Advanced cell culture technology for generation of in vivo-like tissue models

Human tissues are mostly composed of different cell types, that are often highly organised in relation to each other. Often cells are arranged in distinct layers that enable signalling and cell-to-cell interactions. Here we describe the application of scaffold-based technology, that can be used to create advanced organotypic 3D models of various tissue types that more closely resemble in vivo-like conditions (Knight et al., 2011). The scaffold comprises a highly porous polystyrene material, engineered into a 200 micron thick membrane that is presented in various ways including multi-welled plates and well inserts, for use with conventional culture plasticware and medium perfusion systems. This technology has been applied to generate numerous unique types of co-culture model. For example: 1) a full thickness human skin construct comprising dermal fibroblasts and keratinocytes, raised to the air-liquid interface to induce cornification of the upper layers (Fig.1) (Hill et al., 2015); 2) a neuron-glial co-culture to enable the study of neurite outgrowth interacting with astroglial cells to model and investigate the glial scar found in spinal cord injury (Clarke et al., 2016); 3) formation of a sub-mucosa consisting of a polarised simple epithelium, layer of ECM proteins simulating the basement membrane, and underlying stromal tissues (e.g. intestinal mucosa). These organotypic models demonstrate the versatility of scaffold membranes and the creation of advanced in vivo-like tissue models. Creating a layered arrangement more closely simulates the true anatomy and organisation of cells within many tissue types. The addition of different cell types in a temporal and spatial fashion can be used to study inter-cellular relationships and create more physiologically relevant in vivo-like cell-based assays. Methods that are relatively straightforward to use and that recreate the organised structure of real tissues will become valuable research tools for use in discovery, validation studies, and modeling disease

A destabilisation domain approach to define the in vivo functional importance of PfHsp70-1 and PfHsp40 in the intraerythrocytic life cycle of Plasmodium falciparum

The apicomplexan malaria parasite, Plasmodium falciparum is capable of invading red blood cells and causes the most virulent form of malaria. The life cycle of P. falciparum involves the migration from the poikilothermic mosquito vector to warm-blooded human host and vice versa. Such transition introduces radical differences between the cellular environments where the parasite resides, imposing physiological stress. The diverse environmental insults in addition to the febrile fever episodes imparts challenge on the proteostasis, resulting in the evolutionary selection of a diverse network of molecular chaperones. In fact, some molecular chaperones are essential for the survival of Plasmodium. Due to the developing resistance of Plasmodium against currently available drugs, heat shock proteins have received extensive research attention as antimalarial targets in recent years. Plasmodium codes for one Hsp90 homologue and a constitutively expressed heat inducible cytosolic Hsp70 known as PfHsp70-1. In general, Hsp70 interacts with co-chaperone Hsp40 initiating the protein folding machinery that finally interacts with Hsp90 to maintain proteostasis in a cell. PfHsp90 has been found to be essential for the intraerythrocytic development of P. falciparum. Although there have been some in vitro studies on the biology of PfHsp70-1, the information on the in vivo essential function of PfHsp70-1 and its interaction with PfHsp40 is limited. In this study, we wanted to identify the in vivo biological importance of PfHsp70-1 and one of its predicted co-chaperones, PfHsp40 by the overexpression of the dominant negative alleles tagged to recently characterised destabilisation domain (dd) to regulate protein level. We expressed a dominant negative PfHsp70-1 possessing a point mutation (E187K), severely affecting normal domain movement important for its function. PfHsp40 was mutated in the conserved HPD motif (D34N) necessary for establishing interaction with PfHsp70-1. Unfortunately, we could not obtain sufficient overexpression of the episomal dominant negative versions to override the function of the endogenous proteins in a competitive manner. The cellular levels of endogenous proteins were higher by several folds compared to that of the episomally expressed dominant negative alleles. The destabilisation strategy has been reported to be successful for studying certain plasmodial proteins. But in contrast, during our work the level of almost none of the candidate chaperones could be controlled by either FKBP or E. coli DHFR derived destabilisation domains in a ligand dependent fashion. Although, the level of wild type PfHsp70-1 could be regulated by this strategy, the dominant negative version with only one amino acid substitution made it non responsive to dd tagging and further ligand treatment. At the same time, the level of control proteins could be efficiently regulated by stabilising ligands. Recently, success of destabilization domain strategy for conditional knockdown of several genes has been reported. But in contrast, our observations in this study unravel the possible drawbacks. We assume that the success of such an approach is greatly protein dependent. Based on the several reports initially this approach appeared to be the most beneficial system. But, the failure to successfully implement this strategy demands careful consideration in selecting an alternative future approach to study the function of essential genes in Plasmodium

