Nicholas E. Timmins

Towards quantitative metabolomics of mammalian cells: Development of a metabolite extraction protocol

Metabolomics aims to quantify all metabolites within an organism, thereby providing valuable insight into the metabolism of cells. To study intracellular metabolites, they are first extracted from the cells. The ideal extraction procedure should immediately quench metabolism and quantitatively extract all metabolites, a significant challenge given the rapid turnover and physicochemical diversity of intracellular metabolites. We have evaluated several quenching and extraction solutions for their suitability for mammalian cells grown in suspension. Quenching with 60% methanol (buffered or unbuffered) resulted in leakage of intracellular metabolites from the cells. In contrast, quenching with cold isotonic saline (0.9% [w/v] NaCl, 0.5 degrees C) did not damage cells and effectively halted conversion of ATP to ADP and AMP, indicative of metabolic arrest. Of the 12 different extraction methods tested, cold extraction in 50% aqueous acetonitrile was superior to other methods. The recovery of a mixture of standards was excellent, and the concentration of extracted intracellular metabolites was higher than for the other methods tested. The final protocol is easy to implement and can be used to study the intracellular metabolomes of mammalian cells.

Hanging-drop multicellular spheroids as a model of tumour angiogenesis

The establishment of a vascular network within tumours is a key step in the progression towards an aggressive, metastatic state, with poor prognosis. We have developed a novel in vitro model to specifically capture the interaction between endothelial cells and solid tumours. Micro-vascularised in vitro tumour constructs were produced by introducing endothelial cells to multicellular spheroids formed in hanging drops. Upon introduction, the endothelial cells migrated into the tumour spheroid, establishing tubular networks and luminal structures. This system relies on the natural pro-angiogenic capacity of multicellular spheroids, and does not require the addition of exogenous angiogenic factors, or use of extracellular-matrix substitutes.

Generation of multicellular tumor spheroids by the hanging-drop method

Owing to their in vivo-like characteristics, three-dimensional (3D) multicellular tumor spheroid (MCTS) cultures are gaining increasing popularity as an in vitro model of tumors. A straightforward and simple approach to the cultivation of these MCTS is the hanging-drop method. Cells are suspended in droplets of medium, where they develop into coherent 3D aggregates and are readily accessed for analysis. In addition to being simple, the method eliminates surface interactions with an underlying substratum (e.g., polystyrene plastic or agarose), requires only a low number of starting cells, and is highly reproducible. This method has also been applied to the co-cultivation of mixed cell populations, including the co-cultivation of endothelial cells and tumor cells as a model of early tumor angiogenesis.

Metabolite profiling of CHO cells with different growth characteristics

Mammalian cell cultures are the predominant system for the production of recombinant proteins requiring post‐translational modifications. As protein yields are a function of growth performance (among others), and performance varies greatly between culture medium (e.g., different growth rates and peak cell densities), an understanding of the biological mechanisms underpinning this variability would facilitate rational medium and process optimization, increasing product yields, and reducing costs. We employed a metabolomics approach to analyze differences in metabolite concentrations of CHO cells cultivated in three different media exhibiting different growth rates and maximum viable cell densities. Analysis of intra‐ and extracellular metabolite concentrations over the course of the cultures using a combination of HPLC and GC‐MS, readily detected medium specific and time dependent changes. Using multivariate data analysis, we identified a range of metabolites correlating with growth rate, illustrating how metabolomics can be used to relate gross phenotypic changes to the fine details of cellular metabolism. Biotechnol. Bioeng. 2012; 109:1404–1414.

Closed system isolation and scalable expansion of human placental mesenchymal stem cells.

Mesenchymal stem cells (MSC) are emerging as a leading cellular therapy for a number of diseases. However, for such treatments to become available as a routine therapeutic option, efficient and cost‐effective means for industrial manufacture of MSC are required. At present, clinical grade MSC are manufactured through a process of manual cell culture in specialized cGMP facilities. This process is open, extremely labor intensive, costly, and impractical for anything more than a small number of patients. While it has been shown that MSC can be cultivated in stirred bioreactor systems using microcarriers, providing a route to process scale‐up, the degree of numerical expansion achieved has generally been limited. Furthermore, little attention has been given to the issue of primary cell isolation from complex tissues such as placenta. In this article we describe the initial development of a closed process for bulk isolation of MSC from human placenta, and subsequent cultivation on microcarriers in scalable single‐use bioreactor systems. Based on our initial data, we estimate that a single placenta may be sufficient to produce over 7,000 doses of therapeutic MSC using a large‐scale process. Biotechnol. Bioeng. 2012; 109:1817–1826.

