Testis organoid as well as construction method and application thereof
A construction method and organoid technology, applied in the field of biomedicine, can solve the problem of unobtained haploid cells, and achieve the effect of simple operation and low cost
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Embodiment 1
[0041] Example 1 Construction of mouse testicular organoids
[0042] A method for constructing mouse testicular organoids, the flow chart of which is as follows figure 1 As shown, the details are as follows:
[0043] (1) Newborn mice (3 days after birth, ICR mice, purchased from Guangdong Provincial Medical Experimental Animal Center) were killed, and the mice were scrubbed with 75% alcohol cotton balls; In a plate with a diameter of 3.5 cm in the MEMα base solution, remove the capsule of the testis tissue; tear the testis into small pieces with tweezers, wash with PBS twice, add 2 mL of collagenase IV (2 mg / mL), and digest at 37 ° C for 3 minutes , then centrifuged at 1000rpm for 3min, discarded the supernatant, washed once with PBS; added 2mL of 0.05% trypsin, digested at 37°C for 5 minutes, added 2mL of DMEM (12800017, Life Technologies) + 10% FBS (SE200-ES, VISTECH) to terminate Digest, centrifuge at 1000rpm for 3min, discard the supernatant to obtain testicular cells. ...
Embodiment 2
[0054] Example 2 Identification of mouse testicular organoids
[0055] 1. Identification of Testicular Organoid Spermatogonia Types
[0056] The testicular organoids (polymeric balls) obtained in Example 1 were fixed with 4% paraformaldehyde, then paraffin-embedded and sectioned, and the tissue sections were subjected to immunofluorescence staining, specifically: adding primary antibodies (anti-PLZF(SC- 28319, Santacruz) and anti-DDX4 (ab13840, abcam), anti-KIT (3074T, Cell Signaling Technology) and anti-DDX4 (a b13840, abcam), anti-STRA8 (ABN1656, Millipore) and PCNA (SC-56, Santacruz ), the antibody dilution ratio was 1:400, and placed in a 4°C refrigerator overnight; add secondary antibodies: Alexa Fluor488 (115-545-003, Jackson), Alexa Fluor 594 (111-585-003, Jackson), antibody dilution ratio 1:400, stand at room temperature for 1 hour; Hoechst33342 (H3570, Thermo Fisher) stained nuclei, dilution ratio l:1000, stained at room temperature for 10 minutes, observed, the resu...
Embodiment 3
[0065] Example 3 Verification of sperm cell function in mouse testicular organoids
[0066] 1. Flow Cytometry Sorting of Sperm Cells in Testicular Organoids
[0067] Use a 1 mL pipette tip to draw the testicular organoid (polymeric ball) obtained in Example 1 into a domestic 3.5 cm dish, wash once with PBS, cut the testicular organoid into small pieces with a 1 mL disposable syringe, add 2 mL of collagenase IV (4 mg / ml), digested for 10 minutes, pipetted halfway, then added 4mL DMEM+10%FBS to stop the digestion, transferred the cell suspension obtained from cell digestion to a 15mL centrifuge tube, centrifuged at 1300rpm for 3min, discarded the supernatant, and obtained the cell pellet (test group).
[0068]At the same time, 6-week-old adult mice (ICR mice, purchased from the Guangdong Provincial Medical Experimental Animal Center) were killed by neck dislocation, one side of the testis was taken and put into PBS, the buffy coat was removed, washed 3 times, and digested with...
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