Isolated culture method of primary jejunum epithelial cells of meat calves

Abstract

The invention belongs to the field of biochemistry, and discloses an isolated culture method of jejunum epithelial cells of primary meat calves, which comprises the following steps: step 1, collecting jejunum tissues of newly born non-milk-sucking calves, cleaning, digesting by using enzyme digestive juice, and collecting the jejunum epithelial cells after digesting; 2, performing purification and passage on the jejunum epithelial cells separated in the step 1 by adopting a cell culture medium through a differential adhesion method; 3, the jejunum epithelial cells obtained through passage in the step 2 are digested and then added into a cell cryopreservation solution for cryopreservation; according to the method, the primary jejunum epithelial cell strain of the meat calf is successfully separated and obtained, and the method has the advantages of being good in separation effect, high in cell passage efficiency, high in cell viability, high in cell survival rate after recovery and the like.

Description

Technical Field

[0001] This invention relates to the field of biology, specifically to a method for isolating and culturing primary jejunal epithelial cells of beef calves. Background Technology

[0002] The intestinal structure of ruminants is more unique than that of monogastric animals. The jejunum, the longest segment of the small intestine in cattle, is the primary site of nutrient digestion and absorption, responsible for absorbing the vast majority of nutrients essential for the growth, weight gain, and reproduction of beef cattle. Jejunal epithelial cells are the most functionally core and structurally specialized cell group in the small intestine of beef cattle, accounting for nearly 80% of the intestinal mucosal epithelial cell population and forming the structural and physiological basis of jejunal absorptive function. The jejunal epithelial cell membrane is embedded with a wide variety of highly specific transport proteins and channel proteins, precisely regulating the absorption of different substances: jejunal epithelial cells possess extremely high metabolic activity and substance transport capacity, ensuring rapid and large-scale nutrient uptake from the continuously flowing chyme. Furthermore, the jejunal epithelial cells and their interconnections together constitute a crucial physiological barrier: preventing large molecules, toxins, and pathogens from freely entering the interstitial spaces and bloodstream. This barrier selectively allows nutrient absorption while preventing the invasion of harmful substances. Impaired barrier function (e.g., due to infection, inflammation, toxins) can trigger local or even systemic inflammatory responses, poor nutrient absorption, and stunted growth, threatening the health and production performance of beef cattle. Therefore, in vitro isolation and culture of jejunal epithelial cells is of great significance for studying small intestinal physiological function, nutrient absorption mechanisms, drug effects, and pathophysiological changes under the influence of various pathogenic factors.

[0003] Currently, there are four methods for isolating jejunal epithelial cells: tissue block culture, mechanical separation, non-enzymatic digestion (using chelation or depolymerization solutions), and enzymatic digestion. Tissue block culture produces highly viable cells, but it also results in a higher proportion of contaminating cells and bacterial contamination, making it difficult to obtain complete intestinal epithelial cells. Mechanical separation uses external force to disrupt intercellular and cell-matrix connections, releasing free cells; this method is suitable for research scenarios involving enzyme sensitivity or where chemical interference must be avoided. Non-enzymatic digestion uses a salt-buffered reagent containing EDTA to treat sample tissue, resulting in relatively intact intestinal epithelial cells; however, high EDTA concentrations can be toxic to cells, causing cell death. Therefore, the concentration of EDTA and the digestion time must be carefully controlled during cell separation. The greatest advantage of enzymatic digestion is its ability to protect cell-cell interactions, but it can damage the integrity of cell surface receptors, affecting subsequent studies on cell receptor function. Furthermore, different digestive enzymes have varying effects on intestinal epithelial cells. Trypsin and streptomycin, when used together, have strong digestive power but low cell viability. Collagenase and neutral dispase, when used together, have weaker digestive power, cause less damage to cells, and have higher cell viability, but require multiple digestion cycles with careful control of enzyme concentration and digestion time. Cells isolated from high concentrations of collagenase and neutral dispase are more dispersed, appearing as single cells or cell clusters, and have poor adhesion. Additionally, when proteases digest intestinal mucosal tissue, they also separate smooth muscle cells and fibroblasts from the outer wall of the intestinal tract, resulting in mixed cells and increasing the difficulty of purification.

[0004] The success rate of in vitro culture of jejunal epithelial cells from adult cattle is extremely low because the intestines of adult cattle contain a large amount of food residue and microbial flora, which can easily cause contamination. Moreover, the intestinal epithelial cells of adult cattle are highly differentiated and can hardly adhere to the wall for growth.

