Imaging the division process in living tissue culture cells

We detail some of the pitfalls encountered when following live cultured somatic cells by light microscopy during mitosis. Principle difficulties in this methodology arise from the necessity to compromise between maintaining the health of the cell while achieving the appropriate temporal and spatial resolutions required for the study. Although the quality of the data collected from fixed cells is restricted only by the quality of the imaging system and the optical properties of the specimen, the major limiting factor when viewing live cells is radiation damage induced during illumination. We discuss practical considerations for minimizing this damage, and for maintaining the general health of the cell, while it is being followed by multi-mode or multi-dimensional light microscopy.     

NMR-based metabolic profiling of human hepatoma cells in relation to cell growth by culture media analysis

Metabolic profiling is a metabolomic approach that allows the characterization of metabolic phenotypes under specific set of conditions. In the present paper we investigated the metabolism of sparse and high density cultures in relation to different cell growth phases. Changes in the metabolome were evaluated by using 1H-NMR spectroscopy, correlation map and Multivariate Data Analysis on the net balances of metabolites in the medium. This approach allowed us to identify two different metabolic profiles in relation to the cell growth phases in subconfluence and confluence cultures. The results have been interpreted on the basis of patterns of correlations obtained in the two physiological cell states. Cells almost arrested in G0/G1 phase by contact dependent growth inhibition underwent changes in the channeling of amino acids utilization from synthetic to energetic purpose and in anaplerosis/cataplerosis regulation of the TCA cycle.     

Role of cell division in the cytodifferentiation of rat heart cells in culture

BrdU and irradiation with visible light eliminates dividing cells from rat heart cell cultures. The lethal effect of light on the cultures is dependent upon the prior integration of BrdU into the DNA. Elimination of dividing cells is shown by the decreased uptake of ³H TdR in the remaining cells. Cultures in which the dividing cells were eliminated displayed a loss in myosin ATPase activity and a decrease in the rate of myosin ATPase activity increase, normally seen in control cultures. These results are consistent with the existence of cells at three stages of development; premyoblasts, which divide and contain no myosin; myoblasts which divide and contain myosin; and myocytes, which cannot divide and contain myosin. The results also indicate that the increase in myosin ATPase activity normally seen in heart cell cultures is a result of an increase in myosin in fully developed cells and in the increase in the number of cells capable of synthesizing myosin.     

Proteomic profiling of recombinant cells from large-scale mammalian cell culture processes

Global expression profiling of mammalian cells used for the production of biopharmaceuticals will allow greater insights into the molecular mechanisms that result in a high producing cellular phenotype. These studies may give insights for genetic intervention to possibly create better host cell lines or even to provide clues to more rational strategies for cell line and process development. In this review I will focus on the contribution of proteomic technologies to a greater understanding of the biology of Chinese hamster ovary cells and other producing cell lines such as NS0 mouse cells.     

Biochemically differentiated clonal human glial cells in tissue culture

A clonal line of astrocytes (designated CHB) derived from a brain tumor of human origin has been established in tissue culture. This cell line is capable of synthesizing an acid protein unique to the nervous system (S100-protein). The S100-protein synthesized by CHB cells is immunologically indistinguishable (by micro-complement fixation) from S100-protein present in human brain.     

Cytoplasmic transfer of chloramphenicol resistance in human tissue culture cells

The cytoplasmic inheritance of human chloramphenicol (cap) resistance has been demonstrated by removing the nuclei of cells of the CAP-resistant HeLa strain 296-1 (enucleation) and fusing them to a CAP-sensitive HeLa strain lacking nuclear thymidine kinase. Plating the fusion products in bromodeoxyuridine and CAP resulted in the growth of about 150 colonies/10(6) parent cells plated. Permanent cell lines (cybrids) grown from such fusions have been designated HEB. A recloned HEB cybrid (HEB7A) has also been enucleated and fused to hypoxanthine phosphoribosyl transferase (HPRT)-deficient HeLa cells (S3AG1) and HPRT-deficient lymphocytes (WAL-2A). Cybrids were selected in thioguanine and CAP. In the fusion of enucleated (en) HEB7A to S3AG1, 1,200 colonies/10(6) parents were observed. Fusion of enHEB7A to WAL-2A was done in mass culture and cybrids were obtained on three separate occasions. In every case the parental controls were negative. All isolates tested from the above fusions have the CAP-resistant characteristics, in vivo and in vitro, of the enucleated parent and the nuclear characteristics of the CAP-sensitive parent, such as chromosome number, morphology, and specific isozyme and chromosome markers. Therefore, it can be concluded that CAP resistance is coded in the cytoplasm and not in the nucleus of 296-1 cells. Furthermore, this resistance can be transferred to cells of widely different origin and differentiated state. These studies represent the first genetic evidence of cytoplasmic inheritance in human cells.     

