Method for culturing colonic epithelial cells of horses
Through sterile operation and specific culture conditions, horse colon epithelial cells were successfully isolated and cultured, solving the problem of lack of equine colon epithelial cell culture methods in the prior art, achieving efficient and low-cost cell isolation and purification, providing a basis for the study of intestinal diseases in equine animals.
Patent Information
- Application Number
- CN202510445369.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-10
- Publication Date
- 2025-06-24
AI Technical Summary
The lack of isolation and culture methods for colonic epithelial cells of equine animals in the prior art makes it difficult to study the colonic epithelial cells of equine animals.
Horse colon tissue was collected by sterile operations, and after oil layer removal, DPBS cleaning, tissue block cutting and culture medium addition, cell culture was cultured at 37°C and 5% CO2, and purified horse colon epithelial cells were obtained by passage and removal of fibroblasts.
An in vitro model of equine colon epithelial cells was successfully established. The procedure was simple and low-cost, which could effectively remove impurity cells and improve separation efficiency, and was suitable for subsequent intestinal disease research.
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Abstract
Description
Technical Field
[0001] The present invention relates to the technical field of cell separation and culture, and more particularly to a method for culturing equine colonic epithelial cells. Background Art
[0002] In recent years, with the improvement of people's living standards and the development of the livestock industry, the breeding of equine animals has been greatly improved. The prevention and treatment of diseases are the key to breeding efficiency, and colitis is one of the major diseases that endanger the health of equine animals. This disease not only has a high incidence but is also difficult to cure, and once it occurs, it will cause huge losses to the breeding industry.
[0003] The intestine is the main digestive organ in the body, which can promote digestion, extract nutrients, participate in immune surveillance, maintain a key symbiotic relationship with the microbiota and affect overall health. The intestine is also an important defense organ of the body, which can prevent harmful molecules from invading the boundary of the mucosal tissue. The intestine is composed of various types of cells, such as epithelial cells, Paneth cells, macrophages and lymphocytes, which constitute the intestinal immune system and play an important role in maintaining intestinal homeostasis by producing antibacterial substances and controlling the host-symbiont balance. Intestinal epithelial cells (IECs) are one of the most highly organized and fastest-renewing cells in the body, and are the first line of defense for mammals to isolate the external environment, belonging to a physical barrier; the intestinal mucosal barrier composed of intestinal epithelial cells can help the body isolate pathogens or symbiotic microorganisms in the intestinal lumen environment, and is also a coordination hub for immune defense and crosstalk between bacteria and immune cells. Therefore, the isolation and culture of intestinal epithelial cells is one of the main means to study the regulatory factors of intestinal epithelial cell proliferation and differentiation, and substance absorption and metabolism, and is an ideal experimental model for studying the biological functions of intestinal mucosal epithelial cells.
[0004] Since Blay et al. isolated rat intestinal epithelial cells more than 40 years ago, the isolation and culture system of intestinal epithelial cells of animals such as humans, cattle, pigs, sheep, and poultry has become increasingly mature in order to obtain highly pure, highly viable and functional intestinal epithelial cells. However, there is no literature report on the culture system of equine animal intestinal epithelial cells.
[0005] In the prior art, the methods commonly used for extracting and separating intestinal epithelial cells are mainly enzyme digestion method and tissue explant method. The enzyme digestion method can obtain primary cells relatively quickly, but the cost of various enzymes is relatively high and it will cause varying degrees of damage to the cells, resulting in slow cell growth and affecting the activity and function of the cells. The enzyme digestion method requires precise control of enzyme concentration and digestion time, and the operation is relatively cumbersome. The tissue explant method has a simple procedure, low cost and the cells are not treated with any enzymes. Currently, the isolation of intestinal epithelial cells is mainly carried out in ruminants and some other small animals. Equidae animals belong to typical monogastric herbivorous large animals with a special digestive system. The posterior segment of the digestive tract includes the cecum and colon, which are very large in volume and are inhabited by a huge number and variety of microorganisms. Therefore, attention should be paid to avoiding microbial contamination when isolating and culturing equine colonic epithelial cells.
