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This work investigates a completely novel and experimental concept of exposing L5178Y cells at the air-agar-interface to mainstream cigarette smoke aerosol (Kentucky reference 3R4F). This study highlights the associated challenges of combining a suspension cell line alongside an in vitro aerosol exposure system. To achieve a monolayer, cells were 'seeded' in a concentrated cell super-mix suspension onto an RPMI/agar-matrix -base. The resulting cell suspension media was adsorbed into the agar base leaving the L5178Y cells lightly suspended on the agar surface, approximating a monolayer. Cells were deemed supportable on the agar-matrix, viable and recoverable. Using Vitrocell VC 10 exposure system and the Ames 4 exposure module, L5178Y cells were successfully exposed to a dynamic cigarette smoke aerosol, recovered and assessed for mutant frequencies, using standard assay procedures. Method development included assessment of flowing air conditions, plating efficiency and recovery of L5178Y cells from the agar-matrix surface. Positive controls MMS and B[a]P were successfully incorporated into the agar-matrix and metabolic activation was achieved by S-9 incorporation into the same agar-base-matrix. B[a]P demonstrated metabolic activation and positive response, suggesting a clear cellular interaction with the agar-matrix. Whole smoke exposed cells in the presence of metabolic activation showed a clear dose response and increasing mutant frequencies, well in excess of the controls (air and incubator) and the global evaluation factor following a 2 or 3 day expression period. This experimental concept demonstrates that L5178Y cells can be exposed to cigarette smoke aerosol, using a completely novel and a previously untested approach. Although this work successfully demonstrates the approach is viable and cells can be plated and maintained on an agar-matrix, more optimisation and robustness assessment is required before it can be considered fully adapted and used alongside other whole aerosol methodologies for the assessment of cigarette smoke and other inhaled aerosols.

Purpose The clonogenic property and radiobiological responses of a fish brain endothelial cell line, eelB, derived from the American eel were studied. Methods Clonogenic assays were performed to determine the plating efficiency of the eelB cells and to evaluate the clonogenic survival fractions after direct irradiation to low-dose low-LET gamma radiation or receiving irradiated cell conditioned medium in the bystander effect experiments. Result eelB had the second highest plating efficiency ever reported to date for fish cell lines. Large eelB macroscopic colonies could be formed in a short period of time and were easy to identify and count. Unlike with other fish clonogenic cell lines, which had a relatively slow proliferation profile, clonogenic assays with the eelB cells could be completed as early as 12 days in culture. After direct irradiation with gamma rays at low doses ranging from 0.1 Gy to 5 Gy, the dose-clonogenic survival curve of the eelB cell line showed a linear trend and did not develop a shoulder region. A classical radio-adaptive response was not induced with the clonogenic survival endpoint when the priming dose (0.1 or 0.5 Gy) was delivered 6 h before the challenge dose (3 or 5 Gy). However, a radio-adaptive response was observed in progeny cells that survived 5 Gy and developed lethal mutations. eelB appeared to lack the ability to produce damaging radiation-induced bystander signals on both eelB and HaCaT recipient cells. Conclusion eelB cell line could be a very useful cell model in the study of radiation impacts on the aquatic health.

In this study, umesu phenolics were purified from the salt extracts of Japanese apricot (Nanko-mume cultivar of Prunus mume Sieb. et Zucc.). Characterization of umesu phenolics revealed that, when added to the culture media of the infected cells, they inhibited the multiplication of influenza and many other RNA and DNA viruses. In addition to these antiviral activities, the phenolics significantly decreased the plating efficiency of influenza virus, if present in the virus inoculum. More drastic effects were observed in terms of virucidal activity; the infectivity of several strains of influenza viruses decreased less than 0.001 when they were incubated with 4 mg/ml phenolics at 30 ℃ for 5 min. The virucidal activity of phenolics was found to be more remarkable in acidic conditions; however, the activity was not merely a result of the acidity of the phenolics. These results clearly support the antiviral and virucidal activities of the umesu phenolics against influenza viruses and suggest their potential pharmacological usefulness as disinfectants or preventive medicine against superficial infections, such as the respiratory infections.

Cholangiopathies, which affect extrahepatic bile ducts (EHBDs), include biliary atresia, primary sclerosing cholangitis, and cholangiocarcinoma. They have no effective therapeutic options. Tools to study EHBD are very limited. Our purpose was to develop an organ-specific, versatile, adult stem cell-derived, preclinical cholangiocyte model that can be easily generated from wild type and genetically engineered mice. Thus, we report on the novel technique of developing an EHBD organoid (EHBDO) culture system from adult mouse EHBDs. The model is cost-efficient, able to be readily analyzed, and has multiple downstream applications. Specifically, we describe the methodology of mouse EHBD isolation and single cell dissociation, organoid culture initiation, propagation, and long-term maintenance and storage. This manuscript also describes EHBDO processing for immunohistochemistry, fluorescent microscopy, and mRNA abundance quantitation by real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). This protocol has significant advantages in addition to producing EHBD-specific organoids. The use of a conditioned medium from L-WRN cells significantly reduces the cost of this model. The use of mouse EHBDs provides almost unlimited tissue for culture generation, unlike human tissue. Generated mouse EHBDOs contain a pure population of epithelial cells with markers of endodermal progenitor and differentiated biliary cells. Cultured organoids maintain homogenous morphology through multiple passages and can be recovered after a long-term storage period in liquid nitrogen. The model allows for the study of biliary progenitor cell proliferation, can be manipulated pharmacologically, and may be generated from genetically engineered mice. Future studies are needed to optimize culture conditions in order to increase plating efficiency, evaluate functional cell maturity, and direct cell differentiation. Development of co-culture models and a more biologically neutral extracellular matrix are also desirable.

No optimal assay for assessing isolated hepatocytes before hepatocyte transplantation (HTx) has been established, therefore reliable and rapid assays are warranted. Isolated rat hepatocytes were dipped in a water bath (necrosis model), and were also cultured with Okadaic acid (apoptosis model) or vehicle, followed by cellular assessment including trypan blue exclusion (TBE) viability, ADP /ATP ratio, plating efficiency (PE), DNA quantity and ammonia elimination. Hepatocytes were transplanted into the liver of analbuminemic rats, subsequently engraftment was assessed by serum albumin and the histology of transplanted grafts. In the necrosis model, the ADP/ATP ratio was strongly and negatively correlated with the TBE (R2 = 0.559, P P

In the present study, the role of monoglucosyl-rutin as a potential radioprotector was investigated using mammalian cell culture models. Cell survival and DNA damage were assessed using colony formation, sister chromatid exchange and γH2AX assays. It was demonstrated that monoglucosyl-rutin was able to increase cell survival when exposed to ionizing radiation, possibly by decreasing the amount of base damage experienced by the cell. However, the present study also demonstrated that, despite monoglucosyl-rutin exhibiting radioprotective effects at low doses, high doses of monoglucosyl-rutin led to a decrease in plating efficiency and an increased doubling time. This effect may be due to double-strand breaks caused by high concentrations of monoglucosyl-rutin.

A new epithelial cell line, WAF was developed from caudal fin of freshwater shark, Wallago attu. The cell line was optimally maintained at 28 °C in Leibovitz-15 (L-15) medium supplemented with 20 % fetal bovine serum. The cell line was characterized by various cytogenetic and molecular markers. The cytogenetic analysis revealed a diploid count of 86 chromosomes at different passages. The origin of the cell lines was confirmed by the amplification of 547 and 654 bp sequences of 16S rRNA and cytochrome oxidase subunit I genes of mitochondrial DNA, respectively. WAF cells were characterized for their growth characteristics at different temperature and serum concentration. Epithelial morphology of the cell line was confirmed using immunocytochemistry. Further cell plating efficiency, transfection efficiency and viability of cryopreserved WAF cells was also determined. Cytotoxicity and genotoxicity assessment of cadmium salts on WAF cells by MTT, NR and comet assay illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The cell line will be further useful for studying oxidative stress markers against aquatic pollutants.

