3D gel skeleton composition and method for culturing organoid in different sections of epididymis

Abstract

The invention relates to the technical field of biological medicine, and discloses a 3D gel skeleton composition and a culture method of organoid in different sections of epididymis. The composition comprises a basement membrane extract BME and organoid liquid. The method comprises the following steps: separating epididymal epithelial cells; mixing the obtained epididymal epithelial cells with the base membrane extract BME in the 3D gel skeleton composition in a sterile environment of lower than 10 DEG C; inoculating a 96-well plate with the mixture to prepare liquid drops, and standing in a carbon dioxide incubator at 34 DEG C for 30-60 minutes until the liquid drops form a colloidal state; after a colloidal state is formed, adding an organoid culture solution, and continuously culturing until an organoid is formed; according to the method, the integrated 3D gel skeleton-cell mixture is constructed, the tedious skeleton construction step is omitted, the epididymal organs of source cells of different sections of the epididymis are constructed for the first time, and the formation of the epididymal organs of different sections provides a proper in-vitro model for researching epididymal functions and related regulation and control mechanisms.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technologies, and particularly relates to a 3D gel scaffold composition and a method for culturing organoids of different segments of the epididymis. Background Art

[0002] The epididymis is the main site for sperm maturation, a complex and coiled tubular organ, mainly divided into the head, body and tail of the epididymis. The epithelial cells in different segments have different functions and mechanisms in regulating sperm motility and maturation. Sperm, as an important carrier of genetic material in males and male animals, enter the epididymal tissue after spermatogenesis and undergo the process of sperm maturation. During this process, the epididymal lumen and the luminal microenvironment it forms jointly regulate sperm motility and fertilization, etc. And there is an inevitable connection between the secretion of epididymal cells and the acquisition of sperm motility and other abilities. Studying the relationship between epididymal epithelium and sperm maturation has become one of the main scientific issues of current concern. Understanding the composition of the epididymal epithelium and developing new research models are important prerequisites for studying the interaction between epididymal epithelium and sperm.

[0003] The constituent cells of the epididymal wall are mainly epithelial cells, forming a pseudostratified epithelium, composed of multiple cell types, including principal cells, basal cells, clear cells, apical cells and narrow cells. Principal cells are the most main cell type, narrow cells and apical cells are mainly present in the initial segment of the epididymis, while other cell types are distributed throughout the epididymal tissue. Epididymal epithelial cells from different species can be successfully isolated and cultured in vitro, and some of them exhibit functional characteristics, especially in enhancing sperm motility. Although there are more and more studies on the relationship between epididymal epithelial cells and sperm maturation, the underlying molecular mechanism is still unclear. This may be because monolayer culture or in vitro passaged cells cannot faithfully replicate the complex biological characteristics obtained by epididymal epithelial cells in vivo, thus limiting our understanding of these complex biological processes. At present, the main research models for sperm maturation are cell line models, but most of them only have cell lines derived from the epididymal head, that is, there is a lack of a suitable epididymal model to simulate the epididymal body and tail. Epithelial cell lines from humans, rats and mice have been used to study the role of cell - cell communication in the epididymis and to evaluate reproductive toxicity. However, these cell lines are derived from a single source and cannot fully represent the comprehensive biological functions of the epididymal tissue.

[0004] As an independent research model between cells and animal models, organoids have gained popularity among researchers in recent years and have achieved remarkable results in market applications. In humans and mammals, many tissue organoids have been reported, including the intestine, lung, liver, brain, prostate, breast, testis, uterus, and ovary, etc. For testicular organoids, a testicular organoid and its construction method and application disclosed in the patent (CN115537376A) first form a solidified matrix gel, then inoculate tens of thousands of cells on the matrix gel, and need to be cultured in an inverted manner to form organoids. This preparation and culture process is complex and prone to uncontrollable situations. The patent (CN114181891A) designed a preparation mold for organoid culture by itself, and on the basis of this mold, a gel mold for organoid culture was prepared, which also led to cumbersome operation steps. At present, there is no public report on the preparation and culture technology of epididymal organoids in China. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies in the prior art and propose a 3D gel scaffold composition and a method for culturing epididymal segment organoids.

