A method for establishing a sustainable cell line of tree shrew spermatogonial stem cells
A technology of spermatogonial stem cells and cell lines, applied in the field of stem cell research, can solve the problems that affect the in-depth study of stem cell culture systems, the establishment of tree shrew spermatogonial stem cells has no achievements, pollution, etc., and achieve long-term in vitro culture and sorting effects Good, high sorting efficiency
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Embodiment 1
[0066] Embodiment 1 (digestion and separation of tree shrew spermatogonial stem cells)
[0067] (1) Tree shrew testis digestion:
[0068] ① Wash the testes of 6-month-old tree shrews with PBS, remove the buffy film, and cut them into pieces (aseptic operation).
[0069] ② Prepare a digestion solution containing 1 mg / mL hyaluronidase, 3 mg / mL collagenase, and 0.2 mg / mL DNaseI, digest the tissue fragments in a 37°C water bath for 20 minutes, and thoroughly mix the tissue and the digestion solution with a pipette every 5 minutes during the period.
[0070] ③ Wash twice with PBS to remove collagenase, centrifuge at 1500rmp for 5min, and discard the supernatant.
[0071] ④ Place the tissue fragments and digested cells in 0.25% trypsin digestion solution and digest the tissue fragments in a 37°C water bath for about 10 minutes. Mix once every 2 minutes with a pipette gun. When most of the cells in the digestive solution are in a single-cell state, the serum stops digestion.
[00...
Embodiment 2
[0093] Embodiment 2 (feeder layer cell culture and processing of tree shrew spermatogonial stem cells)
[0094] The invention provides a method for making feeder cells specially used for culturing tree shrew spermatogonial stem cells in vitro, comprising the following steps:
[0095] (1) Digest into single cells according to the above tree shrew testis digestion method.
[0096] (2) Cells were sorted according to the above tree shrew spermatogonial stem cell sorting method, and Thy1-negative cells were collected by magnetic bead sorting.
[0097] (3) Use DMEM medium as the base medium, add 10% NCS to culture the cells, remove the non-adherent cells after culturing for one day, and use the remaining cells as the feeder layer cells.
[0098] The processing method of the above feeder layer cells comprises the following steps:
[0099] (1) Petri dishes are pre-coated with gelatin;
[0100] (2) Digest the cells obtained by the method for making the feeder layer with 0.025% tryps...
Embodiment 3
[0105] Embodiment 3 (the preparation of the special culture medium of tree shrew spermatogonial stem cells)
[0106] Prepare 10mL of special medium for tree shrew spermatogonial stem cells, add 5μL GDNF (100ug / ml), 1.2mL FBS, 15μL EGF (10ug / mL), 0.05μL bFGF (20ug / ml), 12μL LIF (1*10 6 U / mL), 8μL Wnt3a (10ug / ml), 8.5mL StemPro34 basal culture medium, stemPro34supplement 250μL, mix evenly.
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