The human epithelial cell cytotoxicity assay for determining tissue specific toxicity

The Human Epithelial Cell Cytotoxicity (HECC) Assay for determining organ specific cytotoxicity uses human epithelial cells from eight different human tissues, including: skin, mammary, prostate, renal, bronchial, oral, ecto-cervix, and liver. Although the initial studies using this assay were conducted using cancer chemopreventive agents, the HECC Assay can also be used to evaluate other types of drugs, personal care products, environmental chemicals, and potential toxicants. Human epithelial cells at an early passage are seeded into multi-well dishes. The cells are exposed to multiple concentrations of a test agent for a three day period. The concentration ranges for test agents in the assay are determined in a preliminary assay using an exposure of five days and log dilutions from the highest soluble concentration. At the end of the exposure period, the cultures are evaluated for inhibition of growth. In the HECC Assay, cultures are exposed for three days. At the end of the exposure period, the cultures are evaluated for inhibition of growth, mitochondrial function, and PCNA expression or albumin synthesis (hepatocytes). Data are analyzed to determine the concentration that inhibited and point by 50 percent (TC₅₀). Values for each agent in each target epithelial cell line or culture and the target tissue specific sensitivity are compared to determine the relative sensitivity of each epithelial cell line to the test agent.     

Studies on primary cell cultures derived from ovarian tissue of Penaeus monodon

As part of a bioassay approach to investigate ovarian development and function, primary cell cultures were derived from Penaeus monodon ovaries at various stages of maturation. These cultures were established in modified Grace's or modified 2× L-15 media. Various supplements including growth factors, vitamins and minerals were trailed. Four morphologically different types of cells (epithelioid, fibroblastic, rounded, and epithelioid with large nuclei) were maintained for up to 17 months. Epithelioid cells grew best in modified Grace's medium but were generally short-lived (less than two months). Fibroblast-like cells formed confluent monolayers in modified 2× L-15 medium, were passaged three times and survived for 17 months. In other cultures, millions of rounded cells migrated from tissue. They survived for prolonged periods (up to ten months), either loosely attached to the flask or suspended in the medium. A change in dominant cell type from fibroblastic to epithelioid was observed in some cultures after three or nine months incubation. These epithelioid cells which had very large nuclei, grew to confluence but could not be sub-cultured. It is noteworthy that the rounded cells and the epithelioid cells with the large nuclei both produced vitellogenin in protein-free media.     

The human epidermal cell asay for chemopreventive agents

Methods are described for a human keratinocyte assay for screening cancer preventive agents. Normal human keratinocytes at the first or second subculture are seeded into multi-well dishes. Cultures are repeatedly exposed to the carcinogen, propane sultone, and nontoxic concentrations of potential chemopreventive agents. At the end of the exposure period, the cultures are evaluated for growth and involucrin expression. Data are analyzed to determine the potential for test agents to inhibit propane sultone induced growth or to induce involucrin expression relative to that in propane sultone treated controls.     

A method for estabilishing primary cultures of human gastric epithelial cells

Long-term culture of human gastric epithelial cells has been difficult, and at present no normal human gastric epithelial cell lines are readily available. As part of our experiments to study pathogenesis of H. pylori, a bacterium that infects the stomach, we developed methods to culture normal human gastric epithelial cells. Primary cultures of human gastric epithelial cells can be established from gastric biopsies taken at upper G.I. endoscopy. Enzymatically isolated gastric epithelial-like cells are present in tight colonies on culture dishes within 24 hours of placing the cells in culture. Cells isolated stain positively for cytokeratin and produce neutral mucins, indicating that they are mucin secreting epithelial cells, consistent with gastric epithelial cells. Epithelial cells can be maintained up to 4 weeks in culture with evidence of DNA synthesis up through the first week of culture.     