[0006] In summary, how to provide a method for isolating and culturing equine colonic epithelial cells is an urgent problem to be solved by those skilled in the art. Summary of the Invention
[0007] In view of this, the present invention provides a method for culturing equine colonic epithelial cells.
[0008] In order to achieve the above object, the present invention adopts the following technical scheme:
[0009] A method for culturing equine colonic epithelial cells, comprising the following steps:
[0010] (1) Collecting equine colonic tissue from 1-year-old horses under sterile operation;
[0011] (2) Removing the grease layer from the colonic tissue and washing it with sterile normal saline;
[0012] (3) Cutting the tissue blocks into 1 cm 2 , and washing with DPBS washing solution;
[0013] (4) Cutting the tissue blocks into 1 mm 3 , and washing with DPBS washing solution;
[0014] (5) Discarding the DPBS washing solution and placing it in a cell culture flask for culture;
[0015] (6) Changing the culture medium every 2 days and observing the cell growth status;
[0016] (7) When the cells grow to 80-90%, subculture is carried out;
[0017] (8) Gradually removing fibroblasts during the culture process to obtain purified equine colonic epithelial cells.
[0018] Furthermore, the components of the culture medium: adding 15% FBS and 1% 1× penicillin & streptomycin to DMEM / F12 medium.
[0019] Further, in the step (5), the culture conditions are 37°C and 5% CO2.
[0020] Further, in the step (5), the cell culture flask is first placed upright for 2 - 3 h and then placed horizontally.
[0021] Further, in the step (7), the specific operation of subculture is as follows: The cells are digested with 0.25% trypsin - EDTA until the cells become round and float up, then the medium is added to terminate the digestion, and the cells are collected and continue to be cultured in a new culture flask.
[0022] Further, in the step (8), the fibroblast cells are removed by mechanical scraping method and differential digestion method.
[0023] It can be seen from the above - mentioned technical solutions that, compared with the prior art, the beneficial effects achieved by the present invention are as follows:
[0024] The present invention first uses the colon tissue of a large monogastric herbivore, the horse, to establish a horse colon epithelial cell model in vitro. This method has a simple procedure and low cost. It can avoid the complex in - vivo environment, study the effects of specific conditions and treatments on the cell growth state, and is not affected by individual differences. At the same time, it can also effectively remove the impurity cells generated during the isolation and culture process, thereby improving the separation efficiency of colon epithelial cells. The establishment of the horse colon epithelial cell in - vitro model can provide a basis for subsequent research on intestinal diseases. Compared with the enzyme digestion method, the present invention can reach about 80% at 13 days of primary cell culture and can perform subculture operations. BRIEF DESCRIPTION OF THE DRAWINGS
[0025] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only the embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained according to the provided drawings without creative efforts.
[0026] Figure 1 It is the observation diagram under an inverted microscope (40×) of the primary horse colon epithelial cells cultured for 6 days in Example 1 of the present invention; Starting from step (5) as the first day, the same below;
[0027] Figure 2 It is the observation diagram under an inverted microscope (40×) of the primary horse colon epithelial cells cultured for 8 days in Example 1 of the present invention;
[0028] Figure 3 It is the observation diagram under an inverted microscope (40×) of the primary horse colon epithelial cells cultured for 13 days in Example 1 of the present invention;
[0029] Figure 4 Observation diagram under an inverted microscope (100×) after culturing primary equine colonic epithelial cells for 13 days in Example 1 of the present invention;
[0030] Figure 5 Observation diagram under an inverted microscope (40×) after culturing the second-generation equine colonic epithelial cells for 24 h in Example 1 of the present invention;
[0031] Figure 6 Growth rate result of the second-generation equine colonic epithelial cells in Example 1 of the present invention;
[0032] Figure 7 Immunofluorescence identification result diagram (100×) of the second-generation equine colonic epithelial cells in Example 2 of the present invention, wherein, (+) represents that the primary antibody is keratin CK-18 antibody; (-) represents that the keratin CK-18 antibody is replaced with PBS;
[0033] Figure 8 Result diagram of fluorescence quantitative PCR analysis of CK-18 and CDH1 gene expression in Example 3 of the present invention;
[0034] Fig. 9 Observation result under an inverted microscope (40×) after culturing the primary cells of equine colonic epithelial cells prepared by the culture method of the present invention and the enzyme digestion method for 13 days, wherein, A represents the culture method of the present invention; B represents the enzyme digestion method. Detailed implementation manners
[0035] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.
