CN116286607A - Method for inducing directional differentiation of primary oviduct epithelial cells into ciliated cells - Google Patents

Method for inducing directional differentiation of primary oviduct epithelial cells into ciliated cells Download PDF

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CN116286607A
CN116286607A CN202310413307.3A CN202310413307A CN116286607A CN 116286607 A CN116286607 A CN 116286607A CN 202310413307 A CN202310413307 A CN 202310413307A CN 116286607 A CN116286607 A CN 116286607A
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oviduct
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朱茂碧
王莎
王娜
袭昊曈
张双
邹家伦
范江霖
陈亚檞
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Wuyi University
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Abstract

The invention discloses a method for inducing directional differentiation of primary oviduct epithelial cells into ciliated cells. The method of the invention comprises the following steps: firstly, primary oviduct epithelial cells are obtained, then mixed with a 5Mix culture medium containing serum to prepare a cell suspension, then inoculated into an upper chamber of a cell culture chamber, and a DMEM/F12 culture medium containing serum is added into a lower chamber of the cell culture chamber to be cultured until the cells are attached to the wall; after the cells are attached, the culture mediums in the upper chamber and the lower chamber are replaced by serum-free 5Mix culture mediums, and the culture is continued until the cells reach more than 90% fusion; and finally, removing the culture medium in the upper chamber and the lower chamber, and adding 5Mix culture medium containing estrogen or follicular fluid into the lower chamber for gas-liquid interface culture for 10-15 days. The method has high cell differentiation efficiency, and the obtained ciliated cells have physiological characteristics similar to in-vivo physiological states.

Description

Method for inducing directional differentiation of primary oviduct epithelial cells into ciliated cells
Technical Field
The invention relates to the field of biotechnology, in particular to a method for inducing directional differentiation of primary oviduct epithelial cells into ciliated cells.
Background
The follicular fluid is a normal physiological fluid in females, contains abundant steroid hormone, cytokines, ROS, prostaglandin, proteolytic enzyme and other substances, has relatively few researches on the follicular fluid acting on fallopian tubes, and mainly focuses on the aspects related to canceration, and almost no research on the physiological characteristics and differentiation of the epithelial cells (fallopian tube epithelial cells, hereinafter referred to as FTECs) of the fallopian tubes by the follicular fluid exists.
Immortalized FTECs and High-grade serous ovarian cancer cells (High-grade serous carcinoma, hereinafter referred to as HGSOC) with severe accumulation of driven mutations are used in related researches to explore the role of follicular fluid in the development of HGSOC, and the result shows that factors such as IGF2, HGF and the like in follicular fluid can cause malignant transformation and proliferation of the FTECs and can be transferred to ovaries and peritoneum to form HGSOC. However, in the related art, studies on how to efficiently induce differentiation of primary oviduct epithelial cells into ciliated cells using follicular fluid or other hormonal substances have not been reported.
Therefore, there is a need for a method that can efficiently induce differentiation of primary oviduct epithelial cells into ciliated cells.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art. Therefore, the invention provides a method for inducing directional differentiation of primary oviduct epithelial cells into ciliated cells, which is cultured to obtain ciliated cells with high differentiation degree, physiological characteristics similar to in-vivo physiological states and long-term maintenance in vitro.
The invention relates to a method for inducing directional differentiation of primary oviduct epithelial cells into ciliated cells, which comprises the following steps:
s1, obtaining primary oviduct epithelial cells;
step S2, mixing the primary oviduct epithelial cells with a 5Mix culture medium containing serum to prepare a cell suspension, then inoculating the cell suspension into an upper chamber of a cell culture chamber, adding a DMEM/F12 culture medium containing serum into a lower chamber of the cell culture chamber, and culturing until the cells adhere to the wall;
s3, after the cells are attached, replacing the culture mediums in the upper chamber and the lower chamber with a serum-free 5Mix culture medium, and continuously culturing until the cells reach more than 90% fusion;
s4, removing culture mediums in the upper chamber and the lower chamber, and adding 5Mix culture mediums containing estrogen or 5Mix culture mediums containing follicular fluid into the lower chamber for gas-liquid interface culture for 10-15 days;
wherein the 5Mix culture medium is obtained by adding a B27 additive, a Glutamax additive, an epidermal growth factor, niacinamide and a TGF-beta receptor kinase inhibitor on the basis of a DMEM/F12 culture medium.
The method according to the embodiment of the invention has at least the following beneficial effects:
(1) The invention can better simulate the growth condition of cells in vivo based on a gas-liquid interface culture method, form a pseudo-multi-layer columnar epithelium with a tissue structure and a physiological function similar to those of in vivo tissues, allow the primary oviduct secretory cells and ciliated cells to be separated and co-cultured, and retain the morphology and biological characteristics of the primary oviduct epithelium.
