Biomedical subjects

G Kretzmer

Publications and source records attributed to G Kretzmer.

14 recordsLinked to original sources

PubMed2003

In vitro comparison of complementary interactions between synthetic linear/branched oligo/poly-L-lysines and tissue plasminogen activator by means of high-performance monolithic-disk affinity chromatography.

The recently discovered serine protease called tissue plasminogen activator (t-PA) enables efficient dissolution of blood clots. t-PA works by converting plasminogen into its active form, plasmin, dissolving the major component of blood clots, fibrin. The activation of plasminogen by t-PA is enhanced by the presence of fibrin, and this is probably due to the fact that both plasminogen and t-PA possess high affinity binding sites for fibrin. Besides fibrin, fibrin monomers and some fibrin(ogen) degradation products, certain synthetic polymers (for instance, poly-L-lysines) can provide the same stimulation of plasminogen activation. The recently developed high-performance monolithic-disk chromatography, HPMDC, could become the most convenient way to study biological pairs of interest. The inherent speed of HPMDC isolation facilitates the recovery of a biologically active product, since the exposure to putative denaturing influences, such as solvents or temperature, is reduced. The better mass transfer mechanism (convection rather than diffusion) allows to consider only the biospecific reaction as time limiting. The step-by-step modeling of hypothetical affinity pairs between t-PA and different types of oligo/polymer forms of linear and branched lysine derivatives obtained both by initiated polycondensation and solid-phase peptide synthesis using HPMDC seemed to be possible and a quite useful tool. The results of quantitative evaluation of such affinity interactions were compared with those established for natural affinity counterparts to t-PA (monoclonal antibodies, plasminogen, fibrinogen). The role of steric structure of lysine ligands was observed and analyzed. The results allowing to make the practical choice of affinity systems will be used for development of fast and efficient analytical and preparative methods for the downstream processes of recombinant production of this valuable enzyme.

Animals↗

PubMed2002

Industrial processes with animal cells.

Industrial processes involving animal cells for the production of useful products still seem to be rather uncommon. Nevertheless, during the last four decades of the last century the number of relevant processes has increased from production of virus vaccines to monoclonal antibodies and finally complex structured glycoproteins. As soon as cell lines became permanent and culture medium changed from purely biological fluids to more or less defined chemical media, large-scale cultivation could begin. The developments of the 1970s - fusion of cells to form hybridomas, and genetic engineering - triggered a second wave of products. Monoclonal antibodies and recombinant proteins for diagnosis and therapy set new challenges for the inventors. Historically, there has been no straightforward process development since the product dictates the process operation. Therefore, the scale of production covers the whole range from small multiple-unit reactors (flasks or roller bottles) up to 10,000-l single-unit batch reactors. Products with high value and small demand can be produced in multiple-unit systems whereas "bulk" products for vaccination and therapy may need large-scale bioreactors to be cost effective. All the different systems have their advantages and disadvantages and significant challenges that curb the development of effective perfusion cultures still remain.

Animals↗

PubMed2001

Dose-dependent olanzapine-associated leukopenia: three case reports.

Leukopenia and agranulocytosis are well reported and dangerous haematological side-effects associated with the use of typical and atypical antipsychotics. These potentially life-threatening phenomena have led to treatment discontinuation and the consequent reemergence of psychiatric symptoms. We report three cases of patients who developed leukopenia during olanzapine treatment. In each case, the leukopenia was dose-dependent. Reduction in the dose of olanzapine was followed by normalization of the white blood count which allowed continuation of the medication. These cases suggest the possibility that, in some patients, leukopenia or agranulocytosis during olanzapine treatment might be dose-related. Thus, olanzapine dose reduction may permit treatment continuation where this is clinically indicated. In our cases, haematological side-effects were satisfactorily controlled by dose reduction without allowing the reemergence of psychiatric symptoms. This clinical management may offer an alternative to treatment suspension. A careful monitoring of the white blood count is obviously recommended. Olanzapine may be considered a potential and safer treatment for a this specific group of patients.

Adolescent↗

PubMed2000

Influence of stress on adherent cells.

