title: 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 Maclura 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 greater than 95%. The final cell yield for adult preparations was 17.8 +/- 2.7 x 10(6)/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 x 10(-3) dpm/10(6) cells/h vs. 0.32 x 10(-3) dpm/10(6) cells/h, P less than .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%).
authors.affiliation: Department of Pediatrics, University of Maryland, Baltimore.
authors.forename: R M
authors.initials: RM
authors.lastname: Viscardi
authors.forename: S
authors.initials: S
authors.lastname: Ullsperger
authors.forename: J H
authors.initials: JH
authors.lastname: Resau
mesh_terms: D000818:Animals; D002469:Cell Separation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D002794:Choline / Q000378:metabolism; D005333:Fetus / Q000033:anatomy & histology; D007472:Iohexol; D008168:Lung / Q000166:cytology* / Q000196:embryology / Q000378:metabolism; D010713:Phosphatidylcholines / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D015203:Reproducibility of Results
chemical_list: D010713:Phosphatidylcholines; D007472:Iohexol; D002794:Choline