First steps of an interdisciplinary approach towards miniaturised cryopreservation for cellular nanobiotechnology.

The only widely used and accepted method for long-term cell preservation is storage below -130 degrees C. The biosciences will make increasing use of preservation and place new demands on it. Currently, cells are frozen in volumes greater than 1 ml but the new cell and implantation therapies (particularly those using stem cells) will require accurately defined freezing and storage conditions for each single cell. Broadly-based, routine freezing of biological samples allows the advantage of retrospective analysis and the possibility of saving genetic rights. For such applications, one billion is a modest estimation for the number of samples. Current cryotechniques cannot handle so many samples in an efficient and economic way, and the need for new cryotechnology is evident. The interdisciplinary approach presented here should lead to a new sample storage and operating strategy that fulfils the needs mentioned above. Fundamental principles of this new kind of smart sample storage are: (i) miniaturisation; (ii) modularisation; (iii) informationsample integration, i.e. freezing memory chips with samples; and (iv) physical and logical access to samples and information without thawing the samples. In contrast to current sample systems, the prototyped family of intelligent cryosubstrates allows the recovery of single wells (parts) of the substrate without thawing the rest of the sample. The development of intelligent cryosubstrates is linked to developments in high throughput freezing, high packing density storage and minimisation of cytotoxic protective agents.

Up-regulation of multidrug resistance P-glycoprotein via nuclear factor-kappaB activation protects kidney proximal tubule cells from cadmium- and reactive oxygen species-induced apoptosis.

Cadmium-mediated toxicity of cultured proximal tubule (PT) cells is associated with increased production of reactive oxygen species (ROS) and apoptosis. We found that cadmium-dependent apoptosis (Hoechst 33342 and annexin V assays) decreased with prolonged CdCl(2) (10 microM) application (controls: 2.4 +/- 1.6%; 5 h: +5.1 +/- 2.3%, 20 h: +5.7 +/- 2.5%, 48 h: +3.3 +/- 1.0% and 72 h: +2.1 +/- 0.4% above controls), while cell proliferation was not affected. Reduction of apoptosis correlated with a time-dependent up-regulation of the drug efflux pump multidrug resistance P-glycoprotein (mdr1) in cadmium-treated cells ( approximately 4-fold after 72 h), as determined by immunoblotting with the monoclonal antibody C219 and measurement of intracellular accumulation of the fluorescent probe calcein +/- the mdr1 inhibitor PSC833 (0.5 microM). When mdr1 inhibitors (PSC833, cyclosporine A, verapamil) were transiently added to cells with mdr1 up-regulation by pretreatment for 72 h with cadmium, cadmium-induced apoptosis increased significantly and to a percentage similar to that obtained in cells with no mdr1 up-regulation (72-h cadmium: 5.2 +/- 0.9% versus 72-h cadmium + 1-h PSC833: 7.2 +/- 1.4%; p < or = 0.001). Cadmium-induced apoptosis and mdr1 up-regulation depended on ROS, since co-incubation with the ROS scavengers N-acetylcysteine (15 mM) or pyrrolidine dithiocarbamate (0.1 mM) abolished both responses. Moreover, cadmium- and ROS-associated mdr1 up-regulation was linked to activation of the transcription factor NF-kappaB; N-acetylcysteine, pyrrolidine dithiocarbamate, and the IkappaB-alpha kinase inhibitor Bay 11-7082 (20 microM) prevented both, mdr1 overexpression and degradation of the inhibitory NF-kappaB subunit, IkappaB-alpha, induced by cadmium. The data show that 1) cadmium-mediated apoptosis in PT cells is associated with ROS production, 2) ROS increase mdr1 expression by a process involving NF-kappaB activation, and 3) mdr1 overexpression protects PT cells against cadmium-mediated apoptosis. These data suggest that mdr1 up-regulation, at least in part, provides anti-apoptotic protection for PT cells against cadmium-mediated stress.

Cell surface and nuclear changes during TNF-alpha-induced apoptosis in WEHI 164 murine fibrosarcoma cells. A correlative light, scanning, and transmission electron microscopical study.

Tumour necrosis factor (TNF)-alpha-induced apoptosis is associated with several nuclear and cell surface alterations, in particular with the condensation of chromatin and the fragmentation of the cell nucleus, formation of blebs on the cell surface and breakdown of the plasma membrane. However, there is little information about the relationship between the cell surface alterations and the nuclear changes during apoptosis. To study this, cultured WEHI cells were exposed to TNF-alpha over different time periods. The cytological changes were studied using a correlative approach, which allowed observation of the same cell consecutively under light, scanning and transmission electron microscopy. The earliest sign of cell alteration was a reduction of the number of microvilli after 15 min of TNF-alpha exposure. This reaction was reversible (reappearance of microvilli) and took place during the first hour, in which neither nuclear alterations nor plasma membrane breakdown were observed. The changes in the nucleus began with condensation of chromatin after approximately 1 h of TNF-alpha-exposure. After 4-5 h the microvilli disappeared again, particularly in areas where the formation of blebs (blebbing) was observed. Strikingly, cell surface alterations (bleb formation) were detected only in those cells that presented with condensed chromatin, and not in cells with a normal chromatin pattern, proving at least a close correlation between nuclear and cell surface changes during the process of apoptosis.