(2) According to the invention, the degree of differentiation of primary oviduct epithelial cells into ciliated cells is effectively improved by adding estrogen or follicular fluid. The fluorescent staining test shows that the invention adds a certain amount of Estrogen (E2) and PFF in the differentiation culture process to help the formation of ciliated cells.
(3) In the method, the 5Mix culture medium containing serum is adopted in the early stage of cell culture, and the serum contains hormone and growth factor which are beneficial to cell growth and proliferation, so that the cell adhesion is facilitated, and the 5Mix culture medium without serum is replaced in the later stage of cell differentiation culture, mainly because the serum-containing culture medium is always adopted for culture, the cells lose the original characteristics and even become fibroblasts, and the influence on the proliferation and differentiation of the subsequent cells can be avoided by removing the serum in the later stage of culture.
In some embodiments of the invention, the method of obtaining primary oviduct epithelial cells comprises: scraping the epithelial cell layer in the lumen of the oviduct, sequentially placing the epithelial cell layer in a DMEM/F12 culture medium containing collagenase and DNase I for digestion treatment, collecting cell suspension, and centrifuging to obtain primary oviduct epithelial cells.
The primary oviduct epithelial cells obtained by the method have high activity, and the scheme of the invention adopts the primary oviduct epithelial cells, so that the integrity of the primary oviduct epithelial cells is better compared with that of the conventional passage FTECs system genes, and the cultured cells have physiological characteristics similar to the physiological state in vivo.
In some embodiments of the invention, the cell culture chamber is a Transwell cell culture chamber or a Millicell cell culture chamber.
In some embodiments of the invention, the upper and lower chambers of the cell culture chamber are separated by a polycarbonate membrane.
In some embodiments of the invention, the collagenase is selected from at least one of collagenase i, collagenase ii, and collagenase iv.
In some embodiments of the invention, the collagenase is at a concentration of 80 to 100U/mL; specifically, the collagenase IV may be at a concentration of 100U/mL.
In some embodiments of the invention, the DNase I concentration is 8-12 μg/mL; specifically, the DNase I concentration may be 10. Mu.g/mL.
In some embodiments of the invention, in step S2, the cell density of the primary oviduct epithelial cells in the cell suspension is 6×10 2 ～8×10 2 Each cell per microliter.
In some embodiments of the invention, in step S2, the serum in the serum-containing 5Mix medium is fetal bovine serum;
preferably, the volume content of the serum is 8-12%.
In some embodiments of the invention, in step S3, the culture is continued until the cells reach more than 95% confluence after changing to serum-free 5Mix medium; preferably, the culture is continued until the cells reach 100% confluence.
If the fusion degree of the cells is too low, the cells are easy to fall off and even die, so that the cells need to be cultured as much as possible in the culture process, and then follow-up experiments such as drug addition and the like are carried out after the cells grow fully.
In some embodiments of the invention, in step S4, the concentration of the estrogen in the estrogen-containing 5Mix medium is 1 to 3ng/mL;
preferably, the concentration of estrogen in the estrogen-containing 5Mix medium is 2ng/mL.
In some embodiments of the invention, the follicular fluid is present in the follicular fluid containing 5Mix medium in an amount of 1 to 10% by volume.
Too high follicular fluid can have adverse effects on cells, such as causing the cells to shed from the chamber and even die, and thus need to be controlled within a range in order to fully exert the functions of inducing cell proliferation and differentiation.
Preferably, the volume content of the follicular fluid in the follicular fluid-containing 5Mix culture medium is 3-8%;
preferably, the volume content of the follicular fluid in the follicular fluid-containing 5Mix culture medium is 1-5%;
more preferably, the follicular fluid is present in the follicular fluid-containing 5Mix medium in an amount of 5% by volume.
In some embodiments of the invention, the primary oviduct epithelial cells are derived from the same species as the follicular fluid.
Preferably, both the primary oviduct epithelial cells and the follicular fluid are derived from at least one of pigs, cattle, horses, sheep.
More preferably, both the primary oviduct epithelial cells and the follicular fluid may be of porcine origin.
The primary oviduct epithelial cells and follicular fluid of the same genus are adopted to reduce rejection reaction, which is helpful for improving differentiation effect.
In some embodiments of the invention, the follicular fluid is a pre-pubertal sow ovarian follicular fluid.
In some embodiments of the invention, in step S4, fresh medium is replaced every 2 to 3 days during the culturing.
Specifically, the fresh culture medium refers to a 5Mix culture medium containing estrogen or a 5Mix culture medium containing follicular fluid; when 5Mix culture medium containing estrogen is adopted for gas-liquid interface culture, the fresh culture medium refers to the 5Mix culture medium containing estrogen; when the gas-liquid interface culture is performed using a 5Mix medium containing follicular fluid, the fresh medium refers to a 5Mix medium containing follicular fluid.