[0005] Therefore, successfully isolating, passaged, and cryopreserving bovine jejunal epithelial cells is a challenging task. Summary of the Invention

[0006] The purpose of this invention is to provide a method for isolating and culturing primary jejunal epithelial cells of beef calves. This method successfully isolates primary jejunal epithelial cell lines of beef calves, and has advantages such as good isolation effect, high cell passage efficiency, strong cell activity, and high cell survival rate after resuscitation.

[0007] Unless otherwise specified in this invention: mM represents millimoles per liter, nM represents nanomoles per liter, and μM represents micromoles per liter;

[0008] To achieve the above objectives, the present invention provides the following technical solution: a method for isolating and culturing primary jejunal epithelial cells of beef calves, comprising the following steps:

[0009] Step 1: Collect jejunal tissue from newborn calves that have not suckled, clean it, digest it with an enzyme digestion solution, and collect the jejunal epithelial cells after digestion;

[0010] Step 2: The jejunal epithelial cells obtained in Step 1 were purified and passaged using cell culture medium via differential adhesion.

[0011] Step 3: Digest the jejunal epithelial cells obtained from step 2 and add them to the cell cryopreservation solution for cryopreservation;

[0012] The enzyme digestion solution includes a basal culture medium and neutral protease II and collagenase I added to the basal culture medium; the cell culture medium includes a basal culture medium and FBS, antibiotics, NEAA and buffer added to the basal culture medium; the cell cryopreservation solution is composed of FBS and DMSO, and the FBS content in the cell cryopreservation solution is 90~92wt%.

[0013] In the above-described isolation and culture method, the concentration of neutral proteinase II in the enzyme digestion solution is 4.5~5 mg / ml, and the concentration of collagenase I is 200~400 U / ml.

[0014] In the above-described isolation and culture method, the concentration of FBS in the cell culture medium is 13-17 wt% FBS, 0.8-1.2 wt% antibiotic, and 0.8-1.2 wt% NEAA.

[0015] In the above-described isolation and culture method, in step 3, a digestion termination solution is used to terminate digestion; the digestion termination solution includes basal culture medium, FBS, and PS; the amount of FBS is 3~7 wt%; and the amount of PS is 0.8~1.2 wt%.

[0016] In the above isolation and culture method, step 1 specifically includes:

[0017] Step 11: Collect jejunal tissue from newborn calves that have not suckled, and rinse repeatedly with PBS solution or physiological saline to remove all intestinal contents until the washing solution is clear; cut the jejunal tissue into segments and place them in PBS solution containing penicillin and streptomycin for further processing.

[0018] Step 12: Longitudinally cut open the jejunum segment, remove the mesentery and adipose tissue, rinse thoroughly with PBS, sterilize, and rinse repeatedly with PBS solution containing penicillin and streptomycin.

[0019] Step 13: Completely immerse the jejunal tissue in a sterile centrifuge tube containing enzyme digestion solution, and slowly shake it on a shaker at 37°C for 20-40 minutes.

[0020] Step 14: Transfer the jejunal tissue into a sterile culture dish, gently wash twice with PBS solution to remove the enzyme digestion solution; scrape the surface of the jejunal lumen until the epithelial layer is significantly reduced, exposing the tougher basement membrane and connective tissue underneath; collect the scrapings, suspend them in cell culture medium, centrifuge and resuspend.

[0021] In the above isolation and culture method, step 2 specifically includes:

[0022] Step 21: Purification. Use differential adhesion to purify jejunal epithelial cells; wash the jejunal epithelial cells obtained in step 1 with PBS 2-3 times, discard the supernatant, add 0.25wt% trypsin and separate and digest the cells at 37℃.

[0023] Step 22: Under a microscope, some cells begin to become round and shiny, with a few cells floating. Discard the digestion solution, add PBS solution to wash, discard the PBS solution, and continue digestion with 0.25 wt% trypsin. Stop digestion when the remaining adherent cells begin to become round and shiny, with a few cells floating, under a microscope. Wash 2-3 times with PBS solution, collect the detached cells, and resuspend the precipitate obtained by centrifugation in complete culture medium. Seed the precipitate into a cell culture plate for culture. Change the medium when the culture medium turns yellow. The cell culture plate contains the cell culture medium.

