Sheep female stem cell separation and in-vitro culture method

Abstract

The invention discloses a sheep female stem cell separation and in-vitro culture method, and relates to the technical field of biologication.According to the technical scheme, the sheep female stem cell separation and in-vitro culture method is characterized in that 0.25% pancreatin is adopted for digestion for 1 hour at the room temperature, mechanical cutting operation is combined, intercellular connexin can be effectively decomposed, and the survival rate of the sheep female stem cells is increased; the ovarian granular cells are quickly released. Medical alcohol and a culture medium containing double antibodies are used for cleaning ovaries for many times, so that the probability of bacterial and fungal contamination is remarkably reduced. A DPBS buffer solution preheated at 37 DEG C and a DMEM / F12 culture medium containing 10% of FBS are used in the whole process, and stress damage to cells caused by temperature shock is avoided. Complete granular cells and tissue fragments are efficiently separated through filtration and centrifugation, and the purity of a cell suspension is improved. The method is developed on the basis of ovaries of sheep, does not depend on feeder layer cells or a complex three-dimensional culture system, can be operated only by conventional laboratory equipment, and is suitable for research scenes with limited resources.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and more particularly to a method for separating and culturing sheep female stem cells in vitro. Background Art

[0002] Germline stem cells are the fundamental source of germ cell initiation, development, and differentiation. Traditionally, it was believed that oocytes in women and most female mammals are produced only during the fetal period and that their number does not increase after birth but rather decreases with each passing year. This suggested that germline stem cells do not exist in the ovaries after birth. However, in 2004, scientists discovered that the ovaries of infant and adult mice contain mitotically competent germ cells that continuously renew the follicle pool, indicating the presence of stem cells in the ovaries. Subsequently, researchers in Texas, USA, and Sun Yat-sen University in China have isolated germline stem cells from mouse and human ovaries and successfully induced them to differentiate into germ cells at different stages. On April 12, 2009, a groundbreaking study by Professor Wu Ji's research team at Shanghai Jiao Tong University was published online in the internationally renowned journal Nature Cell Biology. The study demonstrated that ovarian stem cells can be reproduced under laboratory conditions and, when transferred to the ovaries, they can develop into viable eggs and, in turn, produce offspring. This significant discovery overturned conventional wisdom and opened up a whole new field of research. This research result not only provides a new source of oocytes for animal biotechnology and humans, but also has far-reaching significance for the creation of transgenic animals through sex cell pathways, the cultivation of excellent animal breeds, the protection of endangered animals, and animal reproduction.

[0003] Current in vitro culturing of female germline stem cells presents significant drawbacks, including significant risk of microbial contamination, inaccurate hormone regulation, and potential tumorigenicity. These shortcomings need to be gradually addressed through optimization of aseptic procedures and the development of dynamic hormone regulation systems. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for isolating and culturing sheep female stem cells in vitro to solve the above technical problems.

[0005] The above technical objectives of the present invention are achieved through the following technical solutions: A method for isolating female sheep stem cells, comprising the following steps:

[0006] S1. Ovarian collection and processing;

[0007] S2. Ovarian cells were isolated using trypsin.

[0008] Furthermore, the specific operation of ovarian collection and processing in S1 is as follows: collect ovaries from one-year-old sheep, rinse the collected ovaries with medical alcohol 2-3 times, then wash them with DPBS buffer preheated at 37°C 2-3 times, cut off the external fascia of the ovary with sterilized scissors, and place the treated ovaries in DMEM / F12 culture medium containing 10% FBS and 1.5% double antibody preheated at 37°C, and wash them 2-3 times.