In some embodiments of the invention, the volume content of the B27 additive in the 5Mix medium is 1-3%; specifically, in the 5Mix medium, the volume content of the B27 additive may be 2%.
In some embodiments of the invention, the volume content of the GlutaMAX additive in the 5Mix medium is 0.5-2%; the volume content of the GlutaMAX additive may be 1%.
In some embodiments of the invention, the concentration of the epidermal growth factor in the 5Mix medium is 8-12 ng/mL; the concentration of the epidermal growth factor may be 10ng/mL.
In some embodiments of the invention, the concentration of the niacinamide in the 5Mix medium is 0.5 to 1.5mmol/L; specifically, the final concentration of niacinamide may be 1mM.
In some embodiments of the invention, the concentration of the TGF-beta receptor kinase inhibitor in the 5Mix medium is 0.2 to 1. Mu. Mol/L; the final concentration of the TGF-beta receptor kinase inhibitor may be 0.5. Mu.M.
In some embodiments of the invention, the TGF- β receptor kinase inhibitor may be SB431542.
In some embodiments of the inventionIn an embodiment, the temperature of the culture is 37+/-2 ℃, and the CO is in the culture process 2 The volume content is 4-6%.
Additional features and advantages of the invention will be set forth in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
Drawings
The invention is further described with reference to the accompanying drawings and examples, in which:
FIG. 1 is a technical roadmap of the invention.
FIG. 2 is a graph of the fluorescent staining of primary FTECs according to the present invention, wherein green is P73 staining, red is EdU staining, and blue is DAPI staining, scale bar: 100 μm.
Fig. 3 is a graph of quantitative P73 staining positive cell numbers according to the invention, wherein the significance difference was compared to Ctrl group using one-way anova, n=10, significance level: * P <0.0001.
Fig. 4 is a graph of the invention for quantifying EdU positive cell numbers, using a one-factor analysis of variance, n=10, with significance differences compared to Ctrl group, significance level: * P <0.0001.
FIG. 5 is a graph of fluorescence staining of FTECs cilia grown at the gas-liquid interface according to the present invention, wherein green is Ac-T stained, red is EdU, and blue is DAPI stained, scale bar: 20 μm.
Fig. 6 is a graph of the invention for quantifying EdU positive cell numbers using one-way anova with n=7, significance difference compared to Ctrl group, significance level: * p <0.05.
Fig. 7 is a graph of the invention quantifying Ac-T positive cell numbers using a one-factor analysis of variance, n=7, significance difference compared to Ctrl group, significance level: * P <0.0001.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.
In the description of the present invention, the descriptions of the terms "one embodiment," "some embodiments," "illustrative embodiments," "examples," "specific examples," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms do not necessarily refer to the same embodiments or examples. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples.
In an embodiment of the invention, the diabody is a Hyclone diabody (penicillin-streptomycin), purchased at Ke Yibo, accession number: SV30010;
b27 additives are purchased from Thermofisher under accession number 17504044;
GlutaMAX additives are purchased from thermosusher under the accession number 35050061;
epidermal growth factor is purchased from PeproTech under the accession number AF-100-15;
nikkamide was purchased from Sigma-Aldrich under the designation 72340;
TGF-beta receptor kinase inhibitors are purchased from Macklin, cat# S863557;
DMEM/Ham's F-12 medium is purchased from Wako, cat: 042-30795;
fetal bovine serum was purchased from Corning, cat: 35-081-CV;
estrogens were purchased from Sigma-Aldrich under the designation E4389;
the specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1
Acquisition of primary oviduct epithelial cells
1. The oviduct is obtained from healthy sows, then the obtained oviduct is placed in a DMEM/Ham's F-12 (hereinafter referred to as DMEM/F12) medium containing 4% of double antibody (penicillin-streptomycin) prepared in advance, and immediately processed after being transported back to a laboratory;
2. washing the oviduct with 75% alcohol and sterile PBS, trimming to remove excessive connective tissue, and washing with sterile PBS for 5 times;
3. longitudinally splitting the cleaned oviduct, putting the oviduct into a DMEM/F12 culture medium containing 100U/mL collagenase IV, and digesting the oviduct in a water bath at 37 ℃ for 1.5 hours;
4. scraping epithelial cells with digested oviduct cells, adding appropriate amount of DNase I (10 μg/mL), and placing at 37deg.C with 5% CO 2 In a cell culture incubator (the conditions of the incubator are 37 ℃ C. And 5% CO unless otherwise specified) 2 ) Digesting for 15 minutes, blowing off digested FTECs into a centrifuge tube for centrifugation after no sticky tissues exist in the cells, centrifuging for 3 minutes at 800rpm, and removing the supernatant to obtain the cells;
6. adding 2-4 mL of prepared DMEM/F12 culture medium containing 10% of fetal calf serum and 2% of double antibodies into the centrifuge tube, blowing and evenly mixing cells, transferring the cells into a new 10 cm cell culture dish (a plurality of dishes can be divided according to the number of the cells in the case of more cells), then adding a proper amount of fresh culture medium (5-7 mL of culture medium of each dish), evenly mixing the cells, and then placing the cells into a cell culture box for culturing until primary FTECs are attached;
7. after FTECs are attached, the culture medium containing serum is changed into a 5Mix culture medium for continuous culture, and after cells are basically overgrown (80-90%), the cells are frozen for standby, wherein the 5Mix culture medium is obtained by adding 2% by volume of B27, 1% by volume of GlutaMAX, 10ng/mL of epidermal growth factor, 1mM nicotinamide and 0.5 mu M SB431542 (TGF-beta receptor kinase inhibitor) on the basis of the DMEM/F12 culture medium.
