Research in mammalian cell biology often relies on developing in vitro models to enable the growth of cells in the laboratory to investigate a specific biological mechanism or process under different test conditions. The quality of such models and how they represent the behavior of cells in real tissues plays a critical role in the value of the data produced and how it is used. It is particularly important to recognize how the structure of a cell influences its function and how co‐culture models can be used to more closely represent the structure of real tissue. In recent years, technologies have been developed to enhance the way in which researchers can grow cells and more readily create tissue‐like structures. Here we identify the limitations of culturing mammalian cells by conventional methods on two‐dimensional (2D) substrates and review the popular approaches currently available that enable the development of three‐dimensional (3D) tissue models in vitro. There are now many ways in which the growth environment for cultured cells can be altered to encourage 3D cell growth. Approaches to 3D culture can be broadly categorized into scaffold‐free or scaffold‐based culture systems, with scaffolds made from either natural or synthetic materials. There is no one particular solution that currently satisfies all requirements and researchers must select the appropriate method in line with their needs. Using such technology in conjunction with other modern resources in cell biology (e.g. human stem cells) will provide new opportunities to create robust human tissue mimetics for use in basic research and drug discovery. Application of such models will contribute to advancing basic research, increasing the predictive accuracy of compounds, and reducing animal usage in biomedical science.

STEM CELLS, a peer reviewed journal published monthly, provides a forum for prompt publication of original investigative papers and concise reviews. STEM CELLS is read and written by clinical and basic scientists whose expertise encompasses the rapidly expanding fields of stem and progenitor cell biology. STEM CELLS welcomes original articles and concise reviews describing basic laboratory investigations of stem cells and the translation of their clinical aspects of characterization and manipulation from the bench to patient care. The journal covers all aspects of stem cells embryonic stem cells/induced pluripotent stem cells; tissue-specific stem cells; stem cell technology: epigenetics, genomics, proteomics, and metabonomics; cancer stem cells; translational and clinical research; and regenerative medicine.

Cultured cells are dramatically affected by the micro‐environment in which they are grown. In this study, we have investigated whether HepG2 liver cells grown in three dimensional (3‐D) cultures cope more effectively with the known cytotoxic agent, methotrexate, than their counterparts grown on traditional two dimensional (2‐D) flat plastic surfaces. To enable 3‐D growth of HepG2 cells in vitro, we cultured cells on 3‐D porous polystyrene scaffolds previously developed in our laboratories. HepG2 cells grown in 3‐D displayed excellent morphological characteristics and formed numerous bile canaliculi that were seldom seen in cultures grown on 2‐D surfaces. The function of liver cells grown on 3‐D supports was significantly enhanced compared to activity of cells grown on 2‐D standard plasticware. Unlike their 2‐D counterparts, 3‐D cultures were less susceptible to lower concentrations of methotrexate. Cells grown in 3‐D maintained their structural integrity, possessed greater viability, were less susceptible to cell death at higher levels of the cytotoxin compared to 2‐D cultures, and appeared to respond to the drug in a manner more comparable to its known activity in vivo. Our results suggest that hepatotoxicity testing using 3‐D cultures might be more likely to reflect true physiological responses to cytotoxic compounds than existing models that rely on 2‐D culture systems. This technology has potential applications for toxicity testing and drug screening.

Recreating the structure of human tissues in the laboratory is valuable for fundamental research, testing interventions, and reducing the use of animals. Critical to the use of such technology is the ability to produce tissue models that accurately reproduce the microanatomy of the native tissue. Current artificial cell‐based skin systems lack thorough characterisation, are not representative of human skin, and can show variation. In this study, we have developed a novel full thickness model of human skin comprised of epidermal and dermal compartments. Using an inert porous scaffold, we created a dermal construct using human fibroblasts that secrete their own extracellular matrix proteins, which avoids the use of animal‐derived materials. The dermal construct acts as a foundation upon which epidermal keratinocytes were seeded and differentiated into a stratified keratinised epithelium. In‐depth morphological analyses of the model demonstrated very close similarities with native human skin. Extensive immunostaining and electron microscopy analysis revealed ultrastructural details such as keratohyalin granules and lamellar bodies within the stratum granulosum, specialised junctional complexes, and the presence of a basal lamina. These features reflect the functional characteristics and barrier properties of the skin equivalent. Robustness and reproducibility of in vitro models are important attributes in experimental practice, and we demonstrate the consistency of the skin construct between different users. In summary, a new model of full thickness human skin has been developed that possesses microanatomical features reminiscent of native tissue. This skin model platform will be of significant interest to scientists researching the structure and function of human skin.

Our current knowledge of how human tissues grow and develop is limited. We need to increase our understanding of tissue formation if we are to fully realize the potential of stem cells as a source of material for research into health and disease and possible therapeutic applications. Transplanted pluripotent human embryonic stem cells (hESCs) provide a potential system to model and investigate cell differentiation in humans. hESCs transplanted into immune-deficient mice form complex teratomas consisting of a range of differentiated somatic tissues, some of which appear highly organized and resemble structures normally identified in the embryo and adult. Analysis of such tumors may provide a unique opportunity to study organogenesis and lead to novel approaches in bioengineering and the growth of functioning structures composed of a range of alternative cell types. However, little has been done to characterize the developmental potential of hESCs after transplantation. This concise review presents evidence for the ability of hESCs to differentiate in vivo and highlights some of the prominent questions that need to be addressed if transplantation is to be used as a research tool to study hESC differentiation.

The Caco-2 monolayer is the most widely used in vitro model of the human intestinal mucosa to study absorption. However, models lack communication from other cells present in the native intestine, such as signals from fibroblasts in the lamina propria. In this study, we have investigated the effects of fibroblasts upon the Caco-2 epithelium through two mechanisms: indirect signaling from fibroblasts and direct contact with fibroblasts. Culture of Caco-2 cells with paracrine signals from fibroblasts, through the use of conditioned media, did not induce a significant change in epithelial cell morphology or function. To examine the effects of direct contact between the epithelium and fibroblasts, we developed novel, humanized three-dimensional (3D) co-culture models whereby Caco-2 cells are grown on the surface of a subepithelial-like tissue construct containing intestinal or dermal fibroblasts. In our models, we observed endogenous extracellular matrix production from the fibroblasts that provides support to the above epithelium. The Caco-2 epithelium displayed morphological changes in 3D co-culture including enhanced polarization and the formation of a basement membrane-like attachment to the underlying stromal compartment. An important structural alteration was the significantly straightened lateral membrane that closely mimics the structure of the in vivo intestinal mucosa. This enhanced lateral membrane phenotype, in correlation with an reduction in TEER to levels more similar to the human intestine, is thought to be responsible for the increased paracellular permeability observed in 3D co-cultures. Our results demonstrate that direct contact between epithelial and mesenchymal cells results in an enhanced epithelial barrier. The in vitro models described herein have the potential to be used for studying intestinal epithelial-fibroblast interactions and could provide more accurate tools for drug permeability studies.