An Efficient Immunomagnetic Purification Method of Alveolar Epithelial Cells and Their Cellular Kinetics during Sepsis-Induced Acute Lung Injury

Abstract

Background: Alveolar epithelial cells are cells lining along highly vascularized alveolar epithelial surface are capable of efficient gas exchange and host defense. Over the past decades, numerous methods have been developed to reliably isolate these delicate cells to study their functional, molecular, biological, and biochemical characteristics. However, methods for alveolar epithelial cells (AECs) purification with extremely reliable, reproducible, and efficient features are still needed. Methods: We developed an optimized protocol to isolate alveolar epithelial cells based on immunomagnetic enrichment. The protocol mainly includes two steps: (1) Immunocytes expressing Fc fragment receptor (FcR) depletion by rat immunoglobulin (IgG), panning to enrich for alveolar epithelial cells;(2) Immunomagnetic capture using magnetic-beads-conjugated monoclonal antibody against specific membrane markers T1α (Type 1 alpha, a specific AEC1s membrane protein) and epithelial cell adhesion molecule, EpCAM) to purify alveolar epithelial type I cells (ATIs), and alveolar epithelial type II cells (ATIIs). Subsequent observations of separated and cultured cells confirmed highly efficient isolation with common ATIs and ATIIs characteristics. Results: As for the cellular kinetics, there was a significant loss of ATIs at the third day after sepsis-induced acute lung injury (ALI), but the number of ATIIs did not significantly decrease at any point in time. Flow cytometry results showed that the percentage of ATIIs increased significantly 3 days after injury. Conclusions: ATIIs were damage-resistant during sepsis-induced ALI and they might be primary cells participating in sepsis-induced ALI and lung tissue repair.