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

Preface Cell Culture Technology: An Overview Recombinant DNA Technology and Cell Line Development Medium Development Cell Metabolism Protein Glycosylation Cell Culture Bioreactors Aeration, Mixing and Hydrodynamics in Bioreactors Instrumentation and Process Control Cell Culture Kinetics and Modeling Fed-Batch Cultivation Of Mammalian Cells For Production Of Recombinant Proteins Optimization of High Density Perfusion Bioreactors Cell Separation And Product Capture Downstream Processing Formulation, Finishing, and Packaging Validation Of Cell Culture Based Processes & Qualification Of Associated Equipment And Facility Facility Design Transient Expression of Proteins Large-Scale Propagation of Insect Cells Advances In Adult Stem Cell Culture Ex Vivo Culture Of Hematopoietic And Mesenchymal Stem Cells For Tissue Engineering And Cell-Based Therapies

https://www.progenericos.org.br/upload/Sadettin_Ozturk_Aug2009_UFRJ.pdf

Cell culture technology for pharmaceutical and cell-based therapies

Chitosan is one of the most used polysaccharides in the design of drug delivery strategies for administration of either biomacromolecules or low molecular weight drugs. For these purposes, it is frequently used as matrix forming material in both nano and micron-sized particles. In addition to its interesting physicochemical and biopharmaceutical properties, which include high mucoadhesion and a great capacity to produce drug delivery systems, ensuring the biocompatibility of the drug delivery vehicles is a highly relevant issue. Nevertheless, this subject is not addressed as frequently as desired and even though the application of chitosan carriers has been widely explored, the demonstration of systems biocompatibility is still in its infancy. In this review, addressing the biocompatibility of chitosan carriers with application in drug delivery is discussed and the methods used in vitro and in vivo, exploring the effect of different variables, are described. We further provide a discussion on the pros and cons of used methodologies, as well as on the difficulties arising from the absence of standardization of procedures.

https://www.mdpi.com/2079-4983/3/3/615/pdf?y=1

Biocompatibility of Chitosan Carriers with Application in Drug Delivery

D-ribose in cells and human serum participates in glycation of proteins resulting in advanced glycation end products (AGEs) that affect cell metabolism and induce cell death. However, the mechanism by which D-ribose-glycated proteins induce cell death is still unclear. Here, we incubated D-ribose with bovine serum albumin (BSA) and observed changes in the intensity of fluorescence at 410 nm and 425 nm to monitor the formation of D-ribose-glycated BSA. Comparing glycation of BSA with xylose (a control for furanose), glucose and fructose (controls for pyranose), the rate of glycation with D-ribose was the most rapid. Protein intrinsic fluorescence (335 nm), Nitroblue tetrazolium (NBT) assays and Western blotting with anti-AGEs showed that glycation of BSA incubated with D-ribose occurred faster than for the other reducing sugars. Protein intrinsic fluorescence showed marked conformational changes when BSA was incubated with D-ribose. Importantly, observations with atomic force microscopy showed that D-ribose-glycated BSA appeared in globular polymers. Furthermore, a fluorescent assay with Thioflavin T (ThT) showed a remarkable increase in fluorescence at 485 nm in the presence of D-ribose-glycated BSA. However, ThT fluorescence did not show the same marked increase in the presence of xylose or glucose. This suggests that glycation with D-ribose induced BSA to aggregate into globular amyloid-like deposits. As observed by Hoechst 33258 staining, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) activity assay, flow cytometry using Annexin V and Propidium Iodide staining and reactive oxygen species (ROS) measurements, the amyloid-like aggregation of glycated BSA induced apoptosis in the neurotypic cell line SH-SY5Y. Glycation with D-ribose induces BSA to misfold rapidly and form globular amyloid-like aggregations which play an important role in cytotoxicity to neural cells.

