Method for isolating and primary culturing chicken caecal epithelial cells

Abstract

The application discloses a method for separating and primary culturing chicken caecal epithelial cells, and belongs to the technical field of cell culture. The method adopts three-stage gradient decreasing double-antibiotic washing liquid to grade wash the caecal tissue of 18-embryonic-age chicken embryos, so that the stress damage of antibiotics to cells is reduced while the microbial contamination is effectively controlled; the method adopts thermophilic bacterial protease gentle digestion, and removes fibroblast cells and other impurity cells by two-stage screen filtration and differential adhesion; the method breaks through the technical prejudice of conventional 41 DEG C culture of poultry cells, and adopts 37 DEG C and 5% CO2 conditions for primary culture, and a unified nutrient culture liquid system is matched, so that the temperature and nutrient supply are cooperatively adapted. The application can quickly obtain chicken caecal epithelial cells with high purity, complete morphology and good tight connection, and the cells can form a dense brick single layer in 48 h, so that a stable in-vitro cell model is provided for the research on the intestinal tract barrier function, pathogenic infection and nutrient metabolism of poultry.

Description

Technical Field

[0001] This invention belongs to the field of cell culture technology, specifically relating to a method for isolating and primary culturing chicken cecal epithelial cells. Background Technology

[0002] The chicken cecum is a core site of intestinal immunity and microbial interaction in poultry, and its epithelial cells play a crucial role in nutrient absorption, mucosal barrier maintenance, mucin secretion, and immune response regulation. Isolating and culturing primary epithelial cells from the chicken cecum is an important in vitro model for studying intestinal pathogen infection mechanisms, probiotic colonization patterns, feed additive efficacy, and intestinal immune regulation. However, due to the unique structure and complex microbiota of the cecum, the isolation and purification of its epithelial cells has long faced technical bottlenecks.

[0003] In existing technologies, research on the isolation and culture of chicken intestinal epithelial cells mainly focuses on the small intestine. CN104818238A (A method for isolating and culturing chicken intestinal epithelial cells) discloses a method of digesting chicken embryo intestinal tissue blocks with proteases, collecting 100-500 mesh cell clumps by filtration with a cell sieve, and collecting cell pellets by centrifugation. However, mature and reproducible isolation methods for cecal epithelial cells are relatively scarce. The few reported methods for cecal epithelial cell isolation currently available mostly directly copy the isolation strategies for small intestinal epithelial cells, with main steps including: tissue washing, enzymatic digestion, filtration, centrifugation, and differential adhesion purification. However, when these methods are directly applied to cecal tissue, they often face the following problems: First, the cecal mucosa is rich in lymphoid follicles and dense connective tissue, exhibiting high mechanical strength, making it difficult to control the conditions for enzymatic digestion. Insufficient digestion results in low cell yield, while over-digestion leads to a sharp decline in cell viability. Commonly used digestive enzymes include collagenase, thermophilic protease, and trypsin, but there is a lack of systematic optimization of the appropriate concentrations and action times for different enzymes specifically for cecal epithelial cells.

[0004] Secondly, the cecal contents have an extremely high microbial load, making them highly susceptible to bacterial or fungal contamination during the sampling process. The standard practice is to wash the cecal contents with a penicillin-streptomycin antibiotic in PBS; however, excessively high concentrations of this antibiotic can induce epithelial cell stress and damage, leading to decreased cell adhesion; while excessively low concentrations cannot effectively inhibit other bacteria, resulting in culture failure.

[0005] Third, the in vitro culture of chicken-derived cells typically follows the physiological temperature of 41°C. However, at this temperature, fibroblasts proliferate faster, further weakening the competitive advantage of epithelial cells and increasing the difficulty of purification. In addition, the solutions and culture conditions used in each step of the existing culture system (such as filtration, centrifugation, and adhesion) are often independent of each other, lacking a systematic and synergistic design, making it difficult to achieve both high purity and high yield.

[0006] To address the aforementioned technical challenges, there is an urgent need in this field to develop a method specifically for the isolation and primary culture of chicken cecal epithelial cells that balances cell viability and purity, providing a stable and reliable cell model for poultry intestinal function research. Summary of the Invention

[0007] To address the problems existing in the prior art, the purpose of this invention is to provide a method for isolating and primary culturing chicken cecal epithelial cells.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, this invention provides a method for isolating and primary culturing chicken cecal epithelial cells. The method comprises: taking chicken embryo cecum, washing it in a three-stage gradient decreasing double antibiotic wash solution, cutting it into small pieces, and digesting it with thermophilic protease digestion solution; diluting the digestion solution with serum-containing culture medium and filtering it sequentially through 50-mesh and 120-mesh sieves, centrifuging to collect the cells; culturing the cell suspension for 70 min to remove adherent contaminating cells, collecting the non-adherent epithelial cells, and culturing them at 37°C and 5% CO2 to obtain a monolayer of epithelial cells.

