From bioreactors for protein therapeutic production to bioreactors for testing efficacy and safety of protein therapeutics

The tremendous successes of mammalian cell culture engineering since the 1980’s made our modern era of protein therapeutics a reality, bringing tremendous new possibilities for targeted intervention, but also additional challenges in pre-clinical development due to the species specificity of biologics. While the efficacy and safety of all drugs intended for humans is often difficult to extrapolate from assays in animal models and traditional cell cultures, the situation is significantly more difficult for biologics. Traditional cell culture modes, even with human cells, often fail to capture the complexity of pathways, which may involve multiple cell types and cross-talk between different organ systems. The species specificity of most biologics precludes testing in most common animal models. Thus, adverse events are observed in the clinic due to lack of adequate predictive models. For example, the anti IL-6 receptor Tocilizumab, developed to treat chronic inflammatory diseases like arthritis, earned a warning label from the FDA after clinical evidence that the metabolism of statins and other drugs was altered by Tocilizumab in ways that were not predicted by pre-clinical models. (Long, Cosgrove et al. 2016). The modern field of “organs-on-chips” or “microphysiological systems (MPS)” is poised to address these gaps, and is coming full circle back to the wealth of knowledge about cell culture bioreactor performance produced by the therapeutic protein field. The field of “organs on chips” had its origins several decades ago with demonstrations by Michael Shuler and others that facets of human drug pharmacokinetics could be replicated by interconnected cultures of various cell lines (liver, fat, etc). These initial demonstrations were powerful but ultimately limited by the simplicity of the cell cultures – cell lines that mimicked only modest facets of the functions of the in vivo organ system. Over the past decades, tremendous advances in microfluidics, biomaterials, and technologies to process and make available primary human cells have dramatically increased the human-ness of in vitro cultures. Interestingly, cell lines are also still commonly used, and there has not yet been a thoughtful appreciation of how the basic metabolic functions of the tissue engineered constructs may affect the performance of these systems. Special challenges exist in formulating common media, for example, in interconnected organ systems where some cells are primary, some are tumor-derived lines, and yet others are from iPS-derived sources. In this talk, I will highlight the past, present and future interplay between these two dynamic, vibrant fields, with illustrations in particular of how the organs-on-chips technologies are poised to aid the therapeutic protein production field. Long, T., P. Cosgrove, R. N. Dunn, D. Stolz, H. Hamadeh, C. Afshari, H. McBride and L. Griffith (2016). Modeling Therapeutic Antibody-Small Molecule Drug-Drug Interactions Using a Three Dimensional Perfusable Human Liver Coculture Platform. Drug Metab Dispos 44: 1940-1948

Surface Tethered Epidermal Growth Factor Protects Proliferating and Differentiating Multipotential Stromal Cells from FasL-Induced Apoptosis

Multipotential stromal cells or mesenchymal stem cells (MSCs) have been proposed as aids in regenerating bone and adipose tissues, as these cells form osteoblasts and adipocytes. A major obstacle to this use of MSC is the initial loss of cells postimplantation. This cell death in part is due to ubiquitous nonspecific inflammatory cytokines such as FasL generated in the implant site. Our group previously found that soluble epidermal growth factor (sEGF) promotes MSC expansion. Furthermore, tethering EGF (tEGF) onto a two-dimensional surface altered MSC responses, by restricting epidermal growth factor receptor (EGFR) to the cell surface, causing sustained activation of EGFR, and promoting survival from FasL-induced death. sEGF by causing internalization of EGFR does not support MSC survival. However, for tEGF to be useful in bone regeneration, it needs to allow for MSC differentiation into osteoblasts while also protecting emerging osteoblasts from apoptosis. tEGF did not block induced differentiation of MSCs into osteoblasts, or adipocytes, a common default MSC-differentiation pathway. MSC-derived preosteoblasts showed increased Fas levels and became more susceptible to FasL-induced death, which tEGF prevented. Differentiating adipocytes underwent a reduction in Fas expression and became resistant to FasL-induced death, with tEGF having no further survival effect. tEGF protected undifferentiated MSC from combined insults of FasL, serum deprivation, and physiologic hypoxia. Additionally, tEGF was dominant in the face of sEGF to protect MSC from FasL-induced death. Our results suggest that MSCs and differentiating osteoblasts need protective signals to survive in the inflammatory wound milieu and that tEGF can serve this function.National Institute of General Medical Sciences (U.S.) (GM069668)National Institute of Dental and Craniofacial Research (U.S.) (DE019523

Growth factor regulation of proliferation and survival of multipotential stromal cells

Multipotential stromal cells (MSCs) have been touted to provide an alternative to conservative procedures of therapy, be it heart transplants, bone reconstruction, kidney grafts, or skin, neuronal and cartilage repair. A wide gap exists, however, between the number of MSCs that can be obtained from the donor site and the number of MSCs needed for implantation to regenerate tissue. Standard methods of MSC expansion being followed in laboratories are not fully suitable due to time and age-related constraints for autologous therapies, and transplant issues leave questions for allogenic therapies. Beyond these issues of sufficient numbers, there also exists a problem of MSC survival at the graft. Experiments in small animals have shown that MSCs do not persist well in the graft environment. Either there is no incorporation into the host tissue, or, if there is incorporation, most of the cells are lost within a month. The use of growth and other trophic factors may be helpful in counteracting these twin issues of MSC expansion and death. Growth factors are known to influence cell proliferation, motility, survival and morphogenesis. In the case of MSCs, it would be beneficial that the growth factor does not induce differentiation at an early stage since the number of early-differentiating progenitors would be very low. The present review looks at the effect of and downstream signaling of various growth factors on proliferation and survival in MSCs