Fluorescent method for albumin assay in human aqueous humour and tear fluid

A new method for albumin fluorophotometry in human ocular fluids (aqueous humour and tear fluid) with a K-35 fluorescent probe is proposed. The method provides measurements of albumin concentration and fluorescence parameters characterising properties of binding centres of the albumin molecule. There was a strong correlation between albumin concentrations determined by fluorescent method proposed by us and by electrophoresis of ocular fluid proteins. This method can be used for the analysis of ocular fluid albumin and the state of the tissue-blood barriers. Translated fromByulleten' Eksperimental'noi Biologii I Meditsiny, Vol. 128, No. 11, pp. 597–600, November, 1999     

Progress in the development of shrimp cell cultures in Thailand

Primary shrimp cell cultures were developed from lymphoid organ and ovaries of black tiger shrimp, Penaeus monodon, in double-strength Leibovitz's L-15 medium supplemented with 15% fetal bovine serum, 1% glucose, 5 g/L NaCl, 15% shrimp meat extract. The optimum conditions for primary culture in vitro were obtained in L-15 medium with an osmolality of approximately 730 ± 10 mmol/kg, a temperature range of 25--28 °C and incubation in a normal atmosphere. However, basal medium supplemented with 0.01% cholesterol could enhance good growth and cells performance initiated from lymphoid organ. Both epithelial-like and fibroblastic-like cells were observed from those organs within 2 days incubation. Within 3 days, 80% confluent monolayers were obtained from the lymphoid organ while cultures from other tissues required 5 days. Cultures were maintained for at least 43 days. Only cells from lymphoid organ could be subcultured and confluent monolayers achieved within 10 days post-spilt. Healthy cultures of the lymphoid cells did not persist beyond the third passage. Application of these primary shrimp cell cultures for studying pathogenic viruses of shrimp in vitro will be discussed.     

Primary cell cultures isolated from Penaeus monodon prawns

We have devised a cell culture system for Penaeus monodon prawn cells that uses a defined synthetic medium. Organs were removed from adult prawns ranging in size from 13--19cm rostrum-to-telson length. Cultures consisted of either a blend of hematopoietic and lymphoid cells or ovarian cells. The cells divide rapidly in culture, doubling on average once per week for 5 to 6 weeks. These cultures continue to survive for at least 5 months but the rates of cell division are low after the first 5--6 weeks. In the literature, unicellular eukaryotic marine organisms such as chytrids may contaminate marine cell cultures. In some cases these eukaryotic contaminants may be difficult to distinguish from prawn cells unless detailed ultrastructure or characteristic developmental stages such as zoospores can be observed. Alternatively, we prepared molecular probes from repeated DNA sequences 100--400 bp in length in the P. monodon genome. These species-specific probes were hybridised to genomic DNA from cell culture to confirm proliferation of P. monodon cells in our cultures.     

Transfection of Lymantria dispar insect cell lines

Lepidopteran cell lines derived from the gypsy moth, Lymantria dispar, have not been widely used in protein expression studies or systems because they are weakly adherent, have specific growth requirements and characteristics, and are generally difficult to transfect. Using lipid-mediated transfection of a reporter plasmid, we modify the standard method for transfection of L. dispar-derived embryonic cell lines IPLB-LdEp and -LdEIta, obtaining transfection efficiencies of 34% and 30%, respectively, as determined by image analysis assays. Using the standard lipid-mediated method, we obtain transfection efficiencies for L. dispar-derived cell line IPLB-Ld652Y of at least 40% with high mean expression levels, indicating the IPLB-Ld652Y cell line may be a superior choice for expression studies or systems requiring L. dispar-derived cells.     