[0009] Furthermore, the specific operation of isolating ovarian cells using the trypsin method in S2 is as follows:

[0010] (1) In a sterile clean bench, remove the ovaries processed in S1 and place them in a 3.5 cm culture dish. Use sterile ophthalmic scissors to cut the ovaries into pieces less than 2 mm. 3 Add 0.25% trypsin solution (containing 0.01% EDTA) to the small fragments and digest at room temperature for 1 hour, shaking once every 10 minutes;

[0011] (2) The digestion fluid containing ovarian fragments was filtered through a 200-mesh filter, and the filtrate was centrifuged at 1200 × g for 5 min. After discarding the supernatant, 5 mL of DMEM / F12 culture medium was added and the suspension was gently pipetted 5-10 times to prepare a pelleted cell suspension. The suspension was cultured in an incubator (37°C, 5% CO2, saturated humidity). The culture medium was replaced every 48 h, and the cells were observed and photographed under an inverted microscope.

[0012] The present invention also provides a method for culturing female sheep stem cells in vitro: After the female sheep stem cells are isolated, they can be cultured in vitro using the following two methods:

[0013] (1) Direct cell passage: When the cell confluence reaches more than 85%, cell passage can be performed;

[0014] (2) Cryopreservation is performed first, and then the cells are cultured after recovery: Cryopreservation is performed when the cell confluence reaches more than 85%.

[0015] Furthermore, the specific operation of the cell passage is as follows:

[0016] Aspirate the original culture medium; add about 2 ml of PBS, gently shake the culture flask to rinse the cells, aspirate the PBS and discard it; add about 1 ml of 0.25% trypsin solution (containing EDTA), and gently shake the culture flask to infiltrate all cells; place it in the incubator for digestion, and stop when the cells in the middle of the cell block are obviously rounded and have gaps under the microscope. Do not tap the culture flask during the whole process; add 3 ml of serum-containing culture medium to stop digestion, blow the cells to detach them from the wall and blow them repeatedly in the liquid to make the cells as single-cell suspension as possible; collect the cell suspension and centrifuge at 1200 rpm / min for 3 minutes. Aspirate the supernatant and discard it after centrifugation; add fresh culture medium, blow until the cells are mixed, inoculate into a new culture flask at a ratio of 1:2, replenish the culture medium, loosen the bottle cap or use a breathable bottle cap for culture.

[0017] Furthermore, the specific operation of the freezing is as follows:

[0018] In a biosafety cabinet, open the culture flask and collect the culture medium in the flask; add 3 ml of sterile 1× PBS to the culture flask, place the culture flask horizontally so that the PBS can penetrate all areas of the bottom surface of the culture flask, and discard the PBS; add 1 ml of digestion solution to the flask, soak the bottom surface, and then incubate in a 37°C CO2 incubator for 1-2 minutes; after incubation, observe under an inverted microscope to see if the cells have become round and floated. If all cells have been digested, add 2 ml of complete culture medium containing 10% FBS directly to the culture flask and transfer the suspension to a 15 ml centrifuge tube; centrifuge at 1000 rpm for 5 minutes; after centrifugation, discard the supernatant, resuspend the cell pellet with 1 ml of freezing solution, and then transfer it to a 1.8 ml cryovial; transfer the cryovial to a programmed cooling box filled with isopropanol, and then transfer it to a -80°C refrigerator to cool overnight; the next day, remove the cryovial from the programmed cooling box and transfer it to a liquid nitrogen tank for storage as soon as possible.

[0019] Furthermore, the specific operation of reculturing the cells after recovery is as follows:

[0020] When cells are reviving, preheat the water in a constant temperature water bath to 37°C; prepare a 15ml centrifuge tube, add 5ml of complete culture medium containing 10% FBS, and place it in a 37°C water bath to preheat; after wearing goggles and thick woolen gloves, remove the cells to be revived from the liquid nitrogen tank, transfer them to a 37°C constant temperature water bath as soon as possible to rewarm and shake the cryopreserved tube to increase the rewarming rate; aspirate the cells in the thawed cryopreserved tube into the pre-prepared centrifuge tube, mix, and centrifuge at 1000rpm for 5min; prepare a T-25 culture flask, write the cell name and date, and then add 4ml of complete culture medium; after centrifugation, discard the supernatant, resuspend the cells with 1ml of complete culture medium, transfer them to a T-25 cell culture flask, mix, and transfer to a CO2 incubator for culture and stasis.