(II) gas-liquid interface culture
1. The amplified FTECs were inoculated into 24-well plates with Transwell chambers and were prepared into cell suspensions using 5Mix medium containing 10% fetal bovine serum, and then the cell suspensions were inoculated into the upper chamber of the Transwell chambers in an amount of 150. Mu.L (whereinThe total number of the oval tube epithelial cells is 1 multiplied by 10 5 Individual cells), 500. Mu.L of DMEM/F12 medium containing 10% serum was added to the lower chamber of the Transwell chamber, and the mixture was placed in a medium containing 5% CO at 37 ℃ 2 Culturing in a cell incubator;
2. after the cells are attached the next day, the culture solution in the upper and lower chambers of the small chamber is replaced by a serum-free 5Mix culture medium, and the culture is continued until the cells grow fully;
3. culturing with serum-free 5Mix culture medium for two days, sucking up the culture medium in the upper chamber of the chamber, adding 5Mix culture medium containing estrogen (E2, final concentration of 2 ng/mL) in the lower chamber of the chamber, and performing gas-Liquid Interface culture (ALI) under the conditions of 37deg.C and 5% CO 2 The culture medium in the lower chamber is replaced every two days, and the culture is carried out for 10 to 15 days.
Example 2
Acquisition of primary oviduct epithelial cells
1. The oviduct is obtained from healthy sows, then the obtained oviduct is placed in a DMEM/Ham's F-12 culture medium containing 4% of double antibody (penicillin-streptomycin) prepared in advance, and immediately processed after being transported back to a laboratory;
2. washing the oviduct with 75% alcohol and sterile PBS, trimming to remove excessive connective tissue, and washing with sterile PBS for 5 times;
3. longitudinally splitting the cleaned oviduct, putting the oviduct into a culture medium with 100U/mL collagenase IV, and digesting the oviduct in a water bath kettle at 37 ℃ for 1.5 hours;
4. scraping epithelial cells with digested oviduct cells, adding appropriate amount of DNase I (10 μg/mL), and placing at 37deg.C with 5% CO 2 In a cell culture incubator (the conditions of the incubator are 37 ℃ C. And 5% CO unless otherwise specified) 2 ) Digesting for 15 minutes, blowing off digested FTECs into a centrifuge tube for centrifugation after no sticky tissues exist in the cells, centrifuging for 3 minutes at 800rpm, and removing the supernatant to obtain the cells;
6. adding 2-4 mL of prepared DMEM/F12 culture medium containing 10% of fetal calf serum and 2% of double antibodies into the centrifuge tube, blowing and evenly mixing cells, transferring the cells into a new 10 cm cell culture dish (a plurality of dishes can be divided according to the number of the cells in the case of more cells), then adding a proper amount of fresh culture medium (5-7 mL of culture medium of each dish), evenly mixing the cells, and then placing the cells into a cell culture box for culturing until primary FTECs are attached;
7. after FTECs are attached, the culture medium containing serum is changed into a 5Mix culture medium for continuous culture, and after cells are basically overgrown (80-90%), the cells are frozen for standby, wherein the 5Mix culture medium is obtained by adding 2% by volume of B27, 1% by volume of GlutaMAX, 10ng/mL of epidermal growth factor, 1mM nicotinamide and 0.5 mu M SB431542 (TGF-beta receptor kinase inhibitor) on the basis of the DMEM/F12 culture medium.