Lymph vessel expansion and function in the development of hepatic fibrosis and cirrhosis.

The process of lymph vessel expansion and function in the development of CCl4-induced hepatic fibrosis and cirrhosis was studied using intravital fluorescence microscopy of the rat liver. The unique aspect of our approach was the use of high molecular fluorescein-isothiocyanate-labeled dextran (MW, 150,000) as fluorescent marker, which allowed for simultaneous assessment of both 1) the macromolecular blood hepatocytic exchange from the sinusoidal microvasculature (extra-/intrasinusoidal gray level intensity at 1, 3, 5, and 10 minutes after intravenous injection) and 2) the hepatic lymph system. In animals exposed with CCl4 up to 4 weeks, macromolecular trans-sinusoidal exchange was found progressively delayed. This was strongly associated with lymph vessel expansion and function, as indicated by a continuous increase of lymph vessel density and area. Delay of macromolecular exchange and lymph vessel expansion was found not further enhanced at fibrotic and cirrhotic stages of 8- and 12-week CCl4-exposed livers. Linear regression analysis revealed a strong negative correlation between lymphatic network density development and macromolecular trans-sinusoidal exchange (r2 = 0.99; P < 0.01). Thus, our study provides for the first time direct evidence for the pivotal role of lymphatic function for macromolecular transport in case of deteriorated sinusoidal hepatocellular exchange capacity.

[A method of phase-contrast, fluorescence and scanning electron microscopy of one and the same cultured cells undergoing cryogenic action]. / Metod fazovo-kontrastnoi, fluorestsentnoi i rastrovoi élektronnoi mikroskopii odnikh i tekh zhe kul'tiviruemykh kletok, podvergnutykh kriovozdeistviiu.

Cells cultured on glass substrates have been investigated by phase contrast method. After freeze-thawing the same cells were stained for identification of damage by fluorescent dyes and studied by the phase contrast method, fluorescence and scanning electron microscopy. The method has revealed correlations between the damage of cell surface and the cell damage manifestation at morphological level.

[A method for the chemical treatment of cells for the purpose of the subsequent study of the same cell culture preparation by light, scanning and transmission electron microscopy]. / Metod khimicheskoi obrabotki kletok s tsel'iu posledovatel'nogo izucheniia odnogo i togo zhe preparata kletochnoi kul'tury s pomoshch'iu svetovoi, rastrovoi i transmissionnoi élektronnoi mikroskopii.

Cells cultured on transparent conductive substrates (glass coated with indium oxide) were fixed with aldehyde and osmium tetroxide and then treated with tannic acid, uranyl acetate and lead citrate. The same cell culture preparation could be sequentially studied by light microscopy (in water immersed condition), SEM (after dehydration and critical point drying) and TEM (after embedding in an epoxy resin). This method ensures the preservation of intact cell morphology, cell surface topography and intracellular structures. The treatments used render the cells conductivity and permit to carry out successfully SEM of uncoated cells cultured on conductive substrates. This method also provides a higher contrast of TEM images.

[The capacity of the cells of a human skin melanoma to be preserved long-term in an intact state in vitro]. / Sposobnost' kletok melanomy kozhi cheloveka dlitel'no sokhraniat'sia v nepovrezhdennom sostoianii in vitro.

Suspension of primary and metastatic skin melanoma cells isolated by the mechanical and enzymic methods was preserved in ampullae in the medium 199 at 4 degrees C. Native cells and cell preparations stained with trypan-blue solution and studied by the scanning electron microscopy method for 12, 20, 36 months have demonstrated the ability to keep their vitality for 3 years. Mechanism of such a "natural" conservation of cells is not yet clear. It may be assumed that the above cells are genetically stimulated mutants able under definite conditions to initiate an active proliferation in healthy tissues. The cells in a similar state may be a promising model in the experimental oncology.

[Cell surface in the contact interaction of normal masto- and lymphocytes and those subjected to freezing]. / Issledovanie poverkhnosti kletok pri kontaktnom vzaimodeistvii normal'nykh i podvergshikhsia zamorazhivaniiu masto- i limfotsitov.

On studying the surface of intact and cryogenic masto- and lymphocytes by the scanning electron microscopy the formation of close membrane contacts was detected between thymocytes and mast cells looking in vitro as mastolymphocyte rosettes (ML-rosettes). During ML-rosette formation changes in cell surface organization were observed. Availability of viable thymocytes is necessary for ML-rosette formation. The surface topography of cells constituting the ML-rosettes is seen changing during rosette formation. It is proposed that the mastocyte may function as an acceptor acquiring immunological specificity through the contact with lymphocytes whose membrane responds actively towards the antigen by changing its surface. Reorganization of the lymphocyte plasma membrane may be perceived by mastocytes as a signal of granule secretion.