Epithelial cells lining the digestive tract represent a highly organized system built up by multipotent stem cells. A process of asymmetric mitosis produces a population of proliferative cells that are rapidly renewed and migrate along the crypt-villus axis, differentiating into functional mature cells before dying and exfoliating into the intestinal lumen. Isolated crypts or epithelial cells retaining high viability can be prepared within a few h after tissue sampling. After cells are cultured in serum-free media, short-term studies (16–48 h) can be conducted for endocrinology, energy metabolism, or programmed cell death. However, long-term primary culture of intestinal cells (up to 10 d) is still difficult despite progress in isolation methodologies and manipulation of the cell microenvironment. The main problem in developing primary culture is the lack of structural markers specific to the stem cell compartment. The design of a microscopic multidimensional analytic system to record the expression profiles of biomarkers all along the living intestinal crypt should improve basic knowledge of the survival and growth of adult crypt stem cells, and the selection of totipotent embryonic stem cells capable of differentiating into intestinal tissues should facilitate studies of the genomic basis of endodermal tissue differentiation.

Epithelial cells from normal pig bladders proliferated when cocultured with lethally irradiated feeder cells of the LA7 rat mammary tumor line. When the bladder cells and feeders were plated together at a confluent density, the bladder cells proliferated as the feeder cells died, resulting in a confluent culture of bladder cells. The bladder cells were successfully subcultured by plating with freshly irradiated LA7 feeder cells. In this way, bladder cells from five pigs were carried to confluency in passages 1, 4, 7, 7, and 13, amounting to at least 6, 18, 24, 26, and 45 doublings in culture, respectively, and none showed signs of slowed proliferation at the time of culture termination. Fibroblasts never became a prominent feature of these cultures, and their frequency was determined to be about 26 fibroblasts per 10⁵ cells in passage 9. Pig bladder cells in 0.5% serum doubled in number in slightly over 3 d, whereas cells in 5.0% serum doubled in about 6 d. In fresh medium without feeder cells only minimal proliferation of bladder cells occurred. In LA7-conditioned medium the bladder cell numbers decreased, leading to the conclusion that the stimulus from LA7 cells is mechanically or physically transmitted. The bladder cells reacted with antibodies to keratins 7 and 18 but not to keratin 14 or vimentin. Tight junctions, visualized with an antibody to the ZO1 protein, connected all the cells to their neighbors. Most cells in passage 9 carried the diploid chromosome number of 38.

Penaeid cell culture has gained much attention as a potential model to facilitate researches on the characterization of the virus and to develop more sophisticated and improved diagnostic procedures for use in the aquaculture industry. However, to date, cell division processes of cultured penaeid cells have not been found, which is suggested as one of the reasons that block the establishment of the continuous penaeid cell lines. We reported here the cell division processes of cultured lymphoid cells of Penaeus japonicus. The culture medium used was based on M199 and was modified by supplementing saline components. Cultures were incubated at 25° C, and 5% CO₂ was supplemented. In primary cultured lymphoid cells, dividing cells in different shapes were found. Cell division processes of 12 dividing lymphoid cells were tracked. After cell division, their daughter cells turned into fibroblast-like or epithelioid cells. These results proved that the culture conditions used were suitable for lymphoid cells of P. japonicus to proliferate in vitro and that cultured lymphoid cells still had the ability to carry out cell division. These findings would give light to the establishment of continuous penaeid cell lines and would also provide us with the knowledge of cell division processes of the penaeid.

Cell therapy may have the potential for the treatment of Type I diabetes. To date, cells suitable for this purpose have not been developed. This study investigates the feasibility of modifying Vero, a cell line that may be considered safe to implant into humans, for this purpose. Stable Vero transfectants containing full-length human preproinsulin complementary deoxyribonucleic acid (cDNA) were generated using a liposomal transfection reagent. Reverse transcriptase–polymerase chain reaction, immunocytochemistry, Western blotting, and enzyme-linked immunosorbent assays were used to assess the resulting cells. Proinsulin was expressed but was not processed to insulin by these cells. Proinsulin cDNA was genetically modified, resulting in a form that is furin sensitive. The resulting stably transfected Vero clones constitutively release approximately 34%/h (32.68 ± 2.21 to 35.62 ± 3.14%) of the product formed, approximately 62% (59.99 ± 6.45 to 64.64 ± 4.57%) of which is mature insulin. These Vero transfectants did not exhibit glucose-stimulated insulin secretion. As GLUT2 and glucokinase (GCK) are not constitutively expressed by these cells, human GLUT2 cDNA and GCK cDNA were cotransfected with furin-sensitive preproinsulin cDNA into Vero cells. Insulin and GCK proteins were detected in the cytoplasmic region of the resulting cells, whereas GLUT2 was predominantly expressed in the nucleus. Coexpression of GLUT2 and GCK did not result in glucose-stimulated insulin secretion. The results from this study demonstrate the feasibility of engineering a relatively “safe” nonbeta cell line to produce human insulin. Coexpression of GLUT2 and GCK, at the levels achieved here, is not adequate enough to induce glucose-stimulated insulin secretion in such cells; the subcellular location of transfected components may be relevant.

