Super7 passaging method to improve Chinese hamster ovary cell fed-batch performance.

Chinese hamster ovary (CHO) cells are widely used to manufacture biopharmaceuticals, most of all monoclonal antibodies (mAbs). Some CHO cell lines exhibit production instability, where the productivity of the cells decreases as a function of time in culture. To counter this, we designed a passaging strategy that, rather than maximizing the time spent in log-growth phase, mimics the first 7 days of a fed-batch production process.

Toxic concentrations of exogenously supplied methylglyoxal in hybridoma cell culture.

Concentrations at which methylglyoxal, a by-product of cellular metabolism, can be toxic to hybridoma cell cultures were determined using exogenously supplied doses. Trypan blue cell counts of 6-well cultures incubated for 24 h with various methylglyoxal concentrations revealed inhibition of cell growth at 300 muM and higher, with a median inhibitory concentration of 490+/-20 muM. The primary mode of death was apoptosis, as assessed by chromatin condensation, and the effects of methylglyoxal were observed to be complete by approximately eight hours.

CD28 costimulation is essential for human T regulatory expansion and function.

The costimulatory requirements required for peripheral blood T regulatory cells (Tregs) are unclear. Using cell-based artificial APCs we found that CD28 but not ICOS, OX40, 4-1BB, CD27, or CD40 ligand costimulation maintained high levels of Foxp3 expression and in vitro suppressive function. Only CD28 costimulation in the presence of rapamycin consistently generated Tregs that consistently suppressed xenogeneic graft-vs-host disease in immunodeficient mice.

Reduced mitochondrial membrane potential and metabolism correspond to acute chloroform toxicity of in vitro hepatocytes.

Chloroform is a non-genotoxic compound that is present in drinking water and ambient air as a result of water chlorination but whose carcinogenic mechanism in humans is unknown. Chloroform targets the liver in humans, where cytochrome P-450-dependent biotransformation to phosgene results in mitochondrial damage and cell death. The purpose of this study is to investigate the relationship between cell death, loss of mitochondrial membrane potential (MMP) and reduction of metabolic rates for in vitro cultured mouse hepatocytes after acute exposure to two doses of chloroform.

96-well plate assay for sublethal metabolic activity.

A 96-well plate assay has been devised for estimation of sublethal metabolic activity for compounds administered to in vitro cell cultures during 6- and 24-h exposures. This screen combines a resazurin reduction assay with lactate production and glucose consumption rate assays to assess effects of compounds on both culture viability and metabolic inhibition. In this article, the assay is demonstrated by determining the extent to which five glycolysis inhibitors, namely, phloretin, 2-deoxyglucose, iodoacetate, fluoride, and oxamate, induce metabolic inhibition without cell death for in vitro fibroblast cell cultures.