Tibetan sheep rumen epithelial cell immortalized cell line and method for establishing the same

Abstract

The application discloses a kind of Tibetan sheep rumen epithelial cell immortalized cell lines and method for establishing same, and is related to the field of animal cell engineering and molecular biology technology.It includes: primary cell acquisition;Cell property identification;Lentivirus transfection;Positive cell screening;Immortalized cell line establishment.The application has clear and unique design in the selection of immortalization vector, positive cell screening reagent, cell identification method and passage digestion process.Compared with prior art, it has significantly improved in cell immortalization efficiency, genetic stability, epithelial property retention and long-term passage ability, and can stably obtain Tibetan sheep rumen epithelial immortalized cell line with high viability, high purity, good growth state and stable passage to P12 generation or more, effectively overcoming the shortcoming of limited in vitro proliferation of primary cells and easy aging, and providing better in vitro experimental material for related research.

Description

Technical Field

[0001] This invention relates to the fields of animal cell engineering and molecular biology, and more specifically to an immortalized Tibetan sheep rumen epithelial cell line and its establishment method. Background Technology

[0002] The rumen, as the primary site of nutrient digestion and absorption in ruminants, plays a crucial role in feed microbial fermentation, volatile fatty acid absorption, and energy metabolism. Rumen epithelial cells are an important component of the rumen barrier function, and their normal structure and function are essential for the growth, development, and health of ruminants. Tibetan sheep, living in a high-altitude, hypoxic environment, possess unique physiological characteristics in their rumen epithelial cells regarding energy metabolism and environmental adaptation. However, these cells are highly sensitive to in vitro culture conditions, and existing universal culture systems struggle to maintain their stable proliferation and functional state over long periods.

[0003] Currently, rumen epithelial cell research mainly relies on primary culture methods. Traditional methods typically involve enzymatic digestion combined with adherent culture to obtain cells. However, these cells have significant limitations in in vitro culture: firstly, their passage capacity is limited, and they usually age within a limited number of passages; secondly, their proliferative capacity gradually declines, making it difficult to meet the needs of long-term experiments; and thirdly, phenotypic changes are prone to occur during in vitro culture, affecting the stability and reproducibility of experimental results. Furthermore, primary cells exhibit significant individual variability and are limited in their availability, making it difficult to support large-scale and standardized research.

[0004] Various cell immortalization strategies have been developed in the present technology, typically involving the introduction of exogenous genes to prolong cell proliferation and lifespan. Among these, immortalization methods based on SV40 large T antigen or telomerase reverse transcriptase (TERT) have been applied to various animal-derived cells. However, these methods are mostly established for conventional laboratory animals or economically viable animals, and their adaptability is still insufficient when directly applied to Tibetan sheep rumen epithelial cells. For example, although some immortalized cells acquire continuous proliferative capacity, the expression of epithelial cell characteristics decreases, and their responsiveness to nutrient metabolism and signal stimulation is weakened, thus affecting their reliability as in vitro models. In addition, existing culture systems are mostly general conditions, lacking optimized design for the characteristics of rumen epithelial cells and the special physiological background of Tibetan sheep, leading to functional degradation or instability of cells during long-term culture.

[0005] Therefore, there is an urgent need to establish a stable and efficient immortalization method for Tibetan sheep rumen epithelial cells to obtain cell models that have both continuous proliferation capacity and maintain physiological functional characteristics, thereby providing a reliable tool for the study of rumen function and related molecular mechanisms. Summary of the Invention

