Isolation of alveolar type II cells by centrifugal elutriation

Abstract

Centrifugal elutriation (counterflow centrifugation) was used to develop a reproducible method for obtaining a nearly pure population of isolated alveolar type II cells. Lung was dissociated into individual cells with recrystallized trypsin, and the type II cells were partially purified by centrifugation on a discontinuous density gradient. The alveolar type II cells were finally purified by centrifugal elutriation. Cells were collected from the elutriator rotor by stepwise increases in flow rates. Cells obtained at flow rates of 7 and 14 ml per min were lymphocytes, other small cells, a few type II cells and cell debris; cells collected at flow rates of 18 and 22 ml per min were mainly type II cells; and cells collected at flow rates of 28, 34 and 43 ml per min were macrophages, some type II cells, other lung cells and cell aggregates. At flow rates of 18 and 22 ml per min, 1.9 +/- 1.0 x 10(6) cells per rat lung (mean +/- S.D., n=30) were recovered of which 86 +/- 6% were type II cells. At these flow rates, 94% of the cells excluded the vital dye erythrosin B from their cytoplasm. They consumed oxygen at a rate of 101 +/- 21 nmol per hr . 10(6) cells (mean +/- S.D., n=4), and their oxygen consumption increased only 10% after 10 mM sodium succinate was added. The cells incorporated [14C]leucine into protein and lipid for 4 hr. Electron micrographs of the cells collected at flow rates of 18 and 22 ml per min show a high percentage of morphologically intact alveolar type II cells. We conclude that centrifugral elutriation is a reproducible method for obtaining nearly pure, metabolically active alveolar type II cells.