[0024] Step 23: Passage the cells at a ratio of 1:2 to 1:3. When the jejunal epithelial cells have adhered to the culture medium and reached 90% confluence, wash 2-3 times with PBS, discard the supernatant, add 0.25 wt% trypsin for digestion. Stop digestion when the remaining adherent cells begin to become round and shiny under a microscope, and a few cells are observed to float. Centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the pellet in cell culture medium, and adjust the cell density to 0.5 × 10⁻⁶ cells / cm². 4 Inoculate at a rate of 1 per mL into a new culture flask and culture.

[0025] In the above isolation and culture method, step 3 is as follows:

[0026] Wash jejunal epithelial cells 2-3 times with PBS, discard the supernatant, digest adherent jejunal epithelial cells with 0.25wt% trypsin, stop digestion when the jejunal epithelial cells become shiny, round and float, centrifuge, discard the supernatant, add cell cryopreservation solution to resuspend the precipitate, transfer to 2 ml cryovials and seal for cryopreservation.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] 1. This invention uses jejunal tissue from newborn, non-sucking calves as the source of cell material. The intestinal environment is relatively sterile, and the epithelial cells have a low degree of differentiation and strong proliferative capacity.

[0029] 2. This invention combines enzymatic digestion and mechanical separation, shortening the enzymatic digestion time, softening connective tissue, and then mechanically separating the cells. This reduces the damage of enzyme reagents to cell surface receptors, better preserving the integrity of cell surface receptors, making the isolated and cultured intestinal epithelial cells more suitable for metabolic research. Furthermore, the entire cell separation process is shorter, which is more beneficial for cell survival. Simultaneously, the combined use of neutral protease II and collagenase I allows for efficient and low-damage separation of jejunal epithelial cells.

[0030] 3. This invention simplifies the composition of the cell culture medium as much as possible, reduces production costs, and more importantly, the cell culture medium does not contain growth factors, which causes less damage to jejunal epithelial cells and helps maintain the viability of jejunal epithelial cells;

[0031] 4. The cryopreservation solution of the present invention is based on FBS, which can significantly reduce the damage to jejunal epithelial cells during cryopreservation.

[0032] By combining the above methods, the vitality of small intestinal cells was maintained, resulting in high cell resuscitation efficiency and strong vitality. Attached Figure Description

[0033] Figure 1A Microscopic image of jejunal epithelial cells isolated and cultured from beef calves. Figure 1A This is the sample that was scraped more heavily in step 4; Figure 1B Microscopic image of jejunal epithelial cells isolated and cultured from beef calves. Figure 1B It is the sample that was scraped relatively lightly in step 4;

[0034] Figure 2 Microscopic image of jejunal epithelial cells from beef calves that have been purified and passaged to the 3rd generation.

[0035] Figure 3 A growth curve of jejunal epithelial cells in beef calves;

[0036] Figure 4 Immunofluorescence identification image of jejunal epithelial cells from beef calves;

[0037] Figure 5 The image shows a cellular micrograph of a bovine jejunum tissue sample obtained by the digestion method in Comparative Example 1.

[0038] Figure 6A Microscopic image of jejunal epithelial cells cultured in cell culture medium in Example 1;

[0039] Figure 6B Microscopic image of jejunal epithelial cells cultured in the cell culture medium of Comparative Example 2;

[0040] Figure 6C Cell growth curves for Comparative Example 2 and Example 1;

[0041] Figure 7A This is a photograph of cells after resuscitation in Example 1;

[0042] Figure 7B Photographs of cells after recovery in Comparative Example 3;

[0043] Figure 8 The cell growth curves for Example 1 and Comparative Example 4 are shown.

[0044] Figure 9 The bar chart shows the cell survival rates of Example 1, Comparative Example 2, and Comparative Example 4. Detailed Implementation

[0045] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0046] Example 1

[0047] Refer to Table 1 for the reagent and liquid formulations used;