[0006] To achieve the above purpose, the present invention is realized through the following technical solutions:

[0007] In the first aspect, the present invention provides a 3D gel scaffold composition for culturing epididymal organoids, and the composition includes basement membrane extract BME and organoid culture medium; the organoid culture medium includes DMEM / F12 basal medium, transferrin, retinol acetate, dihydrotestosterone, testosterone, and epidermal growth factor EGF;

[0008] When in use, the final concentration of the basement membrane extract BME is 4 - 6 mg / mL, and the volume ratio of the basement membrane extract BME to the organoid culture medium is 1:1.

[0009] Preferably, the concentration of transferrin in the organoid culture medium is 5 μg / mL, the concentration of retinol acetate is 1 μg / mL, the concentration of dihydrotestosterone is 1 μM, the concentration of testosterone is 200 nM, and the concentration of epidermal growth factor EGF is 10 ng / mL.

[0010] In the second aspect, the present invention provides a method for culturing epididymal segment organoids, and the method includes the following steps:

[0011] Step S1, isolation of epididymal epithelial cells;

[0012] Step S2, mixing the epididymal epithelial cells obtained in step S1 with the basement membrane extract BME in the 3D gel scaffold composition in a sterile environment below 10°C;

[0013] Step S3, inoculating the mixture obtained in step S2 into a 96-well plate to form droplets, and placing the droplets in a carbon dioxide incubator at 34° C. for 30-60 minutes until the droplets form a gel;

[0014] Step S4, after the gel is formed, the organoid culture medium is added to the 3D gel skeleton composition, and the culture is continued until the organoid is formed.

[0015] Preferably, the isolation of epididymal epithelial cells in step S1 specifically comprises the following steps: obtaining the epididymis, dispersing the tissue by digestion with trypsin and collagenase I, obtaining epididymal suspension cells, and temporarily storing them in epididymal cell culture medium.

[0016] Preferably, the epididymal cell culture medium comprises RPMI 1640 basal culture medium, pyruvate, insulin, hydrocortisone, transferrin, retinol acetate, dihydrotestosterone, testosterone, fetal bovine serum, antibiotics and fibroblast inhibitors.

[0017] Preferably, the epididymal cell culture medium has a pyruvate concentration of 1 mM, an insulin concentration of 100 nM, a hydrocortisone concentration of 200 nM, a transferrin concentration of 5 μg / mL, a retinol acetate concentration of 1 μg / mL, a dihydrotestosterone concentration of 1 μM, a testosterone concentration of 200 nM, a concentration of 10%, an antibiotic concentration of 1%, and a fibroblast inhibitor concentration of 30 μg / mL.

[0018] Preferably, in step S3, the total volume of each well in the 96-well plate does not exceed 20 μL.

[0019] Preferably, in step S3, the number of cells in each well of a 96-well plate is 2000-4000.

[0020] Preferably, in step S4, the organoid formation time is 4-15 days.

[0021] The present invention has the following beneficial effects: the present invention saves the tedious skeleton construction steps by constructing an integrated 3D gel skeleton composition. This method is used to construct epididymal organoids derived from cells from different segments of the epididymis for the first time. The formation of epididymal organoids from different segments will provide a suitable in vitro model for studying epididymal function and related regulatory mechanisms.

[0022] At present, the models for studying mouse epididymis include primary cells, immortalized cell lines and animal tissues, none of which are sufficient to replace and simulate the functions of the entire epididymis (head, body and tail). The technology of the present invention can effectively culture mouse epididymal organoids, which can lay the foundation for studying the relationship between epididymal epithelial cells and sperm maturation function. The organoid culture system provided by the present invention can allow organoids to maintain their complete morphology continuously, and the organoids can be used to study the functions of epididymal epithelial cells and screen drugs. Brief Description of the Drawings

[0023] Figure 1 It is a schematic diagram for the production of mouse epididymal organoids;

[0024] Figure 2 It is for the isolation and identification of mouse epididymal epithelial cells (A shows epithelial cells stained with AQP9 and DAPI; B shows the positive statistical chart of AQP9; C is the epithelial cells stained with KRT5 and DAPI; D shows the positive statistical chart of KRT5);

[0025] Figure 3 It is for the morphology of mouse epididymal organoids (A shows principal cells and epithelial cells labeled with AQP9 and E-cadherin in the organoids; B shows basal cells labeled with KRT5 in the organoids; C shows epithelial cells labeled with ZO-1 and E-cadherin in the organoids);

[0026] Figure 4 It is for the identification of mouse epididymal organoids. Detailed Embodiments

[0027] The present invention will be further described below in conjunction with embodiments, but it is not used as a basis for limiting the present invention.