[0036] The medicaments required for the present invention are conventional experimental medicaments, purchased from commercial channels; the experimental methods not mentioned are conventional experimental methods, which will not be elaborated here one by one.
[0037] The materials and instruments used in the following examples are as follows:
[0038] (1) Samples
[0039] Equine colonic tissue (collect the colonic tissue of a horse about 1 year old from the slaughterhouse, place it in a thermos cup containing sterile physiological saline and 1×PSN (penicillin & streptomycin) for preservation, and quickly bring it back to the laboratory for processing).
[0040] (2) Main reagents
[0041] Table 1 Main Reagents
[0042]
[0043]
[0044] (3) Main Instruments
[0045] Table 2 Main Instruments
[0046] name factory ELISA reader BioTek Corporation Inverted microscope Olympus Fluorescence quantitative PCR detection system Bio-Rad Transfer decolorization shaker Qilin Bell Instrument Manufacturing Co., Ltd. Sterile operating table OptiMAIR Cell culture incubator OptiMAIR Constant temperature water bath BluePard Benchtop centrifuge Sigma 24-well round cell slide Shanghai Wohong Biotechnology Co., Ltd.
[0047] Example 1
[0048] Primary Isolation and Culture Method of Equine Colonic Epithelial Cells
[0049] (1) Collect about 10 cm of colonic tissue from a horse around 1 year old at the slaughterhouse, place it in a thermos cup containing sterile saline and 1×PSN (penicillin & streptomycin) for preservation, and quickly bring it back to the laboratory for processing (collect the colonic segment with less intestinal content to reduce the contamination ratio). 2 The colonic tissue, with the fat layer removed, is washed 1 - 2 times with sterile saline, cut into 1 cm
[0050] (2) small pieces with surgical scissors, and placed in a 50 mL sterile centrifuge tube containing sterile saline and quickly transferred to a laminar flow hood. 2 The small pieces of colonic tissue after removing the fat layer are transferred to a 15 mL sterile centrifuge tube containing DPBS washing solution (containing 1×PSN), shaken vigorously, left standing for 5 minutes, the tissue pieces are taken out and transferred to a new tube, and this operation is repeated again.
[0051] (3) After removing the fat layer, the 1 cm 2 small pieces of colonic tissue are transferred to a 15 mL sterile centrifuge tube containing DPBS washing solution (containing 1×PSN), shaken vigorously, left standing for 5 minutes, the tissue pieces are taken out and transferred to a new tube, and this operation is repeated again.
[0052] (4) After washing, the 1 cm 2 small pieces of colonic tissue are transferred to a 1.5 mL centrifuge tube and further cut into 1 mm 3 fragments, then transferred to a 15 mL centrifuge tube, added with DPBS washing solution (containing 1×PSN), pipetted for 1 minute, left standing for 5 minutes, the supernatant is discarded, and this operation is repeated 3 times.
[0053] (5) Discard the DPBS washing solution, evenly spread the 1 mm 3 small pieces of colonic tissue after washing into a T25 cell culture flask, add 5 mL of medium (15% FBS, 1% 1× penicillin & streptomycin, and DMEM / F12 medium) to the T25 cell culture flask, place the T25 cell culture flask upright in the incubator for 2 - 3 hours, then place the T25 cell culture flask horizontally for continued culture (placing it upright in the incubator for 2 - 3 hours is to make the tissue pieces firmly adhere to the bottom of the cell culture flask), and the culture conditions are 37°C and 5% CO2.
