200 times after 24 hours of culture.
FIG. 3 is a result of identification of negative staining after 24 hours of incubation and trypsinization into single cells under observation at a microscope magnification of 200 times (control group, no staining). FIG. 4 shows the results of positive staining identification after incubation for 24 hours under a microscope at 200-fold magnification and after trypsinization into single cells.
Comparative example 1
The isolated culture of chicken intestinal epithelial muscle cells was carried out according to the method provided in the literature (isolated culture and identification of primary chicken intestinal epithelial cells in vitro, teacher, Yang Xia, Chen Lu, etc., Jiangsu agricultural science, 2013,41 (1): 34-37.):
aseptically taking out 15-day-old chick embryo intestine tissue, repeatedly washing with PBS, removing mesentery, and cutting into pieces smaller than 1mm3The tissue block is prepared by digesting intestinal tissue with type I neutral protease and type XI collagenase, and stirring at 37 deg.C in water bath at low speed for 30min at a speed of 80 r/min. After digestion was completed, the cells were centrifuged at 1000r/min for 10min, a small amount of DMEM/F12 medium was added to the cell pellet to suspend the cells, and after three times of centrifugal washing, the collected cells were suspended in a 10% FBS-containing cell culture solution (DMEM/F12(pH7.0)12g/L + 20. mu.g/L epidermal growth factor +100mg/L heparin sodium +110mg/L sodium pyruvate +2.5mg/L insulin +200mmol/L glutamic acid ammonia +100U/L penicillin + 100. mu.g/L streptomycin) and seeded into glass cell flasks. After 2.5 hours, the culture medium and nonadherent cells were transferred to a centrifuge tube, centrifuged at 1000r/min for 10min, and the cell pellet was added with a cell culture medium containing 2.5% FBS (DMEM/F12(pH7.0)12g/L + 20. mu.g/L epidermal growth factor +100mg/L heparin sodium +110mg/L sodium pyruvate +2.5mg/L insulin +200mmol/L (2mmol/L) glutamic acid ammonia +100U/L penicillin + 100. mu.g/L streptomycin) and inoculated into a 20% FBS-coated disposable T25 cell bottle, and after 24 hours, the medium was observed and replaced. The isolation culture method of comparative example 1 was compared with that of example 1, and the results are shown in Table 1.
TABLE 1
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Claims (3)
1. A chicken intestinal epithelial cell separation culture method is characterized by comprising the following steps:
step 1: soaking the chicken embryo intestinal tissue block in a DMEM/F12 culture medium, and then performing shake digestion on the chicken embryo intestinal tissue block at 37 ℃ for 25-35min by using type I collagenase and hyaluronidase to obtain a digestion product, wherein the dosage ratio of the type I collagenase to the hyaluronidase is 1-2:1, the concentration of the type I collagenase in a digestion system is 0.8-1.6mg/mL, and the concentration of the hyaluronidase is 0.4-1.6 mg/mL;
step 2: filtering the digestion product by using a cell sieve, and collecting cell blocks with the size of 100-500 meshes;
and step 3: centrifuging the cell mass for 5-15min at the speed of 800-1500r/min, and collecting cell sediment;
and 4, step 4: adding DMEM/F12 containing 20% fetal calf serum into the cell precipitate, performing rotary blowing to obtain cell suspension, inoculating the cell suspension into a rat tail collagen-coated culture plate, and placing at 37 deg.C and 5% CO2Culturing in an incubator for 24-48 hours, and replacing DMEM/F12 culture medium with fetal calf serum concentration of 10% to continue culturing after adherence.
2. The method of claim 1, wherein the chick embryo intestinal tissue mass has a size of 0.5-1mm3。
3. The method of claim 2, wherein the chicken embryo intestinal tissue mass is taken from 15 day old chicken embryos.
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| CN106884002B (en) * | 2015-12-15 | 2021-03-19 | 上海市针灸经络研究所 | Stable colon epithelial cell culture method |
| CN107217040A (en) * | 2017-06-16 | 2017-09-29 | 中国农业大学 | One kind immortalizes rabbit intestinal epithelial cell line and its construction method |
| CN110396496B (en) * | 2018-09-30 | 2023-06-20 | 湖北省农业科学院畜牧兽医研究所 | Culture method and application of duck small intestine epithelial cells |
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| CN104357393A (en) * | 2014-11-14 | 2015-02-18 | 中国农业科学院饲料研究所 | Isolated culture method of chicken intestinal tract epithelial GammaDeltaT cells |
| CN105483078A (en) * | 2015-12-24 | 2016-04-13 | 山西农业大学 | Isolation and primary culture methods of chicken small intestinal epithelial cells |
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| CN104357393A (en) * | 2014-11-14 | 2015-02-18 | 中国农业科学院饲料研究所 | Isolated culture method of chicken intestinal tract epithelial GammaDeltaT cells |
| CN105483078A (en) * | 2015-12-24 | 2016-04-13 | 山西农业大学 | Isolation and primary culture methods of chicken small intestinal epithelial cells |
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| Isolation of intestinal epithelial cells and evaluation of transport functions;KIMMICH G.A.;《Methods in Enzymology》;19901231;第192卷(第1期);摘要、第324页第2段-第326页第3段 * |
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