[0028] 1. Isolation and Identification of Mouse Epididymal Epithelial Cells

[0029] Adult male C57BL / 6 mice were fed in a SPF environment with free access to food. The epididymis of adult male mice was exposed in a sterile environment, and fat and connective tissues were removed. After washing 2 - 3 times with PBS, the epididymis was mechanically dissected into a paste-like tissue of 1 - 3 mm3 with ophthalmic scissors. After washing several times with PBS, 0.25% trypsin and collagenase I (containing DNase I and antibiotics) were added, and after digestion for about 1 hour, single cells from different segments of the epididymis (epididymal head, epididymal body, and epididymal tail) were obtained by centrifugation. The cells were fixed at room temperature with 4% paraformaldehyde, washed with PBS, and then blocked at room temperature with 1% BSA (diluted with PBS) for 60 min. The primary antibodies (AQP-9, 1:200, Biorbyt; KRT5, 1:200, abcam) were incubated overnight at 4°C. After washing 3 times with PBS, the secondary antibody (Alexa 488 donkey anti-rabbit IgG(H+L), 1:500, invitrogen) was incubated for 60 min. 2 μg / mL DAPI (diluted with PBS) was incubated at room temperature for 10 min. The cells were stored temporarily in the epididymal cell culture medium.

[0030] 2. Construction of 3D Gel Skeleton Composition

[0031] The basement membrane extract BME was purchased from R&D systems with the catalog number 3433 - 005 - 01. The 10 mg / mL basement membrane extract was taken out from the -80 °C refrigerator and thawed overnight on ice. In a 4 °C environment, 50 μL of organoid culture medium was mixed with 50 μL of basement membrane extract, and the final concentration of the basement membrane extract was 5 mg / mL.

[0032] 3. Mouse epididymal organoid formation

[0033] As Figure 1 shown, 2000 mouse epididymal epithelial cells were counted and placed at room temperature for standby (for a short time). Then, 10 - 15 μL of basement membrane extract was added and mixed in a sterile environment below 10 °C. 10 μL of the cell suspension was taken with a pipette and inoculated into a 96 - well plate to make a mixed liquid drop for 3D stereoscopic culture, and it was left standing in a 5% carbon dioxide incubator at 34 °C for 30 - 60 minutes until the liquid drop formed a gel; 100 μL of organoid culture medium was added and continuously cultured until organoids formed.

[0034] 4. Identification of mouse epididymal organoids

[0035] After the mouse epididymal organoids were formed, the organoids were separated from the liquid drop and transferred to a glass slide for pasting. After fixation with 4% paraformaldehyde, permeabilization, blocking, and incubation with primary and secondary antibodies were carried out in sequence, and finally, the nuclei were labeled with DAPI dye. The marker proteins for principal cells (AQP9), basal cells (KRT5), epithelial cells (E - cadherin), and the marker protein for the blood - epididymis barrier (ZO - 1) were examined.

[0036] Test results:

[0037] 1. Isolation and identification of mouse epididymal epithelial cells

[0038] As Figure 2 shown, mouse epididymal epithelial cells expressed the marker protein AQP9 for principal cells, and the cell labeling rate of AQP9 was approximately 60 - 70%. Mouse epididymal epithelial cells expressed the marker protein KRT5 for basal cells, and the cell labeling rate of KRT5 was approximately 15 - 20%.

[0039] Figure 2 Identification of mouse epididymal epithelial cells. The head, body, and tail of the epididymis represent three segments of the epididymis. AQP9 labels principal cells, and KRT5 labels basal cells.

[0040] 2. Mouse epididymal organoid formation

[0041] As Figure 3As shown, epithelial cells in different segments of the mouse epididymis can all form organoids. Obvious spheroids can be observed starting from the fourth day, and as time goes by, the spheroids continuously expand to form organoids with a lumen-like shape.

[0042] Figure 3 Morphology of mouse epididymal organoids. The head, body, and tail of the epididymis represent three segments of the epididymis. Starting from droplet culture is day 0, and so on, the changes in organoids from day 4 to day 15 were observed.

