Isolation and purification of spermatogonia from long white pigs
Isolation and purification of spermatogonia from long white pigs
This experiment was mainly used to isolate and purify boar spermatogonial cells of the Long White breeding line for molecular level studies.
Operation method
Isolation and purification of spermatogonia from long white pigs
Principle
Single-cell suspensions of testicular tissue from 2-month-old immature long white pigs were prepared by enzymatic digestion, and spermatogonia were isolated by gravity sedimentation combined with cell-adherence culture using a continuous gradient of 2 % to 4 % bovine serum albumin (BSA) as the isolation medium.
Materials and Instruments
Long White Breeding Line Boar Move I. Materials and methods 1、Experimental animals Long white breeding line boars, 2 months old, were provided by Beijing Pig Breeding Center, Weigou Modern Pig Farm. Live testes were taken and immediately put into 1640 nutrient solution, to be separated from the cells. 2、Main reagents and materials Collagenase (CLS, Sigma), trypsin (Sigma), deoxyribonuclease (DNase, Promega), bovine serum albumin (BSA, Tianjin Institute of Hematology, Chinese Academy of Medical Sciences), fetal bovine serum (FBS, Gibco), RPMI 1640 medium (Gibco, prepared prior to use, and streptomycin and penicillin were added to the final concentration of 100 mg L and 80 mg L, respectively). (Gibco, prepared before use, with streptomycin and penicillin to the final concentration of 100mg L and 80IU ml, respectively, adjusted pH to 7 3), and all other chemical reagents were domestically produced analytically pure. 800 ml capacity of the cell sedimentation tank (customized). Metal and glassware for experiments were cleaned and autoclaved before use. 3、Preparation and staining of paraffin sections Fresh testes were removed from the peritoneum and white membrane, cut into small pieces of 0.5~1.0cm3, immediately put into Bouin's fixative and fixed at 4℃ for 24 h. Paraffin sections were made according to the routine steps and stained with HE. 4、Preparation of single cell suspension Aseptically place the testis in PBS solution, carefully incise the peritoneum, remove fat, epididymis and leucomalacia, cut about 1g of testicular tissue, cut into 1mm3 size pieces with curved scissors, transfer to a centrifuge tube, add 5ml of PBS containing 0.5g/L collagenase, incubate at 33℃ for 15min, during which time, constantly shaking and blowing with a pipette for several times. Let it stand for 4~5min, and then discard the supernatant after the tissues and cells settled to the bottom of the tube. Repeat the above steps 1 time. Add 5 ml of PBS containing 0.5 g L trypsin and 1.0 mg/LDNase, incubate at 33°C for 15 min, and gently blow with a pipette for 3 min until the field of view was filled with scattered single cells and a few small cell clusters when the drop was examined microscopically. centrifuge at 1000 r/min for 10 min, discard the supernatant, and wash it twice with PBS, and the cell precipitates were finally resuspended in 0.5% BSA in PBS, sieved through 100 mesh. The cell precipitate was finally resuspended in PBS containing 0.5% BSA and filtered through a 100-mesh sieve to make a single-cell suspension and cell counting. 5、Sedimentation and separation of cells Fix the sedimentation cell and keep the horizontal state, add tens of silica glass beads around the bottom mouth of the cell. 10 ml of PBS and 10 ml of single-cell suspension to be separated were loaded into the sedimentation cell from the bottom mouth successively. The 4% and 2% BSA solutions of 250 ml each were prepared with 1640 culture solution and poured into the two containers of the gradient generator respectively at the same time. The outlet of the gradient generator was connected directly to the bottom pointed lower port of the settling tank with a rubber hose. The piston was opened and the flow rate was adjusted so that the BSA solution entered the settling tank from the bottom at a rate of 15 ml/min. Eventually the liquid in the settling tank formed a continuous gradient of 2%~4% BSA from top to bottom. The whole settling system was left at 4°C for 3h in order to stratify the cells according to different sizes by natural gravitational settling in the BSA continuous gradient medium. The stratified cell solution was collected from the lower port at the bottom of the settling cell with an automatic collector at a controlled flow rate of 5 ml/min, and 10 ml tubes were used as 1 portion. 1000 r/min centrifugation was performed for 10 min to precipitate the cells, and the supernatant was discarded. Cells were resuspended in 0.5 ml of 1640 culture medium containing 0.5% BSA. The cells in each tube were titrated separately, HE stained, and the morphological and structural characteristics and degree of homogeneity of the cells were examined under the light microscope to determine the type and purity of the cells. Similar cells were merged, and 1 drop of cell suspension was taken and 10 μl of 0.4% Taipan blue was added to count the total number of cells and the percentage of dead cells. Finally, the cell concentration was adjusted to 106 cells/ml. drop slides were stained. Select any 5 fields of view under the microscope and calculate the cell purity. 