Isolation and characterization of human alveolar type II cells and phenotypes mintaining study

Abstract

Objective To establish a method of isolate,purify,primary culture and identify human alveolar type Ⅱ cells (AT Ⅱ ) in vitro,as well as its possible maintaining phenotype characteristics.Methods The marginal lung tissue was collected.AT Ⅱ cells were isolated with trypsin and elastase,purified by a series of steps,such as,cell sieve filtration,differential adhesion,gradient separation and anti-CD14 beads separation.AT Ⅱ cells were identified with immunofluorescence of human pro-surfactant-associated protein C (pro-SP-C),Green DND-26 probe and electron microscope.The purity of AT Ⅱ cells was measured by immunofluorescence of human pro-SP-C and Green DND-26 probe.The viability of AT Ⅱ cells was measured by trypan blue staining.The phenotypes (SP-A,SP-B,SP-C,SP-D ) were monitored with reverse transcription-polymerase chain reaction ( RT-PCR ) at different time points.Results The output of AT Ⅱ cells from lung tissue was (5-10) × 105/g,and the cell viability was (93 ± 2 )％ with trypan blue staining,the cell purity was about 98％ with pro-SP-C immunofluorescence and Green DND-26 fluorescent probe,the lamellar bodies were clearly observed with transmission electron microscope.In the aspect of phenotypes maintaining,the time of surfactant expression was about 24 days [ SP-A:0.52 ± 0.03 (day 16),0.35 0.02 ( day 20),0.26 ± 0.01 (day 24),0.10 ± 0.08 (day 28 );SP-C:0.68 ± 0.16 (day16),0.31 ± 0.04 (day 20),0.18 ± 0.06 (day 24),0.14 ±0.09 (day 28)],and the longest one was more than 28 days [SP-B:1.05 ±0.17 (day 16),0.76 ±0.35 (day 20),0.55 ± 0.15 (day 24),0.36 ± 0.19 (day 28);SP-D:0.52 ± 0.19 (day 16),0.33 ± 0.12 (day 20),0.31 ±0.04 (day 24),0.23 ± 0.02 (day 28)].Conclusion We successfully established a procedure to separate,purify,identify of AT Ⅱ cells,which retain primary phenotypic characteristics over long period. Key words: Alveolar type Ⅱ cell ; Primary cell culture ; Surfactant ; Lamellar body ; Human ;