Design considerations of benchtop fluid flow bioreactors for bio-engineered tissue equivalents in vitro

One of the major aims of bio-engineering tissue equivalents in vitro is to create physiologically relevant culture conditions to accurately recreate the cellular microenvironment. This often includes incorporation of factors such as the extracellular matrix, co-culture of multiple cell types and three-dimensional culture techniques. These advanced techniques can recapitulate some of the properties of tissue in vivo, however fluid flow is a key aspect that is often absent. Fluid flow can be introduced into cell and tissue culture using bioreactors, which are becoming increasingly common as we seek to produce increasingly accurate tissue models. Bespoke technology is continuously being developed to tailor systems for specific applications and to allow compatibility with a range of culture techniques. For effective perfusion of a tissue culture many parameters can be controlled, ranging from impacts of the fluid flow such as increased shear stress and mass transport, to potentially unwanted side effects such as temperature fluctuations. A thorough understanding of these properties and their implications on the culture model can aid with a more accurate interpretation of results. Improved and more complete characterisation of bioreactor properties will also lead to greater accuracy when reporting culture conditions in protocols, aiding experimental reproducibility, and allowing more precise comparison of results between different systems. In this review we provide an analysis of the different factors involved in the development of benchtop flow bioreactors and their potential biological impacts across a range of applications

An in vitro model to study immune activation, epithelial disruption and stromal remodelling in inflammatory bowel disease and fistulising Crohn’s disease

At present, preclinical models of inflammatory bowel disease (IBD) are insufficient, limiting translation between research and new therapeutics. This is especially true for fistulising Crohn’s disease (CD), as the severe lack of relevant models hinders research progression. To address this, we present in vitro human IBD mucosal models that recapitulate multiple pathological hallmarks of IBD simultaneously in one model system - immune cell infiltration, stromal remodelling and epithelial disruption. Stimulation of models induces epithelial aberrations common in IBD tissue including altered morphology, microvilli abnormalities, claudin gene expression changes and increased permeability. Inflammatory biomarkers are also significantly increased including cytokines and chemokines integral to IBD pathogenesis. Evidence of extracellular matrix remodelling, including upregulated matrix-metalloproteinases and altered basement membrane components, suggests the models simulate pathological stromal remodelling events that closely resemble fistulising CD. Importantly, MMP-9 is the most abundant MMP and mimics the unique localisation observed in IBD tissue. The inflamed models were subsequently used to elucidate the involvement of TNF-α and IFN- γ in intestinal stromal remodelling, in which TNF-α but not IFN- γ induced MMP upregulation, specifically of MMP-3 and MMP-9. Collectively, our results demonstrate the potential of the IBD models for use in preclinical research in IBD, particularly for fistulising CD

”CyberWorld” as a Theme for a University-wide First-year Common Course

Nowadays we all live in a cyber world and use the internet for emailing, banking, streaming video, shopping, reading news, or other activities. Given all the time people spend online, it is important that all students (regardless of their major) learn some basics about living in a cyber world, e.g., strategies for online safety, impact of artificial intelligence, digital forensics or ancestry.com. To facilitate students from many majors to learn about important issues related to the internet, eight faculty from a variety of disciplines at the University of New Haven integrated the theme of Cyber World into our team-taught, first-year experience course, also referred to as the “Common Course.” The Common Course’s primary purpose is to enable students to develop evidence-based arguments and to challenge their own and others’ assumptions in relation to that evidence. Each Common Course class focuses on a broad topic (e.g., Justice, Happiness, or Identity) that instructors use as a touch point to facilitate critical thinking. In Cyber World, however, the topic is given stronger focus, and all students in the class are expected to come away with specific cyber-related knowledge. A special challenge is that the majority of the 160 students are from non-STEM majors. Given the varied background of students, this course covers a variety of topics such as sharing DNA with ancestry.com, protecting against identity theft, detecting fake news, and oversharing personal information. The course is taught by eight faculty members from four different colleges having expertise in a variety of disciplines. An important side effect of this faculty diversity is that interdisciplinary collaborations among faculty are promoted. Our paper has three significant contributions: (1) We present the eight topics related to living in a cyber world that we chose for this course, including our rationale for why they are appropriate and relevant; (2) We summarize how we integrated the Cyber World topics into the structure of the Common Course, which includes a discussion of the challenges we faced; and (3) We summarize some initial results on how students perceived their experience as well as how they performed compared to other common course sections / topics

