Tissue culture of normal and cystic fibrosis sweat gland duct cells. I. Alterations in dome formation.

The elucidation of the underlying defect in fluid secretion by cystic fibrosis (CF) sweat glands is hindered by the unavailability of an experimental model for investigating this disease. As a potential model system, a serum-free growth medium was developed that supports the explant growth of epithelial cells from fragments of human skin. Immunohistochemical analysis demonstrated that these epithelial cell outgrowths originated from the duct of the sweat gland. By electron microscopy, the cells were demonstrated to possess keratinocyte-like morphology as noted by the presence of a multilayered outgrowth of cells containing well-defined keratin bundles. Identical outgrowths from skin biopsies of CF patients were compared to normal outgrowths and alterations were noted to occur in dome formation and in the number of intercellular spaces between cells. Doming alterations were also noted to occur in the CF heterozygous state. No differences in cell fine structure or in growth factor requirements for cell proliferation were noted between normal and CF cells. The potential use of this system as a model for CF research is discussed.

Tissue culture of epithelial cells from urine. I. Serum-free growth of cells from newborn infants.

The growth of epithelial cells from the urine of newborn infants was improved by the use of serum-free medium and a collagen type 1 matrix present on the growth surface of the culture vessel. The optimal concentrations and components of the serum-free medium consisted of a 1:1 mixture of Dulbecco's Modified Eagles' medium and Ham's F-12 medium supplemented with insulin (5 micrograms/ml), transferrin (5 micrograms/ml), selenium (5 ng/ml), and hydrocortisone (1 X 10(-7) M). The use of this medium allowed clonal isolates to undergo 25 generations and 5 passages with a doubling time of 24-36 hr with retention of original cell morphology. The culture of epithelial cells from the urine of newborn infants may provide a simple, reproducible system for the study of inborn errors of metabolism, especially those not expressed in fibroblast cultures.

Tissue culture of epithelial cells from urine. II. Application to patients with cystic fibrosis.

The urine from patients with cystic fibrosis (CF) was screened for the presence of epithelial cells that could be serially propagated in vitro. The urine from two newborn infants with meconium ileus was an excellent source of epithelial cells, and over 400 cultures were initiated in each case. The urine of older CF patients hospitalized for pulmonary complications also yielded cells but in fewer numbers. No cells were obtained from older CF patients without pulmonary complications or from healthy volunteers. Hypoxia may be the factor stimulating cell exfoliation in the urine of sick CF patients and in a larger percentage of newborn infants.

Effect of amiloride on potential difference across rectal mucosa in cystic fibrosis patients.

This study investigated whether the altered epithelial cell function in cystic fibrosis (CF) concerns channels for passive diffusion of sodium across cell membranes. For this purpose, the potential difference (PD), which arises across the colonic mucosa from electrogenic sodium transport, was compared in CF and non-CF subjects, together with the effect on this PD from bathing the mucosa with the specific Na+ channel blocker, amiloride. The resting rectal mucosal PD did not differ significantly in the two groups. Amiloride caused a dose-dependent decrease in the PD (lumen negative to skin) in all subjects. The mean amiloride effect was less in the CF population at 10(-8), 10(-7), 10(-6), 10(-5), and 10(4) M amiloride. The decrease of transmucosal PD in CF patients was significantly less when compared with controls at 10(6) M and 10(-5) M and suggestive of a similar trend at 10(-4) M.