Kinetic physico-chemical model for cell culture processes – applications and opportunities

Cell culture processes for production of recombinant proteins can be modeled to provide critical insights into the interrelationship between several parameters that impact the process performance and control. For the first time, we describe a model that incorporates pH control in combination with gas transfer to provide a more complete description of the physicochemical processes that occur during the entire course of the cell culture process. The model includes multi-component chemical equilibria involving carbonate, lactate and sodium hydroxide coupled with electroneutrality for calculation of pH. Further, the pH feedback control has been incorporated depending on levels of carbonate and lactate in the culture. Additionally, pH ramp, and pH dead-band controls have been included to facilitate simulation of real process conditions. For oxygen transfer, cascaded control including agitation, air flow and oxygen flow are implemented. Very limited actual data from at-scale or small scale studies are required for the model, essentially requiring only cell density profiles and lactate profiles. Other specific rates are readily calculated based on measurements. Simulations based on these models provide key relationships that provide a clear basis for designing control strategies for the entire process. Several scenarios, including the choice of base, the impact of lactate consumption and production, the impact of cascaded controls for oxygen transfer, as well as the buffer composition of the media have been simultaneously evaluated through simulations, resulting in valuable approaches to scale-up and scale-down design of cell culture processes. Case studies will be presented demonstrating some of the applications. Potential improvements and opportunities will also be presented

Improving bioreactor design through pH mapping of bioreactors employing Computational Fluid Dynamics coupled with equilibrium calculations

Computational Fluid Dynamics has proven to be a very valuable tool in predicting spatial heterogeneities in mixing systems and other vessels used in bioprocess operations. These calculations are performed by typically obtaining steady state velocity profiles and subsequently using tracer type of studies to understand mixing times and heterogeneities. Bioreactor operations involve semi-continuous addition of base of different types of relatively high concentrations and the rate of addition varies constantly as the cell culture process progresses. These additions cause the local pH profiles to be different from the bulk, given the heterogeneities in mixing as well as the position and rate of base addition. In this modeling and simulation study, CFD simulations of a bioreactor will be coupled with equilibrium calculations to predict pH profiles in bioreactors. Impact of addition point, agitation rates, impeller position as well as type and concentration of base used will be presented. Transient simulations assessing the impact of semi-continuous and bolus feeds will be assessed. Overall, this study provides the next level of understanding and control of bioreactor processes, with the potential to improve the processes as well as potentially improve product quality

Proteomic Profiling Across Breast Cancer Cell Lines and Models

We performed quantitative proteomics on 60 human-derived breast cancer cell line models to a depth of ~13,000 proteins. The resulting high-throughput datasets were assessed for quality and reproducibility. We used the datasets to identify and characterize the subtypes of breast cancer and showed that they conform to known transcriptional subtypes, revealing that molecular subtypes are preserved even in under-sampled protein feature sets. All datasets are freely available as public resources on the LINCS portal. We anticipate that these datasets, either in isolation or in combination with complimentary measurements such as genomics, transcriptomics and phosphoproteomics, can be mined for the purpose of predicting drug response, informing cell line specific context in models of signalling pathways, and identifying markers of sensitivity or resistance to therapeutics

High-Entropy 2D Carbide MXenes: TiVNbMoC3 and TiVCrMoC3

Two-dimensional (2D) transition metal carbides and nitrides, known as MXenes, are a fast-growing family of 2D materials. MXenes 2D flakes have n + 1 (n = 1–4) atomic layers of transition metals interleaved by carbon/nitrogen layers, but to-date remain limited in composition to one or two transition metals. In this study, by implementing four transition metals, we report the synthesis of multi-principal-element high-entropy M4C3Tx MXenes. Specifically, we introduce two high-entropy MXenes, TiVNbMoC3Tx and TiVCrMoC3Tx, as well as their precursor TiVNbMoAlC3 and TiVCrMoAlC3 high-entropy MAX phases. We used a combination of real and reciprocal space characterization (X-ray diffraction, X-ray photoelectron spectroscopy, energy dispersive X-ray spectroscopy, and scanning transmission electron microscopy) to establish the structure, phase purity, and equimolar distribution of the four transition metals in high-entropy MAX and MXene phases. We use first-principles calculations to compute the formation energies and explore synthesizability of these high-entropy MAX phases. We also show that when three transition metals are used instead of four, under similar synthesis conditions to those of the four-transition-metal MAX phase, two different MAX phases can be formed (i.e., no pure single-phase forms). This finding indicates the importance of configurational entropy in stabilizing the desired single-phase high-entropy MAX over multiphases of MAX, which is essential for the synthesis of phase-pure high-entropy MXenes. The synthesis of high-entropy MXenes significantly expands the compositional variety of the MXene family to further tune their properties, including electronic, magnetic, electrochemical, catalytic, high temperature stability, and mechanical behavior

Human Embryonic and Rat Adult Stem Cells with Primitive Endoderm-Like Phenotype Can Be Fated to Definitive Endoderm, and Finally Hepatocyte-Like Cells