[0009] Preferably, the method includes the following steps: (1) Remove the cecum: Disinfect the outer shell of the chicken embryo that has been incubated for 18 days, remove the chicken embryo, pick out the cecum, and temporarily store it in cecum washing solution No. 1; (2) Remove mesentery and blood: After separating the chicken cecum and removing the mesentery, place it in cecum washing solution No. 2; (3) Washing: After removing the mesentery, wash with No. 3 cecal washing solution. After the supernatant is clear, cut the cecal tissue into a paste. Then wash with No. 3 cecal washing solution and PBS in sequence and set aside. (4) Tissue digestion: Digest with thermophilic protease digestion solution at 37℃ and 80-200 r / min for 5-30 min with shaking. (5) Filtration and centrifugation: After digestion, blow the enzyme digestion solution for 3-5 minutes, dilute the digestion solution with DMEM / F12 culture medium containing 10% fetal bovine serum preheated to 37°C, and pass it through 50-mesh and 120-mesh cell filter screens in sequence. Collect the filtered cell suspension, centrifuge and retain the cell pellet. (6) Remove adhering cells by adhering culture: The precipitate obtained by filtration and centrifugation is mixed with chicken primary cecal epithelial cell culture medium and the cell suspension is inoculated into a culture flask and cultured in a 37°C, 5% CO2 incubator to remove adhering cells. (7) Collect chicken cecal epithelial cells: Take out the culture flask, collect the cell suspension, centrifuge, discard the supernatant, and then mix the cell pellet with chicken primary cecal epithelial cell culture medium. Dilute the cell suspension with trypan blue and count the number of cell clusters without blue staining using a hemocytometer for later use. (8) Primary culture of chicken cecal epithelial cells: Chicken cecal epithelial cells were cultured at a density of 1×10⁻⁶ cells / year. 6 / mL was inoculated into primary chicken cecal epithelial cell culture medium and cultured in a 37℃, 5% CO2 incubator. The medium was changed every 2 days. After 48 h, the cells grew into a "brick-shaped" monolayer of epithelial cells with a density of 80%-90%.

[0010] The ratio of PBS to penicillin antibody in the No. 1 cecal washing solution is 9:1; the ratio of PBS to penicillin antibody in the No. 2 cecal washing solution is 19:1; and the ratio of PBS to penicillin antibody in the No. 3 cecal washing solution is 39:1. The bispecific antibody consists of penicillin and streptomycin sulfate.

[0011] The thermophilic protease digestion solution is prepared by dissolving thermophilic protease in HEPES buffer and stirring thoroughly until completely dissolved, adjusting the pH to 7.4, and then filtering to remove bacteria.

[0012] The chicken primary cecal epithelial cell culture medium was based on DMEM / F12, supplemented with 10% fetal bovine serum, 1% 50 mmol / L L-glutamine solution, 1% 0.11 g / mL sodium pyruvate solution, 1% 10 g / L sodium heparin solution, 1% 250 mg / L insulin solution and 1% penicillin-dextrin solution.

[0013] The primary culture cell seeding density was 1×10⁻⁶. 6 per mL.

[0014] In a second aspect, the present invention provides the application of chicken cecal epithelial cells isolated and cultured by the above method in the preparation of in vitro cell models for the study of intestinal barrier function, pathogen infection or nutritional metabolism in poultry.

[0015] In a third aspect, the present invention provides a kit for the isolation and primary culture of chicken cecal epithelial cells, comprising: cecal washing solution No. 1, cecal washing solution No. 2, cecal washing solution No. 3, thermophilic protease digestion solution, and chicken primary cecal epithelial cell culture medium.

[0016] The beneficial effects of this invention are: 1. Addressing the dual challenges of high microbial load and fragile mucosa in chicken cecum, this invention proposes and employs a three-stage gradient of decreasing PBS:antibiotic wash (PBS:antibiotic ratios of 9:1, 19:1, and 39:1, respectively) for graded washing of cecum tissue. This application, through a systematic design of rapid inhibition of contaminating bacteria with high concentrations in the early stage and reduction of cellular stress with low concentrations in the later stage, solves the long-standing technical challenge of simultaneously achieving antibacterial activity and cell viability preservation.

