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6. If the cells are dividing, try several different subculture regimens, for example, low and high density, frequent versus less often, trypsin versus collagenase"Cdispase. Keep watching the plates for several weeks; sometimes surprising things occur (for example, see protocol for primary culture of nonciliated cells, below).
7. Characterize the cultures. Determine the percentage of the cell type of interest in the culture and how this changes over the duration of the life of the cultures. If the cells can be subcultured or can be frozen and thawed for later use, then characterize the secondary or thawed cultures as well.
8. If none of the above has worked, try a different species (or strain) of animal, a different age of animal, or purposely coculture the cell type of interest with nearby cells or related (or not so related) feeder cell lines.
Primary culture of rat embryonic e9 neuroepithelial cells requires coculture or conditioned medium for survival. (A) All cells in the culture are dead 24 hr after plating in all supplements tried. (B) Culture supplemented with hormones and cocultured with an embryonic Schwann cell line. (The colony in the center is neuroepithelial and the surrounding cells, easily distinguished morphologically, are the ESC cells.)
9. If you have had success and the cultures are satisfactory for the need at hand, then publish. Publishing a complete description of the method used to derive a cell line is important for all those investigators who may later use the line. This initial publication should always be cited in any later publications using the line.
Primary Culture of 20-Day-Old Rat Sertoli Cells
In this protocol, the tissue dissociation conditions are chosen to minimize damage to Sertoli cell surface receptors while obtaining relatively pure preparations of testicular tubules through sequential chemical, enzymatic, and mechanical separation of the various cell types in the testis. The major contaminating cell type, the germ cell, can be removed easily from the cultures after the first day, if necessary. While yield is less important than purity in this protocol, a good yield is important, since the Sertoli cells are postmitotic at this age. The cells can be used for studies within 24 hr of plating and the cultures vary little over the first 1"C2 weeks in vitro when maintained in serum-free, hormone-supplemented conditions. This procedure is described for use with day 20 rat tissue, but it has been used for rats 5"C60 days old and for obtaining Sertoli cell cultures from mice, hamster, dog, pig, monkey, and human tissue with variations of the enzyme amount and time of digestion.
1. Five p20 Sprague-Dawley rats
2. 70% ethanol
3. Dumont #7 curved fine forceps
4. 4-inch iris scissors, straight and curved
5. Solution A: Glycine solution: 1 M glycine, 2 mM EDTA, 20 IU DNase (highly purified), and STI (0.002%) made up in Ca2+, Mg2+ -free PBS. Adjust the pH to 7.2 and filter sterilize.
6. Note: The 1 M glycine solution is hyperosmotic, and the EDTA is a chelator (e.g., binds calcium and magnesium). The DNase is not enzymatically active in the absence of di-
valent cations, but seems to bind to any released DNA and becomes active when the cells are returned to the medium. The STI protects against proteolysis. The alternation of hyperosmotic and normal osmotic solutions will lyse some cell types while leaving others viable. It also seems to frequently make it easier to subsequently remove cells from basement membranes during enzymatic dissociation. We have applied this procedure to good effect in isolating some types of cells from other tissues, but the results must be carefully monitored (Mather and Phillips, 1984).
7. Solution B: Collagenase'Cdispase solution: Collagenase'Cdispase (50 mg/ml, Boehringer-Mannheim) in medium with 20% (v/v), STI (1 mg/ml stock, Sigma); filter sterilize
8. 50-ml conical centrifuge tubes
9. F12'CDME medium supplemented with insulin (5 ^g/ml), transferrin (5 ^g/ml), and EGF (5 ng/ml) immediately before use
10. F12"CDME wash medium with 100 ^g/ml gentamycin
11. Five 100-mm tissue culture dishes
12. Nitex nylon mesh 100-^m mesh size; cut into 6-inch squares and autoclave to sterilize Procedure
1. Kill the animals by CO2 asphyxiation on dry ice.
2. Soak the outside of the fur in ethanol and remove the testis.
3. Decapsulate the testis and remove as much of the testicular vein as possible.
4. Place the tubules in a 100-mm plate and add 15"C20 ml of solution A.
5. Carefully disperse the tubules with the forceps while incubating for 10 min at room temperature. Gently pipette the tubule mass through a large-bore pipette (3-mm opening: use a 5-ml Pipetman pipettor with the tip cut off). The tubules will "unravel" to form a cloudy mass of tubules. Interstitial tissue is released (and severely damaged since interstitial cells collected at this point will not grow in culture).
6. Pipette tubules with a minimum amount of solution A into 50 ml F12"CDME wash medium. Wash 3 x, allowing tubules to settle by unit gravity. Discard wash.