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

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

Fully biodegradable and biocompatible emulsion template polymer scaffolds by thiol-acrylate polymerisation of polycaprolactone macropolymers

The emulsion templating process offers a route to highly porous polymers with well-defined morphologies. This study describes the preparation of such porous polymers (polyHIPEs) via the photopolymerization of a multi-functional thiol and polycaprolactone macromonomer. The resulting materials have nominal porosities of 90% and 95%, and are seen to have an interconnected pore morphology, with an average pore diameter of approximately 60 μm. Initial biocompatibility assessments with fibroblast cells (L929) have shown that the polymers are capable of supporting cell growth over 7 days and degradation products are non-toxic to cells up to a concentration of 0.1 mg ml−1

Synthesis and applications of 2,4-disubstituted thiazoles derivatives as small molecule modulators of cellular development

Understanding how the structure of molecules relates to their function and biological activity is essential in the development of new analogues with targeted activity. This is especially relevant in mediating developmental processes in mammalian cells and the regulation of stem cell differentiation. In this study, thiazole-containing small molecules were synthesised and investigated for their ability to induce the differentiation of human pluripotent stem cells and their derivatives. Analyses of cell morphology, cell viability, expression of cell surface markers and ability to induce cell differentiation and regulate neurite formation identified the analogue with the longest and most bulky hydrophobic side chain as possessing comparable or enhanced activity to all-trans-retinoic acid (ATRA). Interestingly, a shorter, less bulky, known thiazole compound reported to be isoform selective for the retinoic acid receptor β2 (RARβ2) agonist did not mediate differentiation under the conditions tested; however, activity could be restored by adjusting the structure to a longer, more bulky molecule. These data provide further insight into the complexity of compound design in terms of developing small molecules with specific biological activities to control the development and differentiation of mammalian cells

Using Advanced Cell Culture Techniques to Differentiate Pluripotent Stem Cells and Recreate Tissue Structures Representative of Teratoma Xenografts

Various methods are currently used to investigate human tissue differentiation, including human embryo culture and studies utilising pluripotent stem cells (PSCs) such as in vitro embryoid body formation and in vivo teratoma assays. Each method has its own distinct advantages, yet many are limited due to being unable to achieve the complexity and maturity of tissue structures observed in the developed human. The teratoma xenograft assay allows maturation of more complex tissue derivatives, but this method has ethical issues surrounding animal usage and significant protocol variation. In this study, we have combined three-dimensional (3D) in vitro cell technologies including the common technique of embryoid body (EB) formation with a novel porous scaffold membrane, in order to prolong cell viability and extend the differentiation of PSC derived EBs. This approach enables the formation of more complex morphologically identifiable 3D tissue structures representative of all three primary germ layers. Preliminary in vitro work with the human embryonal carcinoma line TERA2.SP12 demonstrated improved EB viability and enhanced tissue structure formation, comparable to teratocarcinoma xenografts derived in vivo from the same cell line. This is thought to be due to reduced diffusion distances as the shape of the spherical EB transforms and flattens, allowing for improved nutritional/oxygen support to the developing structures over extended periods. Further work with EBs derived from murine embryonic stem cells demonstrated that the formation of a wide range of complex, recognisable tissue structures could be achieved within 2-3 weeks of culture. Rudimentary tissue structures from all three germ layers were present, including epidermal, cartilage and epithelial tissues, again, strongly resembling tissue structure of teratoma xenografts of the same cell line. Proof of concept work with EBs derived from the human embryonic stem cell line H9 also showed the ability to form complex tissue structures within this system. This novel yet simple model offers a controllable, reproducible method to achieve complex tissue formation in vitro. It has the potential to be used to study human developmental processes, as well as offering an animal free alternative method to the teratoma assay to assess the developmental potential of novel stem cell lines

The action of all-trans-retinoic acid (ATRA) and synthetic retinoid analogues (EC19 and EC23) on human pluripotent stem cells differentiation investigated using single cell infrared microspectroscopy

All trans-retinoic acid (ATRA) is widely used to direct the differentiation of cultured stem cells. When exposed to the pluripotent human embryonal carcinoma (EC) stem cell line, TERA2.cl.SP12, ATRA induces ectoderm differentiation and the formation of neuronal cell types. We have previously generated synthetic analogues of retinoic acid (EC23 and EC19) which also induce the differentiation of EC cells. Even though EC23 and EC19 have similar chemical structures, they have differing biochemical effects in terms of EC cell differentiation. EC23 induces neuronal differentiation in a manner similar to ATRA, whereas EC19 directs the cells to form epithelial-like derivatives. Previous MALDI-TOF MS analysis examined the response of TERA2.cl.SP12 cells after exposure to ATRA, EC23 and EC19 and further demonstrated the similarly in the effect of ATRA and EC23 activity whilst responses to EC19 were very different. In this study, we show that Fourier Transform Infrared Micro-Spectroscopy (FT-IRMS) coupled with appropriate scatter correction and multivariate analysis can be used as an effective tool to further investigate the differentiation of human pluripotent stem cells and monitor the alternative affects different retinoid compounds have on the induction of differentiation. FT-IRMS detected differences between cell populations as early as 3 days of compound treatment. Populations of cells treated with different retinoid compounds could easily be distinguished from one another during the early stages of cell differentiation. These data demonstrate that FT-IRMS technology can be used as a sensitive screening technique to monitor the status of the stem cell phenotype and progression of differentiation along alternative pathways in response to different compounds

Isolation of human embryonal carcinoma stem cells by immuno-magnetic sorting

Embryonal carcinoma cells are pluripotent stem cells derived from germ cell tumors and can be used to study cell differentiation in vitro. This report describes an approach designed to isolate pluripotent stem cells from primary/parent stock cultures of explanted tumor material. Cells expressing the pluripotent stem cell marker, SSEA-3, were isolated from heterogeneous stock cultures of the human teratoma line, TERA2, using immunomagnetic isolation. Single cell selection was performed on isolated SSEA-3+ cells and clonal lines were established. Each line was ultimately grown as a homogeneous monolayer, independent of feeder cells and expressed high levels of markers for pluripotent stem cells. In response to retinoic acid, clone TERA2.cl.SP-12 cells displayed enhanced neural differentiation compared to previously isolated TERA2 sublines and formed both neurons and glia. Deriving human pluripotent stem cell lines that differentiate into a range of cell types will provide useful tools to understand the molecular mechanisms controlling cell differentiation in a manner pertinent to human embryonic development