[0021] In summary, the present invention has the following beneficial effects:

[0022] 1. Efficient dissociation of ovarian tissue: Digestion with 0.25% trypsin (containing EDTA) at room temperature for 1 hour, combined with mechanical shearing, can effectively decompose intercellular junction proteins and quickly release ovarian granulosa cells.

[0023] 2. Reduce the risk of microbial contamination: Use medical alcohol and culture medium containing double antibiotics (penicillin / streptomycin) to clean the ovaries multiple times during pretreatment, which significantly reduces the probability of bacterial and fungal contamination.

[0024] 3. Maintain cell activity: Use DPBS buffer and DMEM / F12 medium preheated at 37°C throughout the process to avoid stress damage to cells caused by sudden temperature changes.

[0025] 4. High degree of operation standardization: Filtration through a 200-mesh filter combined with 1200×g centrifugation can efficiently separate intact granular cells and tissue fragments, thereby improving the purity of the cell suspension.

[0026] 5. Wide applicability: This method was developed based on sheep ovaries, but its enzymatic hydrolysis parameters (concentration, time) and culture conditions can be adapted to the isolation needs of ovarian tissues from other mammals.

[0027] 6. Cost control: It does not rely on feeder cells or complex three-dimensional culture systems and can be operated with only conventional laboratory equipment, making it suitable for research scenarios with limited resources. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a real picture taken under a fluorescence microscope in Example 3 of the present invention;

[0029] Figure 2 This is the qRT-PCR electrophoresis diagram in Example 3 of the present invention; DETAILED DESCRIPTION

[0030] The following is combined with Figure 1-2 The present invention is described in further detail.

[0031] Example 1. A method for isolating female sheep stem cells

[0032] S1. Ovarian collection and processing: Ovaries were collected from one-year-old sheep. The collected ovaries were rinsed 2-3 times with medical alcohol, and then washed 2-3 times with DPBS buffer preheated at 37°C. The outer fascia of the ovary was cut off with sterilized scissors. The processed ovaries were placed in DMEM / F12 culture medium containing 10% FBS and 1.5% double-antibody preheated at 37°C and washed 2-3 times.

[0033] S2. Isolation of ovarian cells using trypsin:

[0034] (1) In a sterile clean bench, remove the ovaries processed in S1 and place them in a 3.5 cm culture dish. Use sterile ophthalmic scissors to cut the ovaries into pieces less than 2 mm. 3 Add 0.25% trypsin solution (containing 0.01% EDTA) to the small fragments and digest at room temperature for 1 hour, shaking once every 10 minutes;

[0035] (2) The digestion fluid containing ovarian fragments was filtered through a 200-mesh filter, and the filtrate was centrifuged at 1200 × g for 5 min. After discarding the supernatant, 5 mL of DMEM / F12 culture medium was added and the suspension was gently pipetted 5-10 times to prepare a pelleted cell suspension. The suspension was cultured in an incubator (37°C, 5% CO2, saturated humidity). The culture medium was replaced every 48 h, and the cells were observed and photographed under an inverted microscope.

[0036] Example 2. A method for culturing female sheep stem cells in vitro

[0037] Once ovine female stem cells are isolated, they can be cultured in vitro using the following two methods:

[0038] (1) Direct cell passage: When the cell confluence reaches more than 85%, cell passage can be performed. Aspirate the original culture medium; add about 2 ml of PBS, gently shake the culture bottle to rinse the cells, aspirate and discard the PBS; add about 1 ml of 0.25% trypsin solution (containing EDTA), gently shake the culture bottle to infiltrate all cells; put it into the incubator for digestion, and stop when the cells in the middle of the cell block are obviously rounded and have gaps under the microscope. Do not tap the culture bottle during the whole process; add 3 ml of serum-containing culture medium to stop digestion, blow the cells to detach them from the wall and blow them repeatedly in the liquid to make the cells as single cell suspension as possible; collect the cell suspension and centrifuge at 1200 rpm / min for 3 minutes. After centrifugation, aspirate and discard the supernatant; add fresh culture medium and blow until the cells are mixed. Inoculate into a new culture bottle at a ratio of 1:2, replenish the culture medium, loosen the bottle cap or use a breathable bottle cap for culture.