Engineering liver

Interest in “engineering liver” arises from multiple communities: therapeutic replacement; mechanistic models of human processes; and drug safety and efficacy studies. An explosion of micro- and nanofabrication, biomaterials, microfluidic, and other technologies potentially affords unprecedented opportunity to create microphysiological models of the human liver, but engineering design principles for how to deploy these tools effectively toward specific applications, including how to define the essential constraints of any given application (available sources of cells, acceptable cost, and user-friendliness), are still emerging. Arguably less appreciated is the parallel growth in computational systems biology approaches toward these same problems—particularly in parsing complex disease processes from clinical material, building models of response networks, and in how to interpret the growing compendium of data on drug efficacy and toxicology in patient populations. Here, we provide insight into how the complementary paths of engineering liver—experimental and computational—are beginning to interplay toward greater illumination of human disease states and technologies for drug development.National Institutes of Health (U.S.) (UH2TR000496)National Institutes of Health (U.S.) (R01-EB 010246)National Institutes of Health (U.S.) (R01-ES015241)National Institutes of Health (U.S.) (P30-ES002109

The food safety culture in a large South African food service complex: Perspectives on a case study

Published ArticleThe purpose of this paper is to assess elements of food safety management and food safety culture within a prominent South African entertainment, hotel and food service complex. Design/methodology/approach In this paper a qualitative case study approach was used. Following a comprehensive literature review, based on factors known to be important in developing a food safety culture, in combination with national and international food safety standards, an interview guide was constructed and utilised in a series of semi-structured interviews. The interviewees represented different management levels involved in food delivery but did not include board level managers. Findings Many of the factors considered important in good food safety management, including the presence of a formal food safety policy and the creation and maintenance of a positive food safety culture, were absent. Although a formal system of internal hygiene auditing existed and food safety training was provided to food handlers they were not integrated into a comprehensive approach to food safety management. Food safety leadership, communication and support were considered deficient with little motivation for staff to practise good hygiene. Originality/value Food safety culture is increasingly recognised as a contributory factor in foodborne disease outbreaks and is the focus of increasing research. However, although every food business has a unique food safety culture there are relatively few published papers concerning its analysis, application and use within specific businesses. This case study has identified food safety culture shortcomings within a large food service facility suggesting there was a potentially significant food safety risk and indicates ways in which food safety could be improved and the risk reduced. The results also suggest further work is needed in the subject of food safety culture and its potential for reducing foodborne disease

Controlling multipotent stromal cell migration by integrating “course-graining” materials and “fine-tuning” small molecules via decision tree signal-response modeling

Biomimetic scaffolds have been proposed as a means to facilitate tissue regeneration by multi-potent stromal cells (MSCs). Effective scaffold colonization requires a control of multiple MSC responses including survival, proliferation, differentiation, and migration. As MSC migration is relatively unstudied in this context, we present here a multi-level approach to its understanding and control, integratively tuning cell speed and directional persistence to achieve maximal mean free path (MFP) of migration. This approach employs data-driven computational modeling to ascertain small molecule drug treatments that can enhance MFP on a given materials substratum. Using poly(methyl methacrylate)-graft-poly(ethylene oxide) polymer surfaces tethered with epidermal growth factor (tEGF) and systematically adsorbed with fibronectin, vitronectin, or collagen-I to present hTERT-immortalized human MSCs with growth factor and extracellular matrix cues, we measured cell motility properties along with signaling activities of EGFR, ERK, Akt, and FAK on 19 different substrate conditions. Speed was consistent on collagen/tEGF substrates, but low associated directional persistence limited MFP. Decision tree modeling successfully predicted that ERK inhibition should enhance MFP on collagen/tEGF substrates by increasing persistence. Thus, we demonstrated a two-tiered approach to control MSC migration: materials-based “coarse-graining” complemented by small molecule “fine-tuning”.National Institutes of Health (U.S.) (NIH grant R01-DE019523)National Institutes of Health (U.S.) (NIH Cell Migration Consortium U54-GM064346)National Institutes of Health (U.S.) (NIH grant R01-GM018336)National Institutes of Health (U.S.) (NIH grant R01-DE019523

In vitro models for liver toxicity testing

Over the years, various liver-derived in vitro model systems have been developed to enable investigation of the potential adverse effects of chemicals and drugs. Liver tissue slices, isolated microsomes, perfused liver, immortalized cell lines, and primary hepatocytes have been used extensively. Immortalized cell lines and primary isolated liver cells are currently the most widely used in vitro models for liver toxicity testing. Limited throughput, loss of viability, and decreases in liver-specific functionality and gene expression are common shortcomings of these models. Recent developments in the field of in vitro hepatotoxicity include three-dimensional tissue constructs and bioartificial livers, co-cultures of various cell types with hepatocytes, and differentiation of stem cells into hepatic lineage-like cells. In an attempt to provide a more physiological environment for cultured liver cells, some of the novel cell culture systems incorporate fluid flow, micro-circulation, and other forms of organotypic microenvironments. Co-cultures aim to preserve liver-specific morphology and functionality beyond those provided by cultures of pure parenchymal cells. Stem cells, both embryonic- and adult tissue-derived, may provide a limitless supply of hepatocytes from multiple individuals to improve reproducibility and enable testing of the individual-specific toxicity. This review describes various traditional and novel in vitro liver models and provides a perspective on the challenges and opportunities afforded by each individual test system.National Institutes of Health (U.S.) (P42 ES005948)National Institutes of Health (U.S.) (R01 ES01524