(II) gas-liquid interface culture
1. The amplified FTECs were inoculated into 24-well plates with a Transwell chamber, and the FTECs were prepared into a cell suspension using 5Mix medium containing 10% fetal bovine serum, and then the cell suspension was inoculated into the upper chamber of the Transwell chamber, and 150. Mu.L of the cell suspension was added (wherein the number of oviduct epithelial cells was 1X 10) 5 Individual cells), 500. Mu.L of DMEM/F12 medium containing 10% serum was added to the lower chamber of the chamber, and the mixture was placed in a medium containing 5% CO at 37 ℃C 2 Culturing in a cell incubator;
2. after the cells are attached the next day, the culture solution in the upper and lower chambers of the small chamber is replaced by a serum-free 5Mix culture medium, and the culture is continued until the cells grow fully;
3. culturing with serum-free 5Mix culture medium for two days, sucking up the culture medium in the upper chamber of the chamber, adding 5Mix culture medium containing follicular fluid (PFF, final concentration 1%) into the lower chamber of the chamber, and performing gas-Liquid Interface culture (ALI) under the conditions of 37deg.C and 5% CO 2 The culture medium in the lower chamber is replaced every two days, and the culture is carried out for 10 to 15 days.
Example 3
Acquisition of primary oviduct epithelial cells
1. The oviduct is obtained from healthy sows, then the obtained oviduct is placed in a DMEM/Ham's F-12 culture medium containing 4% of double antibody (penicillin-streptomycin) prepared in advance, and immediately processed after being transported back to a laboratory;
2. washing the oviduct with 75% alcohol and sterile PBS, trimming to remove excessive connective tissue, and washing with sterile PBS for 5 times;
3. longitudinally splitting the cleaned oviduct, putting the oviduct into a culture medium with 100U/mL collagenase IV, and digesting the oviduct in a water bath kettle at 37 ℃ for 1.5 hours;
4. scraping epithelial cells with digested oviduct cells, adding appropriate amount of DNase I (10 μg/mL), and placing at 37deg.C with 5% CO 2 Digesting for 15 minutes in a cell culture box, blowing off digested FTECs into a centrifuge tube for centrifugation after no viscous tissues exist in cells, and removing supernatant to obtain cells;
6. adding 2-4 mL of prepared DMEM/F12 culture medium containing 10% of fetal calf serum and 2% of double antibodies into the centrifuge tube, blowing and evenly mixing cells, transferring the cells into a new 10 cm cell culture dish (a plurality of dishes can be divided according to the number of the cells in the case of more cells), then adding a proper amount of fresh culture medium (5-7 mL of culture medium of each dish), evenly mixing the cells, and then placing the cells into a cell culture box for culturing until primary FTECs are attached;
7. after FTECs are attached, the culture medium containing serum is changed into a 5Mix culture medium for continuous culture, and after cells are basically overgrown (80-90%), the cells are frozen for standby, wherein the 5Mix culture medium is obtained by adding 2% by volume of B27, 1% by volume of GlutaMAX, 10ng/mL of epidermal growth factor, 1mM nicotinamide and 0.5 mu M SB431542 (TGF-beta receptor kinase inhibitor) on the basis of the DMEM/F12 culture medium.
(II) gas-liquid interface culture
1. The amplified FTECs were inoculated into a 24-well plate having a Transwell chamber, and the FTECs were prepared into a cell suspension using 5Mix medium containing 10% serum, and then the cell suspension was inoculated into the upper chamber of the Transwell chamber, and 150. Mu.L of the cell suspension was added (wherein the number of oviduct epithelial cells was 1X 10) 5 Individual cells), 500. Mu.L of DMEM/F12 medium containing 10% serum was added to the lower chamber of the chamber, and the mixture was placed in a medium containing 5% CO at 37 ℃C 2 Culturing in a cell incubator;
2. after the cells are attached the next day, the culture solution in the upper and lower chambers of the small chamber is replaced by a serum-free 5Mix culture medium, and the culture is continued;
3. culturing with serum-free 5Mix culture medium for two days, sucking the culture medium in the upper chamber of the chamber, adding follicular fluid (PFF, final concentration of 5%) containing 5Mix culture medium in the lower chamber of the chamber, and performing gas-liquid interface culture at 37deg.C under 5% CO 2 The culture medium in the lower chamber is replaced every two days.