/pdf/rapid-glycation-with-d-ribose-induces-globular-amyloid-like-vra7uvgpws.pdf

Rapid glycation with D-ribose induces globular amyloid-like aggregations of BSA with high cytotoxicity to SH-SY5Y cells

The invention provides methods for producing mixtures of antibodies from a single host cell clone. Thereto a nucleic acid sequence encoding a light chain, and nucleic acid sequences encoding different heavy chains are expressed in a recombinant host cell. The antibodies in the mixtures according to the invention suitably comprise identical light chains paired to different heavy chains capable of pairing to the light chain, thereby forming functional antigen binding domains. Mixtures of antibodies are also provided by the invention. Such mixtures can be used in a variety of fields.

Recombinant production of mixtures of antibodies

This study reports the use of lactate dehydrogenase release to monitor changes in culture viability in flask culture and fixed bed, porosphere bioreactor systems. Lactate dehydrogenase release shows good agreement with increase in non-viable cell numbers and decline in glucose utilisation in flask cultures. Studies with the immobilised system show that lactate dehydrogenase release can detect loss of viability which is not always indicated by a decrease in glucose utilisation. The data show that culture viability in a repeated-feed-and-harvest system is influenced markedly by both a) the medium change regime itself and b) the use of an immobilised bioreactor compared to a flask system for the same medium change regime.

Use of lactate dehydrogenase release to assess changes in culture viability.

Immobilization of animal cells in porous carrier culture

Process intensity of fixed bed glass sphere culture systems is increased considerably by replacing solid glass spheres with open pore glass spheres. This technique demonstrates the possibility of having a system capable of both volumetric and cell density scale up and being suitable for substrate attached and suspension cells. The yields achieved for a number of attached cell lines (approximately 107/ml) demonstrate an increase approaching one order of magnitude over solid glass spheres (approximately 106/ml). Also suspension cells were successfully entrapped in the open pore structure with similar yields.

Fixed bed porous glass sphere (porosphere) bioreactors for animal cells.

In this work a recombinant BHK21 clone producing a fusion protein with potential application in tumour target therapy was adapted to five different serum-free media (SFM) and to a protein-free medium (PFM). Only the PFM did not require a gradual adaptation to cell growth in the absence of serum. All tested SFM required a gradual adaptation (up to 35 days). For the majority of the SFM tested, cell specific productivity was not affected by the decrease in serum concentration during adaptation; however, cell growth was significantly affected by the serum decrease. Both cell growth and productivity were increased when PFM SMIF6 was used instead of the control medium. Long term measurements (approximately 100 days) of cell specific productivity for PFM and the two best SFM showed that productivity was maintained. This indicates the media capability to be used in long term production processes.

/pdf/adaptation-of-bhk-cells-producing-a-recombinant-protein-to-togpglntun.pdf

Adaptation of BHK cells producing a recombinant protein to serum-free media and protein-free medium.

Recombinant fusion proteins offer important new therapeutic approaches for the future. This report describes the use of three different genetic strategies (i.e. “mono-”, “bi-” and “tri-cistronic” vectors) to achieve stable secretion from BHK cells of a glycosylated antibody-cytokine fusion protein designed for use in anti-tumour therapy. It describes selection of a robust and effective production cell line based on stability of secretion of the product, quality of mRNA and protein products and performance in in vitro bioassays for potency. The data obtained at this stage were utilised in the selection of a suitable candidate production cell line. The relative productivity and general performance of the cells in stirred tank and fixed bed culture systems indicated that a variety of cell culture technologies provided robust tools for production of a highly selected cell clone. Consistency of the product glycosylation was determined by analysis of released oligosaccharides using matrix-assisted laser desorption ionisation – time of flight mass spectrometry and high-performance anion exchange chromatography. These investigations showed consistent expression of three glycoforms of the fusion protein which varied in their relative proportions in different culture systems and at different time points in a fixed bed reactor with continuous perfusion. In conclusion, this study dealt with a range of important scientific and technical issues which are essential for regulatory approval and commercial success of a recombinant protein and elucidates some useful markers for process development for similar recombinant biologicals.

An integrated strategy for the process development of a recombinant antibody-cytokine fusion protein expressed in BHK cells.

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