[0054] (6) After 2 - 3 days of culture, add 1 mL of medium (15% FBS, 1% 1× penicillin & streptomycin, and DMEM / F12 medium) to the T25 cell culture flask. Then change the medium every 2 days, and observe the cell growth status daily. When the tissue blocks are all attached to the bottom of the cell flask and cells crawl out around them and grow rapidly, it indicates that the primary culture is initially successful.
[0055] (7) When the cells grow to 80% - 90%, subculture is carried out. The specific operation is as follows:
[0056] Cell cryopreservation: Discard the medium, wash with DPBS washing solution (containing 1×PSN) 1 - 2 times, add 1 mL of 0.25% trypsin - EDTA and incubate in the incubator for 1 - 2 minutes. Observe under the microscope. When a large number of cells become round and float up, add an equal volume of medium (15% FBS, 1% 1× penicillin & streptomycin, and DMEM / F12 medium) to terminate digestion. Collect the digestion solution in a 15 mL centrifuge tube, centrifuge at 1500 rpm / min for 3 min, discard the supernatant, resuspend and continue to culture in a new culture flask or culture dish; or add 1 mL of cell cryopreservation solution, transfer to a 2 mL cryotube, label it, place it in a programmable cooling cryopreservation box and keep it overnight in an - 80°C refrigerator, and transfer it to liquid nitrogen for long - term storage on the second day.
[0057] Cell resuscitation: Take out the cryotube containing equine colonic epithelial cells from liquid nitrogen, quickly put it into a 37°C water bath and shake to melt it; after melting, aspirate the cryopreservation solution into a 15 mL centrifuge tube, then add 5 mL of medium (15% FBS, 1% 1× penicillin & streptomycin, and DMEM / F12 medium), centrifuge at 1500 rpm / min for 3 min; discard the supernatant, add 1 mL of medium, pipette evenly and inoculate into a cell culture dish containing 2 mL of medium or a cell culture flask containing 4 mL of medium, and culture in an incubator at 37°C and 5% CO2, and change the medium every 2 days.
[0058] (8) Use mechanical scraping method or differential digestion method to gradually remove fibroblasts during the culture process to obtain purified equine colonic epithelial cells;
[0059] If the distribution of equine colonic epithelial cells and fibroblasts is relatively clear, a pipette tip or a cell scraper can be used to scrape up the fibroblasts (when scraping, it should be gentle to avoid serious damage to the bottom of the culture flask and affecting cell growth);
[0060] The differential digestion method utilizes the characteristic of the different tolerances of epithelial cells and fibroblasts to enzymatic digestion. Add 0.25% trypsin-EDTA to the culture flask and observe under a microscope. When the fibroblasts become round and float up, add an equal volume of culture medium to terminate digestion. At this time, discard the digestion solution to remove the fibroblasts (it is necessary to observe the cell condition at all times to avoid over-digestion causing the equine colonic epithelial cells to float up).
[0061] Figure 1 to Figure 5 It is the observation diagram under an inverted microscope during cell culture. It can be seen that on the 6th day, some colonic epithelial cells crawled out and grew mixed with fibroblasts ( Figure 1 ). After purification, on the 8th day, the culture flask mainly contained colonic epithelial cells ( Figure 2 ). On the 13th day of culture, the cells had basically covered the bottom of the culture flask and could be subcultured ( Figure 3 、 Figure 4 ). The equine colonic epithelial cells after cryopreservation and resuscitation culture for 24 h were oval, long strip-shaped or island-shaped. A large number of cells adhered to the wall and proliferated and divided, and the cell state was good ( Figure 5 ).
[0062] Digest the equine colonic epithelial cells in the good growth period and use the CCK-8 method to draw the cell growth curve. Inoculate the equine colonic epithelial cells into a 96-well plate at a concentration of 5×10 4 cells / mL, divided into 8 groups in total, and culture in an incubator at 37°C and 5% CO2; add 10 μL of CCK-8 reagent to each well at 1, 2, 3, 4, 5, 6, 7, and 8 d after inoculation respectively, measure the absorbance (OD) value at 450 nm with an enzyme-linked immunosorbent assay (ELISA) reader, calculate the average value of each group, use the culture time as the abscissa and the average OD value as the ordinate to draw the growth curve of equine colonic epithelial cells.