[0039] (2) Cryopreservation is performed first, and then the cells are cultured after recovery: Cryopreservation is performed when the cell confluence reaches more than 85%.

[0040] In a biosafety cabinet, open the culture flask and collect the culture medium in the flask; add 3 ml of sterile 1× PBS to the culture flask, place the culture flask horizontally so that the PBS can penetrate all areas of the bottom surface of the culture flask, and discard the PBS; add 1 ml of digestion solution to the flask, soak the bottom surface, and then incubate in a 37°C CO2 incubator for 1-2 minutes; after incubation, observe under an inverted microscope to see if the cells have become round and floated. If all cells have been digested, add 2 ml of complete culture medium containing 10% FBS directly to the culture flask and transfer the suspension to a 15 ml centrifuge tube; centrifuge at 1000 rpm for 5 minutes; after centrifugation, discard the supernatant, resuspend the cell pellet with 1 ml of freezing solution, and then transfer it to a 1.8 ml cryovial; transfer the cryovial to a programmed cooling box filled with isopropanol, and then transfer it to a -80°C refrigerator to cool overnight; the next day, remove the cryovial from the programmed cooling box and transfer it to a liquid nitrogen tank for storage as soon as possible.

[0041] When the cells are reviving, preheat the water in the constant temperature water bath to 37°C; prepare a 15ml centrifuge tube, add 5ml of complete culture medium containing 10% FBS, and place it in a 37°C water bath to preheat; after wearing goggles and thick woolen gloves, take out the cells to be revived from the liquid nitrogen tank, transfer them to a 37°C constant temperature water bath as soon as possible to rewarm and shake the cryopreserved tube to increase the rewarming rate; aspirate the cells in the thawed cryopreserved tube into the pre-prepared centrifuge tube, mix, and centrifuge at 1000rpm for 5min; prepare a T-25 culture flask, write the cell name and date, and then add 4ml of complete culture medium; after centrifugation, discard the supernatant, resuspend the cells with 1ml of complete culture medium, transfer them to a T-25 cell culture flask, mix, and transfer to a CO2 incubator for stasis.

[0042] Example 3. Isolation and identification of primary cells

[0043] Immunofluorescence was used to identify the isolated and cultured cells. PLA pretreated coverslips were placed in 24-well plates. Cells were digested with trypsin and counted. Appropriate cell density was selected and seeded into the culture plate. The next day, intervention treatment was performed. The treated cell slides were transferred to 24-well plates and soaked in cold PBS for no more than 3 minutes. 4% paraformaldehyde was added to each slide, fixed on ice for 15 minutes, and evenly covered horizontally. Cold phosphate buffer was used to fix the cells. The slides were washed with saline (PBS) buffer for 5 minutes, repeated twice, and the edges of the slides were wiped dry; blocking serum was added, and the slides were placed in a humidified box at 37°C for 60 minutes. The blocking solution was wiped off with filter paper; standard diluted primary antibodies were added to the slides and incubated in a humidified box at 4°C overnight, and then washed with PBS for 3 minutes, repeated 5 times, to wash away the primary antibodies (OCT4, SOX2, Nanog), and the PBS outside the specimen was wiped off with filter paper; fluorescent secondary antibodies were added, and the slides were incubated in a humidified box at 37°C for 1 hour in the dark, and then washed with PBS for 3 minutes, repeated 3 times, to wash away the secondary antibodies, and the PBS outside the specimen was wiped off with filter paper; DAPI was added and incubated in the dark for 2 minutes to visualize the nuclei of the specimens, and the blue fluorescence was excellent. Wash with PBS, repeat 3 times for 5 minutes to remove DAPI (4',6-diamidino-2-phenylindole), wipe off the PBS outside the specimen with filter paper; seal the slide with anti-fluorescence quencher, and immediately observe and photograph under a fluorescence microscope. Figure 1 shown.