[0017] 2. This application overcomes the technical bias of the "41℃ physiological temperature" for avian cecal cells by pioneering a 37℃ co-culture system. This invention employs 37℃ and 5% CO2 conditions during differential adhesion and subsequent primary culture stages. In the isolation and primary culture system of this invention, lowering the culture temperature from the conventional 41℃ for poultry to 37℃ significantly inhibits excessive fibroblast proliferation, reduces heat stress damage to epithelial cells, and thus simultaneously improves the purity, viability, and barrier function integrity of epithelial cells.

[0018] 3. This invention focuses on the physiological characteristics of chicken cecal epithelial cells, employing graded filtration (50 mesh + 120 mesh) and precisely optimizing the differential adhesion time (70 min). Using this method, chicken cecal epithelial cells can form a dense, uniform "brick-shaped" monolayer of cells after 48 h of culture (e.g., Figure 2 As shown), the positive rate reached over 90%. Figure 3 ), and the tight junction protein E-cadherin was expressed continuously and uniformly. Figure 4 This indicates that the obtained cell model highly mimics the living cecal epithelial barrier in both morphology and function, and can be directly used for research on intestinal pathogen infection, probiotic colonization, nutrient metabolism, and drug screening in poultry. Attached Figure Description

[0019] Figure 1 This is a flowchart of the present invention.

[0020] Figure 2 A light microscopic image (200×) showing the morphological characteristics and growth status of primary cecal epithelial cells in chickens.

[0021] Figure 3 Immunofluorescence identification image of chicken primary cecal epithelial cells CK18 (630×).

[0022] Figure 4 Immunofluorescence image of E-cadherin in primary chicken cecal epithelial cells (630×). Detailed Implementation

[0023] It should be noted that the following detailed descriptions are illustrative and intended to provide further explanation of this application. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0024] The specific embodiments of the present invention will be described in further detail below with reference to examples. The following detailed descriptions are illustrative and intended to provide further explanation of this application, rather than limiting the scope of the invention.

[0025] Example 1: Isolation and culture of chicken cecal epithelial cells using the method of the present invention 1. Experimental Materials Experimental animals: White Leghorn chicken embryos, incubated to 18 days old.

[0026] Preparation of washing solution and culture medium: The first cecal wash solution consisted of 36 mL PBS + 4 mL of penicillin antibody (PBS: penicillin antibody = 9:1), the second cecal wash solution consisted of 38 mL PBS + 2 mL of penicillin antibody (PBS: penicillin antibody = 19:1), and the third cecal wash solution consisted of 39 mL PBS + 1 mL of penicillin antibody (PBS: penicillin antibody = 39:1).

[0027] The bispecific antibody consists of penicillin and streptomycin sulfate, with a working concentration of 100 U / mL for penicillin G sodium salt and a working concentration of 0.1 mg / mL for streptomycin sulfate.

[0028] The thermophilic protease digestion solution was prepared by dissolving 25 mg of thermophilic protease in 100 mL of HEPES buffer and stirring thoroughly until completely dissolved. The pH was adjusted to 7.4, and the solution was filtered to remove bacteria and then stored at -20°C.

[0029] The culture medium for primary cecal epithelial cells of chickens was prepared as follows: 1% 50 mmol / L L-glutamine solution + 1% 0.11 g / mL sodium pyruvate solution + 1% 10 g / L sodium heparin solution + 1% 250 mg / L insulin solution + 1% penicillin-dextrin solution + 10% fetal bovine serum + DMED / F12, and was prepared fresh before use.

[0030] The preparation of trypan blue is as follows: Weigh 2 g of trypan blue, add a small amount of ddH2O and grind, add double-distilled water to 50 mL, filter with filter paper, and store at 4℃.

