Gene therapy: challenges in cell culture scale-up.

Gene therapy is designed to cure various diseases resulting from genetic defects. Currently, recombinant adeno-associated viral vectors (rAAV) are the vehicles of choice for therapeutic gene delivery in vivo. To date, manufacturing sufficient rAAV product to meet rapidly expanding clinical demand remains a bottleneck in the industry. In the past decade, multiple production platforms have been rapidly implemented with encouraging improvements in productivity and scalability. In this review, we discuss the advantages and limitations of the most popular production platforms in the industry with a focus on the cell culture process scale-up.

Induced pluripotent stem cells can utilize lactate as a metabolic substrate to support proliferation.

Human-induced pluripotent stem cells (iPSCs) hold the promise to improve cell-based therapies. Yet, to meet rising demands and become clinically impactful, sufficient high-quality iPSCs in quantity must be generated, a task that exceeds current capabilities. In this study, K3 iPSCs cultures were examined using parallel-labeling metabolic flux analysis (13 C-MFA) to quantify intracellular fluxes at relevant bioprocessing stages: glucose concentrations representative of initial media concentrations and high lactate concentrations representative of fed-batch culture conditions, prior to and after bolus glucose feeds. The glucose and lactate concentrations are also representative of concentrations that might be encountered at different locations within 3D cell aggregates. Furthermore, a novel method was developed to allow the isotopic tracer [U-13 C3 ] lactate to be used in the 13 C-MFA model. The results indicated that high extracellular lactate concentrations decreased glucose consumption and lactate production, while glucose concentrations alone did not affect rates of aerobic glycolysis. Moreover, for the high lactate cultures, lactate was used as a metabolic substrate to support oxidative mitochondrial metabolism. These results demonstrate that iPSCs have metabolic flexibility and possess the capacity to metabolize lactate to support exponential growth, and that high lactate concentrations alone do not adversely impact iPSC proliferation.

High extracellular lactate causes reductive carboxylation in breast tissue cell lines grown under normoxic conditions.

In cancer tumors, lactate accumulation was initially attributed to high glucose consumption associated with the Warburg Effect. Now it is evident that lactate can also serve as an energy source in cancer cell metabolism. Additionally, lactate has been shown to promote metastasis, generate gene expression patterns in cancer cells consistent with "cancer stem cell" phenotypes, and result in treatment resistant tumors. Therefore, the goal of this work was to quantify the impact of lactate on metabolism in three breast cell lines (one normal and two breast cancer cell lines-MCF 10A, MCF7, and MDA-MB-231), in order to better understand the role lactate may have in different disease cell types. Parallel labeling metabolic flux analysis (13C-MFA) was used to quantify the intracellular fluxes under normal and high extracellular lactate culture conditions. Additionally, high extracellular lactate cultures were labelled in parallel with [U-13C] lactate, which provided qualitative information regarding the lactate uptake and metabolism. The 13C-MFA model, which incorporated the measured extracellular fluxes and the parallel labeling mass isotopomer distributions (MIDs) for five glycolysis, four tricarboxylic acid cycle (TCA), and three intracellular amino acid metabolites, predicted lower glycolysis fluxes in the high lactate cultures. All three cell lines experienced reductive carboxylation of glutamine to citrate in the TCA cycle as a result of high extracellular lactate. Reductive carboxylation previously has been observed under hypoxia and other mitochondrial stresses, whereas these cultures were grown aerobically. In addition, this is the first study to investigate the intracellular metabolic responses of different stages of breast cancer progression to high lactate exposure. These results provide insight into the role lactate accumulation has on metabolic reaction distributions in the different disease cell types while the cells are still proliferating in lactate concentrations that do not significantly decrease exponential growth rates.