PriCells: Introduction of Isolation and Culture of Human Alveolar Epithelial Cells

PriCells: Introduction of Isolation and Culture of Human Alveolar Epithelial Cells

1.1. BackgroundThe human lung consists of more than 40 different cell types. The morphology and function of constituent cells of the proximal, conducting airway epithelium differ drastically from those of the more distal, alveolar epithelium.

No cell lines that possess significant functional properties of alveolar epithelial cells (AEC) are reported to date. Primary culture of AEC is, therefore, used for most in vitro studies of alveolar epithelial function (e.g., transport and various metabolic pathways). The primary culture of human alveolar epithelial cells involves isolation, purification, and culture of alveolar epithelial type II (ATII) cells from human tissue obtained after lung resections. These ATII cells, when plated on permeable supports or plasticware, acquire the type 1 cell-like phenotype and morphology under appropriate culture conditions. Owing to the lack of availability of human tissue and some ethical issues pertaining to use of human tissues in certain countries, most studies were based on isolation and culture of cells from the lungs of small laboratory animals including mouse, rat, and rabbit. However, not much information on species differences in this specific area of cellular research has been systematically studied yet.

1.2. AECsATII cells constitute about 60% of AECs and about 15% of all lung parenchymal cells, although they cover less than 5% of the alveolar air spaces of adult human lungs. It is well known that ATII cells synthesize, secrete, and recycle some components of the surfactant that regulates alveolar surface tension in the distal airspaces of mammalian lungs. ATII cells govern extracellular surfactant transformation by regulating, for example, pH and Ca2+ of the hypophase, and play various roles in alveolar fluid balance, coagulation/fibrinolysis, and contribute to host defense. ATII cells proliferate, differentiate into type I (ATI) cells, and remove apoptotic ATII cells by phagocytosis. thus, contributing to epithelial repair following lung injury. ATII cells are thought to be progenitor cells for type I cells, especially in injury/repair of epithelial tract lining the distal airspaces of the lung. For a summary of the latest state in type II cell research, the reader is encouraged to read Fehrenbach’s excellent review that appeared recently.

Compared with its neighbor, the ATII cell, the ATI cell has received little attention. The general functions of ATI cells are relatively unexplored, because specific marker molecules that could be used for definitive identification of ATI cells were identified only very recently. Several ATI cell-specific gene products described below were used to address many problems in alveolar cell biology/molecular biology. Nonetheless, the identification of several proteins expressed by this cell and their presumed activities suggest more-sophisticated cell functions than mere gas exchange. The putative functions of type I cells include control of proliferation of peripheral lung cells, metabolism and/or degradation of peptides and peptide growth factors, generation of cyto/chemokines, regulation of alveolar fluid balance, and transcellular ion and water transport.

1.3. Differentiation Markers for AECsThe study of differentiation of type II cells into type I cells crucially depends on the possibility to distinguish both cell types. Beside the pure morphological characterization (e.g., presence of lamellar bodies, cuboidal shape), a number of alternative approaches to distinguish ATII from other cell types have been developed, such as modified Papanicolaou staining, cell-type-specific lectins, and immunohistochemical and immunocytochemical markers. The expression of markers, however, may be altered because of the specific culture conditions and the situation is further complicated by the transient appearance of an inter- mediate phenotype during differentiation, in that both ATI and ATII cell-like features may coexist in such cells. It has to be taken into account that so far there is no clear evidence showing that the differentiation of ATII cells definitively yield terminally differentiated ATI cells, necessitating the use of a term, “type I cell-like phenotype” to reflect this fact in the literature.

It has been reported that specific lectins label apical membranes of either type I or type II cells. The lectins Ricinus communis 1, Bauhinia purpurea, and Lycopersicon esculentum bind to ATI cells, but not ATII cells, while Maclura pomifera binds to ATII, but not ATI cells. These findings strongly suggested that ATI cells express membrane glycoproteins (and/or glycolipids) that are distinctly different from those expressed on the apical cell membranes of ATII cells.

A number of molecular markers, specific for type I cells, have been described in the recent past. The first one was T1α, a 36-kDa glycoprotein found in rodent lungs. Antibodies have been developed against lung proteins that specifically label type I cells in human lung with patterns that match those of rodent T1α. However, the human data are unclear, because antibodies to rodent T1α do not recognize ATI cell antigens in normal adult human lung. This likely indicates that O-glycosylation of the human protein(s), differs significantly from that of the rodent proteins.

Aquaporin-5 (AQP-5), a second ATI cell marker, is a member of the large family of aquaporin proteins, most of which are water channels. AQP-5 is a transmembrane protein of approx 27–34-kDa that resides in the ATI cell apical plasma membrane. Immunohistochemical and immunocytochemical studies by several investigators with various antibodies, as well as Northern and Western analyses of isolated cells, have shown that AQP-5 is uniquely expressed by ATI cells in the peripheral regions of the lung.

It was shown many years ago that ATI cells contain numerous, small, flask-shaped membrane invaginations, or caveolae, that open to the alveolar lumen or interstitial space. The presence of such caveolar structure in ATII cells is not clear yet. In addition to these caveolae at the cell membranes, numerous small vesicles are also noted in both ATI (but not ATII) cells and pulmonary vascular endothelial cells. Caveolin-1, a 21–24 kDa protein, is the major scaffolding protein that forms the vesicular skeleton of the caveolae. In the alveolar epithelium, caveolin-1 expression appears limited to ATI cells, as ATII cells normally express none or very little. Thus for many in vitro studies, caveolin-1 expression was used to discriminate between ATI and ATII cell phenotypes.

Several monoclonal antibodies, that discriminate between unidentified ATI and ATII cell surface epitopes, have been produced. Some of these antibodies may recognize the markers described above. The epitope of some antibodies is not yet identified, an example being the rat type I cell-specific antibody, VIIIB2.

Type I cells are also reported to express carbopeptidase membrane-bound enzyme (CP-M), intercellular adhesion molecule-1 (ICAM-1), β2-adrenergic receptor, insulin-like growth factor receptor-2 (IGFR-2), γ-glutamyl transferase (γ-GT), AQP-4, and VAMP-2, a membrane protein associated with caveolae. Whether or not these molecules are expressed in ATI cells exclusively or may also be present in other cell types needs still to be elucidated.

PriCells: Primary cell products:1. Primary cell extract2. Primary cell RNA3. Primary cell DNA4. Primary cell base medium5. Primary cell growth supplement6. Primary cell isolation kit7. Primary cell identification kit8. Primary cell transfection kit