[0031] 2. Isolation and Culture Steps (1) Remove the cecum: Disinfect the outer shell of the chicken embryo that has been incubated for 18 days, remove the chicken embryo, pick out the cecum, and temporarily store it in cecum washing solution No. 1; (2) Remove mesentery and blood: After separating the chicken cecum and removing the mesentery, place it in cecum washing solution No. 2; (3) Washing: After removing the mesentery, wash three times with No. 3 cecal washing solution. After the supernatant is clear, cut the cecal tissue into a paste, then wash three times with No. 3 cecal washing solution, and then wash three times with PBS before use. (4) Tissue digestion: Use thermophilic bacterial protease digestion solution with a concentration of 50 µg / mL at 37℃ and shake at 80-200 r / min for 5-30 min; (5) Filtration and centrifugation: After digestion, blow the enzyme digestion solution for 3-5 min, dilute the digestion solution with DMEM / F12 culture medium containing 10% fetal bovine serum preheated to 37℃, and pass it through 50 mesh and 120 mesh cell filter screens in sequence. Collect the filtered cell suspension, centrifuge at 1000 r / min for 5 min, and retain the cell pellet. (6) Remove adherent cells by adhering culture: The precipitate obtained by filtration and centrifugation is mixed with chicken primary cecal epithelial cell culture medium and the cell suspension is inoculated into a culture flask and cultured at 37°C in a 5% CO2 incubator for 70 min to remove adherent cells. (7) Collect chicken cecal epithelial cells: Take out the culture flask, collect the cell suspension, centrifuge at 1000 r / min for 5 min, discard the supernatant, and then mix the cell pellet with chicken primary cecal epithelial cell culture medium. Dilute the cell suspension with trypan blue and count the number of cell clusters without blue staining using a hemocytometer for later use. (8) Primary culture of chicken cecal epithelial cells: Chicken cecal epithelial cells were cultured at a density of 1×10⁻⁶ cells / year. 6 / mL was inoculated into primary chicken cecal epithelial cell culture medium and cultured in a 37℃, 5% CO2 incubator. The medium was changed every 2 days. After 48 h, the cells grew into a "brick-shaped" monolayer of epithelial cells with a density of 80%-90%.

[0032] 3. Cell morphology observation After 48 hours of culture, cell morphology was observed under an inverted phase-contrast microscope. Figure 2 As shown, the cells grow in a typical "paving stone" or "brick" monolayer pattern, with clear cell boundaries, tight intercellular connections, and a clean background, without obvious fibroblast contamination. The cell density can reach 80%-90%, indicating that the cells obtained by this method have excellent adhesion and proliferation capabilities.

[0033] Example 2: Immunofluorescence identification of primary epithelial cells 1. CK18 immunofluorescence identification of epithelial-derived [substances / types] Coverslips containing primary chicken cecal epithelial cells (cultured for 48 h) were removed, washed three times with PBS for 3 min, and then fixed with 4% paraformaldehyde for 20 min. The cells were then washed three times with PBS for 3 min, permeabilized with 0.5% Triton X-100 at room temperature for 15 min, washed three times with PBS for 3 min, and blocked with 5% goat serum for 30 min. The cells were then incubated overnight at 4°C with CK18 antibody (1:350, bs-1339R, Bioss, China). The cells were then washed three times with PBS for 3 min, incubated with FITC-labeled secondary antibody (1:100, AB0101, Abways, China) at 37°C for 1 h, washed three times with PBS for 3 min, and the cell nuclei were stained with DAPI. After washing four times with PBS for 5 min each time, the samples were observed using a confocal microscope.

[0034] The results are as follows Figure 3 As shown: CK18 is expressed as a green fluorescent positive expression in the cytoplasm, and the cell nucleus appears blue when stained with DAPI. Merge images show that all cells stained with DAPI express CK18, confirming that the obtained cells are epithelial cells with high purity.

[0035] 2. E-cadherin immunofluorescence identification of tight junctions Coverslips containing primary cecal epithelial cells cultured for 48 h were removed and washed three times with PBS for 3 min each. Cells were then fixed with 4% paraformaldehyde for 20 min. The cells were washed three times with PBS for 3 min each, permeabilized with 0.5% Triton X-100 at room temperature for 15 min, washed three times with PBS for 3 min each, and blocked with 5% goat serum for 30 min. The cells were then incubated overnight at 4°C with E-cadherin antibody (1:350, bs-1016R, Bioss, China). The cells were then washed three times with PBS for 3 min each, incubated with FITC-labeled secondary antibody (1:100, AB0101, Abways, China) at 37°C for 1 h, washed three times with PBS for 3 min each, and the cell nuclei were stained with DAPI. After washing four times with PBS for 5 min each, the samples were observed using a confocal microscope.

[0036] The results are as follows Figure 4As shown, E-cadherin was used to identify cultured primary chicken cecal epithelial cells. Fluorescence microscopy revealed that E-cadherin expressed green fluorescence in the cytoplasm and cell membrane, with high expression levels and a uniform and continuous distribution. DAPI-stained cell nuclei were observed in the same field of view. Merge images show the expression level of E-cadherin in the field of view. These results indicate that the barrier of primary chicken cecal epithelial cells maintained good tightness after 48 h of culture.

[0037] The above description is merely a preferred embodiment of this application and is not intended to limit the application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications made within the spirit and principles of this application are not permitted. Equivalent substitutions, improvements, etc., should all be included within the scope of protection of this application.