Establishment of cell culture systems from penaeid shrimp and their susceptibility to white spot disease and yellow head viruses

Monolayer cultures were established from ovary, heart, lymphoid tissue and peripheral hemocytes of penaeid shrimps including Penaeus monodon, P. japonicus and P. penicillatus. The most favorable conditions for the culture of penaeid shrimp cells in vitro was in CMRL and L-15 tissue culture media when used within an osmolarity range of 620--760 mmol/kg. The optimal maintenance temperature was 25 °C for tissues of P. japonicus and 28 °C for tissues of P. monodon and P. penicillatus. Among the four tissues tested, lymphoid tissue, or 'Oka organ', was superior to the other tissues for the formation of confluent cell monolayers. Cell cultures from lymphoid tissue and ovary have been subcultured up to three times. When peripheral hemocytes and heart were cultured, a maximum survival of 4 days was obtained. In contrast, cell cultures derived from ovary and lymphoid tissue were maintained alive for at least 20 days in appropriate culture systems. Neither confluent cell sheet nor adherence of cells was obtained in cultivation of hepatopancreas using the present culture systems. The results obtained from the present study also revealed that ovary extract, muscle extract and lobster hemolymph enhanced the survival of the cultured cells of penaeid shrimp in vitro. When the 'Oka organ' cell monolayer was incubated with either white spot disease virus (WSDV) or yellow head virus (YHV), no cytopathic effect (CPE) was obtained. However, at 5--7 days after establishment, significant CPE (a few foci) was observed in cell monolayers derived from WSDV- and YHV-infected Oka tissue. By electron microscopy, virions of WSDV and YHV were observed in the nuclei and cytoplasm of cultured cells. The CPE foci developed further with increased incubation time.     

A new method to obtain epithelial and stromal explants from human Corneo-Scleral Discs for the routine culture of corneal epithelial and fibroblast cells

Acquisition of human corneal cells for culture is hindered not only by the scarcity of donor tissues but also by some of the standard enzymatic and mechanical isolation techniques. Good yields have been reported from full-thickness explant and sclero-limbal pieces. However, due to their greater proliferative capacity, fibroblasts will encroach and subsequently overwhelm epithelial cultures whichever technique is used. The novel approach presented here is to minimise this by removal of the whole stroma from the epithelial layers at the outset. This is achieved by selective sectioning with the Webb mini-microtome developed in the Norwich Eye Research Laboratory. The microtome can be sterilised by alcohol spraying or autoclaving and is small enough to use in the culture hood. A selective cut in the region of the Bowmans membrane results in the isolation of the epithelium from the stroma and thus exposed, the basal epithelial layers are released from contact inhibition to allow growth. The stroma is further cut to produce multiple sections for the culture of fibroblasts. Both pure epithelial and stromal fibroblast cultures have been successfully generated in serum-enriched medium as well as defined serum-free media with growth supplements, from the corneo-scleral discs of donors of all ages.     

Mechanical properties of human skeletal muscle fromin vitro studies of biopsies

The steady-state and small-scale dynamic mechanical properties of human rectus abdominis and intercostal muscle have been investigated by testing small biopsiesin vitro, taken during normal surgery. It has proved possible to obtain valid data on the tension/length/stimulation-rate relationship at body temperature for up to 24 hours after removal. The twitch and tetanus parameters, i.e. maximum tension, rise time and decay time, show comparability with those found by other workersin vivo. The steady-state tension/length/stimulation-rate relationship is similar to those found for other mammalian muscles; the tension/stimulation-rate relationship being dependent on muscle length. The responses to small-scale dynamic inputs show the importance of dynamic properties at physiological rates of change of parameters. Responses to single-parameter inputs appear to add when both inputs are applied simultaneously.     

Transplantation of human fetal tissue as a promising method in the treatment of diabetes mellitus

Experience accumulated in Russia during the last century in the treatment of diabetes mellitus by transplantation of human fetal tissues is analyzed from the historical and geographical viewpoint. Over the last 15 years about 3000 patients have been treated using this method. Such treatment has mainly resulted in stabilization of the labile forms of insulin-dependent diabetes: in 80% of recipients the exogenous insulin requirements have been reduced by 20–85%, and in some cases a short-term insulin independence has been established. Discontinuation and partial regression of secondary diabetic complications have been observed: pain and paresthesia in the extremities have diminished or disappeared; in the case of angiopathy of the lower extremities the incidence of indications for amputation due to gangrene has been reduced; the pathological process in the fundus of the eye has been arrested and visual acuity has increased in 45–65% of patients with diabetic retinopathy. At the prenephrotic stage of diabetic nephropathy transplantation has been attended by a reduction or disappearance of proteinuria and normalization of arterial pressure in 40–50% of patients. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 4, pp. 350–355, April, 1994     