[0043] 3. Successful identification of mouse epididymal organoids

[0044] As Figure 4 shown, the results of immunofluorescence staining indicate that AQP-9, KRT5, E-cadherin, and ZO-1 are all expressed in the organoids formed in different segments of the epididymis, indicating the successful establishment of epididymal organoids. And the staining result of ZO-1 suggests that it may possess the protective function of the blood-epididymis barrier.

[0045] Figure 4 Immunofluorescence staining of mouse epididymal organoids. Immunofluorescence staining map, AQP-9 represents the marker protein of principal cells; KRT5 represents the marker protein of basal cells; E-cadherin represents the marker protein of epithelial cells; ZO-1 represents the marker protein of the blood-epididymis barrier.

[0046] The above shows and describes the basic principles, main features, and advantages of the present invention. However, the above are only specific embodiments of the present invention, and the technical features of the present invention are not limited thereto. Any other implementation manners obtained by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. A 3D gel scaffold composition for epididymal organoid culture, characterized in that, The composition comprises a basement membrane extract BME and an organoid culture medium; the organoid culture medium comprises a DMEM / F12 basal culture medium, transferrin, retinol acetate, dihydrotestosterone, testosterone, and epidermal growth factor EGF; When in use, the final concentration of the basement membrane extract BME is 4 - 6 mg / mL, and the volume ratio of the basement membrane extract BME to the organoid culture medium is 1:

1.

2. The 3D gel scaffold composition for epididymal organoid culture according to claim 1, wherein In the organoid culture medium, the concentration of transferrin is 5 μg / mL, the concentration of retinol acetate is 1 μg / mL, the concentration of dihydrotestosterone is 1 μM, the concentration of testosterone is 200 nM, and the concentration of epidermal growth factor EGF is 10 ng / mL.

3. A method for culturing organoids of different segments of the epididymis, characterized in that, The method comprises the following steps: Step S1, isolation of epididymal epithelial cells; Step S2, mixing the epididymal epithelial cells obtained in step S1 with the basement membrane extract BME in the 3D gel scaffold composition according to claim 1 in a sterile environment at a temperature below 10°C; Step S3, inoculating the mixture obtained in step S2 into a 96-well plate to form droplets, and leaving them standing in a carbon dioxide incubator at 34°C for 30 - 60 minutes until the droplets form a gel; Step S4, after the gel is formed, adding the organoid culture medium in the 3D gel scaffold composition according to claim 1, and continuously culturing until organoids are formed.

4. The method for culturing epididymal segment-specific organoids according to claim 3, wherein, The isolation of the epididymal epithelial cells in step S1 is specifically as follows: obtaining an epididymis, dissociating the tissue by digestion with trypsin and collagenase I to obtain suspended cells of the epididymis, and temporarily storing them in an epididymal cell culture medium.

5. The method for culturing epididymal different-segment organoids according to claim 4, wherein The epididymal cell culture medium in step S9 comprises an RPMI 1640 basal culture medium, pyruvate, insulin, hydrocortisone, transferrin, retinol acetate, dihydrotestosterone, testosterone, fetal bovine serum, antibiotics, and a fibroblast inhibitor.

6. The method for culturing epididymal different-segment organoids according to claim 5, wherein, In the epididymal cell culture medium, the concentration of pyruvate is 1 mM, the concentration of insulin is 100 nM, the concentration of hydrocortisone is 200 nM, the concentration of transferrin is 5 μg / mL, the concentration of retinol acetate is 1 μg / mL, the concentration of dihydrotestosterone is 1 μM, the concentration of testosterone is 200 nM, the concentration is 10%, the concentration of antibiotics is 1%, and the concentration of the fibroblast inhibitor is 30 μg / mL.

7. The method for culturing epididymal different-segment organoids according to claim 3, wherein In step S3, the total volume in each well of the 96-well plate does not exceed 20 μL.

8. The method for culturing epididymal different-segment organoids according to claim 7, wherein, In step S3, the number of cells in each well of the 96-well plate is 2000 - 4000.

9. The method for culturing epididymal different-segment organoids according to claim 3, characterized in that, In step S4, the organoid formation time is 4 - 15 days.

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