Schizothorax richardsonii, commonly called snowtrout, is an important indigenous coldwater fish of the Himalayas, India with high commercial values as food and game fish. A cell line named as SRCF was developed from the caudal fin tissue of S. richardsonii. The cell line has been maintained in Leibovitz's L-15 medium supplemented with 10% fetal bovine serum (FBS) at 24°C. The cells showed fibroblastic morphology, high plating efficiency and cell doubling time of 48h. Chromosomal analysis of SRCF cells revealed a diploid count of 98 chromosomes. The origin of the cell line was confirmed by the amplification of 655 and 578bp of cytochrome oxidase subunit I (COI) and 16S rRNA of mitochondrial DNA (mt-DNA) genes, respectively. Transfection of SRCF cells with pEGFP-C1 plasmid resulted in bright fluorescent signals, suggesting the application of cell line in transgenic and genetic improvement programme. In addition, genotoxicity assessment illustrated the utility of the cell line as an in vitro model for aquatic toxicological studies.

Current long term cryopreservation of cell stocks routinely requires the use of liquid nitrogen (LN2), because commonly used cryopreservation media containing cell membrane permeating cryoprotectants are thermally unstable when frozen at higher storage temperatures, e.g. −80 °C. This instability leads to ice recrystallization, causing progressive loss of cell viability over time under the storage conditions provided by most laboratory deep freezers. The dependency on LN2 for cell storage significantly increases operational expense and raises issues related to impaired working efficiency and safety. Here we demonstrate that addition of Ficoll 70 to cryoprotectant solutions significantly improves system thermal stability at the working temperature (~−80 °C) of laboratory deep freezers. Moreover, a medium comprised of Ficoll 70 and dimethyl sulfoxide (DMSO) in presence or absence of fetal bovine serum (FBS) can provide reliable cryopreservation of various kinds of human and porcine pluripotent stem cells at −80 °C for periods that extend up to at least one year, with the post-thaw viability, plating efficiency, and full retention of pluripotent phenotype comparable to that achieved with LN2 storage. These results illustrate the practicability of a promising long-term cryopreservation method that completely eliminates the need for LN2.

Liver transplantation, the only effective treatment for end-stage disease, is limited by donor availability. Cell transplantation offers the possibility to restore liver mass and function. Cryopreserved primary hepatocytes that can be thawed as needed might address this problem. Hepatocytes were harvested from a male Sprague Dawley rat, weighing approximately 250 g, using a 2-step in situ collagenase perfusion technique modified from the method described by Seglen. Hepatocytes were immobilized using a 100-mmol/L calcium with 1.5% alginate solution. Primary, immobilized hepatocytes transferred to various cryopreservation solutions containing 15% dimethyl sulfoxide were immediately placed into an isopropanol progressive freezing container at −80°C. We analyzed 4 cryopreservation solutions: Hormonally defined medium, histidine-tryptophan-ketoglutarate (HTK), fetal bovine serum (FBS), and HTK-modified cryopreservation solution (JH). After thawing, we measured viability, plating efficiency, urea synthesis, and albumin secretion to assess the effects of cryopreservation. Primary hepatocytes in HTK solution showed the better results in hepatocytes viability and urea synthesis after thawing. Cryopreserved immobilized hepatocytes in FBS maintained their viability and urea synthesis function. However, JH seemed to be the most effective medium for albumin secretion by both cyropreserved primary and immobilized hepatocytes.Our study suggested that HTK and JH cryopreservation solutions without FBS can be used to develop a bioartificial liver system.

Puntius (Tor) chelynoides, commonly known as dark mahseer, is a commercially important coldwater fish species which inhabits fast-flowing hill-streams of India and Nepal. Cell culture systems were developed from eye, fin, heart and swim bladder tissues of P. chelynoides using explant method. The cell culture system developed from eye has been maintained towards a continuous cell line designated as PCE. The cells were grown in 25cm(2) tissue culture flasks with Leibovitz' L-15 media supplemented with 20 % fetal bovine serum (FBS) at 24°C. The PCE cell line consists of predominantly fibroblast-like cells and showed high plating efficiency. The monolayer formed from the fin and heart explants were comprised of epithelial as well as fibroblast-like cells, a prominent and rhythmic heartbeat was also observed in heart explants. Monolayer formed from swim bladder explants showed the morphology of fibroblast-like cells. All the cells from different tissues are able to grow at an optimum temperature of 24°C and growth rate increased as the FBS concentration increased. The PCE cell line was characterized using amplification of mitochondrial cytochrome oxidase subunit I (COI) & 16S rRNA genes which confirmed that the cell line originated from P. chelynoides. Cytogenetic analysis of PCE cell line and cells from fin revealed a diploid count of 100 chromosomes. Upon transfection with pEGFP-C1 plasmid, bright fluorescent signals were observed, suggesting that this cell line can be used for transgenic and genetic manipulation studies. Further, genotoxicity assessment of PCE cells illustrated the utility of this cell line as an in vitro model for aquatic toxicological studies. The PCE cell line was successfully cryopreserved and revived at different passage levels. The cell line and culture systems are being maintained to develop continuous cell lines for further studies.

Proton beam therapy offers the advantage of precise delivery with limited damage to the healthy tissue and is being tested in the management of exudative age-related macular degeneration (AMD). However, the dosages tested are empirical and not based on preclinical studies.In this study we evaluated the effects of varying doses of proton beam radiation on choroidal endothelial cells (CECs) and retinal ganglion cells (RGCs) using clonogenic assay to determine differential sensitivity.Each cell type has different efficiency to replicate (plating efficiency (PE)). PE of CEC (RF/6A) and RGC (RGC-5) grown in culture flasks was determined by plating 250 cells each (without any treatment) and counting the number of colonies after 13 days. Radiation induced sensitivity was determined by exposing the semi-confluent RF/6A and RGC-5 cells to proton beam at the doses of 0 (control), 2, 4, 8 and 12 cobalt gray equivalent (CGE). The ability of the cells to repair and replicate to form colonies were analyzed 13 days after radiation with crystal violet stain and the survival ratio was calculated. The significance of survival was analyzed using ANOVA (Graphpad Instat.3).The PE of CEC and RGC was 12.96 ± 0.29% and 40.7 ± 1.48%, respectively. A survival ratio of CEC at 2, 4, 8 and 12 CGE proton radiation was 66.0 ± 8.6%, 44.3 ± 6.5%, 7.6 ± 0.3% and 1.14 ± 0.06% on exposure to 2, 4, 8 and 12 CGE proton radiation, respectively, p 0.01). Survival ratio of RGC was 71.1 ± 22.4% (p = 0.05), 40.2 ± 7.9%, 8.89 ± 2.6% and 0.78 ± 0.31% at 2, 4, 8 and 12 CGE dosages (p 0.001).CEC showed dose-dependent decrease in survival rate with values attaining significance at all radiation dosages. In contrast, RGC was comparatively radio resistant and were able to replicate at lower doses and sensitive at higher doses after proton beam radiation.Since CECs proliferate during neovascularization, this clonogenic assay is a useful assay to assess the sensitivity of CEC to radiation. This study identified that CEC were more sensitive to proton beam radiation than RGC at all doses. This may provide a therapeutic window for administration of proton beam radiation in the management of AMD.