A quantitative method for thein vitro study of sounds produced by prosthetic aortic heart valves Part III: an experimental comparative study of the sounds produced by normal and simulated-abnormal Smeloff aortic prostheses

Anin vitro study was made of the sounds produced by a normal, Smeloffaortic-valve prosthesis and compared for the entire cardiac cycle with the same valve having a simulated overgrowth on the upstream struts. Time and amplitude information, power-density spectra, power-distribution functions, and three-dimensional power-frequency-time surfaces were compared. Power-density spectra and power-distribution functions were compared in more detail for those portions of the cycle corresponding to the opening, systolic, and closing sounds of the valve. Parameters for the acoustical model were estimated from these power-density spectra. All results were then compared and discussed in terms of the physical changes in the valve. Each comparison gave useful information about the simulated malfunction. Opening, systolic, and closing sounds were different for each case. More power was displayed in the opening sound of the abnormal valve and the systolic sound was early. The closing sound of the abnormal valve had a lower frequency content, and a diastolic sound was observed only for the abnormal valve. Use of the frequency domain gave additional understanding of differences in performance.     

Viability of Cancer Cells Exposed to Pulsed Electric Fields: The Role of Pulse Charge

The goal of this study was to collect a comprehensive set of data that related lethal effects of electric fields to the duration of the pulse. Electric pulses of different strengths and durations were applied to a suspension of HEp-2 cells (epidermoid carcinoma of the human larynx) using a six-needle electrode array connected through an autoswitcher to a square wave generator. Pulse durations varied from 50 s to 16 ms and the ranges of electric field were adjusted for each duration to capture cell viabilities between 0% and 100%. After pulsation, cells were incubated for 44 h at 37 ×C, and their viability was measured spectrophotometrically using an XTT assay. For each pulse duration (d), viability data were used to determine the electric field that killed half of the cells (E ₅₀). When plotted on logarithmic axes, E ₅₀ vs. d was a straight line, leading to a hyperbolic relationship: E ₅₀=const/d. This relationship suggests that the total charge delivered by the pulse is the decisive factor in killing HEp-2 cells.© 2003 Biomedical Engineering Society.     

Model forin vitro investigation of bone mineralization

Anin vitro model was developed for the investigation of bone cell mineralization processes. Out-grown cells of explanted human nasal bone fragments were selected into three lineages and one of them, the osteoblast-enriched line, was partially characterized. A large number of cells of this line stained readily for alkaline phosphatase. They produced prostanoids, PGl₂, PGF₂ and PGE₂, and osteocalcin in the presence of 1,25(OH)₂D₃. Cells synthesized collagen and mineralized the developed extracellular matrix. This latter line seems to be appropriate as a model system to find effective compounds which increase bone calcification.     

Effect of Bacillus anthracis virulence factors on human dendritic cell activation

Bacillus anthracis possesses three primary virulence factors: capsule, lethal toxin (LT), and edema toxin (ET). Dendritic cells (DCs) are critical to innate and acquired immunity and represent potential targets for these factors. We examined the ability of B. anthracis spores and bacilli to stimulate human monocyte-derived DC (MDDC), primary myeloid DC (mDC), and plasmacytoid DC (pDC) cytokine secretion. Exposure of MDDCs and mDCs to spores or vegetative bacilli of the genetically complete strain UT500 induced significantly increased cytokine secretion. Spores lacking genes required for capsule biosynthesis stimulated significantly higher cytokine secretion than UT500 spores from mDCs, but not MDDCs. In contrast, bacilli lacking capsule stimulated significantly higher cytokine secretion than UT500 bacilli in both MDDCs and mDCs. Spores or bacilli lacking both LT and ET stimulated significantly higher cytokine secretion than UT500 spores or bacilli, respectively, in both mDCs and MDDCs. pDCs exposed to spores or bacilli did not produce significant amounts of cytokines even when virulence factors were absent. In conclusion, B. anthracis employs toxins as well as capsule to inhibit human MDDC and mDC cytokine secretion, whereas human pDCs respond poorly even when capsule or both toxins are absent.     