[0063] The results of the cell growth rate are as Figure 6 shown. The growth curve of equine colonic epithelial cells is in an "S" shape. During the culture for 1-4 d, it is in the latent period and the cell growth is relatively slow; during the culture for 4-6 d, it is in the logarithmic growth phase and the cell proliferation rate significantly accelerates; after the culture for 6-8 d, it enters the plateau phase and the cell proliferation rate slows down. Therefore, when the cells proliferate to a certain number, subculture should be carried out in time, otherwise it will affect the subsequent experiments.
[0064] Example 2
[0065] Immunofluorescence staining identification
[0066] The equine colonic epithelial cells used in this example are the second-generation cells cultured to more than 80%.
[0067] ① Prepare a coverslip: Use forceps to place the coverslip soaked in alcohol into a 24-well plate, digest the equine colonic epithelial cells, resuspend them, and then inoculate them at 5×10 4Add to a 24-well plate at [number] cells / mL. After the cell density reaches 60% - 70%, discard the culture medium and wash 3 times with DPBS washing solution.
[0068] ② Fixation: Fix with 4% paraformaldehyde for 25 min. After discarding the liquid, wash 3 times with DPBS washing solution.
[0069] ③ Permeabilization: Add 0.1% Triton permeabilization solution and let stand for 30 min. After discarding the liquid, wash 3 times with DPBS washing solution.
[0070] ④ Blocking: Add 3% goat serum to block for 1 h.
[0071] ⑤ Incubate with primary antibody: After discarding the goat serum, add mouse-derived cytokeratin 18 primary antibody and incubate overnight at 4°C.
[0072] ⑥ Incubate with secondary antibody: Take out the next day, wash 3 times with DPBS washing solution on a shaker, then add donkey anti-mouse fluorescent secondary antibody, incubate for 1 h on a shaker in the dark, and wash 3 times with DPBS.
[0073] ⑦ DAPI nuclear staining: Add DAPI and stain in the dark for 10 min, then wash 3 times with DPBS washing solution on a shaker.
[0074] ⑧ Mounting: Drop the anti-fluorescence quencher onto the glass slide, pick out the cell culture insert and invert it onto the glass slide to complete the mounting. Observe immediately under the microscope or store in a 4°C slide box in the dark.
[0075] Figure 7 The result is the immunofluorescence staining. Cytokeratin CK-18 is one of the cytokeratins that make up the cytoskeleton in epithelial cells. The expression of cytokeratin CK-18 in equine colonic epithelial cells was analyzed using immunofluorescence staining technology. The results showed that red fluorescence was emitted around the detected cells, indicating that after positive staining, the cells could normally express cytokeratin CK-18.
[0076] Example 3
[0077] Real-time fluorescence quantitative PCR identification
[0078] In this example, the second-generation equine colonic epithelial cells and the third-generation fibroblasts were cultured to more than 80%.
[0079] The total RNA of equine colonic epithelial cells and fibroblasts was extracted using the Trizol method. After measuring its concentration and purity using an enzyme-linked immunosorbent assay (ELISA) reader, the RNA was reverse transcribed into cDNA using a reverse transcription kit. According to the gene design primer sequences (the primer sequence information is shown in Table 3), GAPDH was used as the internal reference gene in both groups for real-time fluorescence quantitative PCR amplification, and the differential expression in the detected genes was monitored in real time using the accumulation of fluorescence signals. After data statistics, the 2- △△Ct Calculate the relative expression levels of CK-18 and CDH1.
[0080] Table 3 Primer sequence information
[0081]
[0082] The results showed that the relative expression levels of CK-18 and CDH1 genes in equine colonic epithelial cells were significantly higher than those in fibroblasts (the expression of CK-18 and CDH1 genes was as shown in Figure 8 ), proving that equine colonic epithelial cells were successfully isolated in this experiment.