[0044] DAPI results showed uniform staining of the cell nuclei, with intact nuclear morphology and a dense, punctate distribution, indicating normal cell status. The green fluorescence signals of OCT4, SOX2, and Nanog were primarily localized in the nuclei, with clear overlap with the DAPI signals (Merge channel), indicating that OCT4, SOX2, and Nanog proteins are specifically expressed in the nucleus.

[0045] Real-time fluorescence quantitative PCR was used to identify cell isolation and culture results. The steps were as follows: transfer the ground sample with 1 ml of Trizol to a 1.5 ml EP tube; add 500 μl of chloroform to the 1.5 ml EP tube, shake and mix thoroughly, and let it rest for 5 minutes; centrifuge at 12,000 rpm at 4°C for 10 minutes, and carefully transfer the supernatant to a new 1.5 ml EP tube; add 700 μl of isopropanol to the separated supernatant and mix thoroughly; centrifuge at 12,000 rpm at 4°C for 10 minutes, and carefully discard the supernatant; wash the precipitate once with 75% ethanol and air dry at room temperature; dissolve the RNA precipitate in 50 μl of DEPC water; and detect by agarose gel electrophoresis. Reverse transcription was performed using the Invitrogen Superscript III reverse transcription kit. Reaction system 1 was established:

[0046] RNA 200ng(10uL) Oligo-dT 1uL

[0047] Mix well and centrifuge. Incubate at 65°C for 5 minutes and place on ice.

[0048] Reaction system 1 12uL dNTP (10uM) 1uL 0.1MDTT 2uL 5XBuffer 4uL RT enzyme 1uL

[0049] Mix thoroughly and centrifuge. Incubate in a 42°C water bath for 60 minutes. Remove and incubate at 85°C for 10 minutes to inactivate the reverse transcriptase. After the reaction, store the product at -20°C until ready for use.

[0050] Real time PCR reaction system establishment:

[0051] cDNA 2uL PCRmix 10uL primerF 1uL primerR 1uL ddH2O 6uL

[0052] After the system is mixed, centrifuge briefly, place on a fluorescent quantitative PCR instrument, and react according to the following conditions: Realtime PCR reaction conditions are as follows:

[0053]

[0054] From the qRT-PCR electrophoresis diagram, we can see that target bands of 100 bp, 208 bp, 445 bp and 343 bp were detected, respectively, indicating that the isolated and cultured female germline stem cells have Oct4, Nanog and Sox2 surface markers.

[0055] This specific embodiment is merely an explanation of the present invention and is not intended to limit the present invention. After reading this specification, those skilled in the art may make non-creative modifications to this embodiment as needed. However, as long as such modifications are within the scope of the claims of the present invention, they are protected by patent law.

Claims

1. A method for isolating female sheep stem cells, characterized by: The following steps are involved: S1. Ovarian collection and processing; S2. Ovarian cells were isolated using trypsin.

2. The method for isolating female sheep stem cells according to claim 1, wherein: The specific operation of ovarian collection and processing in S1 is as follows: collect ovaries from one-year-old sheep, rinse the collected ovaries with medical alcohol 2-3 times, then wash them with DPBS buffer preheated at 37°C 2-3 times, cut off the external fascia of the ovary with sterilized scissors, place the treated ovaries in DMEM / F12 culture medium containing 10% FBS and 1.5% double antibody preheated at 37°C, and wash them 2-3 times.

3. The method for isolating female sheep stem cells according to claim 1, wherein: The specific operation of using the trypsin method to separate ovarian cells in S2 is as follows: (1) In a sterile clean bench, remove the ovaries processed in S1 and place them in a 3.5 cm culture dish. Use sterile ophthalmic scissors to cut the ovaries into pieces less than 2 mm. 3 Add 0.25% trypsin solution (containing 0.01% EDTA) to the small fragments and digest at room temperature for 1 hour, shaking once every 10 minutes; (2) The digestion fluid containing ovarian fragments was filtered through a 200-mesh filter, and the filtrate was centrifuged at 1200 × g for 5 min. After discarding the supernatant, 5 mL of DMEM / F12 culture medium was added and the suspension was gently pipetted 5-10 times to prepare a pelleted cell suspension. The suspension was cultured in an incubator (37°C, 5% CO2, saturated humidity). The culture medium was replaced every 48 h, and the cells were observed and photographed under an inverted microscope.

