title: A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals.
abstract: The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation-6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung.
authors.affiliation: Department of Thoracic Medicine and Surgery, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.|Center for Inflammation, Translational and Clinical Lung Research, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.|CENTRe: Collaborative for Environmental and Neonatal Therapeutics, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.
authors.forename: Mariola M
authors.initials: MM
authors.lastname: Marcinkiewicz
authors.affiliation: Department of Thoracic Medicine and Surgery, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.|Center for Inflammation, Translational and Clinical Lung Research, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.|CENTRe: Collaborative for Environmental and Neonatal Therapeutics, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.
authors.forename: Sandy T
authors.initials: ST
authors.lastname: Baker
authors.affiliation: Department of Thoracic Medicine and Surgery, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.|Center for Inflammation, Translational and Clinical Lung Research, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.|CENTRe: Collaborative for Environmental and Neonatal Therapeutics, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.
authors.forename: Jichuan
authors.initials: J
authors.lastname: Wu
authors.affiliation: Department of Thoracic Medicine and Surgery, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.|Center for Inflammation, Translational and Clinical Lung Research, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.|CENTRe: Collaborative for Environmental and Neonatal Therapeutics, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.
authors.forename: Terrence L
authors.initials: TL
authors.lastname: Hubert
authors.affiliation: Department of Thoracic Medicine and Surgery, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.|Department of Physiology, Pediatrics and Medicine, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.|Center for Inflammation, Translational and Clinical Lung Research, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.|CENTRe: Collaborative for Environmental and Neonatal Therapeutics, Katz School of Medicine at Temple University, Philadelphia, PA, United States of America.
authors.forename: Marla R
authors.initials: MR
authors.lastname: Wolfson
mesh_terms: D056809:Alveolar Epithelial Cells / Q000166:cytology*; D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation; D018496:Hyperoxia / Q000473:pathology; D037661:Pulmonary Surfactant-Associated Proteins / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012756:Sheep
chemical_list: D037661:Pulmonary Surfactant-Associated Proteins; D012333:RNA, Messenger