6. Cell attachment culture and purification The spermatogonial cells separated by sedimentation were mixed with a small number of supporting cells and mesenchymal stromal cells, the cell suspension was inoculated into disposable aseptic culture flasks, cultured at 37℃, 5% CO2 and saturated humidity for 6-8h, the supporting cells and mesenchymal stromal cells were attached to the wall while the spermatogonial cells had not yet been attached to the wall, the flasks were gently shaken to make the spermatogonial cells fully suspended, and then the culture medium was sucked out, and then the purity and the number of cells were detected under the microscope and the mean number was calculated. The purity and number of cells were tested again under the microscope, and the mean value was calculated. Results and discussion 1、Observation of paraffin section Observe the paraffin section of testicular tissue of immature boars at 2 months old under 400 times optical microscope. At this time, the testicular tissue mainly consisted of interstitium and newly developed seminiferous tubules. The mesenchymal tissue was mainly composed of mesenchymal cells; the epithelium of the seminiferous tubules was mainly composed of spermatogonia which were close to the basement membrane, and supportive cells, both of which were differentiated from primitive spermatogonia, and the primary spermatogonia had not yet been seen. The lumen of the vas deferens had not yet developed in 2-month-old pigs. 2、Classification and purity of cells separated by gravity sedimentation After the single cell suspension of immature porcine testicular tissue was separated by continuous gradient sedimentation of BSA, the collected cells could be combined into four categories according to their morphological characteristics. The 2nd-15th portions were combined as class I cells, 20-33th portions were combined as class II, 38-44th portions were combined as class III, and 46-50th portions were combined as class IV. Their main composition, cell purity and proportion of live cells are detailed in the attached table. Among them, class Ⅱ was the spermatogonia we wanted, with a purity of 91%. Cell compositioncells purity (%) purity (%) viability (%) viability (%) Ⅰ cell clumps (cellclumps)-95 Ⅱ spermatogonia (spermatocytes) 9197 Ⅲ supporting cells (sertolicells) 9597 Ⅳ cell debris and noncellular structures (cytoplasm) - - The class IV fraction obtained in this experiment belongs to a kind of non-cellular structural material with a diameter of only 2~3 μm, with dark coloring and without nucleus and cytoplasmic inclusions.Bellve et al. also isolated a material with similar structure and characteristics when isolating spermatogonial cells of juvenile mice and named it cytoplasm.We also obtained this kind of material in the isolation of testicular germ cells of adult mice and pigs, suggesting that it is present in the germ cells of adult mice and pigs, and that it is not present in the germ cells of adult mice and pigs. This suggests that its presence is not animal species or developmental stage specific between pigs and mice. Its origin, function and ultrastructure have not been reported.3 Cell purity after wall culture Mesenchymal and supporting cells are easily adherent to the wall when cultured in vitro. We inoculated spermatogonia obtained after BSA gravity sedimentation separation (with a few stray cells mainly support cells and individual mesenchymal cells) in culture flasks, and after 6~8h, the support cells and mesenchymal cells adhered to the wall and grew, while the spermatogonia remained in suspension. The spermatogonia could be further purified by this selective wall attachment method. The suspended spermatogonia were collected and stained with HE staining, and the purity of the cells was 93%, 94%, 96%, 93% and 95% in five selected fields of view under the light microscope, with an average value of 94.2%, which was higher than that of 91% after sedimentation and separation. 4、Morphological identification of various types of cells in 2-month-old pig testicular tissue under optical microscope Under the microscope, the spermatogonial cells were round or oval, with a diameter of 11~13μm, little cytoplasm, large nucleus, the nucleus was mostly euchromatin, and there were 2~4 nucleoli near the nuclear membrane. Supporting cells were irregular in shape, with much cytoplasm and light coloring, small nuclei, little chromatin in the nucleus, more chromatin near the nuclear membrane, and 1~2 nucleoli, which were first attached to the wall of spermatogonial cells in culture, and then spread into a pike shape or polygonal shape after attachment to the wall. The above cell morphology was completely consistent with that of the cells in paraffin sections from testicular tissues of pigs at the age of 2 months. We have used different methods and media for the isolation of juvenile porcine spermatogonia in an attempt to find an optimal isolation method. The establishment of this experimental method has important reference value for obtaining mammalian spermatogenic cells with high purity, further in-depth study of the molecular mechanism of spermatogenesis, as well as the isolation and cultivation of spermatogenic stem cells. Caveat 1. the experimental procedure should be aseptic. 2. The entire operation should be completed in as short a time as possible to avoid increasing the number of dead cells. Common Problems Source "Isolation and purification of spermatogonial cells from long white pigs". For more product details, please visit Aladdin Scientific website.
Collagenase, Trypsin, Deoxyribonuclease, Bovine Serum Albumin, Fetal Bovine Serum, RPMI 1640, HE Stain, PBS.
Cell settling tanks Tubes Suction tubes Sieves Gradient generators Centrifuges Optical microscopes