A Novel Ovine Primary Alveolar Type II Cell Culture Approach

Abstract

The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary development; however, a developmental ovine primary ATII cell culture model is lacking. The objective of this study to develop and optimize submerged and air-liquid interface ovine ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. Lung tissue was harvested from mechanically ventilated preterm (128-132 d gest.), juvenile (4-6 months), adult (2-4 yrs) sheep and then immediately processed or frozen until processing for 7 day cell culture methods: submerged (7 d) or submerged (3 d) followed by air-liquid interface cell culture (4 d). Cells viability (>95%) was determined by Trypan blue exclusion; isolated ATII cell purity (>90%) was determined by modified Papanicolaou stain, ATII cells were identified by the presence of lamellar bodies, and ATII yield was normalized per gram tissue (preterm: 2.6 x 106; juvenile: 1.9 x 106; adult: 0.70 x 106). Presence, abundance, and mRNA expression of surfactant protein B (SP-B) was assessed by immunocytochemistry, Western Blot, and quantitative PCR, respectively, on the day of isolation, at midpoint and at the end of the study period. In cells derived from all age-groups from either fresh or frozen tissue, all biomarkers of SP-B were significantly greater in air-liquid interface as compared to submerged culture conditions at all time points. These data demonstrate an optimized cell culture methodology using fresh or frozen tissue to support study of ovine primary ATII cell function and responses to further understanding of mechanisms that contribute to in vivo function of the lung. DOD/ONR;N000141210597;N000141210810.