Analysis of mammalian viable cell biomass based on cellular ATP

Analysis of cellular ATP as a means of measuring viable biomass loading was investigated in hybridoma cell culture. ATP analysis by the luciferin-luciferase assay was compared with trypan blue-stained hemocytometer counts. The cell-specific ATP content varied between 2 and 6 fmol per viable cell over a batch culture. ATP levels were highest during exponential growth, and decreased during the stationary and decline phases. Electronic counting and volume measurements were performed to assay the viable cell biomass. Cell sorting, using fluorescein diacetate, was used to separate viable and nonviable cells in cultures with between 35% and 90% viable cells. Viable cells contained over 2 orders of magnitude greater cell-specific ATP than nonviable cells. Cell-specific ATP correlated directly with the viable cell volume rather than viable cell numbers. Over the range of batch culture conditions, ATP analysis should provide a more accurate measurement of hybridoma viable biomass than hemocytometer counts.     

Cell separator operation within temperature ranges to minimize effects on Chinese hamster ovary cell perfusion culture

A cell retention device that provides reliable high-separation efficiency with minimal negative effects on the cell culture is essential for robust perfusion culture processes. External separation devices generally expose cells to periodic variations in temperature, most commonly temperatures below 37 degrees C, while the cells are outside the bioreactor. To examine this phenomenon, aliquots of approximately 5% of a CHO cell culture were exposed to 60 s cyclic variations of temperature simulating an acoustic separator environment. It was found that, for average exposure temperatures between 31.5 and 38.5 degrees C, there were no significant impacts on the rates of growth, glucose consumption, or t-PA production, defining an acceptable range of operating temperatures. These results were subsequently confirmed in perfusion culture experiments for average exposure temperatures between 31.6 and 38.1 degrees C. A 2(5-1) central composite factorial design experiment was then performed to systematically evaluate the effects of different operating variables on the inlet and outlet temperatures of a 10L acoustic separator. The power input, ambient temperature, as well as the perfusion and recycle flow rates significantly influenced the temperature, while the cell concentration did not. An empirical model was developed that predicted the temperature changes between the inlet and the outlet of the acoustic separator within +/-0.5 degrees C. A series of perfusion experiments determined the ranges of the significant operational settings that maintained the acoustic separator inlet and outlet temperatures within the acceptable range. For example, these objectives were always met by using the manufacturer-recommended operational settings as long as the recirculation flow rate was maintained above 15 L day(-1) and the ambient temperature was near 22 degrees C.     

Screening and Evaluation of Human Intestinal Lactobacilli for the Development of Novel Gastrointestinal Probiotics

The aim of this study was to screen intestinal lactobacilli strains for their advantageous properties to select those that could be used for the development of novel gastrointestinal probiotics. Ninety-three isolates were subjected to screening procedures. Fifty-nine percent of the examined lactobacilli showed the ability to auto-aggregate, 97% tolerated a high concentration of bile (2% w/v), 50% survived for 4 h at pH 3.0, and all strains were unaffected by a high concentration of pancreatin (0.5% w/v). One Lactobacillus buchneri strain was resistant to tetracycline. None of the tested strains caused lysis of human erythrocytes. Six potential probiotic strains were selected for safety evaluation in a mouse model. Five of 6 strains caused no translocation, and were considered safe. In conclusion, several strains belonging to different species and fermentation groups were found that have properties required for a potential probiotic strain. This study was the first phase of a multi-phase study aimed to develop a novel, safe and efficient prophylactic and therapeutic treatment system against gastrointestinal infections using genetically modified probiotic lactobacilli.     

Scale-up and optimization of an acoustic filter for 200 L/day perfusion of a CHO cell culture

Acoustic cell retention devices have provided a practical alternative for up to 50 L/day perfusion cultures but further scale-up has been limited. A novel temperature-controlled and larger-scale acoustic separator was evaluated at up to 400 L/day for a 10(7) CHO cell/mL perfusion culture using a 100-L bioreactor that produced up to 34 g/day recombinant protein. The increased active volume of this scaled-up separator was divided into four parallel compartments for improved fluid dynamics. Operational settings of the acoustic separator were optimized and the limits of robust operations explored. The performance was not influenced over wide ranges of duty cycle stop and run times. The maximum performance of 96% separation efficiency at 200 L/day was obtained by setting the separator temperature to 35.1 degrees C, the recirculation rate to three times the harvest rate, and the power to 90 W. While there was no detectable effect on culture viability, viable cells were selectively retained, especially at 50 L/day, where there was a 5-fold higher nonviable washout efficiency. Overall, the new temperature-controlled and scaled-up separator design performed reliably in a way similar to smaller-scale acoustic separators. These results provide strong support for the feasibility of much greater scale-up of acoustic separations.     

Glycolipid membrane anchored recombinant protein production from CHO cells cultGred on porous microcarriers

Recombinant proteins were harvested from Chinese hamster ovary (CHO) cells by a controlled release process, which increased the purity and concentration of the harvested protein. Recombinant human melano-transferrin (p97) was expressed linked to the outer surface of CHO cells by a glycosyl-phosphatidylinositol (GPI) membrane anchor. Cells were grown to confluence in T-flask culture, and the p97 harvested by replacing the growth medium for 30 min with phosphate-buffered saline (PBS) containing 10 mU/mL phosphatidylinositol-phospholipase C (PI-PLC). The GPI anchor was selectively cleaved by PI-PLC. In fresh medium, the CHO cells regained over 95% of their p97 expression within 40 h. The process was repeated for eight harvests. Harvested protein concentrations varied from 1.5 to 3.8 mug/mL due to difficulties in maintaining stable confluent T-flask cultures. Harvesting from cells growing on porous microcarriers was investigated to increase p97 product concentrations and to overcome culture stability problems. Semicontinuous cultures were maintained in spinners for up to 76 days with average bioreactor cell densities of over 10(7) cell/mL. The p97 was harvested at up to 100 mug/mL and 30% purity with protein production remaining stable for 4 harvest cycles. Production of high levels of p97 from CHO cells was maintained at 0.5% serum. (c) 1994 John Wiley & Sons, Inc.     

