Cryopreservation, hepatocyte monolayers and DMSO shock

Cryopreservation of primary hepatocyte monolayers may provide a means of long-term storage of the cells for in vitro studies of xenobiotic metabolism and toxicity. This talk focuses on cryopreservation, hepatocyte monolayers and DMSO shock

Cryopreserved hepatocyte monolayers

Successful cryopreservation of hepatocytes would be useful to the pharmaceutical industry and for bioartificial liver support systems. Unfortunately, cryopreservation of hepatocytes in suspension results in low attachment efficiency in culture upon thawing. To circumvent this problem, we have frozen rat hepatocytes as monolayers on collagen substrates. Hepatocytes prepared from male Sprague Dawley rats by collagenase perfusion were cultured (3*106 cells/60mm Petri dish) on collagen films (30mg/cm2) in 2ml Chee's medium containing 5% v/v foetal calf serum (FCS). 24h cultures were frozen in Chee's medium with 10% Dimethyl Sulphoxide and between 0-90% FCS at -708C for 24h

Cryopreservation of rat hepatocyte monolayers: cell viability and cytochrome P450 content in post-thaw cultures

Cryopreservation of primary hepatocyte monolayers may provide a means of long-term storage of the cells for in vitro studies of xenobiotic metabolism and toxicity. Rat hepatocytes can be stored at −70 °C as simple monolayers attached to collagen-coated dishes, and post-thaw cultures can be continued for up to 72 h. Throughout this post-thaw period viability of the cells was demonstrated by retention of intracellular fluorescence after exposure to carboxyfluorescein diacetate (CFDA) and examination by confocal laser scanning microscopy (CLSM). CLSM images revealed an uneven distribution of CFDA-derived fluorescence within hepatocytes post-thaw, particularly in Williams' E medium, indicating generation and retention of carboxyfluorescein within the intracellular organelles. The membranes of the intracellular organelles appear to be less sensitive to freeze/thaw damage than the cell membrane. Viability was not compromised with storage for up to 28 days at −70 °C. Cytochrome P450 content was retained in post-thaw culture to a similar extent as in non-frozen cultures. Cryopreserved rat hepatocyte monolayers may provide a useful in vitro model for studying xenobiotic metabolism and toxicity

Growth and viability of osteoblasts on hydroxyapatite and tri-calcium phosphate ceramics: the effect of collagen coating

Summary of paper describing how Hydroxyapatite (HA) and tricalcium phosphate (TCP) ceramics form the basis for bioartificial bone replacement materials. They lack mechanical strength and cannot at present be considered as replacements for loadbearing areas. We have investigated the characteristics of two ceramic materials, HA and a 60:40 composite of HA:TCP, which are being developed for facial bone reconstruction. The growth and viability of an immortalized cell line, FFC, derived from neonatal rat calvaria, was investigated when cultured on the materials for 7 days

Metabolism and distribution of phenanthridine trypanocides in trypanosome Brucei brucei

Phenanthridine trypanocides (isometamidium chloride hydrochloride, ISM, and Ethidium bromide, EBr) have been widely used to treat African trypanosomiasis in livestock for more than 40 years. Their main action is to inhibit nucleic acid synthesis in trypanosome parasites, by intercalation between the DNA base pairs. They can also linearise selectively kinetoplast DNA minicircles; a form of mitochondrial DNA unique to this group of parasites. However, the metabolism of these compounds by trypanosomes has not been reported. Indeed, it is not known whether or not their metabolism by the parasite contributes to their activity, selective toxicity for these parasites or to the development of chemoresistance. Therefore, we studied the metabolism of EBr and ISM, and their distribution in Trypanosoma brucei (TREU 927) using high performance liquid chromatography (HPLC), liquid chromatography combined with mass spectrometry (LC-MS) and confocal laser scanning microscopy (CLSM). Incubation of EBr with trypanosomes led to the formation of a small amount (0.606±0.191%) of one metabolite (MI). Ion chromatograms extracted from an LC-MS analysis using electrospray ionisation (ESI), showed that the difference in mass between the parent compound and its metabolite was 30. This may correspond to the addition of a hydroxyl and a methyl group. No metabolites could be detected for ISM. The distribution of the two drugs in trypanosomes was investigated by CLSM, using their intrinsic fluorescence. ISM and EBr showed differences in their distribution in trypanosomes. ISM had a greater affinity for the kinetoplast than EBr and it stained other organelles like the flagellum; in contrast the distribution of EBr was more diffuse