Isolation and Culture of Type II Pulmonary Epithelial Cells

"I. IntroductionThe alveolar surface of the lung is covered by two populations of pneumocytes, type I and type II epithelium. The type I cell, which accounts for most of the alveolar surface area and provides a highly specialized barrier for gas exchange (Rannels et al., 1991), is poorly understood. Although approaches to type I cell purification have been discussed, they have not been applied widely because of the lack of phenotypic markers to monitor isolation procedures (Dobbs et al., 1988; Danto et al., 1992). In contrast, the type II cell has been studied extensively, largely because of the development of reliable procedures for its isolation and primary culture. Investigations based on the use of purified populations of cultured type II cells have provided convincing evidence to support a role for the pneumocytes both in the maintenance of normal lung function and in the response of the lung to injury (for review, see Rannels and Rannels, 1989).While type II cells can be isolated by a variety of approaches, the protocols used most commonly are modifications of the gradient method of Dobbs and Mason (1979) or of the panning method of Dobbs et al. (1986). Purification of the cells based on density gradient centrifugation is reliable and consistent, with yields of 30 to 40 X 106 type II cells per rat of 230 g body weight. Isolation procedures applied to a minimum of three to four rats provide the best results, as lower cell yields are common with the use of fewer animals. The protocol below is designed to obtain a maximal cell yield from the lungs of six rats.II. Materials and InstrumentationMaterialsCalbiochem: DNase I (Cat. No. 260912); Corning: culture flask, 75 cm2 #25115; Costar: culture plate, 24-well #3424; disposable serological pipette #4101; Elastin Products Inc: elastase #E-134; Falcon: cellulose nitrate membrane filter, 0.22 µm, #7105; conical tube, 50 ml, #2070; culture dish, 100 mm, #3003; culture plate, sixwell, #3046; Fisher: BaS04 , #B68-500; NaCl, #5271-3; NaHC03 , #5233-50; Fort Dodge Laboratories: pentobarbital, #1206-lE; Gibco: Dulbecco's minimal essential medium (DMEM), #430-1600EC; Hanks' balanced salt solution (HBSS), #310- 4185AG; Joklik's modified Eagle's medium (JMEM), #410-2300EC; HyClone Laboratories Inc: fetal calf serum (FCS ), # A-1115-L; I CN Biochemicals (Flow Labora tories): amphotericin B, #16-723-48; newborn calf serum (NCS), #29-121-54; try pan blue, # 19 5 5 32; Na/gene: Erlenmeyer flask, # 100-4116C; Pharmacia: Percoll, #17-0891-01; density marker beads, #17-0549-01; Sigma: gentam1cm sulfate, #G3632; penicillin/streptomycin, #P3539; trypsin inhibitor, #T9128; Tetko: HC- 160 nylon mesh, #3-160-53."