Reproducible Isolation of Type II Pneumocytes from Fetal and Adult Rat Lung Using Nycodenz Density Gradients

Abstract

Isolating fresh, relatively pure type II pneumocytes from the lung, particularly of fetal origin, is a difficult process. Separation by buoyant density gradient centrifugation has been used successfully to isolate adult type II cells. There is concern, however, that Percoll, a gradient medium that is commonly used for type II cell isolation, may be toxic to cells. We evaluated a new gradient medium, Nycodenz, that is (1)a true solution, (2) transparent, (3) not metabolized by cells, and (4) nontoxic to cells. Type II pneumocytes were isolated from 19- and 21-day gestation fetal and adult rat lung by elastase digestion and separated on preformed isotonic Nycodenz gradients (2 mL each of 27.6, 20.7, 13.8, and 4.6 (w/v) solutions). Type II pneumocytes were recovered from the density range 1.057–1.061 and identified by binding of FITC-conjugated and gold-complexed Madura pomifera lectin. Cells derived from 19-day fetal lung contained abundant glycogen and reacted with a monoclonal antibody to the cytokeratins 8 and 18, which are markers of the fetal type II cell. Adult type II cells reacted with antibodies to cytokeratins 8, 18, and 19. Type II cell purity was 79.7 ± 2.4%, 83.8 ± 2.8%, and 82.6 ± 1.8% (means ± SEM). for 19- and 21-day gestation fetal and adult lung preparations, respectively. Cell viability was >95%. The final cell yield for adult preparations was 17.8 ± 2.7 × 10⁶/rat (means ± SEM). To determine if the freshly isolated type II pneumocytes were functionally active, the incorporation of [³H]choline into phosphatidylcholine was measured. The percent saturation of phosphatidylcholine was high for both populations of freshly isolated cells. However, adult type II pneumocytes incorporated [³H]choline into phosphatidylcholine more rapidly than 21-day gestation fetal cells (5.97 × 10⁻³ dpm/10⁶ cells/h vs. 0.32 × 10⁻³ dpm/10⁶ cells/h, P < .005). We have demonstrated that, using the Nycodenz isolation method, it is possible to obtain a high yield of relatively pure viable type II cells from fetal and adult lung that can be used for immediate study or cultured with an excellent plating efficiency (39 ± 6.3%).