[0006] In view of this, the present invention provides an immortalized cell line of Tibetan sheep rumen epithelial cells and a method for establishing it, in order to obtain a cell model with stable proliferative capacity, good genetic stability, and typical epithelial cell functional characteristics.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] A method for establishing an immortalized cell line of Tibetan sheep rumen epithelial cells includes the following steps: (1) Primary cell acquisition: Take rumen tissue from healthy Tibetan sheep, aseptically scrape the mucosa, wash and cut it into pieces, digest it with trypsin in several steps, filter and centrifuge to collect cells and resuspend and inoculate them, and finally purify them by differential adhesion to obtain Tibetan sheep rumen epithelial cells. (2) Cell characterization: Immunofluorescence was used to detect cell-specific keratin to confirm cell purity and viability; (3) Lentiviral transfection: SV40 overexpressing lentivirus was transfected into the qualified Tibetan sheep rumen epithelial cells identified in step (2). (4) Screening of positive cells: After transfection, cells were screened and cultured with puromycin to obtain positive cells that stably express the SV40 gene; (5) Establishment of immortalized cell line: Seed the positive cells obtained in step (4) and expand and passage them; control the degree of cell fusion during passage, digest and passage them in proportion; after continuous passage, obtain an immortalized cell line of Tibetan sheep rumen epithelial cells that can be stably passaged.

[0009] Preferred method: Step (2) uses CK19 antibody for immunofluorescence detection.

[0010] Preferred: Step (3) includes: co-incubating SV40 overexpressing lentivirus with Tibetan sheep rumen epithelial primary cells; controlling the multiplicity of infection to 10-30; adding 5 μg / mL polybrene during transfection; and transfection conditions of 37°C and 5% CO2 incubation for 48-72 h.

[0011] Preferred: In step (4), the screening concentration is 1~7 μg / mL, and the culture system containing the corresponding concentration of puromycin is replaced every 2 days during the continuous screening period.

[0012] Preferred: Step (5) includes: inoculating the positive cells obtained from step (4) with a special culture medium for Tibetan sheep primary rumen epithelial cells, and culturing them at 37°C and 5% CO2, replacing the culture medium with fresh medium every 2 days; when the cell confluence reaches 80%~90%, discard the old culture medium, wash with PBS buffer, add 0.25% trypsin for digestion, stop digestion after the cells shrink and detach from the cell wall, pipette to make a single cell suspension, centrifuge at 1000r / min for 5min, discard the supernatant and passage at a ratio of 1:2~1:4, and after continuous passage culture, obtain the immortalized cell line of Tibetan sheep rumen epithelial cells.

[0013] The present invention also provides immortalized Tibetan sheep rumen epithelial cells obtained by any of the above methods.

[0014] This invention also provides the application of any of the above methods, or the above-described immortalized Tibetan sheep rumen epithelial cells, in Tibetan sheep breed improvement, ruminant nutrition and metabolism research, rumen barrier function research, and feed development.

[0015] As can be seen from the above technical solution, compared with the prior art, this invention discloses an immortalized cell line of Tibetan sheep rumen epithelial cells and its establishment method. The technical effect achieved is that this invention adopts a unique combination scheme of "primary cell differential adhesion and impurity removal + SV40 overexpressing lentivirus infection and transfection + puromycin screening", which specifically solves the technical pain points of Tibetan sheep rumen epithelial primary cells being difficult to immortalize and limited in passage. This invention specifically selects SV40 overexpressing lentivirus as the immortalization-mediating vector, rather than conventional immortalization gene vectors such as hTERT, and combines it with puromycin for specific screening of positive cells. The screening concentration is precisely controllable, which can not only efficiently obtain positive cells that stably express SV40, but also completely remove untransfected cells and impurity cells, significantly improving cell purity, especially suitable for the species specificity and growth characteristics of Tibetan sheep rumen epithelial cells.

[0016] This invention utilizes complete epithelial culture medium for primary and passage culture, combined with immunofluorescence identification of cell surface-specific marker proteins. This allows for precise confirmation of the epithelial cell characteristics of the obtained cells and enables monitoring of cell characteristic stability throughout the immortalization process. Furthermore, during passage, the cell confluence is strictly controlled at 80%–90%. A standardized procedure is employed: digestion with 0.25% trypsin containing 0.02% EDTA, followed by a single rinsing with PBS buffer and termination of digestion with complete culture medium. This simplifies the digestion process while minimizing damage to cell viability from trypsin, preventing morphological abnormalities or functional loss in immortalized cells. This provides a more reliable cell model for rumen function research in Tibetan sheep, such as nutritional metabolism and rumen barrier function in high-altitude environments.