Stem cell-derived hepatocytes may be an alternative cell source to treat liver diseases or to be used for pharmacological purposes. We developed a protocol that mimics mammalian liver development, to differentiate cells with pluripotent characteristics to hepatocyte-like cells. The protocol supports the stepwise differentiation of human embryonic stem cells (ESC) to cells with characteristics of primitive streak (PS)/mesendoderm (ME)/definitive endoderm (DE), hepatoblasts, and finally cells with phenotypic and functional characteristics of hepatocytes. Remarkably, the same protocol can also differentiate rat multipotent adult progenitor cells (rMAPCs) to hepatocyte-like cells, even though rMAPC are isolated clonally from cultured rat bone marrow (BM) and have characteristics of primitive endoderm cells. A fraction of rMAPCs can be fated to cells expressing genes consistent with a PS/ME/DE phenotype, preceding the acquisition of phenotypic and functional characteristics of hepatocytes. Although the hepatocyte-like progeny derived from both cell types is mixed, between 10–20% of cells are developmentally consistent with late fetal hepatocytes that have attained synthetic, storage and detoxifying functions near those of adult hepatocytes. This differentiation protocol will be useful for generating hepatocyte-like cells from rodent and human stem cells, and to gain insight into the early stages of liver development

Scalable expansion of multipotent adult progenitor cells as three-dimensional cell aggregates

Many applications of stem cell technologies require a large quantity of cells for which scalable processes of cell expansion and differentiation are essential. Multipotent adult progenitor cells (MAPCs) are adult stem cells isolated from the bone marrow with extensive self-renewal and broad differentiation capabilities. MAPCs are typically cultured surface adherent (2D) and at low cell density, making the large surface required for cell expansion a hindrance for many applications. This study demonstrates that MAPCs can be cultivated as aggregates in an undifferentiated state for at least 16 days, as levels of a number of transcripts, including Oct4, remained similar, Oct4 protein was unchanged, and differentiation to neural progenitor, endothelial cell and hepatocyte like cells was retained. Cultivation of these aggregates in stirred bioreactor lead to a 70-fold expansion in 6 days with final cell densities of close to 10⁶/mL. Importantly, the MAPC aggregates recovered from stirred bioreactors could be differentiated to hepatocyte-like cells that expressed albumin, alpha-1-antitrypsin (AAT), and tyrosine amino transferase (TAT) transcripts and also secreted albumin and urea. This method of scalable expansion combined with differentiation of MAPCs can potentially be used for generating large numbers of MAPC and MAPC-derived differentiated cells.status: publishe

Potential Role of a Bistable Histidine Kinase Switch in the Asymmetric Division Cycle of <i>Caulobacter crescentus</i>

<div><p>The free-living aquatic bacterium, <i>Caulobacter crescentus</i>, exhibits two different morphologies during its life cycle. The morphological change from swarmer cell to stalked cell is a result of changes of function of two bi-functional histidine kinases, PleC and CckA. Here, we describe a detailed molecular mechanism by which the function of PleC changes between phosphatase and kinase state. By mathematical modeling of our proposed molecular interactions, we derive conditions under which PleC, CckA and its response regulators exhibit bistable behavior, thus providing a scenario for robust switching between swarmer and stalked states. Our simulations are in reasonable agreement with <i>in vitro</i> and <i>in vivo</i> experimental observations of wild type and mutant phenotypes. According to our model, the kinase form of PleC is essential for the swarmer-to-stalked transition and to prevent premature development of the swarmer pole. Based on our results, we reconcile some published experimental observations and suggest novel mutants to test our predictions.</p></div

Dynamical Localization of DivL and PleC in the Asymmetric Division Cycle of <i>Caulobacter crescentus</i>: A Theoretical Investigation of Alternative Models

<div><p>Cell-fate asymmetry in the predivisional cell of <i>Caulobacter crescentus</i> requires that the regulatory protein DivL localizes to the new pole of the cell where it up-regulates CckA kinase, resulting in a gradient of CtrA~P across the cell. In the preceding stage of the cell cycle (the “stalked” cell), DivL is localized uniformly along the cell membrane and maintained in an inactive form by DivK~P. It is unclear how DivL overcomes inhibition by DivK~P in the predivisional cell simply by changing its location to the new pole. It has been suggested that co-localization of DivL with PleC phosphatase at the new pole is essential to DivL’s activity there. However, there are contrasting views on whether the bifunctional enzyme, PleC, acts as a kinase or phosphatase at the new pole. To explore these ambiguities, we formulated a mathematical model of the spatiotemporal distributions of DivL, PleC and associated proteins (DivJ, DivK, CckA, and CtrA) during the asymmetric division cycle of a <i>Caulobacter</i> cell. By varying localization profiles of DivL and PleC in our model, we show how the physiologically observed spatial distributions of these proteins are essential for the transition from a stalked cell to a predivisional cell. Our simulations suggest that PleC is a kinase in predivisional cells, and that, by sequestering DivK~P, the kinase form of PleC enables DivL to be reactivated at the new pole. Hence, co-localization of PleC kinase and DivL is essential to establishing cellular asymmetry. Our simulations reproduce the experimentally observed spatial distribution and phosphorylation status of CtrA in wild-type and mutant cells. Based on the model, we explore novel combinations of mutant alleles, making predictions that can be tested experimentally.</p></div