Constitutive activation of Signal Transducers and Activators of Transcription 3 (STAT3) is frequently detected in osteosarcoma, and hence, may serve as a therapeutic target. In order to target STAT3, we tested two new STAT3 inhibitors, LLL12 and FLLL32. LLL12 and FLLL32 inhibit STAT3 phosphorylation and STAT3 downstream targets. LLL12 and FLLL32 also inhibit IL-6 induced STAT3 phosphorylation. The inhibition of STAT3 by LLL12 and FLLL32 resulted in the induction of apoptosis, reduction of plating efficiency, and migration in osteosarcoma cells. Furthermore, LLL12 and FLLL32 inhibited SJSA osteosarcoma cells and OS-33 tumor growth in murine xenografts. These results provide evidence that constitutive STAT3 signaling is required for osteosarcoma survival and migration in vitro and tumor growth in vivo. Blocking persistent STAT3 signaling by LLL12 and FLLL32 may be a novel therapeutic approach for osteosarcoma.

The establishment and partial characterization of a continuous cell line from the dorsal fin of red sea bream, Pagrus major, are described. The cell line, designated RSBF-2, has been subcultured for more than 100 passages since its initiation in November 2000. It was optimally maintained at 28 degrees C in Leibovitz L-15 medium with 10% foetal bovine serum. Propagation of RSBF-2 cells was serum dependent and exhibited low plating efficiency (

Retinoblastoma-like cells grew in colonies on the bottom of tissue culture flasks. The population doubling time was 19 h. Tumour cells from cell cultures had a 39% plating efficiency, and fresh tumour cells from intraocular tumours had a 32% plating efficiency in colony forming assays. Inoculation of 1.5 times 104 tumour cells in the vitreous of F-344 rats resulted in a 100% tumour take and regularly growing tumours with a doubling time of 3 days. The tumour take-rate was not changed in wholebody immunosuppressed animals. The tumour volume was assessed under a stereo microscope, and it was possible to divide the tumours into 4 groups according to the number of intraocular tumour cells. Tumour growth caused eye perforation in 89% of the inoculated eyes. Spontaneous tumour regression was not seen in non-perforation groups. Immunosuppression with whole-body irradiation and dense traumatic cataract had no significant effect on the growth. It is concluded that this animal retinoblastoma-like tumour is suitable for quantitative therapy studies in vivo and in vitro.

Object An understanding of single glioma cell invasion has been limited by the static picture provided by histological studies. The ability to nondestructively assess cell invasion dynamically in a full 3D volume would improve the quality and quantity of information available from both in vivo and in vitro experiments. The purpose of this study was to observe glioma cell invasion in a 3D in vitro model using a microimaging protocol at 1.5 tesla and to assess the uptake of micron-sized particles of iron oxide (MPIO) and the consequent effects on cell function. Methods Rat C6 glioma cells were labeled with MPIO to a sufficient extent to allow single cell detection in vitro without significant effects on cell proliferation or plating efficiency. When placed on agar-coated plates, the cells formed stable multicellular tumor spheroids (MCTSs), which were embedded in collagen type I gel and serially visualized using magnetic resonance (MR) imaging and phase-contrast microscopy over 8 days. The MCTSs initially appeared as large susceptibility artifacts on MR images, but within 2 days, as cells moved away from the main MCTS, small discrete areas of signal loss, possibly due to single cells, could be observed and tracked. Conclusions Glioma cell invasion can be nondestructively observed using MR imaging. The sensitivity of MR imaging, along with its ability to represent full 3D volumes noninvasively over time, makes it ideal for longitudinal in vivo cell tracking studies.

The relative sensitivities of different tumor cells to photodynamic therapy (PDT) with benzoporphyrin derivative monoacid ring A (BPD-MA) were compared in the four tumor cells. A good correlation was observed between the cell survival at 0.1 μg/ml of BPD-MA and sensitizer uptake/10 6 cells ( r = −0.99) or the plating efficiency of cells ( r = 0.99). At 3 h after the irradiation, a significant difference was observed in the proportion of apoptotic cells among the four tumor cells ( p = 0.024). In conclusion, cell responses to PDT depend on the several factors such as the cell line, photosensitizer dose, and fluence.

This lab reported previously that the plating efficiency of a herpes simplex virus type 1 ICP0-null mutant was enhanced upon release from an isoleucine block which synchronizes cells to G 1 phase (W. Cai and P. A. Schaffer, J. Virol. 65:4078-4090, 1991). Peak plating efficiency occurred as cells cycled out of G 1 and into S phase, suggesting that the enhanced plating efficiency was due to cellular activities present in late G 1 /early S phase. We have found, however, that the enhanced plating efficiency did not occur when cells were synchronized by alternative methods. We now report that the plating efficiency of ICP0 − viruses is not enhanced at a particular stage of the cell cycle but rather is enhanced by specific cellular stresses. Both the plating and replication efficiencies of ICP0 − viruses were enhanced as much as 25-fold to levels similar to that of wild-type virus when monolayers were heat shocked prior to infection. In addition to heat shock, UV-C irradiation but not cold shock of monolayers prior to infection resulted in enhanced plating efficiency. We further report that the effect of cellular stress is transient and that cell density rather than age of the monolayers is the primary determinant of ICP0 − virus plating efficiency. As both cell stress and ICP0 are required for efficient reactivation from latency, the identification of cellular activities that complement ICP0 − viruses may lead to the identification of cellular activities that are important for reactivation from neuronal latency.

The eel pathogen Vibrio vulnificus biotype 2 comprises at least three serovars, with serovar E being the only one involved in both epizootics of eel vibriosis and sporadic cases of human infections. The virulent strains of this serovar (VSE) have only been recovered from clinical (mainly eel tissue) sources. The main objective of this work was to design and validate a new protocol for VSE-specific isolation from environmental samples. The key element of the new protocol is the broth used for the first step (saline eel serum broth [SEB]), which contains eel serum as a nutritive and selective component. This approach takes advantage of the ability of VSE cells to grow in eel serum and thus to separate themselves from the pool of competitors. The growth yield in SEB after 8 h of incubation was 1,000 times higher for VSE strains than for their putative competitors (including biotype 1 strains of the species). The selective and differential agar Vibrio vulnificus medium (VVM) was selected from five selective media for the second step because it gave the highest plating efficiency not only for the VSE group but also for other V. vulnificus groups, including biotype 3. The entire protocol was validated by field studies, with alkaline peptone water plus VVM as a control. V. vulnificus was isolated by both protocols, but serovar E was only recovered by the new method described here. All selected serovar E isolates were identified as VSE since they were virulent for both eels and iron-overloaded mice and resisted the bactericidal action of eel and iron-overloaded human sera. In conclusion, this new protocol is a suitable method for the isolation of VSE strains from environmental samples and is recommended for epidemiological studies of the pathogenic serovar E.

Manganese superoxide dismutase (MnSOD) levels have been found to be low in human pancreatic cancer [Pancreas 26, (2003), 23] and human pancreatic cancer cell lines [Cancer Res. 63, (2003), 1297] when compared to normal human pancreas. We hypothesized that stable overexpression of pancreatic cancer cells with MnSOD cDNA would alter the malignant phenotype. MIA PaCa-2 cells were stably transfected with a pcDNA3 plasmid containing sense human MnSOD cDNA or containing no MnSOD insert by using the lipofectAMINE method. G418-resistant colonies were isolated, grown and maintained. Overexpression of MnSOD was confirmed in two selected clones with a 2-4-fold increase in MnSOD immunoreactive protein. Compared with the parental and neo control cells, the MnSOD-overexpressing clones had decreased growth rates, growth in soft agar and plating efficiency in vitro, while in vivo, the MnSOD-overexpressing clones had slower growth in nude mice. These results suggest that MnSOD may be a tumor suppressor gene in human pancreatic cancer.

