Optimization and control of perfusion cultures using a viable cell probe and cell specific perfusion rates

Consistent perfusion culture production requires reliable cell retention and control of feed rates. An on-line cell probe based on capacitance was used to assay viable biomass concentrations. A constant cell specific perfusion rate controlled medium feed rates with a bioreactor cell concentration of ∼5 × 10⁶ cells mL^-1. Perfusion feeding was automatically adjusted based on the cell concentration signal from the on-line biomass sensor. Cell specific perfusion rates were varied over a range of 0.05 to 0.4 nL cell^-1 day^-1. Pseudo-steady-state bioreactor indices (concentrations, cellular rates and yields) were correlated to cell specific perfusion rates investigated to maximize recombinant protein production from a Chinese hamster ovary cell line. The tissue-type plasminogen activator concentration was maximized (∼40 mg L^-1) at 0.2 nL cell^-1 day^-1. The volumetric protein productivity (∼60 mg L^-1 day^-1 was maximized above 0.3 nL cell^-1 day^-1. The use of cell specific perfusion rates provided a straightforward basis for controlling, modeling and optimizing perfusion cultures. This revised version was published online in July 2006 with corrections to the Cover Date.     

Purified human pancreatic duct cell culture conditions defined by serum-free high-content growth factor screening

The proliferation of pancreatic duct-like CK19+ cells has implications for multiple disease states including pancreatic cancer and diabetes mellitus. The in vitro study of this important cell type has been hampered by their limited expansion compared to fibroblast-like vimentin+ cells that overgrow primary cultures. We aimed to develop a screening platform for duct cell mitogens after depletion of the vimentin+ population. The CD90 cell surface marker was used to remove the vimentin+ cells from islet-depleted human pancreas cell cultures by magnetic-activated cell sorting. Cell sorting decreased CD90+ cell contamination of the cultures from 34±20% to 1.3±0.6%, yielding purified CK19+ cultures with epithelial morphology. A full-factorial experimental design was then applied to test the mitogenic effects of bFGF, EGF, HGF, KGF and VEGF. After 6 days in test conditions, the cells were labelled with BrdU, stained and analyzed by high-throughput imaging. This screening assay confirmed the expected mitogenic effects of bFGF, EGF, HGF and KGF on CK19+ cells and additionally revealed interactions between these factors and VEGF. A serum-free medium containing bFGF, EGF, HGF and KGF led to CK19+ cell expansion comparable to the addition of 10% serum. The methods developed in this work should advance pancreatic cancer and diabetes research by providing effective cell culture and high-throughput screening platforms to study purified primary pancreatic CK19+ cells.     

Controlled shear affinity filtration (CSAF): a new technology for integration of cell separation and protein isolation from mammalian cell cultures

Up-flow anaerobic sludge blanket (UASB) reactors are being used with increasing regularity all over the world, especially in India, for a variety of wastewater treatment operations. Consequently, there is a need to develop methodologies enabling one to determine UASB reactor performance, not only for designing more efficient UASB reactors but also for predicting the performance of existing reactors under various conditions of influent wastewater flows and characteristics. This work explores the feasibility of application of an artificial neural network-based model for simulating the performance of an existing UASB reactor. Accordingly, a neural network model was designed and trained to predict the steady-state performance of a UASB reactor treating high-strength (unrefined sugar based) wastewater. The model inputs were organic loading rate, hydraulic retention time, and influent bicarbonate alkalinity. The output variables were one or more of the following, effluent substrate concentration (Se), reactor bicarbonate alkalinity, reactor pH, reactor volatile fatty acid concentration, average gas production rate, and percent methane content of the gas. Training of the neural network model was achieved using a large amount of experimentally obtained reactor performance data from the reactor mentioned above as the training set. Training was followed by validation using independent sets of performance data obtained from the same UASB reactor. Subsequently, simulations were performed using the validated neural network model to determine the impact of changes in parameters like influent chemical oxygen demand (COD) concentration and hydraulic retention time on the reactor performance. Simulation results thus obtained were carefully analyzed based on qualitative understanding of UASB process and were found to provide important insights into key variables that were responsible for influencing the working of the UASB reactor under varying input conditions.     

Intensive local surveys can complement rapid survey techniques to provide insights into the population size and ecology of lichenised fungi

We propose that insights to population ecology of lichenised fungi can be efficiently obtained by combining rapid biodiversity surveys, which representatively sample large areas, with intensive studies in selected populations discovered. To illustrate this approach, we compared results from an Estonian rapid survey scheme with an intensive local population survey of the poorly known epiphytic crustose lichen, Lecanora thysanophora. In contrast to what the data from rapid surveys suggested, the intensive survey revealed that this typically sterile species can occur in remarkably dense populations obviously limited by host tree availability; we also recorded emerging sexual reproduction in the population centre. Our results imply that the detection of even poorly identifiable species may mostly depend on total field effort.     

Novel guanidine-based inhibitors of inosine monophosphate dehydrogenase

A series of novel guanidine-based small molecule inhibitors of inosine monophosphate dehydrogenase (IMPDH) was explored. IMPDH catalyzes the rate determining step in guanine nucleotide biosynthesis and is a target for anticancer, immunosuppressive and antiviral therapy. The synthesis and the structure-activity relationships (SARs), derived from in vitro studies, for this new series of inhibitors is given.     