Comparative example 1
Acquisition of primary oviduct epithelial cells
1. The oviduct is obtained from healthy sows, then the obtained oviduct is placed in a DMEM/Ham's F-12 (hereinafter referred to as DMEM/F12) medium containing 4% of double antibody (penicillin-streptomycin) prepared in advance, and immediately processed after being transported back to a laboratory;
2. washing the oviduct with 75% alcohol and sterile PBS, trimming to remove excessive connective tissue, and washing with sterile PBS for 5 times;
3. longitudinally splitting the cleaned oviduct, putting the oviduct into a culture medium with 100U/mL collagenase IV, and digesting the oviduct in a water bath kettle at 37 ℃ for 1.5 hours;
4. scraping epithelial cells with digested oviduct cells, adding appropriate amount of DNase I (volume determined by tissue amount), and adding 5% CO at 37deg.C 2 In a cell culture incubator (the conditions of the incubator are 37 ℃ C. And 5% CO unless otherwise specified) 2 ) Digesting for 15 minutes, blowing off digested FTECs into a centrifuge tube for centrifugation after no sticky tissues exist in the cells, and removing the supernatant to obtain the cells;
6. adding 2-4 mL of prepared DMEM/F12 culture medium containing 10% of fetal calf serum and 2% of double antibodies into the centrifuge tube, blowing and evenly mixing cells, transferring the cells into a new 10 cm cell culture dish (a plurality of dishes can be divided according to the number of the cells in the case of more cells), then adding a proper amount of fresh culture medium (5-7 mL of culture medium of each dish), evenly mixing the cells, and then placing the cells into a cell culture box for culturing until primary FTECs are attached;
7. after FTECs are attached, the culture medium containing serum is changed into a 5Mix culture medium for continuous culture, and after cells are basically overgrown (80-90%), the cells are frozen for standby, wherein the 5Mix culture medium is obtained by adding 2% by volume of B27, 1% by volume of GlutaMAX, 10ng/mL of epidermal growth factor, 1mM nicotinamide and 0.5 mu M SB431542 (TGF-beta receptor kinase inhibitor) on the basis of the DMEM/F12 culture medium.
(II) gas-liquid interface culture
1. The amplified FTECs were inoculated into a 24-well plate having a Transwell chamber, and the FTECs were prepared into a cell suspension using 5Mix medium containing 10% serum, and then the cell suspension was inoculated into the upper chamber of the Transwell chamber, and 150. Mu.L of the cell suspension was added (wherein the number of oviduct epithelial cells was 1X 10) 5 Individual cells), 500. Mu.L of DMEM/F12 medium containing 10% serum was added to the lower chamber of the chamber, and the mixture was placed in a medium containing 5% CO at 37 ℃C 2 Culturing in a cell incubator;
2. after the cells are attached the next day, the culture solution in the upper and lower chambers of the small chamber is replaced by a serum-free 5Mix culture medium, and the culture is continued;
3. culturing with serum-free 5Mix culture medium for two days, sucking up the culture medium in the upper chamber of the chamber, adding 5Mix culture medium containing progestogen (P4, final concentration of 10 nM) in the lower chamber of the chamber, and performing gas-liquid interface culture at 37deg.C under 5% CO 2 The culture medium in the lower chamber is replaced every two days.
Comparative example 2
Acquisition of primary oviduct epithelial cells
1. The oviduct is obtained from healthy sows, then the obtained oviduct is placed in a DMEM/Ham's F-12 culture medium containing 4% of double antibody (penicillin-streptomycin) prepared in advance, and immediately processed after being transported back to a laboratory;
2. washing the oviduct with 75% alcohol and sterile PBS, trimming to remove excessive connective tissue, and washing with sterile PBS for 5 times;
3. longitudinally splitting the cleaned oviduct, putting the oviduct into a culture medium with 100U/mL collagenase IV, and digesting the oviduct in a water bath kettle at 37 ℃ for 1.5 hours;
4. scraping epithelial cells with digested oviduct cells, adding appropriate amount of DNase I (10 μg/mL), and placing at 37deg.C with 5% CO 2 Digesting for 15 minutes in a cell culture box, blowing off digested FTECs into a centrifuge tube for centrifugation after no viscous tissues exist in cells, and removing supernatant to obtain cells;
6. adding 2-4 mL of DMEM/F12 culture medium containing 10% serum and 2% double antibody prepared in advance into the centrifuge tube, blowing and evenly mixing cells, transferring the cells into a new 10 cm cell culture dish (a plurality of dishes can be divided according to the number of the cells under the condition of more cells), then adding a proper amount of fresh culture medium (5-7 mL of culture medium of each dish), evenly mixing the cells, and then placing the cells into a cell culture box for culturing until primary FTECs are attached to the wall;
7. after FTECs are attached, the culture medium containing serum is changed into a 5Mix culture medium for continuous culture, and the cells are frozen for standby after being basically overgrown (80-90%), wherein the 5Mix culture medium is prepared by adding 2% of B27, 1% of Glutamax, epidermal growth factor with the final concentration of 10ng/mL, 1mM of niacinamide with the final concentration and 0.5 mu MSB431542 into a DMEM/F12 culture medium.