Germination initiation and outgrowth of spores ofBacillus brevis strain Nagano and its gramicidin S-negative mutant

Bacillus brevis strain Nagano and its gramicidin S-negative mutant, BI-7, were compared with respect to germination of their spores produced in several media. Germination initiation occurred in the presence of nutrient broth orL-alanine but not with inosine, glucose, glycerol or fructose; the process was activated by heat. Parental and mutant spores behaved similarly in these experiments. During outgrowth, parental spores remained in this phase of germination much longer than did mutant spores, but only when the parental spores had been harvested from a sporulation medium where significant gramicidin S synthesis had occurred. When parental spores were extracted or treated with an enzyme that hydrolyzes gramicidin S, rapid outgrowth occurred. Adding exogenous gramicidin S or the extract from parental spores to mutant spores lengthened the outgrowth in a dose-dependent manner. The uptake of labeledL-alanine by parental spores was delayed compared to mutant spores in the presence or absence of chloramphenicol. These data suggest a mechanism of action for gramicidin S whereby it interferes in membrane function, such as transport or energy metabolism, in outgrowing spores.Abbreviations GS Gramicidin S - CFU colony-forming units     

Allosteric Effect of Adenosine Triphosphate on Peptide Recognition by 3′5′-Cyclic Adenosine Monophosphate Dependent Protein Kinase Catalytic Subunits

The allosteric influence of adenosine triphosphate (ATP) on the binding effectiveness of a series of peptide inhibitors with the catalytic subunit of 3′5′-cyclic adenosine monophosphate dependent protein kinase was investigated, and the dependence of this effect on peptide structure was analyzed. The allosteric effect was calculated as ratio of peptide binding effectiveness with the enzyme-ATP complex and with the free enzyme, quantified by the competitive inhibition of the enzyme in the presence of ATP excess, and by the enzyme-peptide complex denaturation assay, respectively It was found that the principle “better binding—stronger allostery” holds for interactions of the studied peptides with the enzyme, indicating that allostery and peptide binding with the free enzyme are governed by the same specificity pattern. This means that the allosteric regulation does not include new ligand–protein interactions, but changes the intensity (strength) of the interatomic forces that govern the complex formation in the case of each individual ligand. We propose that the allosteric regulation can be explained by the alteration of the intrinsic dynamics of the protein by ligand binding, and that this phenomenon, in turn, modulates the ligand off-rate from its binding site as well as the binding affinity. The positive allostery could therefore be induced by a reduction in the enzyme’s overall intrinsic dynamics.     

High-throughput analysis of single hematopoietic stem cell proliferation in microfluidic cell culture arrays

Heterogeneity in cell populations poses a major obstacle to understanding complex biological processes. Here we present a microfluidic platform containing thousands of nanoliter-scale chambers suitable for live-cell imaging studies of clonal cultures of nonadherent cells with precise control of the conditions, capabilities for in situ immunostaining and recovery of viable cells. We show that this platform mimics conventional cultures in reproducing the responses of various types of primitive mouse hematopoietic cells with retention of their functional properties, as demonstrated by subsequent in vitro and in vivo (transplantation) assays of recovered cells. The automated medium exchange of this system made it possible to define when Steel factor stimulation is first required by adult hematopoietic stem cells in vitro as the point of exit from quiescence. This technology will offer many new avenues to interrogate otherwise inaccessible mechanisms governing mammalian cell growth and fate decisions.     

Musashi expression in ��-cells coordinates insulin expression, apoptosis and proliferation in response to endoplasmic reticulum stress in diabetes

Diabetes is associated with the death and dysfunction of insulin-producing pancreatic β-cells. In other systems, Musashi genes regulate cell fate via Notch signaling, which we recently showed regulates β-cell survival. Here we show for the first time that human and mouse adult islet cells express mRNA and protein of both Musashi isoforms, as well Numb/Notch/Hes/neurogenin-3 pathway components. Musashi expression was observed in insulin/glucagon double-positive cells during human fetal development and increased during directed differentiation of human embryonic stem cells (hESCs) to the pancreatic lineage. De-differentiation of β-cells with activin A increased Msi1 expression. Endoplasmic reticulum (ER) stress increased Msi2 and Hes1, while it decreased Ins1 and Ins2 expression, revealing a molecular link between ER stress and β-cell dedifferentiation in type 2 diabetes. These effects were independent of changes in Numb protein levels and Notch activation. Overexpression of MSI1 was sufficient to increase Hes1, stimulate proliferation, inhibit apoptosis and reduce insulin expression, whereas Msi1 knockdown had the converse effects on proliferation and insulin expression. Overexpression of MSI2 resulted in a decrease in MSI1 expression. Taken together, these results demonstrate overlapping, but distinct roles for Musashi-1 and Musashi-2 in the control of insulin expression and β-cell proliferation. Our data also suggest that Musashi is a novel link between ER stress and the compensatory β-cell proliferation and the loss of β-cell gene expression seen in specific phases of the progression to type 2 diabetes.