Impact of high extracellular lactate on induced pluripotent stem cell metabolism and pluripotency

Induced pluripotent stem (iPS) cells hold the potential to drastically improve cell-based therapies in the near future. However, in order for stem cell therapies to become clinically feasible, these cells must be generated in sufficient quantity and quality. This aim will require a comprehensive understanding of how environmental conditions affect iPS cell metabolism and pluripotency. Rapidly proliferating cells, including cancer and iPS cells, consume glucose and secrete lactate at high rates, even in the presence of sufficient oxygen, a process referred to as the Warburg effect. In cancer cell metabolism, lactate accumulation is associated with cancer stem cell-like gene expression, drug-resistance, metastasis, and poor prognosis in breast cancer patients. Elevated lactate conditions have also been shown to preferentially cause iPS cells to differentiate into cardiomyocytes in glucose-deficient media. Yet, there remains an incomplete understanding of the role of lactate in stem cell metabolism and pluripotency in glucose containing media. This study examined the impact of extracellular lactate on the metabolic activity and pluripotency of iPS K3 cells grown with sufficient glucose. Extracellular glucose, lactate, and amino acid concentrations were monitored throughout the experiment to determine the extracellular consumption or production fluxes. High extracellular lactate resulted in altered cell metabolism, including decreased lactate production while glucose consumption remaining unchanged. These results support hypotheses that there is a possible redistribution of carbons within metabolism under high extracellular lactate, with a larger portion of carbon entering the tricarboxylic acid cycle. The implications of these findings towards understanding iPS cell metabolism and designing cell culture conditions to limit lactate accumulation will be discussed

Boundary of oxidative and overflow metabolism (boom) controller for CHO cell feed control

There is limited literature for CHO cell cultures with low batch glucose concentrations (Gowtham et al. 2017; Lu et al. 2005; Wong et al. 2005). Work like Xu et al. (2016) and Berry et al. (2016) have shown positive results for controlled fed-batch cultures at low glucose concentrations following standard high glucose (5-6 g/L) batch cultures. However, the Xu et al. (2016) and Berry et al. (2016) approaches still accumulate lactate. Controlling glucose earlier could potentially avoid lactate accumulation and lead to even greater improvements in culture outcomes. The objective of this project was to develop an advanced feed controller for CHO cell cultures that maximizes cell growth by maintaining the culture in a state of maximal oxidative metabolism while minimizing overflow metabolism. The Boundary of Oxidative and Overflow Metabolism (BOOM) controller periodically manipulates the feed rate while monitoring online signals to gauge the remaining oxidative “space”, in order to decide whether feed can be increased while remaining in oxidative metabolism. The Oxygen Uptake Rate (OUR) is the primary signal of interest, since it plateaus when a culture shifts from oxidative to overflow metabolism, encoding vital information about metabolic state. This project’s approach is different from past work in that the batch glucose concentrations is much lower (on the order of 1 g/L), the glucose and/or glutamine feeding begins very early in the process, and glucose feed is triggered/controlled by the off-gas sensing of the metabolic state instead of a targeted glucose concentration. During early runs several chemistry effects were observed directly due to the bolus feed additions interfering with the media-dissolved gas equilibrium. For example, a bolus feed that only contained 5 mM bicarbonate, resulted in an observed short sharp decrease in CO2 off-gas as the feed absorbed CO2 from the 5% CO2 sparge gas. Continuous feeding was introduced in subsequent runs as a means to mitigate disrupting the media-dissolved gas-equilibrium and disturbing the off-gas sensing. In order to have effective continuous feeding, the feed pump used a pulse width modulation (PWM) with a 10-minute period to allow extremely low effective feed rates required for the 1-L vessel. Two runs were used to demonstrate that the PWM feed pump could provide these very low pump feed rates for the 1-L vessel containing as little as 500 mL media. Initial glucose concentrations between 0.6 to 2.0 g/L were used (compared to 8 g/L glucose in the standard media formulation). Feedings have started between 6- and 20-hour post-inoculation. Distinct qualitative and quantitative differences have been observed in the corresponding oxygen uptake rate (OTR) responses due to the feeding spikes, suggesting that metabolic state can be detected. The development of the state estimator to control glucose feeding will be presented

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

Towards a universal CHO reference platform with epigenome characterization for the biotechnology community

Chinese Hamster ovary (CHO) cells are widely used both by academic researchers and in the biotechnology industry. However, comparing studies across a wide spectrum of labs in the CHO community has been challenging due to the different variants of host cell line, culture media and proteins of interest used in the individual laboratories. Unfortunately, unlike other communities, there is no standard CHO platform that can be used as a baseline for experimentation and evaluation, leading to a limited understanding of how a result or innovation from one group may be applied to another group. This limits the pace at which innovations in cell line development are achieved by the community. As a result, there is a growing need to create and establish a common platform with the goal of comparability and compatibility across the CHO bioprocessing community. The Advanced Mammalian Biomanufacturing Innovation Center (AMBIC) is a US based academic-industrial-government collaborative initiative dedicated to developing improved upstream biomanufacturing methods. Together, AMBIC’s five academic and sixteen industrial members are working together to implement a newly developed CHO based reference platform that has performance characteristics similar to what is used in the industry. Our initial goals have been to identify reference production and host cell lines together with a common platform medium used in the production of model recombinant protein targets, including antibodies and other targets. These cell lines are also being used to develop standardized processes that can be comparable across AMBIC sites and within the CHO community. In concert, these reference platforms are being applied to evaluate and understand CHO cell line capabilities and processing parameters for improving the production platform. Our progress in establishing a reference cell host and the partner media and processing parameters will be described, and the role of such a reference standard in helping to define the scope of other AMBIC research endeavors will also be delineated. We believe such a reference CHO platform will facilitate a robust and dynamic CHO research and development environment and hasten progress in cell culture engineering in coming decades