[0017] In summary, this invention features a clear and unique design in terms of the selection of immortalization carriers, positive cell screening reagents, cell identification methods, and passage digestion processes. Compared with existing technologies, it significantly improves cell immortalization efficiency, genetic stability, preservation of epithelial characteristics, and long-term passage capability. It can stably obtain Tibetan sheep rumen epithelial immortalized cell lines with high viability, high purity, and excellent growth status, and can be stably passaged to P12 or higher generations. It effectively overcomes the shortcomings of primary cells in vitro proliferation and easy aging, providing higher quality in vitro experimental materials for related research. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0019] Figure 1 The attached figure shows primary cells obtained from the isolation and culture of Tibetan sheep rumen epithelial cells provided by the present invention, wherein both Figure A and Figure B are 40X.

[0020] Figure 2 The attached figure shows the immunofluorescence identification results of Tibetan sheep rumen epithelial cells provided by the present invention, wherein A: DAPI; B: CK19; C: Merger staining.

[0021] Figure 3 The attached figure shows the cell culture results of Tibetan sheep rumen epithelial cells transfected with SV40 overexpressing lentivirus provided by the present invention, where A and B are both 40X.

[0022] Figure 4 The attached figure is a diagram of the immortalized P13 generation cells provided by the present invention, wherein A: fold 40X, B: fold 100X.

[0023] Figure 5 The attached figure shows a primary rumen epithelial cell cultured to the 7th generation provided by this invention.

[0024] Figure 6 The attached figure is a bar chart showing the expression levels of short-chain fatty acid-related transport genes in primary cells and cell lines provided by this invention.

[0025] Figure 7 The attached figure is a spectrum of the carrier provided by the present invention. Detailed Implementation

[0026] 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.

[0027] This invention discloses an immortalized rumen epithelial cell line from Tibetan sheep and its establishment method. Detailed information on the instruments, equipment, reagents, and consumables required for this embodiment is shown in Tables 1 and 2 below.

[0028]

[0029]

[0030] Example 1 A method for establishing an immortalized cell line of Tibetan sheep rumen epithelial cells includes the following steps: (1) Primary cell acquisition: 1) Primary isolation and culture of Tibetan sheep rumen epithelial cells: Remove the received rumen (retroventral sac) tissue from healthy Tibetan sheep and transfer it to a biosafety cabinet. Under aseptic conditions, place the tissue in a culture dish and gently scrape the rumen mucosal epithelium with ophthalmic forceps. Then, repeatedly wash the collected mucosal tissue in PBS buffer until the washing solution is clear and transparent to remove residual impurities and contents from the tissue surface. 2) The collected mucosal tissue was thoroughly minced using sterile scissors and repeatedly washed with PBS buffer to further remove tissue debris and impurities; 3) After settling, discard the supernatant and retain the tissue precipitate; 4) Add at least 3 times the volume of 0.25% Trypsin digestion solution to the tissue pellet and digest it under shaking conditions in a 37°C water bath. Collect the digestion solution every 30 minutes and add complete culture medium to terminate the digestion reaction. At the same time, add fresh digestion solution to the remaining tissue and continue digestion for 3 to 4 consecutive times to improve cell acquisition efficiency. 5) After mixing the digestive fluids collected from each batch, filter them through a 100μm cell filter to remove incompletely digested tissue fragments and larger impurities; 6) Collect the filtrate after filtration, centrifuge at 300g for 5 minutes, discard the supernatant and retain the cell pellet; 7) Resuspend the cell pellet in complete epithelial culture medium and gently pipette to mix and form a cell suspension; 8) Inoculate the cell suspension into cell culture flasks and incubate them in a 37°C, 5% CO2 cell culture incubator; 9) Differential Adhesion: After 1 hour of culture, differential adhesion treatment is performed. The supernatant containing the non-adhered cells is gently transferred to a new culture flask for continued culture to reduce contamination by fibroblasts and other contaminating cells, thereby improving the purity of rumen epithelial cells. The resulting Tibetan sheep rumen epithelial cells are as follows: Figure 1 As shown.