Galactose-Functionalized PolyHIPE Scaffolds for Use in Routine Three Dimensional Culture of Mammalian Hepatocytes

Three-dimensional (3D) cell culture is regarded as a more physiologically relevant method of growing cells in the laboratory compared to traditional monolayer cultures. Recently, the application of polystyrene-based scaffolds produced using polyHIPE technology (porous polymers derived from high internal phase emulsions) for routine 3D cell culture applications has generated very promising results in terms of improved replication of native cellular function in the laboratory. These materials, which are now available as commercial scaffolds, are superior to many other 3D cell substrates due to their high porosity, controllable morphology, and suitable mechanical strength. However, until now there have been no reports describing the surface-modification of these materials for enhanced cell adhesion and function. This study, therefore, describes the surface functionalization of these materials with galactose, a carbohydrate known to specifically bind to hepatocytes via the asialoglycoprotein receptor (ASGPR), to further improve hepatocyte adhesion and function when growing on the scaffold. We first modify a typical polystyrene-based polyHIPE to produce a cell culture scaffold carrying pendent activated-ester functionality. This was achieved via the incorporation of pentafluorophenyl acrylate (PFPA) into the initial styrene (STY) emulsion, which upon polymerization formed a polyHIPE with a porosity of 92% and an average void diameter of 33 μm. Histological analysis showed that this polyHIPE was a suitable 3D scaffold for hepatocyte cell culture. Galactose-functionalized scaffolds were then prepared by attaching 2′-aminoethyl-β-D-galactopyranoside to this PFPA functionalized polyHIPE via displacement of the labile pentafluorophenyl group, to yield scaffolds with approximately ca. 7−9% surface carbohydrate. Experiments with primary rat hepatocytes showed that cellular albumin synthesis was greatly enhanced during the initial adhesion/settlement period of cells on the galactose-functionalized material, suggesting that the surface carbohydrates are accessible and selective to cells entering the scaffold. This porous polymer scaffold could, therefore, have important application as a 3D scaffold that offers enhanced hepatocyte adhesion and functionality

Comparison of photodamage in non-pigmented and pigmented human skin equivalents exposed to repeated ultraviolet radiation to investigate the role of melanocytes in skin photoprotection

Introduction: Daily solar ultraviolet (UV) radiation has an important impact on skin health. Understanding the initial events of the UV-induced response is critical to prevent deleterious conditions. However, studies in human volunteers have ethical, technical, and economic implications that make skin equivalents a valuable platform to investigate mechanisms related to UV exposure to the skin. In vitro human skin equivalents can recreate the structure and function of in vivo human skin and represent a valuable tool for academic and industrial applications. Previous studies have utilised non-pigmented full-thickness or pigmented epidermal skin equivalents to investigate skin responses to UV exposure. However, these do not recapitulate the dermal-epidermal crosstalk and the melanocyte role in photoprotection that occurs in vivo. In addition, the UV radiation used in these studies is generally not physiologically representative of real-world UV exposure.Methods: Well-characterised pigmented and non-pigmented skin equivalents that contain human dermal fibroblasts, endogenous secreted extracellular matrix proteins (ECM) and a well-differentiated and stratified epidermis have been developed. These constructs were exposed to UV radiation for ×5 consecutive days with a physiologically relevant UV dose and subsequently analysed using appropriate end-points to ascertain photodamage to the skin.Results: We have described that repeated irradiation of full-thickness human skin equivalents in a controlled laboratory environment can recreate UV-associated responses in vitro, mirroring those found in photoexposed native human skin: morphological damage, tanning, alterations in epidermal apoptosis, DNA lesions, proliferation, inflammatory response, and ECM-remodelling.Discussion: We have found a differential response when using the same UV doses in non-pigmented and pigmented full-thickness skin equivalents, emphasising the role of melanocytes in photoprotection