Established cancer cell lines are useful in the study of various cancers. We established a human gastric carcinoma cell line TMC-1 derived from the lymph node of a moderately differentiated adenocarcinoma of the stomach. TMC-1 cells grew in vitro as a mixture of attached and suspension cells, and exhibited spindle or ovoid morphology. They had a population doubling time of 15 h, a plating efficiency of 61%, formed colonies in semisolid agar, secreted the tumor marker CA 19-9, and were tumorigenic in athymic nude mice. The cells expressed E-cadherin and β-catenin. The karyotypic analysis demonstrated hyperdiploid features with a modal chromosome of 53. The cell had the deletion at chromosome 18q and gains at chromosome 2p13–25, 5p15, 5q21–35, 7, 8q24, 9q, 11, 12p, 14q24–32 and 20. Analysis by fluorescence in situ hybridization showed the deletion at 7qtel and duplication at 7q11.2 at the rearranged chromosome 7. Growth of TMC-1 cells was inhibited by 27–32% by interferon-α (2,000 U/ml) and by interferon-γ with an IC50 of 125 U/ml. The cell line is tumorigenic in vivo, and its growth is moderately inhibited by interferon-α and interferon-γ. It can be used to develop new modalities of human gastric cancer treatment.

Development of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, requires a large amount of functional hepatocytes as needed. To achieve this development, establishing an excellent cryopreservation method of hepatocytes is an extremely important issue. Therefore, we performed a comparative review of cryoprotective effects of various cryopreservation solutions using primarily isolated porcine hepatocytes. Porcine hepatocytes were isolated with a four-step dispase and collagenase perfusion method. The obtained hepatocytes with the initial viabilities of 76%, 84%, and 96% were assigned to the following four groups for cryopreservation at −80°C: Dulbecco's modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) + 12% dimethyl sulfoxide (DMSO) (group A), University of Wisconsin (UW) solution + 12% DMSO (group B), Cell Banker 1 (group C), and Cell Banker 2 (group D). The hepatocytes in each group were thawed at 3 days, 10 days, and 5 months of cryopreservation and subjected to comparative analyses, including viability, plating efficiency, LDH release, ammonia removal test, and lentiviral gene transfer. These parameters were the most favorable in the hepatocytes cryopreserved with UW solution. Approximately 5% of thawed cryopreserved porcine hepatocytes expressed LacZ activity after lentiviral transduction. Intrasplenic transplantation of UW solution-cryopreserved hepatocytes improved the survival of rats treated with D-galactosamine. UW solution maintained the functions of cryopreserved porcine hepatocytes.

Normal human hepatocytes are an ideal source of liver-targeted cell therapies, such as hepatocyte transplantation and bioartificial livers, but availability of human donor livers for liver cell isolation is severely limited. To effectively utilize scarce donor organs for cell therapies, it is of extreme importance to establish an efficient isolation technique and an effective cold preservation solution for transportation of isolated cells. A lateral segment of the liver was surgically resected from pigs weighing 10 kg and a four-step collagenase and dispase digestion was conducted. Isolated hepatocytes were subjected to 8-h cold storage on ice. The following preservation solutions were tested: 1) University of Wisconsin (UW) solution, 2) UW with 100 μg/ml of ascorbic acid-2 glucoside (AA2G), 3) 100% fetal bovine serum (FBS), and 4) Dulbecco's modified Eagle's medium (DMEM) supplemented with 100% FBS. The mean viability of porcine hepatocytes was 95.5 ± 2.5% when isolated in three independent experiments. Viability, plating efficiency, membrane stability, and ammonia metabolic capacity of cold-preserved hepatocytes were significantly better maintained by the use of UW solution. When AA2G (100 μg/ml) was combined with UW solution, such parameters were further improved. It was explained by inhibition of caspase-3 activation and retention of ATP at high levels of hepatocytes preserved with UW solution containing AA2G. The present work demonstrates that a combination of UW solution with AA2G (100 μg/ml) would be a useful cold preservation means for the development of cell therapies.

Schwann cell (SC) transplantation is a promising strategy for axonal regeneration in the nervous system. Identifying the grafted SCs is an important aspect of this approach. The current study sought to establish a simple, reliable, fluorescent labeling method for SCs with a lipophilic molecule, 5-(and-6)-carboxyfluorescein diacetate succinimidyl ester (CFSE). Human SCs were incubated with varying concentrations of CFSE for different time periods. Based on the viability of labeled SCs and its plating efficiency, 1 min incubation with 5 microM CFSE at 37 degrees C was selected as the optimal labeling condition. Flow cytometric analysis and fluorescence microscopy demonstrated that the fluorescence of labeled SCs would fade over 4 weeks. Immunostaining for the phenotypic expression of SC markers, including S100, GFAP, P75, and MHC-I/II at 1 and 4 weeks after incubation with CFSE showed no difference between labeled and non-labeled SCs. Mixed cultures of labeled human SCs and rat SCs for 48 h were performed in triplicate and demonstrated that no leakage of dye from labeled SCs in cell culture occurred across species. A total of 14 injections of 2x10(5) labeled SCs were performed within the spinal cord at T8 and/or L1 level in 9 nude rats. The animals were euthanized at 1 (6 injections) and 4 weeks (8 injections). Grafted labeled SCs survived for at least 4 weeks, and could be easily recognized in the nude rat spinal cord without leakage of dye to surrounding cells. The SCs migrated in white and gray matter 3-6 mm away from the injection and in the central canal for up to 12 mm. These results suggest that CFSE can be used as a fluorescent tracer of human SCs for both in vitro and in vivo studies, for a period of at least 4 weeks.

OBJECTIVE: The aim of our study was to use (1H) nuclear magnetic resonance (NMR) spectroscopy as a tool to assess metabolic functions of hepatocytes and to monitor major metabolic pathways of these cells during culture following hypothermic preservation. METHODS: After isolation, 2 x 10(7) primary porcine hepatocytes were preserved at 4 degrees C in supplemented Leibovitz L-15 medium for 48 h. Viability was assessed at isolation, 24 and 48 h. At isolation and at 48 h cells were plated and cultured with serum free supplemented Williams E medium. 1H NMR spectroscopy was used to assess indices of glucose metabolism, ammonia clearance indices and ketone bodies precursors at 48 h post-plating. Peak integration was applied with sodium 3-(trimethylsilyl-2,2,3,3-2H4)-1-propionate as an internal standard to obtain quantitative results. RESULTS: Results were obtained from six isolations. Viability was 78.1 +/- 1.2% at isolation, 69 +/- 3.4% at 24 h and 58.9 +/- 3.8% at 48 h of hypothermia. Plating efficiency was 87 +/- 4% for freshly isolated cells and 33.6 +/- 7.6% for hypothermically preserved cells. Glucose consumption was comparable in both groups. Hypothermically preserved cells consumed more threonine, produced more pyruvate and alanine but less lactate. They also produced less acetate and consumed less tyrosine. Glutamate and glutamine concentrations were similar under both conditions. CONCLUSION: 1H NMR spectroscopy is a valid method for assessing metabolic pathways of cultured primary porcine hepatocytes. Although hypothermically preserved cells had a reduced plating efficiency, they were still metabolically active. Thus, hypothermia can be used as a temporary preservation technique for primary porcine hepatocytes.

Bioartificial liver support systems, which use freshly isolated primary hepatocytes (PH), present severe logistical difficulties. Stored frozen PH that are thawed as required could answer this problem. The aim of this study was to develop a cryopreservation protocol for large-scale preparation of porcine PH. We cryopreserved single and spheroid hepatocytes. Harvested hepatocytes were cryopreserved in various concentrations of dimethyl sulfoxide (DMSO) using hormonally defined medium (HDM) and various presentation solutions, such as University of Wisconsin (UW) and fetal bovine serum. After thawing the hepatocytes, we measured the viability, plating efficiency, ammonia removal, urea synthesis, and albumin secretion. UW solution was most effective for cryopreservation, as evidenced by the viability and liver-specific functions of the thawed hepatocytes. The optimal DMSO concentration for porcine hepatocyte cryopreservation was 15%. After cryopreservation, spheroid hepatocytes maintained greater viability and functional activity compared with single hepatocytes. Moreover, spheroid hepatocytes showed native cell structures and maintained high levels of liver-specific functions. In conclusion, cryopreserved spheroid hepatocytes were superior to cryopreserved single hepatocytes in terms of viability and liver-specific functions.