[0048] Table 1 Reagent List

[0049] Reagent Name Manufacturers Item number DMEM / F12 culture medium Gibco 10565-018 Fetal bovine serum (FBS) Gibco 10091-155 Penicillin / Streptomycin (P / S) Gibco 15140-122 Dimethyl sulfoxide (DMSO) Sigma-Aldrich D4540 Neutral protease II (Dispase II protease) Sigma-Aldrich D4693 Collagenase Type 1 Worthington LS004196 0.25% pancreatic enzyme Invitrogen 252000-056 Non-essential amino acids (NEAA, 100×) Beijing Solarbio Technology Co., Ltd. N1250 Hepes (1M) Beijing Solarbio Technology Co., Ltd. H1095 PBS buffer (pH 7.2-7.4, 1×) Beijing Solarbio Technology Co., Ltd. P1020 Penicillin / Streptomycin / Amphotericin B (100×) Beijing Solarbio Technology Co., Ltd. P7630 CK 18 (keratin 18) primary antibody Huaan Bio ET1603-8 iFluor™ 488 Goat Anti-Rabbit Secondary Antibody Huaan Bio HA1121 Alexa Flour 647-tagged goat anti-rabbit IgG Shanghai Beyotime Biotechnology Co., Ltd. A0468 4% tissue cell fixative Beijing Solarbio Technology Co., Ltd. P1110 Immunostaining blocking solution Shanghai Beyotime Biotechnology Co., Ltd. P0102 Diluent for Immunostaining Primary Antibody Shanghai Beyotime Biotechnology Co., Ltd. P0262 Diluent for Immunostaining Primary Antibody Shanghai Beyotime Biotechnology Co., Ltd. P0265 DAPI solution (ready-to-use) Shanghai Beyotime Biotechnology Co., Ltd. C1005

[0050] The formulations for each reagent are as follows:

[0051] Preparation of enzyme digestion solution: DMEM / F12 + neutral protease II (4.8 mg / ml) + collagenase I (300 U / ml);

[0052] The cell culture medium was prepared as follows: DMEM / F12 + 15wt% FBS + 1wt% triple antibiotics (penicillin-streptomycin B) + 1wt% NEAA + Hepes. The liquid was filtered through a 0.22-micron filter membrane and placed in a sterile centrifuge tube. It was placed in an incubator for 30 minutes before use to preheat and balance the pH.

[0053] Preparation of the final digestion solution: DMEM / F12 + 5wt% FBS (fetal bovine serum) + 1wt% PS. The liquid is used after being filtered through a 0.22-micron filter membrane.

[0054] The cryopreservation solution was prepared as follows: 90wt% FBS (fetal bovine serum) + 10wt% DMSO (dimethyl sulfoxide).

[0055] The method for isolating and culturing primary jejunal epithelial cells from beef calves is as follows:

[0056] I. Separation of jejunal epithelial cells:

[0057] 1. Collect jejunal tissue from newborn, non-sucking calves at a beef cattle farm. Gently and repeatedly rinse the jejunal tissue with PBS solution using a syringe until all intestinal contents are flushed out and the solution is clear. Cut the intestinal tissue into small segments of about 1 cm in length and place them in PBS solution containing 3 wt% penicillin and streptomycin. Transport the tissue back to the laboratory at low temperature and perform further processing in a clean bench.

[0058] 2. Longitudinally cut open the jejunum segment, remove as much mesentery and adipose tissue as possible, rinse thoroughly with PBS, immerse in 75 vol% alcohol for 2 min to sterilize, and rinse repeatedly with PBS solution containing 1 wt% penicillin and streptomycin (about 10 times).

[0059] 3. Completely immerse the jejunal tissue in a sterile centrifuge tube containing enzyme digestion solution and gently shake it on a shaker at 37°C for 30 minutes;

[0060] 4. Transfer the jejunal tissue to a sterile culture dish and gently wash twice with PBS solution to remove the enzyme digestion solution. Use a scalpel blade or sterile scalpel flap or other sterile instruments to gently scrape the surface of the jejunal lumen at a 45° angle until the epithelial layer is significantly reduced, exposing the underlying tougher basement membrane and connective tissue;

[0061] 5. Collect the scraped material and suspend it in cell culture medium. Transfer it to a sterile culture tube, centrifuge at 500g for 5 min, discard the supernatant, resuspend the cells in cell culture medium, spread them evenly in a sterile culture flask, and incubate at 37℃ in a 5% CO2 incubator. Change the medium after 48 h, and thereafter every 72 h. Observe the cell growth status daily. The isolated and cultured jejunal epithelial cells of calves for beef production are shown in the figure. Figure 1A and Figure 1B ; Figure 1A The sample scraped more heavily in step 4 above shows the presence of bovine small intestinal epithelial cells and fibroblasts. Figure 1B This is a sample that was scraped relatively lightly in step 4 above. It is evident that it consists mostly of bovine small intestinal epithelial cells. In subsequent experiments, [the following samples were selected]. Figure 1B The sample shown was subjected to subsequent purification procedures.