Stress is a broad term often used with animal cells. Frequently mechanical forces are meant using this term but chemical stress is also important cultivating animal cells. The chemical environment of the cell in a reactor have to be considered very carefully. The complexity of the medium requirements and the metabolic pathway cause very often growth limitations. Studying these limitations in order to find the reasons showed to be difficulty because of the complexity of the system. Nevertheless, glucose, glutamine, lactate and ammonia are found to be critical parameter as well as the osmotic pressure. The influence of mechanical forces on cell viability is of great importance when growing the cells in agitated systems. By far the greatest amount of work reported in the literature has been done on suspension cells but adherent cells also experience shear forces not only in bioreactors also in vivo. Therefore, most research has be done on endothelial cells but studies exists done on non-endothelial cells. The influence of shear forces on cell growth, morphology and productivity will be discussed as well as possibilities of making the cells more resistant.

Animals↗

PubMed1997

Influence of the medium composition and plasmid combination on the growth of recombinant Escherichia coli JM109 and on the production of the fusion protein EcoRI::SPA.

Plasmid-free and plasmid-harbouring E. coli JM109 strains were investigated in shaken flasks, stirred tanks in batch and continuous operation. The shaken flask cultivations were performed in M9 minimal medium and in media with various protein supplements. The host hardly grows on M9 minimal medium as opposed to the plasmid-harbouring cells, which grow well on this medium. All of the investigated cells propagate well on protein-containing media. The influence of the combinations of repressor plasmid pRK248cI, the protection plasmid EcoR4 and the production plasmid pMTC48 were determined on the initial specific growth rate of the E. coli JM109 without gene expression, on the yield coefficient of cell growth, acetate concentration and acetate yield coefficient in the yeast extract-containing (HM) medium. The influence of various media on the induction of the gene expression were evaluated. In cultivation media with protein supplement, the growth rate and yield coefficient increased. The variation of the volumetric and specific beta-lactamase activities with the cultivation time were determined in a stirred tank reactor in HM medium. With increasing dilution rate the process performance decreased. Simple relationships exist between the substrate uptake rate and the specific growth rate of the continuous cultivated cells in M9 and HM media. The influence of the dilution rate on the cell mass concentration, colony forming units, acetate formation, yield coefficients of growth and acetate formation, substrate uptake rate, CO2 production rate, ammonium formation rate and beta-lactamase activity in M9 and HM media were determined as well. Carbon balances of the batch and continuous cultivations indicated high carbon recoveries. On account of the higher growth rate of plasmid-harbouring cells than than of the plasmid-free cells, the behaviour of the investigated plasmid-free and plasmid-harbouring E. coli JM109 cells deviates from the published properties of other plasmid-free and plasmid-harbouring E. coli cells.

Acetates↗

PubMed1994

Problems with serum-free production of antithrombin III regarding proteolytic activity and product quality.

Human antithrombin III (AT-III) was produced using a recombinant BHK-21 cell line with a microcarrier culture in spinner flasks. Cells were cultivated for the first 4 days in a medium containing 10% fetal calf serum (FCS). Afterwards, the medium was exchanged and production of AT-III occurred at high cell numbers in a serum-free medium. The product was determined by an immunoassay and further analysed after isolation from the culture medium. During cultivation, high proteolytic activity was detected which caused a considerable product decomposition. Furthermore, a higher level of non-glycosylated AT-III was found after serum-free production.

Animals↗

PubMed1994

Low temperature cultivation--a step towards process optimisation.

Adherent recombinant BHK cells were cultivated at temperatures between 30 and 37 degrees C. Batch and repeated-batch-cultivations in a 2-litre bioreactor showed a significant influence on metabolism and cell growth. The low-temperature-cultivations showed a lower growth rate and a lower glucose consumption rate and, therefore, less lactate production. On the other hand, the maximum cell density and productivity seemed not to be affected by the temperature reduction.

Animals↗

PubMed1993

Effect of cholesterol addition on growth kinetics and shear stress sensitivity of adherent mammalian cells.

The growth kinetics of adherent baby hamster kidney cells cultivated with additional cholesterol as well as the effect of cholesterol addition on shear stress sensitivity were investigated. The influence of various cholesterol preparations was tested, whereby dimethylsulfoxide and ethanol show negative effects at higher concentrations. With addition of cholesterol in the range of 90 micrograms ml-1, a positive effect on the shear stress resistance was achieved.