Hepatocyte transplantation is a potential alternative to whole organ liver transplantation. To realize this procedure, a hepatocyte bank system capable of supplying large numbers of hepatocytes must be established. The aim of this study was to develop a cryopreservation protocol using controlled rate freezers (CRF) for the application of a bioartificial system of porcine hepatocytes. Hepatocytes were harvested from 3- to 4-week-old male pigs weighing 11–14 kg. Liver cell preparations were prepared and the spheroid hepatocytes were cryopreserved using University of Wisconsin (UW) solution in controlled freezing. After thawing, viability, plating efficiency, urea synthesis, and ammonia removal were measured to assess the effects of freezing methods. Freezing methods had effects on the viability and specific functions of hepatocytes after thawing. About 80% of the cell viability could be obtained with an optimal computer programming method (−1°C slow cooling rate with shock cooling, using UW solution with 15% dimethyl sulfoxide [DMSO]). The cryopreservation method for hepatocytes was significantly improved by using the above cryopreservation conditions. However, research on application to large-scale cryopreservation is needed for practical use.

Since N-nitroso compounds as strong carcinogens are closely related to food and nutrition, the cytotoxic effects of N-nitrosomorpholine (NMOR) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) and their reduction by vitamins C and E were investigated in hamster V79 cells and human colon carcinoma Caco-2 cells. Cytotoxicity was evaluated by the trypan blue exclusion technique in Caco-2 cells and by the plating efficiency assay in V79 cells. NMOR caused a dose-dependent decline of viable cells in both cell lines; MNNG induced a dose-dependent cytotoxic effect only in V79 cells. Pretreatment of cells with vitamin C and vitamin E significantly reduced the cytotoxicity of NMOR, however, both vitamins had not effect on cytotoxicity induced by MNNG. These results suggest that different N-nitroso compounds react differently with cellular macromolecules. Measurement of the level of NMOR-induced DNA strand breaks and alkali-labile sites in both cell types using the alkaline comet assay also indicates a protective effect of both vitamins against the genotoxic effects of NMOR.

BACKGROUND Acute poisoning is a contraindication for organ and tissue donation. In this study the suitability of skin from a methanol-poisoned (MP) donor for future grafting and keratinocytes culturing was investigated. METHODS A patient was admitted with a methanol blood level of 2.7 mg/mL, which became undetectable after 4 days of treatment with 4-methylpyrazole (fomepizole). Upon declared brain death and family consent, organs and skin were harvested. For approving MP skin for grafting, the following parameters were studied: viability and plating efficiency of MP keratinocytes, integrity of MP skin after cryopreservation, and its performance as xenografts on wounds in a pig model. Nonpoisoned (NP) controls included skin of matching age, cryopreservation period, and NP keratinocytes. RESULTS No significant differences were observed for any parameter between NP and MP samples. Furthermore, in vitro exposure of NP keratinocytes and fibroblasts to

The ras proto-oncogene is frequently mutated in human tumors and functions to constitutively stimulate signal transduction cascades, resulting in unchecked proliferation and malignant transformation. In certain cells, superoxide functions as a signal-transduction messenger, mediating the downstream effects of ras and rac. We demonstrated previously that v-Ha-ras–transfected rat kidney epithelial cells (RECs) overproduced superoxide anion and that this superoxide production was mediated by ras. In the present study, we further demonstrated that v-Ha-ras overexpression transformed immortal nonmalignant RECs into malignant cancer cells; v-Ha-ras–transfected cells formed clones in soft agar, had high plating efficiency, and formed tumors in nude mice. Our data suggest that superoxide radical plays a role in ras-induced transformation; modulation of intracellular superoxide level by overexpression of manganese-containing superoxide dismutase or copper- and zinc-containing superoxide dismutase inhibited ras-induced transformation, as evidenced by in vitro studies of plating efficiency and by in vivo studies of tumor formation in nude mice. Overexpression of catalase (CAT) alone was found to have little effect on tumor cell growth, but overexpression of glutathione peroxidase 1 (GPx1) completely suppressed tumor cell growth in nude mice. This finding suggests that peroxides removed by GPx1, but not by CAT, are also involved in ras-induced transformation. 2002 Wiley-Liss, Inc.

The variability in embryonic stem (ES) cell culture is due primarily to serum. Serum is typically produced in large batches from many animals. However, samples may differ depending on the age and diet of the animals, the country of origin, and other factors creating lot-to-lot variations. Some vendors test FBS lots for compatibility with ES cell culture. Many laboratories prefer to test serum batches themselves to identify the lot giving optimal growth. In this protocol, small quantities of specific serum batches are obtained from different suppliers and tested for their ability to support ES cells in an undifferentiated state. A complete test includes the serum batches’ influence on plating efficiency, cell morphology, toxicity, and, if possible, their ability to support generation of chimeras.

We developed a method for culturing normal and malignant human bladder epithelial cells for many generations.Cells from bladder washes or surgical specimens were plated in culture with lethally irradiated cells of the LA7 rat mammary tumor line (American Type Culture Collection, Bethesda, Maryland) at a confluent density and fed regularly. After cells from surgical specimens became confluent they were diluted and subcultured with fresh irradiated LA7 cells. The growth rate was measured by cell counts and cell sorter analysis. Expression of intermediate filaments was determined by immunocytochemical testing.All 5 normal and 3 of the 4 tumor specimens developed into long-term cell strains. A single normal strain was carried through passage 11, amounting to 37 cell doublings. Cell numbers doubled in 2 days in medium with 0.5% serum and in 5.6 days in 5% serum. Plating efficiency was almost 100% and cloning efficiency was approximately 9%. LA7 conditioned medium did not stimulate bladder cell proliferation. Two tumor strains were carried through passage 9, amounting to 20 and 27 doublings, respectively. No cell strains expressed signs of senescence at culture termination. Normal and tumor strains expressed keratins 7, 10, 11, 18 and 19, and ZO1 tight junction protein but not vimentin or keratin 14. Umbrella cells comprised the uppermost cell layer in cultures from normal bladder.LA7 feeder cells stimulate human bladder cell proliferation for many generations in culture by a juxtacrine mechanism and promote the expression of differentiated traits.

The plating efficiency of neurons in culture is highly dependent on the concentration of cells used to establish the monolayer. A discontinuous iodixanol gradient permits both the production of a viable concentrated suspension of neurons and purification from other brain tissue elements. The gradient that is described in this Protocol Article is applicable to brain tissue from rat and also from human biopsy specimens.