[0062] The enzyme digestion solution includes a basal culture medium (DMEM / F12) and neutral protease II and collagenase I added to the basal culture medium. The concentration of neutral protease II in the enzyme digestion solution is 4.8 mg / ml, and the concentration of collagenase I is 300 U / ml.

[0063] The cell culture media mentioned above include basal medium (DMEM / F12) and FBS (15wt%), 1wt% triple antibiotics (penicillin-streptomycin-amphoteric B mixed solution 100×), 1wt% NEAA (non-essential amino acids 100×) and Hepes (1M) added to the basal medium.

[0064] (II) Purification, passage, cryopreservation and identification of jejunal epithelial cells

[0065] 1. Purification: Differential adhesion method was used to purify jejunal epithelial cells of beef cattle.

[0066] Differential adhesion is a separation technique based on the difference in cell adhesion time. Proposed by Kreider and Pleasure, it utilizes mediators such as poly-L-lysine to separate cells with varying degrees of adhesion, including fibroblasts and Schwann cells. The principle is that fibroblasts adhere for a shorter time, astrocytes require a longer time, and olfactory ensheathing cells require even longer. The target cells can be obtained by collecting the non-adhered cells in stages.

[0067] Wash the intestinal epithelial cells obtained in step (I) 2-3 times with PBS, discard the supernatant, add 0.25% trypsin, and separate and digest the cells at 37°C, gently shaking the culture dish. Under a microscope, some cells begin to become round and shiny, and a few cells float; immediately discard the trypsin. Carefully rinse with an appropriate amount of PBS solution and discard. Continue to add 0.25% trypsin for digestion. When the remaining adherent cells begin to become round and shiny and a few cells float under a microscope, stop digestion, wash 2-3 times with PBS solution, collect the detached cells, and centrifuge at 1000 rpm for 5 min. Resuspend the obtained precipitate in cell culture medium, adjust the cell density to 50 cells / ml, and seed it into a new cell culture plate for culture. Change the medium when the culture medium turns yellow. The purified jejunal epithelial cells of beef calves passaged to level 3 are shown in the image. Figure 2 .

[0068] 2. Passaging: Passage at a ratio of 1:2 to 1:3 (i.e., passaging from 1 dish to 2-3 dishes). When the purified bovine jejunal epithelial cells have adhered to the culture medium and reached 90% confluence, wash 2-3 times with PBS, discard the supernatant, add an appropriate volume of 0.25% trypsin for digestion. Stop digestion when the remaining adherent cells begin to become round and shiny under a microscope, and a few cells are observed to float. Centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the pellet in cell culture medium, and adjust the cell density to 0.5 × 10⁶ cells / mL. 4 Inoculate at a rate of 1 per mL into a new culture flask and culture.

[0069] 3. Cryopreservation: Wash cells 2-3 times with PBS, discard the supernatant, digest adherent cells with 0.25% trypsin until the cells become bright, round and float, then stop digestion. Centrifuge at 1000 rpm for 5 min, discard the supernatant, add cell cryopreservation solution and gently resuspend the pellet. Transfer the resuspended solution to 2 ml cryovials, seal and label them, place them in a programmed cooling box and freeze at -80℃ overnight. The next day, transfer them to a liquid nitrogen tank for long-term storage.

[0070] The cell cryopreservation solution consists of FBS and DMSO, wherein FBS (fetal bovine serum) is 90 wt% and DMSO (dimethyl sulfoxide) is 10 wt%.

[0071] 4. Determination of bovine jejunal epithelial cell growth curve: When the purified cell density reaches 80%-90%, the cells are digested with 0.25% trypsin for 5 minutes, followed by cell counting. The cells are then divided into groups of 10 × 10⁻⁶ cells. 4 Cells were seeded at a density of [number] cells / mL in 24-well plates and cultured. The number of cells in three wells was counted every other day, and the average value was calculated. This counting was repeated for 8 days. A growth curve was plotted with the number of culture days on the x-axis and the number of cells on the y-axis. The growth curve for jejunal epithelial cells of beef calves is shown below. Figure 3 .