Animals↗

PubMed1993

Shear stress induced variation of cell condition and productivity.

Anchorage-dependent recombinant BHK-21 cells, which produce beta-D-galactosidase, were subjected to shear stress levels in the range between 0.1 and 1.55 N m-2 over a period of three days. To investigate cell constitution and productivity, the cell number and viability, as well as the lactate dehydrogenase and beta-D-galactosidase activity, were determined. Low (0.25-0.75 N m-2) and intermediate (0.75-1.25 N m-2) shear stress rates stimulated; higher shear stress rates (more than 1.25 N m-2) diminished productivity. Cell number and general condition were influenced negatively by all but the lowest levels of shear stress. An optimal shear stress level between 0.4 and 0.6 N m-2 can be deduced.

Animals↗

PubMed1992

Influence of the temperature on the shear stress sensitivity of adherent BHK 21 cells.

The influence of temperature on the shear sensitivity of anchorage-dependent baby hamster kidney (BHK) cells was investigated. The temperature effect in general was compared for stressed and unstressed cells. Both the growth rate as well as the shear sensitivity are temperature-dependent. Decreasing the temperature lowered the growth rate and increased the ability of the BHK cells to withstand shear stress.

Animals↗

PubMed1992

Determination of a "critical shear stress level" applied to adherent mammalian cells.

An apparatus for the detailed investigation of the influence of shear stress on adherent BHK cells was developed. Shear forces between 0.0 and 2.5 N m-2 were studied. The influence on cell viability, cell morphology, cell lysis, and cell size was determined. Increasing shear forces as well as increasing exposure duration caused increasing changes in cell morphology and cell death. A "critical shear stress level" was determined.

Animals↗

PubMed1992

Lactate dehydrogenase (LDH) activity of the cultured eukaryotic cells as marker of the number of dead cells in the medium [corrected].

One significant problem in monitoring a culture's evolution is to assess change in cell viability. We have demonstrated that LDH release could be a good indicator of cellular damage of many cell lines, especially during shear stress or sonication. Moreover, we have found a significant correlation between the number of dead cells, determined by Trypan Blue staining, and LDH activity measurements in the supernatant of hybridoma strains, whatever the culture conditions. We have also shown that when viability is still near 100% no LDH is released even at high cell concentrations. Therefore, LDH should serve as a potential marker of cell injury and death.

Animals↗

PubMed1991

Response of mammalian cells to shear stress.

The influence of shear forces on adherent mammalian cells was investigated by means of a developed flow chamber. The viability of the cells decreased with increasing exposure level and duration. Additional, changes in the morphology of the cells due to the shear forces were observed.

Animals↗

PubMed1991

Metabolism of hybridoma cells and antibody secretion at high cell densities in dialysis tubing.

The experimental setup, consisting of a bundle of dialysis tubing 2.5 mm in diameter [10-15 kD cutoff, mean pore size 25 A, 20 microns (dry) and 40 microns (wet) wall thickness] inserted into a 1-l glass bioreactor supplied with oxygen and pH electrodes, a porous gas distributor, a sampling tube, and a holder for the eight pieces of dialysis tubing, was developed to investigate the properties and the microenvironment of hybridoma cells enclosed in the tubing during their batch cultivation. The concentrations of low-molecular-weight medium components were the same inside and outside the tubing, and it was possible to control the microenvironment of the cells in the tubing easily. The cell damage caused by mechanical stress was less in the dialysis tubing than in stirred spinner flasks. The influence of the initial cell density in the range from 4 X 10(5) to 1 X 10(8) cells ml-1 and the cultivation time were evaluated according to the total and viable cell concentrations and the cell/cell fragment size distributions. Furthermore, the cell membrane properties, glucose consumption rate, lactate, ammonia and lipid storage material, and the monoclonal antibody production rates as well as intracellular enzyme activities in the culture medium were measured and compared to those in reference cultures in spinner flasks with the same inoculum at low initial cell densities. In dialysis tubing in a concentration range of 5 X 10(6) to 10(8) cells ml-1, the total and viable concentrations of cells remained the same during cultivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