A Multi-Omics Analysis of Recombinant Protein Production in Hek293 Cells

Hek293 cells are the predominant hosts for transient expression of recombinant proteins and are used for stable expression of proteins where post-translational modifications performed by CHO cells are inadequate. Nevertheless, there is little information available on the key cellular features underpinning recombinant protein production in Hek293 cells. To improve our understanding of recombinant protein production in Hek293 cells and identify targets for the engineering of an improved host cell line, we have compared a stable, recombinant protein producing Hek293 cell line and its parental cell line using a combination of transcriptomics, metabolomics and fluxomics. Producer cultures consumed less glucose than non-producer cultures while achieving the same growth rate, despite the additional burden of recombinant protein production. Surprisingly, there was no indication that producer cultures compensated for the reduction in glycolytic energy by increasing the efficiency of glucose utilization or increasing glutamine consumption. In contrast, glutamine consumption was lower and the majority of genes involved in oxidative phosphorylation were downregulated in producer cultures. We observed an overall downregulation of a large number of genes associated with broad cellular functions (e.g., cell growth and proliferation) in producer cultures, and therefore speculate that a broad adaptation of the cellular network freed up resources for recombinant protein production while maintaining the same growth rate. Increased abundance of genes associated with endoplasmic reticulum stress indicated a possible bottleneck at the point of protein folding and assembly.

Mammalian cells as biopharmaceutical production hosts in the age of omics

Mammalian cells are important hosts for the production of a wide range of biopharmaceuticals due to their ability to produce correctly folded and glycosylated proteins. Compared to microbes and yeast, however, the productivity of mammalian cells is low because of their comparatively slow growth rate, tendency to undergo apoptosis, and low production capacities. While much effort has been invested in the engineering of mammalian cells with superior production characteristics, the success of these approaches has been limited to date. One factor responsible for this lack of success is our limited understanding of the cellular basis for high productivity, and of how discrete mechanisms within a cell contribute to the overall phenotype. Aiming to measure and characterize all cellular components at different functional levels, omics technologies have the potential to improve our understanding of mammalian cell physiology, elucidating new targets for the generation of a superior host cell line. This review provides a comprehensive analysis of recent examples of omics studies in the context of mammalian cells as production hosts, highlighting both the challenges and successes in the application of these powerful technologies.

Method for the generation and cultivation of functional three-dimensional mammary constructs without exogenous extracellular matrix

During puberty, pregnancy, lactation and post-lactation, breast tissue undergoes extensive remodelling and the disruption of these events can lead to cancer. In vitro studies of mammary tissue and its malignant transformation regularly employ mammary epithelial cells cultivated on matrigel or floating collagen rafts. In these cultures, mammary epithelial cells assemble into three-dimensional structures resembling in vivo acini. We present a novel technique for generating functional mammary constructs without the use of matrix substitutes.

Clinical scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells

Dose‐intensive chemotherapy results in an obligatory period of severe neutropenia during which patients are at high risk of infection. While patient support with donor neutrophils is possible, this option is restricted due to donor availability and logistic complications. To overcome these problems, we explored the possibility of large scale ex vivo manufacture of neutrophils from hematopoietic progenitor cells (HPC). CD34+ HPC isolated from umbilical cord blood (UCB) and mobilized peripheral blood (mPB) were expanded in serum‐free medium supplemented with stem cell factor, granulocyte colony stimulating factor, and a thrombopoietin peptide mimetic. After 15 days of cultivation a 5,800‐fold expansion in cell number was achieved for UCB, and up to 4,000‐fold for mPB, comprising 40% and 60% mature neutrophils respectively. Ex vivo expanded neutrophils exhibited respiratory burst activity similar to that for donor neutrophils, and were capable of killing Candida albicans in vitro. These yields correspond to a more than 10‐fold improvement over current methods, and are sufficient for the production of multiple neutrophil transfusion doses per HPC donation. To enable clinical scale manufacture, we adapted our protocol for use in a wave‐type bioreactor at a volume of 10 L. This is the first demonstration of a large scale bioprocess suitable for routine manufacture of a mature blood cell product from HPC, and could enable prophylactic neutrophil support for chemotherapy patients. Biotechnol. Bioeng. 2009; 104: 832–840

Manufactured RBC - Rivers of blood, or an oasis in the desert?

Red blood cell (RBC) transfusion is an essential practice in modern medicine, one that is entirely dependent on the availability of donor blood. Constraints in donor supply have led to proposals that transfusible RBC could be manufactured from stem cells. While it is possible to generate small amounts of RBC in vitro, very large numbers of cells are required to be of clinical significance. We explore the challenges facing large scale manufacture of RBC and technological developments required for such a scenario to be realised.