Maintenance of the differentiated type II cell characteristics by culture on an acellular human amnion membrane.
We have developed a Culture system for guinea pig alveolar type II cells using an epithelium-denuded human amnion membrane as a substratum. The differentiated morphology was maintained for 3 wk by both air-interface feeding and immersion feeding when type II cells were cultured on the basement membrane side of the amnion with fibroblasts on the opposite side (coculture). Functionally high levels of surfactant protein B (SP-B) and C (SP-C) messenger ribonucleic acids (mRNAs) were expressed even after the 3-wk cultivation and surfactant protein A mRNA was detected on day 10 of the culture. The differentiation was also maintained when fibroblasts were cultured on lower chambers of the culture plates (separate culture). In contrast, culture of type II cells without fibroblasts (monoculture) could not preserve the mature morphology. When the monoculture was supplemented with keratinocyte growth factor or hepatocyte growth factor, a monolayer of rather cuboidal type II cells with apical microvilli was maintained. However, the percent area of lamellar bodies in these cells was significantly less than that in freshly isolated type II cells, and mRNA expressions of SP-B and SP-C were also considerably suppressed. These findings suggest that other growth factors or combinations of these factors are necessary for the maintenance of the differentiated phenotype. As substratum, a permeable collagen membrane or a thin gel layer of Engelbreth-Holm-Swarm mouse sarcoma extracts did not preserve the mature characteristics. This culture system using an acellular human amnion membrane may provide novel models for research in type II cells.
Sakamoto
T
T
Department of Pulmonary Medicine, Institute of Clinical Medicine, University of Tsukuba, Ibarakii, Japan. t-saka@md.tsukuba.ac.jp
Hirano
K
K
Morishima
Y
Y
Masuyama
K
K
Ishii
Y
Y
Nomura
A
A
Uchida
Y
Y
Ohtsuka
M
M
Sekizawa
K
K
D000650:Amnion / Q000378:metabolism / Q000648:ultrastructure*; D000818:Animals; D001485:Basement Membrane; D018929:Cell Culture Techniques; D002454:Cell Differentiation; D002462:Cell Membrane; D018920:Coculture Techniques; D003470:Culture Media; D004848:Epithelium / Q000502:physiology; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D006168:Guinea Pigs; D006801:Humans; D008854:Microscopy, Electron; D011510:Proteolipids / Q000235:genetics; D011650:Pulmonary Alveoli / Q000166:cytology* / Q000378:metabolism; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D013997:Time Factors
D003470:Culture Media; D011510:Proteolipids; D037661:Pulmonary Surfactant-Associated Proteins; D011663:Pulmonary Surfactants; D012333:RNA, Messenger