[0072] 5. Detection of marker proteins in bovine jejunal epithelial cells: When the purified cell density reaches 80%-90%, the cells are digested with 0.25% trypsin and counted. Cells are then divided into groups of 5 × 10⁻⁶ cells. 4 Cells were seeded at a density of 10 cells / mL in 12-well plates and cultured. After cell adhesion, immunofluorescence detection was performed.

[0073] Fixation: Discard the culture medium, wash 3 times with PBS buffer, add pre-cooled 4% paraformaldehyde solution to cover the entire well, incubate at room temperature for 10 min, and wash 3 times with cold 1×PBS buffer.

[0074] Blocking: Add immunofluorescence blocking solution and block at room temperature for 1 hour. Wash three times with PBS buffer, 5 minutes each time.

[0075] Primary antibody incubation: Prepare the primary antibody working solution using primary antibody dilution buffer. Add the primary antibody to the wells and incubate overnight at 4°C. Wash three times with 1×PBS buffer, 5 min each time.

[0076] Secondary antibody incubation: Prepare the working solution of the secondary antibody using secondary antibody dilution buffer. Add the secondary antibody to the culture chamber and incubate at room temperature for 1 hour. Wash three times with PBS buffer, 5 minutes each time.

[0077] Nuclear staining (cell localization): Add 10 µg / mL DAPI and incubate at room temperature in the dark for 10 min. Wash three times with 1×PBS buffer, 5 min each time.

[0078] Observation: Add an anti-fluorescence quencher and cover with a coverslip. Observe protein expression and localization under a fluorescence microscope. Immunofluorescence identification of jejunal epithelial cells from beef calves is shown in [reference needed]. Figure 4 .

[0079] Comparative Example 1

[0080] The method is largely the same as in Example 1, except that the digestive enzyme used is thermophilic protease, with a specification of 50-100u / mg, manufactured by Shanghai Yuanye Biotechnology Co., Ltd., catalog number S10236.

[0081] The enzyme digestion solution was prepared as follows: thermophilic protease was dissolved in Hepes buffer, filtered through a 0.22 μm filter, and added to DMEM / F12 medium; DMEM / F12 + thermophilic protease (50 mg / L).

[0082] The specific digestion method is as follows:

[0083] Jejunal tissue was collected from newborn, non-sucking calves at a beef cattle farm and processed in the same manner as in Example 1. The mesentery and adipose tissue were removed in a clean bench, rinsed with PBS, sterilized in 75% alcohol for 2 minutes, and repeatedly rinsed with PBS solution containing 1% penicillin and streptomycin (about 10 times).

[0084] After the sample was allowed to stand for 10 min, the PBS was removed, and the jejunal tissue was completely immersed in a sterile centrifuge tube containing thermophilic protease digestion solution. The tissue block was slowly digested by shaking at 37°C and low speed (100 r / min). After 1 h of digestion, the supernatant of the digestion solution was aspirated, and the undigested portion was further digested with the aforementioned digestive enzyme for another 1 h. The tissue fluid from both digestions was placed in a 50 mL centrifuge tube and centrifuged at 300g for 5 min. The supernatant was discarded after centrifugation. The sample was then washed once with PBS and once with DMEM medium until the supernatant was clear. The sample was resuspended in complete culture medium and inoculated into T25 culture flasks. The culture medium was changed after 48 h at 37°C and 5% CO2, and then changed every 2–3 days thereafter.

[0085] Experimental results reference Figure 5 Thermophilic protease was used to separate bovine jejunal tissue samples, yielding a large amount of basement membrane tissue, connective tissue, and cell debris. The connective tissue was fibrous, and there were a small number of intestinal crypt units, some tissue fragments, and single cells.

[0086] The above results indicate that the use of enzyme digestion solution for isolation and culture is ineffective and unsuitable as a method for isolation and culture.

[0087] Comparative Example 2

[0088] The results are largely the same as in Example 1, except for the cell culture medium formulation. The cell culture medium used in this comparative example was: DMEM / F12 with 10% FBS + 20 ng / ml EGF + 1 mmol / L NEAA + 2.5 ug / ml insulin (or 200 IU / L) + 1 M Hepes + 100 mM sodium pyruvate + 100 μg / ml heparin + 100 IU / ml penicillin / streptomycin. The cell culture medium was sterilized by positive pressure filtration through a 0.22 μm microporous membrane and stored at 4°C.