Development and validation of a cell culture based assay for in vitro assessment of anticryptosporidial compounds

In vitro culture of Cryptosporidium parvum oocysts in HCT-8 cells was combined with immunofluorescent labelling and digital image analysis to quantify the development of the parasite by detecting and measuring the labelled area in the respective cell cultures. The number of inoculated oocysts and the labelled area correlated reliably and significantly (R ², 0.98–0.99). The effects of various concentrations of halofuginone bromide (0.00039 to 50 μM) and monensin (0.00225 to 0.144 μM) on in vitro parasite development were determined in further trials in cultures inoculated each with 10⁵ oocysts. Monensin reduced the detected area in a dose-dependant manner. In comparison to the untreated controls, the area positive for C. parvum in the cultures treated with 0.144 to 0.009 μM monensin reached a maximum of 17%, and inhibition of 40% was observed at 0.0045 μM. Halofuginone bromide also efficiently inhibited parasite in vitro reproduction, albeit at higher concentrations. At 12.5 μM or more, inhibition was at least 90%; 0.05 μM still yielded 80% inhibition, whereas at concentrations below 0.00625 μM, labelled areas abruptly increased. Both drugs appeared efficient under in vitro conditions; the applied system is suited to screen drugs for their anti-cryptosporidial capacity.     

A method for pneumatically inserting an array of penetrating electrodes into cortical tissue

The goal of this research was to find a practical means by which an array of 100 needle-shaped electrodes could be implanted into the cerebral cortex with minimal brain tissue trauma. It was found that insertion of these structures into cortical tissues could only be performed using high insertion speeds. A pneumatically actuated impact insertion system has been developed that is capable of inserting an electrode array into feline brain tissue at speeds from about 1 to 11 m/s. We found that a minimum array insertion speed of 8.3 m/s was necessary for a complete, safe insertion of all 100 electrodes in the array to a depth of 1.5 mm into feline cortex. The performance of the impact insertion system is discussed in terms of a simplified representation of cortical tissue.     

A method for the isolation of human gastric mucous epithelial cells for primary cell culture: A comparison of biopsy vs surgical tissue

We have developed a method for the isolation and growth of normal human gastric mucous epithelial cells using biopsies or surgically resected tissues as the source of the cells. The attachment and growth of cells were dependent upon: (1) cell planting density, 50,000 cells/cm²; (2) extracellular matrix (fibronectin); and (3) and the use of a porous filter. In all experiments we found better cells attachment and growth of human gastric mucous cells isolated from surgical specimens compared with those gastric mucous cells isolated from gastric biopsies. The initial cell viability (as measured by Trypan-blue) was the same in both populations of gastric mucous epithelial cells isolated from either gastric biopsies or surgical specimens. After 4–5 days in culture one could detect various amounts of mucin in all the cells using either periodic acid Schiff (PAS) staining or a specific anti-mucin antibody. A similar pattern of much straining was also found in primary cultures of guinea pig gastric mucous epithelial cells. Immunohistochemical staining for chief cells (anti-pepsinogen) or parietal cells (anti-H⁺/K⁺ ATPasc) in the gastric mucous cuboidal-like epithelial cells with tight junctions, desmosomes,short microvilli, a filamentous terminal web, mucous granules, and basal lamina-like structure. We could not detect the presence of fibroblasts during the 7–9 days that the primary cells were in culture. This cell culture method will prove useful in the isolation of normal human gastric mucous epithelial cells for in vitro studies of gastric mucosal injury and repair.