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7. Spin down tubules at 800 xg. Place tubules in a 100-mm dish with a minimal amount of medium. Chop to approximately 1- to 2-mm pieces with the curved scissors.
8. Add 15 ml of solution B and incubate at room temperature 10"C20 min, until the peritubular myoid cell are seen to peel off the outside of the tubules and the tubules are in short pieces (1"C2 mm long) and small cell clumps (20"C30 cells). Place the digested tissue in a conical 50-ml tube, add wash medium, and let the clumps settle by unit gravity. Discard the single cells in the supernate.
9. Filter the tubular fragments through a 100-^M Nitex mesh cloth into a 50 ml tube.
10. Wash the fragments 3"C5 x with wash medium.
11. Plate the cells in five 100-mm tissue culture dishes in medium supplemented with insulin, transferrin, and EGF. Cells should be attached and spread by 24 hr of culture.
Primary Culture of Nonciliated Lung Epithelial Cells
The following protocol provides an example of primary culture of a normal organ (rat lung). The tissue dissociation conditions are chosen to minimize cell damage and the culture conditions used to eventually select a single cell type, in this case a nonciliated bronchiolar epithelial cell (Clara cells), from the multiple cell types present in the primary lung explant (Fig. 9.3). Since the Clara cells are still dividing and differentiating at this age, one
Primary cultures of newborn rat lung. (a) Defined medium selects for Clara cell survival. These cells can then be continuously cultured with the addition of (c) pituitary extract to form a cell line. Using other supplements such as (e) serum or (b, d) other mixtures of hormones, yields mixtures cultures of other cell types from the lung. (f) A 200x magnification of the established cell line.
can select for and expand these cells in the culture. While Clara cells do not become the predominant cell type in these cultures until the third week of culture, the cells can be passaged indefinitely and will form immortal cell lines of pure Clara cells (Roberts et al., 1990, 1992).
1. Ten p. 1-p4 Sprague-Dawley rats
2. 70% ethanol
3. Dumont #5 fine forceps
4. 4-inch iris scissors, straight and curved
5. Microdissection scissors (if dissecting out the bronchioles is desired)
6. Stereodissection microscope (if dissecting out the bronchioles is desired)
7. 50-ml conical centrifuge tubes
Table 9.1 Factors Used for Selecting Clara Cells from Primary Culture (11 F) and Minimal Factors Required to Grow the Established Cell Line (7F)
Factor Insulin Transferrin Epidermal growth factor 7F 1 |ig/ml 10 ^g/ml 11F 10 ^g/ml 10 ^g/ml 5 ng/ml
Ethanolamine 1 x 10'C4 M 1 x 10'C6 M
Phosphoethanolamine 1 x 10'C4 M 1 x 10'C6 M
Selenium 2.5 x 10'C8 M 2.5 x 10'C8 M
Hydrocortisone 2.5 x 10'C7 M 1 x 10'C9 M
Forskolin 5 |iM 1 |iM
Progesterone 1 x 10'C8 M
Triiodothyronine 5 x 10 12 M
Bovine lipoprotein (HDL) 0.5%
Bovine pituitary extract (15 mg/ml protein) 10 |il/ml 10 |il/ml
8. 11F isolation medium (see Table 9.1)
9. F12"CDME wash medium with 100 ^g/ml gentamycin
10. Ten 60-mm tissue culture dishes
11. Fibronectin (human, 1 mg/ml stock, Collaborative Research)
12. Collagenase'Cdispase (50 mg/ml, Boehringer-Mannheim) in medium with 20% (v/v) STI (1 mg/ml stock, Sigma)
13. DNase (1 mg/ml stock)
14. Bovine pituitary extract (commercially available, or prepared as in Appendix 3).
1. Kill the animals by CO2 asphyxiation on dry ice, or decapitate.
2. Rinse in ethanol.
3. Excise the lungs.
4. If dissecting out the bronchioles, the lungs should first be perfused by cannulating the trachea with a 25-g needle and perfusing with 1"C2 ml of PBS. The periphery of the lobes can be cut into parallel 1mm sections with a scalpel, and the bronchioles can then be dissected on a cold plate under a dissecting microscope.
5. If the bronchioles are not going to be dissected, remove the peripheral portion of the lobes to a 60-mm tissue culture dish, dissecting away the trachea and large bronchii.
6. Mince this tissue with the curved iris scissors until discrete chunks of tissue are not easily discernable (< 1 mm). The tissue should remain moist but not diluted with medium to facilitate mincing.
7. Add 3 ml of wash medium and 50 ^l of Collagenase'Cdispase solution per lung.
8. Place in a 37jaC incubator for 50 min, removing every 10 min to resuspend with a 1-ml Pipetman and to observe the dissociation under a phase contrast microscope.
9. The fibronectin-coated dishes should be prepared at least 1 hr before and up to 24 hr prior to use. If they are not prepared prior to dissection, they may be coated during the enzyme incubation step.
10. After the incubation period, the cells will still appear clumped in the mass of tissue, but many cells will have been released. Add 50 ^l of DNase solution to the dish, QS to 5
ml with wash medium, and add to a 50 ml conical tube. Repeat this step with each lung dissociated in this way.