[0089] The above-mentioned cell culture medium was added during cell culture, and the adhesion time and cell number of jejunal epithelial cells were observed. (See...) Figure 6B ;

[0090] As a control, jejunal epithelial cells were cultured using the same method in the cell culture medium from Example 1, and the results were referenced. Figure 6A ;

[0091] Under the two culture medium conditions, the adhesion time of jejunal epithelial cells was roughly the same. After 3 days of culture, the number of cells obtained in Comparative Example 2 was slightly higher than that in Example 1, but the difference was not significant.

[0092] After cell passage culture, cell growth curves were plotted and compared with the cell growth curves in Example 1. Figure 6C ;

[0093] The experimental results showed that the adhesion time of jejunal epithelial cells was approximately the same under both culture medium conditions. After 3 days of culture, the number of cells obtained in Comparative Example 2 was slightly higher than that in Example 1, but the difference was not significant. It is speculated that although cell growth was not affected after 3 days, the use of a large amount of growth factors in Comparative Example 2 would have an adverse effect on growth from 4 to 8 days.

[0094] The above results indicate that the cell culture effect using this culture medium is relatively poor and it is not suitable as a formula for culturing jejunal epithelial cells of beef calves.

[0095] Comparative Example 3

[0096] The general process is the same as in Example 1, except that the cryopreservation solution is different.

[0097] The cryopreservation solution used in this comparative example was formulated as follows: DMEM / F12 + 10wt% FBS + 10wt% DMSO;

[0098] The specific methods for cell cryopreservation and thawing are as follows:

[0099] Cell cryopreservation: Wash cells 2-3 times with PBS, discard the supernatant, digest adherent cells with 0.25% trypsin until the cells become shiny, round, and float, then stop digestion. Centrifuge at 1000 rpm for 5 min, discard the supernatant, gently resuspend the pellet in cell cryopreservation solution, transfer to 2 ml cryovials, seal, label, and place in a programmed cooling box at -80°C overnight. The next day, transfer to a liquid nitrogen tank for long-term storage.

[0100] Cell thawing: Immediately place the cryopreserved cells removed from liquid nitrogen into a 37°C water bath and shake vigorously for approximately 1-2 minutes to thaw. After thawing, transfer to a centrifuge tube, add 10 times the volume of culture medium, mix well, centrifuge at 1000 rpm for 5 minutes, discard the supernatant, and collect the cell pellet. Add freshly prepared jejunal epithelial cell culture medium, resuspend and mix well, then add the cell suspension to a T25 culture flask, adding an appropriate amount of culture medium (6-8 mL for a T25 flask). Mix well and incubate at 37°C in a 5% CO2 incubator. Observe the jejunal epithelial cell adhesion time and cell confluence.

[0101] refer to Figure 7A and Figure 7B , Figure 7A This is a photograph of the patient after recovery in Example 1. Figure 7B This is a photograph of the cells after resuscitation in Comparative Example 3, obtained through... Figure 7A and Figure 7B As can be seen, the cryopreservation solution described in Example 1 can effectively maintain cell number and improve cell viability, and the survival rate after thawing in Example 1 reached 67.7%.

[0102] Comparative Example 4

[0103] The formula is largely the same as in Example 1, except that the enzyme digestion solution used is: DMEM / F12 + 0.4% neutral protease II + 0.3% collagenase I, with the two enzymes added in a V:V ratio of 1:1, and mixed well for later use.

[0104] After cell passage culture, cell growth curves were plotted and compared with the cell growth curves in Example 1. Figure 8 ;

[0105] Experimental results showed that this ratio of digestive enzymes achieved good digestion, but the growth rate was slightly lower than in Example 1. Specifically, cell proliferation in the exponential growth phase was slower, and the cells reached the plateau phase and entered death earlier. It is speculated that this ratio of digestive enzymes may have caused some damage to the cells, having a slightly adverse effect on cell growth performance.

[0106] The above results indicate that the cell culture effect of the enzyme digestion solution with this ratio is relatively poor, and it is not suitable as a digestion solution formula for isolating jejunal epithelial cells of beef calves.

[0107] Furthermore, the present invention also statistically analyzed the cell viability of Example 1, Comparative Example 2, and Comparative Example 4, and the results are as follows: Figure 9 As shown.