Growth of dissociated beating human heart cells in tissue culture

Fetal human heart cells have been dissociated by trypsinization and successfully grown in tissue culture on two occassions. The characteristics of the growth and mitotic activity were quite similar to those previously found for rodent heart cells. The maximum beating rates seen in the cultures from each specimen were 33 and 75 per minute.     

Maintenance of fetal human pancreatic beta cells in tissue culture

Large quantities of viable human islet tissue (beta cells) are required for transplant and for investigations of the autoimmune basis of Type I diabetes. Fetal pancreas offers a potential advantage over other possible sources of beta cells in that it retains some capacity for growth in vitro. We have cultured a total of 45 human pancreata from fetuses of gestational ages from 18 to 23 weeks. Each pancreas was obtained within minutes after delivery and usually cultured within 30 minutes. Pancreata were dispersed and cultured for up to 32 days. Maintenance and growth of the beta cells was assessed by the content of insulin in extracts of cultured tissue. As has been reported by others, fetal human beta cells survived in vitro for over 4 weeks. In three experiments in which a direct comparison was made, collagenase digestion of the fetal pancreas resulted in a significantly greater loss of insulin content compared to minced tissue cultured without digestion. Storage of three pancreata in medium overnight at 4 degrees C significantly reduced the insulin content of the pancreas compared to pancreata cultured immediately. During culture, the majority of the beta cells (based on insulin content) were found in small, macroscopic clumps attached to the surface of the culture dish, and surrounded by a nearly confluent monolayer of fibroblastoid cells. There was a marked decrease in the insulin content of the tissue during culture, most of it (to less than 25% of the original) occurring over the first 4-6 days of culture.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bipolar resistivity profiling of 3D tissue culture

We describe a new low-cost technique for continuous monitoring of the thickness of biofilms and tissue cultures, and we demonstrate the advantage of using electrodes of different dimensions to probe different depths of a sample. We have used electric impedance spectroscopy to monitor keratinocyte stem cells (YF29) growing on an array of Ti/Pt coplanar microelectrodes. The thickness of the sample was reconstructed by fitting the measurements to theoretical curves. We have developed an algorithm for the rapid calculation of the resistance through a multilayered sample. This algorithm is based on conformal mapping and the serial partial capacitance technique. The validity of the technique was tested by measuring the sedimentation rate of an alumina powder. Sample thicknesses between 10 and 80 microm could be measured with a resolution of a few microns using the device.     

Studies of human achondroplasia: oxidative metabolism in tissue culture cells

Mitochondria prepared from the first growth of cells (fibroblasts) from skin biopsies from homozygous (but not heterozygous) achondroplastic human subjects were unable to carry out oxidative phosphorylation. However, successive crops of cells gained the ability to phosphorylate with normal P:O ratios with pyruvate-malate and succinate as substrates. Concentrations of cytochromes a + a3 were markedly and significantly lower in homogenates of homozygous achondroplastic tissue culture cells than in homogenates of normal cells. Levels of cytochromes a + a3 in the heterozygous achondroplastic cells were intermediate between the levels in normal cells and the homozygous achondroplastic cells. Activities of the mitochondrial oxidative systems (NADH, succinic and cytochrome oxidases) were not significantly lower in the achondroplastic cell preparations than in normal cell preparations under standard assay conditions (saturation levels of oxygen).