There are only few primary endothelial cell cultures developed from fishes to date, but in this work the development of an endothelial cell line from Channa striatus is described. The vascular explants were plated into fibronectin (5 µg ml(-1)) and anti-CD31 antibody (100 ng ml(-1))-coated flask; after 60 h incubation explants were removed from the flask. The flask contained only endothelial and blood cells. Blood cells were cleared out after subsequent passages. The culture medium used was Leibovitz's L-15 supplemented with 20 % serum and antibiotics. The cultures were incubated at 28 °C in a normal atmosphere incubator. The plating efficiency was high (53.72 %). The endothelial cells were cryopreserved at different passage levels and revived successfully with 75-85 % survival. Polymerase chain reaction amplification of mitochondrial 16S rRNA using primer specific to C. striatus confirmed the origin of C. striatus cardiovascular endothelial (CSCVE) cell line from C. striatus. This cell line was further characterized for chromosome number, transfection, mycoplasma, cell cycle distribution, mitochondrial staining, and phagocytic activity. Cells were analyzed according to morphological appearance and expression of specific endothelial markers by fluorescent staining (von Willebrand Factor, anti-platelet endothelial cell adhesion molecule-1, and anti-Endoglin). The formation of tubules in the Matrigel and endothelial co-cultured with fibroblast like cells was observed. The cytotoxicity of ciprofloxacin on the CSCVE cell line was determined by MTT, AB, and R-123 cytotoxicity end points. Susceptibility of CSCVE cell line to nodavirus was confirmed by cytopathic effect and reverse transcriptase-polymerase chain reaction.

Reactive oxygen species have been shown to play important roles in v-Ha-Ras mitogenic signaling. We hypothesized that v-Ha-Ras overexpression would induce superoxide production, and therefore modify expression of the primary antioxidant enzyme system. We have demonstrated that immortal rat kidney epithelial cells stably transduced with constitutively active v-Ha-ras produced significantly larger amounts of superoxide radical than wild-type or vector-transfected control cells. The levels of the primary antioxidant enzymes copper- and zinc-containing superoxide dismutase, manganese-containing superoxide dismutase, catalase, and glutathione peroxidase were increased in the superoxide-overproducing cells. DNA-binding activities of the transcription factors activator protein-1, activator protein-2, and nuclear factor-kappaB were all enhanced in the superoxide-overproducing cells. These v-Ha-ras transduced cells also had a shortened cell doubling time and higher plating efficiency, and displayed greater constitutive levels of phosphorylated mitogen-activated protein kinases. These data demonstrate that v-Ha-Ras overexpression increases superoxide production and this apparently affects a wide variety of cell signaling and redox systems.

To explore the possibility to employ 99mTc-MIBI to monitor biological response of tumor cells after irradiation and to observe the relation between the radiation doses and the uptake levels of 99mTc-MIBI in tumor cells, the cells were irradiated with a single dose of 2 Gy, 10 Gy and 20 Gy respectively. The uptake of 99mTc-MIBI in each dosage group was determined before and 24, 48, 72 h after irradiation respectively. Apoptosis index (AI), plating efficiency (PE) of tumor cells was simultaneously determined. There was a positive correlation between uptake levels of 99mTc-MIBI and AI(r = -0.91, P0.05). A negative correlation was noted between the uptake levels and PE (r = -0.86, P0.05). It is suggested that 99mTc-MIBI may be used as a tracer to monitor the change of viability state of tumor cells after being irradiated with different doses.

Introduction Primary porcine hepatocytes are commonly, used in bioartificial liver devices and for in vitro studies of hepatocyte function. Although in vivo isolation of porcine hepatocytes can give high yield and viability, such methods are time-consuming and expensive, requiring specialist surgical facilities. Aim To develop a simple, low-cost, high viability, high yield, reproducible ex vivo method for obtaining functional porcine hepatocytes for use in bioartificial liver systems. Methods Weanling piglets (12 kg) were killed with pentobarbitone sodium, the infra-hepatic inferior vena cava was clamped and the supra-hepatic inferior vena cava cannulated. The whole liver was retrogradely perfused in situ with cold saline and excised, followed by an ex vivo open-loop and re-circulating perfusion method (at 37 degrees C) in five steps. The liver was disrupted, sequentially filtered in washing buffer, purified by centrifugation and resuspended in Williams E medium. Viability and cell number were assessed using trypan blue exclusion. The cells were subsequently cultured in serum-free chemically-defined medium and function was assessed. Results The time interval from when the animals were killed to the final cell wash was 105+/-5 min (n = 20). Cell viability was 85+/-6% with a yield of (2.4+/-0.5) x 10(10) from 12+/-1 kg piglets using 0.03% (w/v) collagenase (n = 20). Hepatocytes from all isolations were successfully plated and grown in monolayer culture. In freshly isolated hepatocytes (day 0) total protein content (TP) was 1.2+/-0.1 mg/10(6) cells (n = 5) and 1.2+/-0.3 mg/10(6) cells (n = 5) for day 2 monolayer cultures, corresponding to approximately 9x10(6) hepatocytes per dish. The percentage of total LDH released into the medium was 13+/-4% for day 0 and 8+/-4% at day 2; conversely, intracellular LDH activities were 87+/-4% and 92+/-4% of the total, respectively. The urea synthesis rate was 196+/-36 nmol/h/mg total protein at day 0 (n = 5) and 292+/-62 nmol/h/mg protein (n = 9) at day 2. The total P450 content was 99+/-11 pmol/mg total protein for fresh cells (n = 5) and maintained at 89+/-35 pmol/mg total protein in day 2 cultures. Conclusions This ex vivo method provides a high viability, high yield, cost-effective and rapid technique for isolating functional porcine hepatocytes with high plating efficiency, which compares favourably with results obtained using complex in vivo techniques.

Our laboratory has been routinely using suspended and cultured human hepatocytes for predicting drug metabolism and enzyme induction by drug candidates to aid drug discovery. Increasing limitation and irregular availability of human tissue has indicated the need for maximizing the use of this valuable resource. Cryopreservation of surplus hepatocytes after isolation would greatly increase the potential of this model. However, cryopreservation of hepatocytes by various methods has resulted in cells with poor metabolic activity and unacceptably low survival rates in culture. Recently, Zaleski et al. (Biochem. Pharmacol. 46 (1993) 111-116) reported that cryopreserved rat hepatocytes retained metabolic capacity similar to fresh hepatocytes when the cells were preincubated for 30 min at 37 degrees C in Krebs Ringer bicarbonate buffer prior to freezing. To further explore this methodology, both the functional capacity of the cells in culture as well as their ability to retain CYP inducibility were investigated with thawed cryopreserved hepatocytes. Although human hepatocytes were used in this study the initial work focused on rat hepatocytes as a cell model. Our results showed that while the preincubation step did not appear to effect the initial viability of cryopreserved hepatocytes, survival of the cells in culture was greatly enhanced. Plating efficiencies for nonpreincubated cryopreserved hepatocytes were decreased to approximately 15% of fresh cells after 48 h in culture. In contrast, cells that had been preincubated prior to freezing had an excellent plating efficiency (approximately 60%) and responded to classical CYP inducers dexamethasone, beta-naphthoflavone and phenobarbital in a manner indistinguishable from that of fresh hepatocytes. Experiments with human hepatocytes have also demonstrated similar results. This is the first time to our knowledge that cryopreserved hepatocytes from both rat and human have been shown to reproducibly respond to CYP inducers in culture.

In vitro changes of normal human keratinocytes (NHKs) derived from the oral mucosa after treatment with the chemical carcinogen 7,12 dimethylbenz[a]anthracene (DMBA; 5, 50, 200 ng/10 ml) were evaluated. NHKs were also treated with chemopreventive nutrient agents that previously had enhanced growth of epidermal and oral keratinocytes or suppressed growth of oral squamous cell carcinoma. These agents included the carotenoids beta-carotene and canthaxanthin and the retinoid retinyl palmitate (60 microM). Plating efficiency, growth in agarose (independent growth), viability [tetrazolium salt (MTT) assay], and proliferation ([3H]thymidine labeling) defined the growth of NHKs. The number of cornified cells and keratin expression (high-molecular-weight keratin) defined differentiation. gamma-Glutamyl transpeptidase, p53 expression, and tumorigenesis in mice defined oxidation and malignant transformation. Treatment with DMBA (50 ng/10 ml) was detected by autofluorescence; it produced an increase in pleomorphism and multinucleation and enhanced plating efficiency and the number of colonies grown in agarose. Chemopreventive treatment enhanced the number of colonies grown in agarose, but the MTT levels and [3H]thymidine incorporation-proliferation (24 h) were reduced. Chemopreventives also increased differentiation defined by the number of cornified cells and the expression of high-molecular-weight keratin-positive cells. Malignant transformation potential was depressed by reducing gamma-glutamyl transpeptidase and mutant p53 expression, whereas tumor suppressor p53 was enhanced. NHKs treated with DMBA and injected into nude mice (nu/nu: 1 x 10(6) cells/0.25 ml) produced tumor masses (3 of 3 animals), whereas the nutrient and DMBA groups produced smaller tumor masses, some with central ulcers (2 of 3 animals). Mock injection of untreated or nutrient-treated NHKs without DMBA treatment did not produce a tumor mass (0 of 3 animals). beta-Carotene, retinyl palmitate, and canthaxanthin increased differentiation and reduced transformation induced by DMBA in oral NHKs.