Claims

1. A method for isolating and primary culturing chicken cecal epithelial cells, characterized in that, The method is as follows: Chicken embryo cecum is taken, washed in a three-level gradient decreasing double antibiotic washing solution, cut into pieces, and digested with thermophilic protease digestion solution; the digestion solution is diluted with serum-containing culture medium and filtered through 50-mesh and 120-mesh sieves in sequence, and the cells are collected by centrifugation; the cell suspension is cultured for 70 min to remove adherent cells, and the non-adherent epithelial cells are collected and cultured at 37℃ and 5% CO2 to obtain a monolayer of epithelial cells.

2. The method for isolation and primary culture according to claim 1, characterized in that, The method includes the following steps: (1) Remove the cecum: Disinfect the outer shell of the chicken embryo that has been incubated for 18 days, remove the chicken embryo, pick out the cecum, and temporarily store it in cecum washing solution No. 1; (2) Remove mesentery and blood: After separating the chicken cecum and removing the mesentery, place it in cecum washing solution No. 2; (3) Washing: After removing the mesentery, wash with No. 3 cecal washing solution. After the supernatant is clear, cut the cecal tissue into a paste. Then wash with No. 3 cecal washing solution and PBS in sequence and set aside. (4) Tissue digestion: Digest with thermophilic protease digestion solution at 37℃ and 80-200 r / min for 5-30 min with shaking. (5) Filtration and centrifugation: After digestion, blow the enzyme digestion solution for 3-5 minutes, dilute the digestion solution with DMEM / F12 culture medium containing 10% fetal bovine serum preheated to 37°C, and pass it through 50-mesh and 120-mesh cell filter screens in sequence. Collect the filtered cell suspension, centrifuge and retain the cell pellet. (6) Remove adhering cells by adhering culture: The precipitate obtained by filtration and centrifugation is mixed with chicken primary cecal epithelial cell culture medium and the cell suspension is inoculated into a culture flask and cultured in a 37°C, 5% CO2 incubator to remove adhering cells. (7) Collect chicken cecal epithelial cells: Take out the culture flask, collect the cell suspension, centrifuge, discard the supernatant, and then mix the cell pellet with chicken primary cecal epithelial cell culture medium. Dilute the cell suspension with trypan blue and count the number of cell clusters without blue staining using a hemocytometer for later use. (8) Primary culture of chicken cecal epithelial cells: Chicken cecal epithelial cells were cultured at a density of 1×10⁻⁶ cells / year. 6 / mL was inoculated into primary chicken cecal epithelial cell culture medium and cultured in a 37℃, 5% CO2 incubator. The medium was changed every 2 days. After 48 h, the cells grew into a "brick-shaped" monolayer of epithelial cells with a density of 80%-90%.

3. The method for isolation and primary culture according to claim 2, characterized in that, The ratio of PBS to penicillin antibody in the No. 1 cecal washing solution is 9:1; the ratio of PBS to penicillin antibody in the No. 2 cecal washing solution is 19:1; and the ratio of PBS to penicillin antibody in the No. 3 cecal washing solution is 39:

1. The bispecific antibody consists of penicillin and streptomycin sulfate.

4. The method according to claim 2, characterized in that, The thermophilic protease digestion solution is prepared by dissolving thermophilic protease in HEPES buffer and stirring thoroughly until completely dissolved, adjusting the pH to 7.4, and then filtering to remove bacteria.

5. The method according to claim 2, characterized in that, The chicken primary cecal epithelial cell culture medium was based on DMEM / F12, supplemented with 10% fetal bovine serum, 1% 50 mmol / L L-glutamine solution, 1% 0.11 g / mL sodium pyruvate solution, 1% 10 g / L sodium heparin solution, 1% 250 mg / L insulin solution and 1% penicillin-dextrin solution.

6. The method according to claim 2, characterized in that, The primary culture cell seeding density was 1×10⁻⁶. 6 per mL.

7. The method according to claim 2, characterized in that, The chicken cecal epithelial cells were cultured in an environment of 37°C and 5% CO2.

8. The application of chicken cecal epithelial cells isolated and cultured by the method of any one of claims 1-7 in the preparation of in vitro cell models for the study of intestinal barrier function, pathogen infection or nutritional metabolism in poultry.

9. A kit for isolating and primary culturing chicken cecal epithelial cells, characterized in that, include: Cecal washing solution No. 1, cecal washing solution No. 2, cecal washing solution No. 3, thermophilic bacteria protease digestion solution, and chicken primary cecal epithelial cell culture medium.

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