The Clara cell is believed to be the progenitor of the peripheral airway epithelium, and it produces the surfactant proteins SP-A and SP-B, in addition to the 10-kDa Clara cell secretory protein (CCSP or CC10). To date, attempts to develop Clara cell lines have been unsuccessful. Most such attempts have involved the in vitro insertion of a transforming viral oncogene. We have reported previously the characterization of a differentiated conditionally immortalized murine lung Type II epithelial cell line, T7, from the H-2K^b-tsA58 transgenic mouse. We have also used this mouse model to derive Clara cell lines. In this model, the need for in vitro gene insertion is circumvented by the creation of a transgene, in which the large tumor antigen of a temperature-sensitive strain (tsA58) of the simian virus 40 (SV40) is fused with the major histocompatibility complex promoter H-2K^b. The promoter is active in a wide range of tissues and is induced by interferons (IFN). From the lungs of animals harboring the hybrid construct, we isolated and characterized Clara cells. The cells contain dense secretory granules and mitochondria typical of Clara cells, and express SP-A, SP-B, SP-D, and the Clara cell secretory protein, CC10. Withdrawal of the IFN and elevation of the incubation temperature permit normal cell differentiation similar to that of Clara cells in vivo. This cell line should be very useful for the investigation of normal Clara cell function and gene expression.

This work reports the isolation and characterization of a line of human, nontransformed and differentiated prostate epithelial cells (EPN) in continuous culture. Primary cultures of epithelial prostate cells were set up using normal tissue isolated from a prostate sample collected after radical prostatectomy for cancer. After 70 passages, EPN cells did not undergo “Hayflikc crisis” and were free of fibroblast contamination and were thus subcloned and characterized. EPN cells in culture, as prostate epithelial cells in vivo, express high–molecular weight cytokeratin and Pyk2, whereas they do not express desmin. EPN cells are nontransformed because they do not form colonies in semisolid medium and do not form tumors once injected into nude mice. EPN cells express the functional androgen receptor, which can mediate the mitogenic activity of testosterone. Finally, clonal production of the prostate-specific antigen could be detected in EPN cells. The availability of a line of epithelial nontransformed prostate cell in culture will be useful in investigating the complex process regulating normal prostate physiology as well as the development and progression of prostate tumors.

A cell line from Trichoplusia ni (TN-CL1) infected with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV-HPP) and a cell line from Helicoverpa zea (BCIRL-HZ-AM1) infected with the Helicoverpa zea single nucleopolyhedrovirus (HzSNPV/BrCL2) were subjected to ultraviolet-B (UV-B) irradiation at a predetermined level of exposure that would inactivate greater than 95% of the virus suspended in the liquid. The working hypothesis was that the homologous insect cells would utilize their inherent deoxyribonucleic acid (DNA) repair mechanism(s) to prevent, repair, or at least mitigate the damaging effects of UV-B light on viral DNA synthesis. We attempted to determine this by using infected cells that were subjected to UV-B irradiation at different postinoculation periods under two experimental conditions of exposure: (1) shielded, and (2) nonshielded. Of the two cell lines infected with their respective homologous viruses, the virus from TN-CL1 cells was the least sensitive to UV-B light because the extracellular virus (ECV) and occlusion body (OB) levels of virus-infected TN-CL1 cells were higher than those of the virus-infected BCIRL-HZ-AM1 cells. Production of ECV and OB from both cell lines was lower in the exposed, nonshielded treatment than in the exposed, shielded treatment. However, AcMNPV-HPP was produced in enough quantity to indicate that TN-CL1 might impart a level of protection to the virus against UV light.

A partnership between the ectodomain of the fibroblast growth factor receptor (FGFR) isotypes and the chains of pericellular matrix heparan sulfate determines the fibroblast growth factor (FGF) and cell-type specificitives of the FGFR signaling complex. The contribution of the FGFR intracellular tyrosine kinase domains to the specificity of FGFR signaling is unclear. This report shows that the quantity and quality of phosphorylation of the FGFR kinase substrate SNT1 (also called FGFR substrate 2, FRS2) is both FGFR isotype and cell-type specific in prostate tumor epithelial cells at different stages of malignancy. Epithelial cell–resident FGFR2 that promotes homeostasis yields a low level of phosphorylated 65-kDa SNT1. Phosphorylation by ectopic FGFR1 that promotes malignancy was much more intense and yielded a phosphorylated 85-kDa SNT1. The amount of the 85-kDa SNT1 increased by 20-fold during proliferative aging of FGFR1-expressing cell populations that is required for FGFR1-stimulated mitogenesis and the malignant phenotype. In addition, the receptor-specific differential phosphorylation of SNT1 by FGFR isotypes, both of which are normally anchored to the cell membrane, occurred only in intact cells. Therefore, similar to kinase subunits within the heparan sulfate–FGFR complex, cell membrane and cytoskeletal context likely determine FGFR isotype– and cell-type–specific conformational relationships between FGFR kinases and external substrates. This determines the quantity and quality of SNT1 phosphorylation and differential signaling.