(II) gas-liquid interface culture
1. The amplified FTECs were inoculated into a 24-well plate having a Transwell chamber, and the FTECs were prepared into a cell suspension using 5Mix medium containing 10% serum, and then the cell suspension was inoculated into the upper chamber of the Transwell chamber, and 150. Mu.L of the cell suspension was added (wherein the number of oviduct epithelial cells was 1X 10) 5 Individual cells), 500. Mu.L of DMEM/F12 medium containing 10% serum was added to the lower chamber of the chamber, and the mixture was placed in a medium containing 5% CO at 37 ℃C 2 Culturing in a cell incubator;
2. after the cells are attached, the culture solution in the upper chamber and the culture solution in the lower chamber of the small chamber are replaced by a serum-free 5Mix culture medium, and the culture is continued until the cells are full;
3. culturing with serum-free 5Mix culture medium for two days, sucking up the culture medium in the upper chamber of the chamber, and adding 5Mix culture medium into the lower chamber of the chamber for gas-liquid interface culture at 37deg.C under 5% CO 2 The culture medium in the lower chamber is replaced every two days,culturing for 10-15 days.
Test case
(one) Effect of different Components on proliferation potency and differentiation potential of Primary FTECs
1. Experimental method
Firstly, putting the climbing sheet into a 24-hole plate, adding 200 mu L Matrigel gel which is diluted by 50 times by a DMEM/F12 culture medium to coat the climbing sheet, putting a constant-temperature incubator at 37 ℃ for 20-30 minutes, sucking the supernatant after the climbing sheet is coated, and washing three times by using sterile PBS for later use; the primary FTECs obtained in the above example were then inoculated onto coated slide plates using a cell suspension prepared from 5Mix medium containing 10% serum and E2 (final concentration 2 ng/mL), P4 (final concentration 10 nM), 1% PFF or 5% PFF were added, respectively (previous studies showed that E2 at final concentration 2ng/mL and P4 at final concentration 10nM were optimal concentrations for inducing cilia formation), 500. Mu.L per well, cell density 1.5X10 5 Every 500. Mu.L of individual cells; the following day, the serum-containing 5Mix medium was removed, followed by incubation with the EdU-containing medium (concentration of EdU 50 μm) for 24h, followed by immunofluorescent staining.
Wherein the immunofluorescence staining specifically comprises the following steps: firstly, taking out the cultured FTECs, washing the FTECs once by using PBS, fixing the FTECs for 10 minutes by using 4% paraformaldehyde at room temperature, and washing the FTECs for 5 minutes by using 1X PBS after removing the 4% paraformaldehyde for 3 times; then 0.1% TritonX-100 plus 5% donkey serum is permeably blocked for 30 minutes and washed once with PBS; incubation of the wet box overnight at 4℃for incubation of primary antibody (P73 (diluted with 1 XPBS, 1:200, purchased from Abcam, cat. No. ab 40658)), washing with 1 XPBS 3 times for 5 minutes after incubation of the primary antibody; the following day, secondary antibodies (purchased from Abcam, cat No. ab 150077) were incubated at room temperature in the absence of light for 1 hour, and after incubation, washed 3 times with 1×pbs for 5 minutes each; after the incubation, edU (purchased from Ruibo organism, cat# C10310-1) staining was performed; finally, the nuclei were stained, stained with DAPI (purchased from GIBCO, cat No. 62248) for 5 minutes, and then washed once with PBS for plate sealing observation.
2. Experimental results
The primary FTECs obtained by the invention are inoculated on a climbing plate and are respectively cultured for 24 hours by using a culture medium containing E2, P4, 1% PFF and 5% PFF, then a new culture medium is added to EdU to be cultured together for 24 hours, and then the cells are subjected to P73 (green), edU (red) and DAPI (blue) staining observation, the results are shown in figure 2, and the results show that compared with other groups, the addition of a certain content of follicular fluid and estrogen in the culture medium is helpful for improving the P73 positive rate and the EdU positive rate.
Further, the quantitative determination of the number of P73-stained positive cells and the number of EdU-stained positive cells is shown in fig. 3 and 4, and the results show that the number of P73-positive cells and the number of EdU-positive cells in the 5% pff group are significantly improved compared with the control group, the E2 group and the P4 group.
The above results indicate that estrogens and follicular fluid have a certain promoting effect on the proliferation and differentiation capacity of primary FTECs, but P4 has no obvious effect on the differentiation of multi-ciliated cells.
(II) detection of degree of differentiation of cell cultured at gas-liquid interface
1. Experimental method
Cells obtained in examples 1 to 3 and comparative examples 1 to 2 were cultured with ALI for 10 days, and then fresh medium was changed and EdU was added thereto, followed by further culturing for 48 hours at 37℃under 5% CO 2 Cells were then stained with Ac-T, edU and DAPI. Wherein EdU staining is used to examine the proliferative capacity of the FTECs after differentiation.
2. Experimental results
The results of the staining observations are shown in FIG. 5, where the scale is 20 μm, and the results show that the FTECs, E2 (example 1) plus 5% PFF (example 3) cells formed a large number of ciliated cells and the control (comparative example 2) and progestogen (comparative example 1) plus cells formed only a small number of ciliated cells, cultured at the gas-liquid interface.