[0083] Comparative Example 1
[0084] Preparation of equine colonic epithelial cells by enzymatic digestion method
[0085] Enzymatic digestion method: Collect small pieces of colonic tissue from a 1-year-old healthy Mongolian horse in a Mongolian horse farm in Hohhot, Inner Mongolia Autonomous Region. Put the colonic tissue into a sterile normal saline thermos cup containing antibiotics and quickly bring it back to the laboratory. Remove the external adipose tissue, cut the remaining tissue into 1 cm 2 small pieces and put them into a 50 mL centrifuge tube containing sterile normal saline, and quickly transfer them to a laminar flow hood. Wash the tissue repeatedly with DPBS washing solution until the liquid is clear, transfer it to a 1.5 mL centrifuge tube and continue to cut it into fragments about 1 mm 3 in size, then repeatedly pipette it with DPBS washing solution in a 15 mL centrifuge tube until it is clear. Discard the supernatant, add 5 mL of separation and digestion solution (3% type I collagenase and DMEM / F12 medium), and digest it in a 37 °C water bath for 40 min, shaking vigorously every 10 min. After digestion, pipette it repeatedly for 2 min until the digestion solution becomes turbid, and let it stand for 1 min. Take the supernatant and put it into a 15 mL centrifuge tube, and add an equal amount of complete medium (15% FBS, 1% 1× penicillin & streptomycin and DMEM / F12 medium). Centrifuge at 1500 rpm for 5 min, discard the supernatant, add 5 mL of complete medium to resuspend and centrifuge at the same speed, repeat twice. Discard the supernatant, resuspend and add it to the culture dish, shake it crosswise to make the cells evenly distributed, and culture it in a 37 °C, 5% CO2 incubator. After 90 min, transfer the liquid in the culture dish to a new dish and continue to culture it in the incubator.
[0086] The equine colonic epithelial cells prepared by the culture method of the present invention and the enzymatic digestion method are as shown in Fig. 9 . The equine colonic epithelial cells cultured by the enzymatic digestion method showed a cobblestone-like shape and grew in patches at 13 d, but were not sufficient for subculture. The cells cultured by the method of the present invention had basically covered the bottom of the culture flask at 13 d of the cultured equine colonic epithelial cells and could be subcultured.
[0087] In the present specification, the various embodiments are described in a progressive manner. Each embodiment focuses on the differences from other embodiments, and the same or similar parts among the various embodiments can be referred to each other.
[0088] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be obvious to those skilled in the art, and the general principles defined herein can be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. A method for culturing horse colon epithelial cells, characterized in that: The steps include: (1) Colon tissues were collected from 1-year-old horses using sterile techniques; (2) Remove the fat layer in the colon tissue and wash with sterile saline; (3) Cut the tissue block into 1 cm 2 , washed with DPBS; (4) Cut the tissue block into 1 mm 3 , washed with DPBS; (5) Discard the DPBS washing solution and place the cells in a cell culture flask for culture; (6) Change the medium every 2 days and observe the cell growth status; (7) When the cells grow to 80-90%, subculture; (8) Fibroblasts were gradually removed during the culture process to obtain purified equine colon epithelial cells.
2. The culture method according to claim 1, characterized in that The composition of the culture medium: 15% FBS and 1% 1× penicillin & streptomycin were added to DMEM / F12 culture medium.
3. The culture method according to claim 1, characterized in that In the step (5), the culture conditions are 37° C. and 5% CO 2 .
4. The culture method according to claim 1, characterized in that In the step (5), the cell culture flask is first placed upright for 2 to 3 hours and then placed horizontally.
5. The culture method according to claim 1, characterized in that In step (7), the specific operation of subculture is: digest the cells with 0.25% trypsin-EDTA until the cells become round and float, then add culture medium to terminate the digestion, collect the cells and continue to culture them in a new culture bottle.
6. The culture method according to claim 1, characterized in that In the step (8), mechanical scraping and phase contrast digestion are used to remove fibroblasts.