bromochloroacetic-acid and maclurin

Here we report two-dimensional cell sheet manipulation (2D CSM) of heterotypically co-cultured lung cell sheets and the maintenance of differentiated phenotypes of lung epithelial cells over prolonged periods of up to 70 days. This was facilitated by poly(N-isopropylacrylamide) (PIPAAm)-grafted tissue culture dishes. PIPAAm-grafted dishes are responsive to temperature changes and offer a unique surface on which cells adhere and multiply like on ordinary tissue culture dishes under the permissive temperature of 37 degrees C, but on lowering of temperature resulting in changes in hydration of the polymer the cells spontaneously detach from the surface without use of enzymes like trypsin which is the common procedure. It has been well documented that type II pneumocytes of the lung lose many of their special features rapidly in culture. The culture system detailed here comprises random co-culture of epithelial and mesenchymal cells of lung. The heterotypic cell culture system promotes cell-cell interactions maintaining a harmonized physiology. When this heterotypic monolayer on PIPAAm-grafted dishes was subjected to lower temperature of 20 degrees C and 2D CSM we were able to transfer the monolayer as a single contiguous sheet with cell-cell connections intact to other surfaces. This non-invasive transfer of cell sheet resulted in shrinkage of the monolayer, enabling the type II cells to regain their cuboidal morphology and specialized characters like Maclura pomifera lectin binding and surfactant protein A (SP-A) expression. The active dome formation also observed subsequent to transfer reaffirms the uniqueness of the culture conditions and 2D CSM in future for developing tissue like architecture in vitro.

Topics: Acrylic Resins; Actins; Animals; Biocompatible Materials; Cell Differentiation; Cells, Cultured; Coculture Techniques; Epithelial Cells; Fibroblasts; Immunohistochemistry; Keratins; Lectins; Lung; Materials Testing; Phenotype; Plant Lectins; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Rats; Temperature; Vimentin

The differentiated phenotype of the alveolar type II cell is rapidly altered in vitro. To evaluate factors that might influence this process, we isolated and plated rat type II cells in serum-supplemented media to promote adherence and then maintained the cells in a simple nutrient medium in the absence (S- cells) or presence (S+ cells) of serum for 5 to 7 d. The type II S- cells remained metabolically active. Despite protein synthesis that was 50% that of S+ cells, S- cells continued to synthesize a broad spectrum of proteins and to express several features of type II cell differentiation. They synthesized an apical integral membrane glycoprotein, Maclura pomifera agglutinin (MPA)-gp200, and a cytokeratin, No. 19, while S+ cells did not. When supplemented with linoleic acid, S- cells contained lamellar and multivesicular bodies, incorporated cell surface MPA into these structures, and secreted their phosphatidylcholine (PC) in response to mastoparan. Despite the relative synthesis of higher levels of total and saturated PC in S- cells supplemented with linoleic acid, phosphatidylglycerol remained diminished. A surfactant protein (SP-A) was present in S- cells, but synthesis was not detected. These studies demonstrate that serum accelerates the loss of type II cell differentiation in vitro and that the expression of type II cell markers of differentiation is not inherently linked.

Topics: Animals; Blood; Cell Differentiation; Cells, Cultured; Culture Media; Electrophoresis, Gel, Two-Dimensional; In Vitro Techniques; Keratins; Lectins; Male; Membrane Glycoproteins; Microscopy, Electron; Plant Lectins; Proteins; Pulmonary Alveoli; Pulmonary Surfactants; Rats; Rats, Inbred Strains; Receptors, Mitogen

Alveolar type II cells were isolated from fetal mouse lung by differential adherence and obtained in monolayer culture. Cultures display a high degree of purity as shown by histochemical and immunocytochemical staining procedures. Seventy-five percent of cells stained positive with specific anti-lavage serum mouse (SALS-M), an antiserum specific for (pre)alveolar type II cells of the mouse, and osmiophilic bodies were present in 82% of cells. These and other characteristics of type II cells in culture correspond to those of alveolar type II cells in fetal mouse lung. The pattern of reactivity of these cells with various anti-cytokeratin antibodies is described, and we show that, in contrast to rat type II cells, they do not exhibit alkaline phosphatase activity. Identity of the type II cell cultures was shown by their specific phospholipid composition and surfactant protein A (SP-A) content. The fetal alveolar type II cells in culture were found to synthesize and express class I but not class II major histocompatibility complex (MHC) antigens. The possibility to culture fetal alveolar type II cells of the mouse and the availability of genetically well-defined inbred and transgenic mouse strains opens ways to study the genetics of type II cell differentiation and function. Also, the in vitro availability of alveolar type II cells, the progenitor cells of mouse lung tumors, will enable us to study in vitro several of the processes involved in lung tumorigenesis in the mouse.

Topics: Alkaline Phosphatase; Animals; Blotting, Western; Cell Differentiation; Fluorescent Antibody Technique; H-2 Antigens; Histocompatibility Antigens Class II; Isoelectric Point; Keratins; Lectins; Mice; Mice, Inbred Strains; Microscopy, Electron; Molecular Weight; Phospholipids; Plant Lectins; Pulmonary Alveoli