4. A method for culturing female sheep stem cells in vitro, characterized by: Once ovine female stem cells are isolated, they can be cultured in vitro using the following two methods: (1) Direct cell passage: When the cell confluence reaches more than 85%, cell passage can be performed; (2) Cryopreservation is performed first, and then the cells are cultured after recovery: Cryopreservation is performed when the cell confluence reaches more than 85%.

5. The in vitro culture method of sheep female stem cells according to claim 4, characterized in that: The specific operation of the cell passage is as follows: Aspirate the original culture medium; add about 2 ml of PBS, gently shake the culture flask to rinse the cells, aspirate the PBS and discard it; add about 1 ml of 0.25% trypsin solution (containing EDTA), and gently shake the culture flask to infiltrate all cells; place it in the incubator for digestion, and stop when the cells in the middle of the cell block are obviously rounded and have gaps under the microscope. Do not tap the culture flask during the whole process; add 3 ml of serum-containing culture medium to stop digestion, blow the cells to detach them from the wall and blow them repeatedly in the liquid to make the cells as single-cell suspension as possible; collect the cell suspension and centrifuge at 1200 rpm / min for 3 minutes. Aspirate the supernatant and discard it after centrifugation; add fresh culture medium, blow until the cells are mixed, inoculate into a new culture flask at a ratio of 1:2, replenish the culture medium, loosen the bottle cap or use a breathable bottle cap for culture.

6. The in vitro culture method of sheep female stem cells according to claim 4, characterized in that: The specific operation of the freezing is as follows: In a biosafety cabinet, open the culture flask and collect the culture medium in the flask; add 3 ml of sterile 1× PBS to the culture flask, place the culture flask horizontally so that the PBS can penetrate all areas of the bottom surface of the culture flask, and discard the PBS; add 1 ml of digestion solution to the flask, soak the bottom surface, and then incubate in a 37°C CO2 incubator for 1-2 minutes; after incubation, observe under an inverted microscope to see if the cells have become round and floated. If all cells have been digested, add 2 ml of complete culture medium containing 10% FBS directly to the culture flask and transfer the suspension to a 15 ml centrifuge tube; centrifuge at 1000 rpm for 5 minutes; after centrifugation, discard the supernatant, resuspend the cell pellet with 1 ml of freezing solution, and then transfer it to a 1.8 ml cryovial; transfer the cryovial to a programmed cooling box filled with isopropanol, and then transfer it to a -80°C refrigerator to cool overnight; the next day, remove the cryovial from the programmed cooling box and transfer it to a liquid nitrogen tank for storage as soon as possible.

7. The in vitro culture method of sheep female stem cells according to claim 4, characterized in that: The specific operation of reculturing the cells after recovery is as follows: When cells are reviving, preheat the water in a constant temperature water bath to 37°C; prepare a 15ml centrifuge tube, add 5ml of complete culture medium containing 10% FBS, and place it in a 37°C water bath to preheat; after wearing goggles and thick woolen gloves, remove the cells to be revived from the liquid nitrogen tank, transfer them to a 37°C constant temperature water bath as soon as possible to rewarm and shake the cryopreserved tube to increase the rewarming rate; aspirate the cells in the thawed cryopreserved tube into the pre-prepared centrifuge tube, mix, and centrifuge at 1000rpm for 5min; prepare a T-25 culture flask, write the cell name and date, and then add 4ml of complete culture medium; after centrifugation, discard the supernatant, resuspend the cells with 1ml of complete culture medium, transfer them to a T-25 cell culture flask, mix, and transfer to a CO2 incubator for culture and stasis.

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