[0031] (2) Cell characteristic identification: Immunofluorescence identification of sheep rumen epithelial cells 1) Cell slides Place a glass slide into a 24-well plate, add cell suspension to each well, with a cell density of 50,000 cells / well, and incubate overnight. 2) Fixed The next day, under a microscope, the cells grew well and the density reached about 40-60%. The culture medium was discarded, the cells were washed three times with PBS, 4% PFA was added, and the cells were fixed at 4°C for 30 min. The cells were then washed three times with PBS for 5 min each time. 3) Membrane rupture and sealing Add membrane breaking and blocking buffer (a mixture of Tritium X-100 and 10% goat serum) and incubate at room temperature for 1-2 hours. Antibody information is detailed in Table 1. 4) Primary antibody incubation After membrane rupture and blocking, add diluted primary antibody to each well (refer to the dilution ratio of primary antibody in the instruction manual) and incubate overnight at 4°C; 5) Secondary antibody incubation On the third day, wash each well three times with PBS for 5 minutes each time, incubate with fluorescent secondary antibody (refer to the instructions for dilution ratio of secondary antibody), incubate at room temperature in the dark for 2 hours, wash three times with PBS for 5 minutes each time, stain with DAPI (DAPI:PBS=1:1000), incubate for 5 minutes, wash three times with PBS for 5 minutes each time. 6) Sealing Add fluorescent blocking solution to a glass slide, and place the side of the slide with cells onto the fluorescent blocking solution; 7) Microscopic examination Microscopic examination and imaging were performed under 10X and 20X objectives, and the signals of each fluorescence channel were recorded. The results show that ( Figure 2 DAPI labeled the cell nucleus (blue), and CK19 labeled the epithelial cell-specific keratin (red). Under 10×10 and 20×10 magnification, almost all DAPI-positive cells also expressed CK19, and the fluorescence signal was mainly located in the cytoplasm, indicating that the isolated and cultured cells were high-purity Tibetan sheep rumen epithelial cells, which can be used for subsequent experiments.

[0032] (3) Lentiviral transfection: Lentiviral virus overexpressing SV40 was used (vector map shown). Figure 7 Transfect the Tibetan sheep rumen epithelial cells obtained in step (2) with the following: 1) Inoculate rumen epithelial cells of Tibetan sheep in good growth condition into 6-well plates, with approximately 1 × 10⁶ cells per well. 5 Individual samples were incubated at 37°C in a 5% CO2 incubator. 2) On the second day, after the cells have adhered to the culture medium, discard the original culture medium and replace it with fresh culture medium; 3) Add 1 mL of complete culture medium to each well, and then add 20 μL of SV40 overexpression lentivirus. The SV40 overexpression lentivirus was constructed by Laibaiha (Shanghai) Biotechnology Co., Ltd. The basic information of the SV40 overexpression lentivirus is shown in Table 3. 4) After gently mixing, continue to incubate in a 37℃, 5% CO2 incubator; 5) After culturing for 12 hours, observe the cell growth status and replace with fresh culture medium to continue culturing; 6) After the cells have grown to the bottom of the well plate, they are digested and passaged with 0.25% trypsin (containing 0.02% EDTA) and then transferred to T25 cell culture flasks for further culture. The total incubation time for all the above steps is 48-72 hours; the multiple of infection (MOI) is controlled at 10-30, and 5 μg / mL polybrene is added during transfection. The results of cell culture after transfection of rumen epithelial cells with SV40 overexpressing lentivirus are as follows: Figure 3 As shown, after transfection with lentivirus, the cells were in good overall condition, exhibiting a typical epithelial-like cobblestone morphology. The cells were tightly packed with a confluence of approximately 90%–100%, and the cytoplasm was uniform with clear boundaries. No significant cytotoxicity was observed, such as obvious shrinkage, vacuolization, or large-area lysis. Only a small number of round, highly refractive cells or cell fragments were scattered in the field of view, suggesting the presence of a very small number of apoptotic or mitotic cells. Overall, the cell viability and tolerance were good, which could meet the requirements for subsequent screening.

