An acellular human amnionic membrane model for in vitro culture of type ii pneumocytes: The role of the basement membrane in cell morphology and function

Abstract

To determine whether a preformed basement membrane contributes to the maintenance of morphology and function of type II pneumocytes, we cultured isolated adult rat type II pneumocytes on the basement membrane and stromal surfaces of an acellular human amnionic membrane and on plastic. The presence of lamellar bodies on transmission electron microscopy and epithelial morphology in culture and a characteristic phospholipid profile after incubation with 3H-acetate identified the cells as type II. When type II cells were cultured on a preexisting basement membrane, they formed a well-organized monolayer with polarity, centrally located surface microvilli, and more basally located nuclei. Individual cells maintained a cuboidal morphology for 8-10 days. Intracellularly, there were numerous mitochondria, endoplasmic reticulum (ER), and lamellar bodies. The cells secreted a new basal lamina of their own. When cultured on the stromal side of the amnion, the cells became flattened within 48-60 hours, formed small lamellar bodies, and had scanty surface microvilli; they formed clumps and appeared less ordered. These cells did not secrete a visible basement membrane, and the majority detached from the stromal surface after 7-8 days in culture. In addition, culture on the basement membrane aspect of the amnion prevented the rapid decline in the percentage of 3H-acetate label incorporated in phosphatidylcholine after 72 hours of culture. We conclude that a preformed basement membrane influences the function and morphology of type II pneumocytes, organizes them into a monolayer in culture, and influences deposition of a visible basal lamina. Thus, the acellular human amnion provides an excellent model for the systematic study of basement membrane influence on the biology and pathology of these cells.

Corneal sensitivity and ocular surface changes following preserved amniotic membrane transplantation for nonhealing corneal ulcers

“…In addition, amniotic membrane consists of a thick basement membrane and an avascular stroma with a high concentration of basic fibroblast growth factor, basement membrane components, and presumed trophic factors, which collectively provide benefits to augment epithelial and stromal wound healing. [11][12][13][14][15][16][17][18][19][20] We expected all these unique properties of the amniotic membrane to improve the epithelialisation in our patients. In addition to taking advantage of these properties, we also took advantage of the amniotic membrane to supplement the collagen by using it as a filling in cases with deep stromal keratolysis.…”

Type II Pneumocytes in Mixed Cell Culture of Human Lung: A Light and Electron Microscopic Study

“…Type II pneumocytes have been shown to retain their cuboidal morphology when cultured on various ECM substrates, in contrast to the loss of morphology and spreading seen on tissue culture plastic. In previous studies the substrates used were pure single components of ECM (9,16) or complete ECM from cells of known origin (15)(16)(17)(18)(19). However, in this study the exact source and composition of the extracellular matrix is unclear.…”

“…2e). Synthesis of basement membrane (16,19), as well as Type IV procollagen and fibronectin has been demonstrated in cultured rat ype II pneumocytes (7)(8)(9) (17,20,24 (9). Thus, the Type II cells themselves remodel the connective tissue core that, in turn, promotes their normal morphology.…”

“…Neither do they divide in isolated cell culture (13,14). The normal morphology of Type II cells in vitro can be prolonged by plating onto exogenous extracellular matrices (ECM), such as laminin (15), collagen gels (16) or basement membrane derived from cultured cells (15,17), and other sources (18,19). Furthermore, macrophage-or fibroblast-derived factors are thought to influence the differentiated function of Type H cells in vitro (17,20).…”

“…Thus, the development of an in vitro model to study lype II pneumocytes must consider the close proximity of the alveolar epithelial cell to mesenchymal and endothelial cells and the ECM produced by these cells in situ. Although some of these factors have recently been investigated using rat Type H cells (7)(8)(9)15,17,(19)(20)(21), there are few similar studies using human tissue (12). In (Fig lb,c,d; Table 1).…”