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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.

Skeletal muscle differentiation within a Wilms' tumor is a well-documented histopathologic entity thought to occur at a relatively low incidence and influence prognosis. A serum-free hormonally defined growth medium has been developed, allowing the long-term growth of the skeletal muscle component of Wilms' tumors. Eight Wilms' tumors have been grown under these conditions. Three cases grew a homogeneous population of cells which ultrastructurally displayed all stages of myogenesis through myotubule formation. They also possessed immunoreactivity for skeletal muscle myosin and myoglobin and synthesized the M and B subunits of creatine kinase. Of interest was the finding that the ability to yield skeletal muscle cultures was limited to those cases which exhibited skeletal muscle fibers in vivo. This technique is also a very sensitive marker for identifying Wilms' tumors possessing a myoid component. A second serum-free hormonally defined medium has also been developed that supports the long-term culture of a unique cell type from Wilms' tumors which contain a myoid component. These cells are spindle-shaped and exhibit all of the characteristics of early myoblasts.

As an approach to investigating the disease cystic fibrosis, attempts were undertaken to culture from human submandibular glands epithelial cells with a potential for manifesting the cystic fibrosis genetic defect. To initiate the culture of submandibular gland epithelial cells, tissue fragments from glands were explanted as a function of both the composition of the serum-free growth medium and of the matrix utilized to coat the culture vessel growth surface. A morphologically homogeneous growth of submandibular gland epithelial cells, uncontaminated by fibroblasts, was obtained, once optimum culture conditions were defined. Light microscopic examination of these explant cultures in a transverse plane of section demonstrated variation in the outgrowth according to distance from the explant. At its outer margin, the outgrowth consisted of one or two layers of viable low cuboidal cells, and more centrally, it was multilayered. Mitotic figures were observed in the periphery of the outgrowth. In the region, a few cells removed from the periphery where the outgrowth consisted of about three to six cell layers, dilated intercellular spaces, indicative of secretion of fluid and ions into the spaces, separated the basal cuboidal cells. Overlying cells were increasingly flattened toward the culture surface and devoid of nuclei. Centrally, near the explant, the multilayer appeared completely involuted throughout. Ultrastructural examination in a transverse plane of the multilayered region with viable basal cells confirmed these observations showing wide spaces separating the cuboidal basal cells, keratinization of midstratum cells, and complete involution of the upper layer of ghost-like cells. These cells cultured from the submandibular gland reacted positively to immunochemical staining for keratin.

A case of childhood small cell undifferentiated sarcoma was grown in vitro in a serum-free culture medium. In vivo the tumor showed no evidence of differentiation by light microscopy, electron microscopy, and immunoperoxidase staining for myoglobin or skeletal muscle myosin. After growth in vitro for two weeks in a defined culture medium, the tumor cells formed myotubes and demonstrated ultrastructural myogenic differentiation. This case demonstrates the use of the tissue culture as a diagnostic aid in the recognition of childhood small cell undifferentiated sarcomas. It also raises questions regarding the origin of these tumors and the ability of in vitro growth to induce differentiation.

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.