[0033]

[0034] (4) Screening for positive cells: 1) Determination of the kill curve Untransfected Tibetan sheep rumen epithelial cells were seeded at a density of 0.05 million per well in 24-well plates and incubated overnight at 37°C in a 5% CO2 incubator to allow for full cell adhesion. The next day, the old culture medium was discarded, and fresh selection medium containing different concentrations of puromycin was added: 1 μg / mL, 2 μg / mL, 3 μg / mL, 4 μg / mL, 5 μg / mL, 6 μg / mL, and 7 μg / mL. During the culture period, the culture medium was replaced with the corresponding concentration of puromycin every two days, and cell growth and survival rates were observed daily under a microscope. The results showed that a puromycin concentration of 1 μg / mL caused the death of all untransfected cells within 3 days. Therefore, 1 μg / mL was determined to be the optimal working concentration for subsequent selection experiments, with a selection time of 3 days. 2) Puromycin screening of transfected cells Subsequently, the cells transfected with SV40 lentivirus were screened using the optimal screening conditions determined above. The transfected cells were seeded at a density of 0.05 million per well in 24-well plates and incubated overnight at 37°C in a 5% CO2 incubator. On the second day, the old culture medium was discarded, and selection medium containing 1 μg / mL puromycin was added for continued culture. During the screening period, the selection medium was replaced with fresh selection medium every 2 days, and cell survival was observed daily. By day 3 of screening, all cells in the untransfected control group had died, while some cells in the transfected group were still able to adhere normally and maintain intact cell morphology, indicating that these cells had successfully integrated the SV40 lentivirus expression vector and acquired puromycin resistance. The surviving positive cells after screening were further expanded and cultured to finally obtain a stable passaged immortalized Tibetan sheep rumen epithelial cell line.

[0035] (5) Establishment of immortalized cell lines: Positive cells that survived the puromycin screening were further expanded and cultured, and the cells obtained after screening were defined as P1 generation cells (seeded in T25 cell culture flasks): 1) During the culture process, Tibetan sheep primary rumen epithelial cells were cultured in a special culture medium (JY-Y413, Shanghai Jinyuan Biotechnology Co., Ltd.) at 37℃ and 5% CO2 constant temperature incubator. The culture medium was replaced with fresh medium every 2 days to maintain stable cell growth. 2) When the cell confluence reaches 80%–90%, discard the old culture medium, gently wash the cells once with PBS buffer, and then add 0.25% trypsin (containing 0.02% EDTA, 1734858, Gibco) for digestion; when the cell edges gradually shrink and begin to detach, immediately add complete culture medium to stop digestion, and gently pipette to form a single-cell suspension; after centrifugation (1000 r / min for 5 min) to collect the cells, passage them at a ratio of 1:2 to 1:4. 3) As the subculture continues, the cells gradually reach a stable proliferative state, with uniform cell morphology, a typical cobblestone arrangement, and good adhesion ability. After continuous expansion and culture to passage P12 and above, the selected cells maintained strong proliferative activity without significant senescence, detachment, or morphological abnormalities, indicating that the established immortalized cells have good in vitro culture stability. Further observation of the morphology of the immortalized P13 generation cells revealed... Figure 4 The results showed that the cells retained typical epithelial cell morphology, were tightly packed, and had clear boundaries, suggesting that this cell line could stably maintain the biological characteristics of rumen epithelial cells during continuous passage; and Figure 5 The primary rumen epithelial cells cultured to passage 7 exhibited obvious signs of aging and functional decline. Cell morphology was irregular, with shrunken cells, blurred boundaries, and some cells appearing rounded and floating. Vacuolation and granular degeneration were observed in the cytoplasm. The cells were sparsely and unevenly distributed, with poor extension and weakened adhesion. Numerous cell fragments and dead cells were also present in the field of view, indicating that these primary cells had entered the aging stage and were biologically unstable, making it difficult to meet the requirements of cell viability and functional uniformity for subsequent experiments. This further highlights the advantages of immortalized cell lines in terms of stability and reproducibility. Simultaneously, the expression of genes related to short-chain fatty acid transport in primary rumen epithelial cells and cell lines was measured, and the results showed (…). Figure 6 Although there were quantitative differences in the expression levels of different genes in the two cell types, the immortalized cell line still stably retained the expression characteristics of short-chain fatty acid transport-related genes, and clear transcriptional signals could be detected. This indicates that the immortalization operation did not destroy the core function of the cell and can be reliably used for subsequent functional assays and mechanism studies, demonstrating the applicability and stability of the cell line in functional studies.