Cell cycle distribution of primitive haematopoietic cells stimulated in vitro and in vivo

A novel approach is used to study the proliferating behaviour of primitive haematopoietic cell populations in response to different stimuli. A mathematical model based on the average proportion of apoptotic, dividing and quiescent cells in primitive haematopoietic cell populations is developed to describe the mitotic history of 5- (and 6-) carboxyfluorescein diacetate succinimidyl ester-labelled cells. The cell cycle distributions in different cytokine-supplemented cultures of primitive human and mouse bone marrow cells are determined and compared with those found in vivo. The results indicate that a combination of flt-3 ligand, Steel factor and interleukin-11 or hyper-interleukin-6 provide a level of mitogenic stimulation similar to that existing in vivo after a myeloablative radiation dose. The comparison of the cell cycle distribution obtained for different cultures of human bone marrow CD34(+)(45RA/71)(-) cells demonstrates that the addition of flt-3 ligand in these cultures decreases apoptosis significantly but does not reduce quiescence. In addition, in vivo and in vitro, it was found that more than 3 days of stimulation are required to recruit a maximum number of quiescent cells into active cell cycle. These kinetics of cell cycle activation are found to be similar to those identified for the haematopoietic stem cells compartment in the same cultures. This mathematical analysis provides a useful tool for the development of haematopoietic stem cell culture processes for clinical applications.     

Relationship between recombinant activated protein C secretion rates and mRNA levels in baby hamster kidney cells

Analysis of 12 baby hamster kidney (BHK) clones in exponential growth revealed a linear relationship between cell-specific recombinant activated protein C (APC) production rates and APC mRNA levels. This correlation indicated that mRNA levels limited APC productivity. Two strategies were employed to increase APC mRNA levels and APC productivity. First, sodium butyrate was added to increase mRNA levels by two- to sixfold in five APC-producing clones to obtain up to 2.7-fold increase in APC production rate. The second strategy was to retransfect an APC-producing BHK cell line with a vector containing additional APC cDNA and a mutant DHFR. This mutant DHFR gene allowed the selection of retransfected clones in higher MTX concentrations. Over two-fold higher mRNA levels were obtained in these retransfected clones and the cell-specific APC production rate increased twofold. At the highest level of APC secretion, increases in mRNA levels did not result in higher rates of APC production. Analysis of the intracellular APC content revealed a possible saturation in the secretory pathway at high mRNA levels. The relation between mRNA level and APC secretion rate was also investigated in batch culture. The levels of total cellular RNA, APC mRNA, and beta-actin mRNA were relatively stable while cells were in the exponential growth phase, but rapidly decreased when cells reached the stationary phase. The decline of cell-specific APC mRNA levels correlated with a decline in APC secretion rates, which indicated that the mRNA levels continued to limit the rates beyond the exponential phase and into the declining growth and stationary phases of batch APC production.     

Thermoreversible gel formulation containing sodium lauryl sulfate as a potential contraceptive device

The contraceptive properties of a gel formulation containing sodium lauryl sulfate were investigated in both in vitro and in vivo models. Results showed that sodium lauryl sulfate inhibited, in a concentration-dependent manner, the activity of sheep testicular hyaluronidase. Sodium lauryl sulfate also completely inhibited human sperm motility as evaluated by the 30-sec Sander-Cramer test. The acid-buffering capacity of gel formulations containing sodium lauryl sulfate increased with the molarity of the citrate buffers used for their preparations. Furthermore, experiments in which semen was mixed with undiluted gel formulations in different proportions confirmed their physiologically relevant buffering capacity. Intravaginal application of the gel formulation containing sodium lauryl sulfate to rabbits before their artificial insemination with freshly ejaculated semen completely prevented egg fertilization. The gel formulation containing sodium lauryl sulfate was fully compatible with nonlubricated latex condoms. Taken together, these results suggest that the gel formulation containing sodium lauryl sulfate could represent a potential candidate for use as a topical vaginal spermicidal formulation to provide fertility control in women.     

Caspase activation precedes PTP opening in TNF-alpha-induced apoptosis in L929 cells

In this work, we studied the apoptotic pathway in murine fibrosarcoma cells L929 exposed to tumor necrosis factor alpha (TNF-alpha). DNA fragmentation, activation of caspases, cytochrome c release and poly (ADP-ribose) polymerase cleavage were demonstrated. We showed that the proapoptotic proteins Bid and Bax as well as caspase 8 are involved in the initiation of this apoptotic pathway triggered by TNF-alpha. Indeed, inhibition of caspase 8 could prevent TNF-alpha-induced DNA fragmentation. Furthermore, Bid and Bax translocation into mitochondria were already evidenced after 6 h. In contrast, permeability transition pore inhibitors did not prevent the DNA fragmentation induced by TNF-alpha. In addition, these events were not associated with changes in the mitochondrial membrane potential nor with the loss of ATP, which only occurred after 16 h. Taken together, these results underline the fact that TNF-alpha is able to induce caspase-dependent apoptosis in L929 in the absence of permeability transition pore opening.     

Optical analysis of perfusion bioreactor cell concentration in an acoustic separator

Automated monitoring of cell concentration in perfusion bioprocesses facilitates the maintenance of constant cell specific perfusion rates. However, most on-line measuring devices are relatively complex and foul as the culture progresses. A simple external optical sensor was developed using the transparent glass walls of acoustic separators for automated optical analysis of their contents. For each measurement, the separator was filled by an automated pumping system with triplicate representative bioreactor samples that were optically analyzed and the device returned to perfusion operation within approximately 1 or 2 min. Chinese hamster ovary cell concentrations, ranging from 5 x 10(5) to 2 x 10(7) cells/mL, were highly correlated (R(2) = 0.99) with the 90 degrees scattered light response. Since the device was operated externally, it did not complicate bioreactor sterilization or cleaning. Viability was not optically analyzed, but this information was not required between manual samples of a properly operated perfusion process. Using single-point recalibration based on routine off-line samples, this external optical system remained effective during a 4-month perfusion run, thus providing a non-invasive and easily maintained on-line cell concentration monitoring system to improve the control of perfusion bioreactors.