The purpose of the present study was to conduct a comparative evaluation of the effect of several serum-free culture conditions on adhesion, population doubling, cryopreservation and PDGF-induced effects on cell proliferation of bovine stromal fibroblasts (BSF). Additionally, these effects were compared to serum-containing cultures. Methods: Only second-passage BSF were used. Cells were cultured using four different culture media (WM/F12, WM/F12 + FCS 1 %, LR-1, DMEM). After 24 h, plating efficiency was determined using a cell-counter system. Subsequently, the cells were seeded at a density of 100 cells/mm2 and cultured for 10 days using the different culture media. Cell number was determined at day 2, 4, 7 and 10 after seeding. Furthermore, the effect of 50 ng/ml PDGF-BB on the proliferation of BSF was tested for these conditions. Cell vitality was determined after cryopreservation of two weeks for each culture medium. Results: The plating efficiency of BSF ranged from 50.2 to 55.5 % for the serum-free culture media in contrast to serum-containing conditions, where plating efficiency was 94.8 %. With WM/F12 + FCS 1 %, a population doubling of 1.27 was observed after an incubation period of 10 days. In contrast, cultivation under serum-free conditions caused neither significant cell proliferation nor cell loss. The stimulation of cell proliferation with PDGF-BB was shown to be 28 % (LR1), 40 % (WM/F12 + FCS 1 %), 76 % (WM/F12) and 95 % (DMEM) compared to the control. While cell vitality after cryopreservation was found to be 62.7 % using WM/F12 + FCS 1 %, cell vitality using serum-free media was 12.6–22.8 %. Conclusions: The results of the present study demonstrate that with respect to optimal cell adhesion and cell vitality after cryopreservation, serum-containing media should be used. BSF cultured under the serum-free conditions used in the present study can be maintained quiescent and vital for at least 10 days. Therefore, these serum-free media are useful for cell-culture studies (e. g., determination of proliferation and cytotoxicity).

Animal models of fungal and other infectious diseases often require that the number of organisms in tissue be quantified, traditionally by grinding organs, plating them on agar and counting colony forming units (CFU). This method is labor intensive, slow as some fungi require two weeks of culture and limited in reliability by poor plating efficiency. To circumvent these problems, we developed a flow cytometric method to quantify yeast. In vitro cultured Blastomyces dermatitidis , Cryptococcus neoformans , Candida albicans and Histoplasma capsulatum yeast were labelled with specific monoclonal or polyclonal antibodies to stain surface determinants or with Calcofluor to stain cell-wall chitin. A defined number of fluorescently labelled beads were added prior to acquisition by flow cytometry as a reference standard for quantitation. Beads were readily distinguished from yeast by forward scatter, side scatter and intensity of fluorescence. Cultured yeast were enumerated by both standard CFU determination and flow cytometry in a range of 10 2 to 10 7 cells. Only flow cytometry enabled discrimination of live and dead yeast by using appropriate fluorescent dyes. The flow cytometric method was applied to murine models of histoplasmosis and blastomycosis to quantify the burden of fungi in the lungs of infected mice. Labelling yeast with Calcofluor alone resulted in unacceptably high levels of nonspecific binding to mouse cell debris. In contrast, labelling H. capsulatum with a rabbit polyclonal antiserum and B. dermatitidis with a monoclonal antibody to the surface protein WI-1 permitted accurate quantitation. We conclude that this flow cytometry technique is rapid, efficient and reliable for quantifying the burden of infection in animal models of fungal disease. The technique also should lend itself to performing cytotoxicity assays that require discrimination of live and dead fungi, or phagocytosis assays that require discrimination of intracellular and extracellular organisms.

To investigate the prolonged reduction in cellular viability (lethal mutations) of surviving cells following irradiation of tumours in vivo and to test the effects of fractionation on the expression of lethal mutations.A mouse mammary carcinoma (CaNT) was treated with single dose or fractionated X-ray treatments in vivo and survival quantified with an in vitro excision assay soon after irradiation and at various times up to 35 days after in vitro propagation of the surviving cells.A dose-dependent reduction in the plating efficiency was observed in cells isolated from irradiated tumours up to 35 days and many cell generations after irradiation. Considerable heterogeneity in plating efficiency was observed in clonal cell lines isolated from individual colonies grown from irradiated tumours. Delayed expression of lethal damage was observed after fractionated irradiation, although recovery of cellular fitness was greater than after irradiation with single doses (reported previously) suggesting that this form of damage is affected by inter-fraction repair. At equi-toxic doses, delayed expression of lethal damage was similar after three compared with two fractions of radiation per day (reported previously).These effects indicate that conventional excision assays of tumour cell viability under-estimate the total lethal damage caused by irradiation and have implications for modelling of the response of tumours to radiotherapy. The effect of fractionation on expression of this type of damage implies the involvement of repair processes. Therefore the repair proficiency may affect the balance between the immediate and delayed reduction of viability in irradiated cells.

Neural progenitor cell populations responsive to epidermal growth factor (EGF) have been shown to have proliferative potential and give rise to neurons, astrocytes, and oligodendrocytes. We have characterized EGF-responsive neural progenitor cells that give rise to bilineage neuronal/glial colonies (colony-forming unit neuron–glia; CFU-NeGl) and unilineage neuronal colonies (CFU-Ne). Clonality was confirmed utilizing mixtures of brain cells from Balb/c and ROSA26 (transgenic for β-galactosidase) mice. With a few exceptions, colonies showed either all blue cells or all clear cells after staining with X-Gal. Clonal growth was analyzed after 10–11 days in relation to cell density by determining colony size and plating efficiency. Growth was density dependent (no growth below 10,000 cell/ml) and thus single cell cloning was not accomplished. An average plating efficiency of 4% was found for EGF-responsive neural cells derived from day 15–18 murine embryos when cultured at 12,500 to 200,000 cells/ml. Similar results were obtained with 1-day-old postnatal neural cells. When colonies were categorized by size, the relative number of colonies over 50 cells appeared to be maximum at 50,000 plated cells/ml. After 11 days in culture, 94, 96, and 78% of the colonies contained cells that expressed nestin, neurofilament, and GFAP, respectively. Double-label experiments revealed that >62% of the colonies contained both GFAP and neurofilament expressing cells. These studies establish the existence of at least two populations of clonal neural progenitors: CFU-Ne and CFU-NeGl in fetal and postnatal murine brain.