[0036] The various embodiments in this specification are described in a progressive manner, with each embodiment focusing on the differences from other embodiments. The same or similar parts between the various embodiments can be referred to each other.

[0037] Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for establishing an immortalized cell line of Tibetan sheep rumen epithelial cells, characterized in that, Includes the following steps: (1) Primary cell acquisition: Take rumen tissue from healthy Tibetan sheep, aseptically scrape the mucosa, wash and cut it into pieces, digest it with trypsin in several steps, filter and centrifuge to collect cells and resuspend and inoculate them, and finally purify them by differential adhesion to obtain Tibetan sheep rumen epithelial cells. (2) Cell characterization: Immunofluorescence was used to detect cell-specific keratin to confirm cell purity and viability; (3) Lentiviral transfection: SV40 overexpressing lentivirus was transfected into the qualified Tibetan sheep rumen epithelial cells identified in step (2). (4) Screening of positive cells: After transfection, cells were screened and cultured with puromycin to obtain positive cells that stably express the SV40 gene; (5) Establishment of immortalized cell line: Seed the positive cells obtained in step (4) and expand and passage them; control the degree of cell fusion during passage, digest and passage them in proportion; after continuous passage, obtain an immortalized cell line of Tibetan sheep rumen epithelial cells that can be stably passaged.

2. The method for establishing as described in claim 1, characterized in that, Step (2) Detection was performed using CK19 antibody by immunofluorescence method.

3. The method for establishing as described in claim 2, characterized in that, Step (3) includes: co-incubating SV40 overexpressing lentivirus with Tibetan sheep rumen epithelial primary cells; controlling the multiplicity of infection to 10-30; adding 5 μg / mL polybrene during transfection; and transfection conditions of 37°C and 5% CO2 incubation for 48-72 h.

4. The method for establishing as described in claim 3, characterized in that, Step (4) screens for concentrations of 1-7 μg / mL, and replaces the culture system containing the corresponding concentration of puromycin every 2 days during the continuous screening period.

5. The method for establishing as described in claim 4, characterized in that, Step (5) includes: inoculating the positive cells obtained from step (4) with a special culture medium for Tibetan sheep primary rumen epithelial cells, and culturing them at 37°C and 5% CO2, replacing the culture medium with fresh medium every 2 days; when the cell confluence reaches 80%~90%, discard the old culture medium, wash with PBS buffer, add 0.25% trypsin for digestion, stop digestion after the cells shrink and detach from the cell wall, pipette to prepare a single cell suspension, centrifuge at 1000r / min for 5min, discard the supernatant and passage at a ratio of 1:2~1:4, and after continuous passage culture, obtain the immortalized cell line of Tibetan sheep rumen epithelial cells.

6. Immortalized Tibetan sheep rumen epithelial cells obtained by the method described in any one of claims 1 to 5.

7. The application of the method described in any one of claims 1 to 5, or the immortalized Tibetan sheep rumen epithelial cells described in claim 6, in the improvement of Tibetan sheep breeds, research on ruminant nutrition and metabolism, research on rumen barrier function, and feed development.

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