Further, the numbers of EdU positive cells and Ac-T positive cells were quantified (one-way anova, n=7, significant difference compared to Ctrl group), and the results are shown in fig. 6 and 7, which indicate that the proliferation rate of cells in both E2 group and PFF group was higher than that in progestogen group and control group, wherein the average proportion of differentiated cilia cells in 5% PFF group was around 22%, which was much higher than that in control group and P4 group.
In summary, the present invention provides a method for inducing directional differentiation of primary oviduct epithelial cells into ciliated cells, which can better simulate the growth condition of cells in vivo, form pseudo-multi-layered columnar epithelium with tissue structure and physiological function similar to that of in vivo tissues, allow separation and co-culture of primary oviduct secretory cells and ciliated cells, and retain the morphology and biological characteristics of the primary oviduct epithelium.
In addition, the method adopts primary FTECs, compared with the conventional passaged FTECs, the gene mutation or deletion sites are fewer, and the cultured cells have physiological characteristics similar to the physiological state in vivo, so that the method has important value for researching the physiological characteristics of the FTECs.
While the embodiments of the present invention have been described in detail, the present invention is not limited to the above embodiments, and various changes can be made without departing from the spirit of the present invention within the knowledge of those skilled in the art. Furthermore, embodiments of the invention and features of the embodiments may be combined with each other without conflict.

Claims (10)

1. A method of inducing directional differentiation of primary oviduct epithelial cells into ciliated cells comprising the steps of:
s1, obtaining primary oviduct epithelial cells;
step S2, mixing the primary oviduct epithelial cells with a 5Mix culture medium containing serum to prepare a cell suspension, then inoculating the cell suspension into an upper chamber of a cell culture chamber, adding a DMEM/F12 culture medium containing serum into a lower chamber of the cell culture chamber, and culturing until the cells adhere to the wall;
s3, after the cells are attached, replacing culture mediums in the upper chamber and the lower chamber with serum-free 5Mix culture mediums, and continuously culturing until the cells reach more than 90% fusion;
s4, removing culture mediums in the upper chamber and the lower chamber, and adding 5Mix culture mediums containing estrogen or 5Mix culture mediums containing follicular fluid into the lower chamber for gas-liquid interface culture for 10-15 days;
wherein the 5Mix culture medium is obtained by adding a B27 additive, a Glutamax additive, an epidermal growth factor, niacinamide and a TGF-beta receptor kinase inhibitor on the basis of a DMEM/F12 culture medium.
2. The method of claim 1, wherein the method of obtaining primary oviduct epithelial cells comprises: scraping the epithelial cell layer in the lumen of the oviduct, sequentially placing the epithelial cell layer in a DMEM/F12 culture medium containing collagenase and DNase I for digestion treatment, collecting cell suspension, and centrifuging to obtain primary oviduct epithelial cells.
3. The method of claim 2, wherein the collagenase is at least one selected from the group consisting of collagenase I, collagenase II, and collagenase IV,
preferably, the final concentration of the collagenase is 80-100U/mL;
preferably, the final concentration of DNase I is 8-12. Mu.g/mL.
4. The method of claim 1, wherein the cell density of the primary oviduct epithelial cells in the cell suspension is 6 x 10 2 ～8×10 2 Each cell per microliter.
5. The method according to claim 1, wherein the temperature of the culture is 37+ -2deg.C, and the CO is during the culture 2 The volume content is 4-6%.
6. The method according to claim 1, wherein in step S2, the serum in the serum-containing 5Mix medium is fetal bovine serum;
preferably, the volume content of the serum is 8-12%.
7. The method according to claim 1, wherein in step S4, the concentration of the estrogen in the estrogen-containing 5Mix medium is 1 to 3ng/mL;
preferably, the volume content of the follicular fluid in the follicular fluid-containing 5Mix medium is 1-10%.
8. The method of any one of claims 1-7, wherein the primary oviduct epithelial cells are derived from the same species as the follicular fluid.
9. The method according to claim 8, wherein in step S4, fresh medium is replaced every 2 to 3 days during the culturing.
10. The method according to claim 9, wherein the volume content of the B27 additive in the 5Mix medium is 1-3%;
preferably, the volume content of the Glutamax additive is 0.5-2%;
preferably, the concentration of the epidermal growth factor is 8-12 ng/mL;
preferably, the concentration of the niacinamide is 0.5-1.5 mmol/L;
preferably, the concentration of the TGF-beta receptor kinase inhibitor is 0.2-1. Mu. Mol/L.
CN202310413307.3A 2023-04-17 2023-04-17 Method for inducing directional differentiation of primary oviduct epithelial cells into ciliated cells Pending CN116286607A (en)

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