The carcinogen 7,12-dimethylbenz(a)anthracene (DMBA) has been used to induce oral carcinogenesis of the hamster buccal mucosa in an experimental model that exhibits many of the genetic, biochemical and pathological features of human oral squamous cell carcinoma. To complement this in vivo process we have established an in vitro transformation procedure that involved the treatment of normal hamster oral mucosal keratinocytes (NHKs) with DMBA. Uptake of DMBA in NHKs was verified by observing autofluorescence of DMBA in the oral mucosal cells. Treatment doses ranged from 5, 50 and 200 ng and the NHKs were generally treated with DMBA for 1-14 days. The 200 ng dose proved to be toxic to these cells. The 5 and 50 ng treatments were found to maintain the viability of the NHKs and demonstrate anchorage-independent agarose growth, producing 18 and 40 colonies, respectively, after 14 days of treatment. Characterisation assays included determinations for cellular growth through plating efficiency, counting of cell colony number, and 3H-thymidine incorporation. Differentiation was ascertained by counting cornified cells, specification of either high or low molecular weight keratins, the percentage of cells expressing gamma glutamyl-transpeptidase (GGT), the level of p53 expression, and a determination of cell cycle. After 24 h the plating efficiency of the NHKs was found to be slightly increased following treatment with a 5 or 50 ng dose of DMBA compared to the untreated NHKs. After 14 days of incubation these doses also enhanced the number of colonies formed by the NHKs (e.g. plating efficiency). In contrast, the number of cornified cells was reduced in these colonies, while immunohistochemistry disclosed an increase in the number of NHKs expressing low molecular weight keratins, significantly lower levels of high molecular weight keratins and high levels for GGT. Flow cytometric analysis verified an increase in p53 expression (e.g. p53 wild type, 19% and p53 mutant, 66%). Cell cycle analysis of NHKs treated with DMBA (5 ng) demonstrated a shift in the number of cells in S phase (17.2%) and G2 + mitosis (11.0%). Cells from this DMBA treatment group were injected into syngeneic hamster recipient buccal pouches (10 x 10(6) cells/0.25 ml). Squamous carcinomas grew in four of six hamster buccal pouches as determined by histopathological analysis. The in vitro assay system will enhance our ability to define the genetic and molecular changes related to chemical carcinogenesis and oral malignant transformation.

Overexpression of human manganese-containing superoxide dismutase (MnSOD) activity has been demonstrated to suppress malignancy in human melanoma and breast carcinoma cells in vitro and in vivo. To study its effects on human oral squamous carcinoma cells, stable transfection and expression of MnSOD in SCC-25 cells have been conducted. The MnSOD-overexpressing cell clones were shown to have approximately two- to five-fold increased MnSOD activity compared to the wild-type parental- or vector control-transfected cell clones, respectively. Plating efficiency with different concentrations of serum was decreased in the high MnSOD activity cell clones. Soft agar assays demonstrated that the clonogenic fractions of high-expressing MnSOD clones were dramatically reduced. When inoculated in nude mice, tumor growth was markedly inhibited in MnSOD overexpressing cell clones compared with the wild-type or vector control transfected cell lines. Thus, gene therapy of human oral cancer by increasing the expression of MnSOD activity in target cells might be used to prevent or reduce human oral tumor malignancy.

Fas/APO-1(CD-95) activation induced rapid apoptotic cell death of primary rat hepatocytes in suspension culture. Activators of cAMP-dependent protein kinase (glucagon and N6-benzoyl-cAMP) protected against apoptosis, whereas the specific cAMP-kinase inhibitor (Rp)-8-Br-cAMPS enhanced Fas-induced death. The latter observation indicated that even the basal cAMP level may provide partial protection against Fas-induced hepatocyte apoptosis. Two-dimensional gel electrophoresis revealed decreased phosphorylation of several proteins in Fas-activated cells. Most of these dephosphorylations were attenuated or not observed in cells simultaneously stimulated by anti-Fas and cAMP, indicating a tight correlation between the dephosphorylations and death. Elevation of cAMP rescued the cells not only from the Fas-induced morphological changes and dephosphorylation, but also from functional deterioration. Whereas cells treated with anti-Fas alone quickly lost plating efficiency, hepatocytes co-treated with glucagon retained their ability to adhere and spread on a collagen substratum.

We evaluated genotoxic and cytotoxic effects of the three non-mutagenic and non-carcinogenic compounds p-nitrophenol, D-menthol and sodium N-lauroyl sarcosine which have previously been shown to induce DNA double strand breaks (DNA dsb) secondary to induced cytotoxicity. We tested whether genotoxic effects in the alkaline single cell gel test (comet assay) may be confounded by cytotoxicity-induced DNA dsb. Cell viability was determined at the end of the treatment using the fluorescein diacetate/ethidium bromide-assay and plating efficiency was used as an indicator of long-term survivability. Experiments with V79 Chinese hamster cells and human white blood cells revealed negative results in the comet assay despite strong cytotoxic effects. However, cells with extremely fragmented DNA ('clouds') occurred but were excluded from the evaluation under the principle that they represent dead cells. We also noticed a significant loss of cells at cytotoxic concentrations that might be attributed to the induction of highly fragmented DNA which is lost during electrophoresis. Since the comet assay allows the determination of DNA effects on the single cell level, a confounding effect of cytotoxicity on test results can be avoided.

The effect of different passage numbers on plating efficiency, doubling time, cell growth, and radiation sensitivity was assessed in Syrian hamster embryo (SHE) cells. Changes in gene expression after UV or gamma-ray irradiation at different passage numbers were also examined. The SHE cells were maintained in culture medium for up to 64 passages. Cells were exposed to 60Co gamma rays or 254-nm UV radiation. Differential display of cDNAs and Northern blots were used for the study of gene expression. With increasing passage number, SHE cells demonstrated decreased doubling time, increased plating efficiency, and a decreased yield in the number of cells per plate. Between passages 41 and 48 a 'crisis' period was evident during which time cell growth in high serum (20%) was no longer optimal, and serum concentrations were reduced (to 10%) to maintain cell growth. Sensitivity to ionizing radiation was no different between early- and intermediate-passage cells. However, after UV exposure at low passages (passage 3), confluent cells were more sensitive to the killing effects of UV than were log-phase cells. At intermediate passages (passages 43, 48), confluent cells were slightly more radioresistant than were log-phase cells. By passage 64, however, both confluent and log-phase cells showed similar patterns of UV sensitivity. Expression of gamma-actin, PCNA, and p53 transcripts did not change following UV exposure. p53 mRNA was induced following gamma-ray exposure of the intermediate (passage 45) epithelial cells. The observed differences in radiation sensitivity associated with increasing passage number may be influenced by radiation-induced gene expression. We are conducting experiments to identify these genes.

A new cell line, Channa striatus gill (CSG), derived from the gill tissue of murrel, was established and characterized. The CSG cell line was maintained in Leibovitz's L-15 supplemented with 10% fetal bovine serum and has been subcultured more than 92 times. This cell line was able to grow in a range of temperatures from 22 to 32°C with optimal growth at 28°C. The plating efficiency was very high (52.21%) and doubling time was approximately 37h. The gill cell line was cryopreserved at different passage levels and revived successfully with 85% survival. Polymerase chain reaction amplification of mitochondrial 16S rRNA using primer specific to C. striatus confirmed the origin of this cell line from murrel. The cell line was further characterized by immunocytochemical analysis, chromosome number, transfection and mycoplasma detection. The cytotoxicity of endosulfan was assessed in CSG cell line using apoptosis assay, comet assay, mitochondrial alteration and five other endpoints such as Rhodamine 123 uptake, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, neutral red assay, Alamar Blue assay and Methylene Blue protein assay. Acute toxicity study on fish was conducted by exposing murrel for 96h to endosulfan under static conditions. Statistical analysis revealed good correlation with r(2)=0.972-0.997 among the five endpoints. Linear correlations between the in vivo lethal concentration 50 (LC50) and each in vitro effective concentration 50 (EC50) were highly significant. The present study highlights the development of a new gill cell line from an air breathing fish that could be used as an alternative in vitro tools for studying pesticide toxicity in fish.