[0108] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

Claims

1. A method for isolating and culturing primary jejunal epithelial cells of beef calves, characterized in that, Includes the following steps: Step 1: Collect jejunal tissue from newborn calves that have not suckled, clean it, digest it with an enzyme digestion solution, and collect the jejunal epithelial cells after digestion; Step 2: The jejunal epithelial cells obtained in Step 1 were purified and passaged using cell culture medium via differential adhesion. Step 3: Digest the jejunal epithelial cells obtained from step 2 and add them to the cell cryopreservation solution for cryopreservation; The enzyme digestion solution includes a basal culture medium and neutral protease II and collagenase I added to the basal culture medium; the cell culture medium includes a basal culture medium and FBS, antibiotics, NEAA and buffer added to the basal culture medium; the cell cryopreservation solution is composed of FBS and DMSO, and the FBS content in the cell cryopreservation solution is 90~92wt%.

2. The isolation and culture method according to claim 1, characterized in that, The concentration of neutral proteinase II in the enzyme digestion solution is 4.5~5 mg / ml, and the concentration of collagenase I is 200~400 U / ml.

3. The isolation and culture method according to claim 1, characterized in that, The cell culture medium contains 13-17 wt% FBS, 0.8-1.2 wt% antibiotics, and 0.8-1.2 wt% NEAA.

4. The isolation and culture method according to claim 1, characterized in that, In step 3, digestion is terminated using a digestion termination solution; the digestion termination solution includes basal culture medium, FBS, and PS; the amount of FBS is 3-7 wt%; and the amount of PS is 0.8-1.2 wt%.

5. The isolation and culture method according to claim 1, characterized in that, Step 1 specifically involves: Step 11: Collect jejunal tissue from newborn calves that have not suckled, and rinse repeatedly with PBS solution or physiological saline to remove all intestinal contents until the washing solution is clear; cut the jejunal tissue into segments and place them in PBS solution containing penicillin and streptomycin for further processing. Step 12: Longitudinally cut open the jejunum segment, remove the mesentery and adipose tissue, rinse thoroughly with PBS, sterilize, and rinse repeatedly with PBS solution containing penicillin and streptomycin. Step 13: Completely immerse the jejunal tissue in a sterile centrifuge tube containing enzyme digestion solution, and slowly shake it on a shaker at 37°C for 20-40 minutes. Step 14: Transfer the jejunal tissue into a sterile culture dish, wash gently twice with PBS solution to remove the enzyme digestion solution; The intestinal lumen surface is scraped clean until the epithelial layer is significantly reduced, exposing the underlying tougher basement membrane and connective tissue; Collect the scraped material, suspend it in cell culture medium, centrifuge, and resuspend.

6. The isolation and culture method according to claim 1, characterized in that, Step 2 specifically involves: Step 21: Purification. Use differential adhesion to purify jejunal epithelial cells; wash the jejunal epithelial cells obtained in step 1 with PBS 2-3 times, discard the supernatant, add 0.25wt% trypsin and separate and digest the cells at 37℃. Step 22: Under a microscope, some cells begin to become round and shiny, with a few cells floating. Discard the digestion solution, add PBS solution to wash, discard the PBS solution, and continue digestion with 0.25 wt% trypsin. Stop digestion when the remaining adherent cells begin to become round and shiny, with a few cells floating, under a microscope. Wash 2-3 times with PBS solution, collect the detached cells, and resuspend the precipitate obtained by centrifugation in complete culture medium. Seed the precipitate into a cell culture plate for culture. Change the medium when the culture medium turns yellow. The cell culture plate contains the cell culture medium. Step 23: Passage the cells at a ratio of 1:2 to 1:

3. When the jejunal epithelial cells have adhered to the culture medium and reached 90% confluence, wash 2-3 times with PBS, discard the supernatant, add 0.25 wt% trypsin for digestion. Stop digestion when the remaining adherent cells begin to become round and shiny under a microscope, and a few cells are observed to float. Centrifuge at 1000 rpm for 5 min, discard the supernatant, resuspend the pellet in cell culture medium, and adjust the cell density to 0.5 × 10⁻⁶ cells / cm². 4 Inoculate at a rate of 1 per mL into a new culture flask and culture.

7. The isolation and culture method according to claim 1, characterized in that, Step 3 is as follows: Wash jejunal epithelial cells 2-3 times with PBS, discard the supernatant, digest adherent jejunal epithelial cells with 0.25wt% trypsin, stop digestion when the jejunal epithelial cells become shiny, round and float, centrifuge, discard the supernatant, add cell cryopreservation solution to resuspend the precipitate, transfer to 2 ml cryovials and seal for cryopreservation.

Images