Cell monolayers have serious limitations for cell biological investigations and for cell-based assays in drug screening and toxicity studies. However, the establishment of three-dimensional cultures as a mainstream approach requires the development of reliable protocols, new cell lines and suitable imaging techniques.

The third dimension bridges the gap between cell culture and live tissue

Classical experiments performed half a century ago demonstrated the immense self-organizing capacity of vertebrate cells. Even after complete dissociation, cells can reaggregate and reconstruct the original architecture of an organ. More recently, this outstanding feature was used to rebuild organ parts or even complete organs from tissue or embryonic stem cells. Such stem cell-derived three-dimensional cultures are called organoids. Because organoids can be grown from human stem cells and from patient-derived induced pluripotent stem cells, they have the potential to model human development and disease. Furthermore, they have potential for drug testing and even future organ replacement strategies. Here, we summarize this rapidly evolving field and outline the potential of organoid technology for future biomedical research.

http://science.sciencemag.org/content/sci/345/6194/1247125.full.pdf

Organogenesis in a dish: modeling development and disease using organoid technologies.

BACKGROUND In adult mammalian organisms, multiple tissues—including the skin, blood, stomach, and intestines—are entrapped in a state of permanent regeneration; older cells are constantly shed, and the tissue is continuously being regenerated from resident stem cells. This phenomenon of “tissue renewal” was appreciated by Leblond in 1956, but the underlying mechanism has been unclear. It is now evident that a class of extracellular developmental signaling proteins, known as Wnt signals, animate the continued renewal of several mammalian tissues by fuelling stem cell activity. If the Wnt pathway is inhibited, tissue renewal is crippled. This signaling pathway is an ancient evolutionary program dating from when Wnt signals arose in the simplest multicellular organisms, in which Wnts acted as primordial symmetry-breaking signals crucial for the generation of patterned tissues during embryogenesis. In vertebrates, these signals also function in pattern maintenance: They sustain tissue renewal, enabling tissues to be continuously replenished and maintained over a lifetime. Multiple adult organs are in a state of continual regeneration. In tissues such as the skin, intestines, brain, and mammary glands, Wnt signaling proteins sustain this constant regeneration by inducing stem cells (green cells in the illustration) to grow. This leads to the robust supply of new cells (green) in order to replenish and maintain the tissue. [Image credits are available in the full article online.] ADVANCES In contrast to traditional “long-range” developmental signals, Wnts seem to act as short-range intercellular signals—acting mostly between adjacent cells. Lending credence to this notion, a membrane-tethered Wnt protein variant can fulfill most functions of a normal Wnt protein in Drosophila . Likely explaining the short-range nature of these signals, Wnt proteins are attached to a lipid and therefore are hydrophobic; they cannot freely traverse the extracellular space by themselves. This provides insight into how tissue renewal is regulated. It implies that Wnt signals emanating from the stem cell microenvironment (the “niche”) may influence adjacent stem cells without affecting a broad field of cells located farther away. The concept of an external niche, however, may have to be refined because it is clear that stem cells can sometimes act as their own niche and have unexpected developmental self-organizing capacities. Last, the widespread importance of Wnt signaling in driving tissue renewal has been revealed by the identification of Axin2 and Lgr5 , genes expressed in cells that are responding to Wnt signals. Genetically labeling Axin2 + or Lgr5 + cells in a variety of tissues has revealed that such cells fuel tissue renewal in the intestines, mammary gland, skin, and brain, among other organs. OUTLOOK The amazing continuous self-regeneration of various mammalian tissues over years and decades continues to be an enigmatic terra incognita in biology. For instance, visualization of stem cells in real-time in vivo (through intravital microscopy) has shown that when some stem cells are ablated, they are replaced by more differentiated cells that are recalled to the stem cell niche, whereupon they regain stem cell identity to effect tissue repair. Therefore, lineage barriers between stem cell and differentiated fates are not always stringent and can be traversed during times of tissue damage. Reactivated Wnt signals may be instrumental in this process, and perhaps such signals could be exploited in order to enkindle tissue regeneration after injury or disease. From a pragmatic perspective, Wnt signals have already found practical use in manipulating stem cells, enabling propagation of stem cells in vitro as self-renewing cell populations and as organoids.

An integral program for tissue renewal and regeneration: Wnt signaling and stem cell control

Bone is a hierarchically structured material with remarkable mechanical performance which may serve as a model for the development of biomimetic materials. Understanding its properties is essential for the assessment of diseases such as osteoporosis. This will lead to a critical evaluation of current therapies and aid in their more targeted development. While the full hierarchical structure of bone is extremely complex and variable, its basic building block, the mineralized collagen fibril, is rather universal. Due to the progress in experimental methods to characterize materials at the nanoscale, new insights have been gained into the structure/mechanical function relation in this nanocomposite. The amount of mineral is usually thought to determine the stiffness of the material, but recent results suggest that the properties of the organic matrix as well as the geometrical arrangement of the two components might have a much larger influence on the properties than traditionally assumed. Some recent results from experiment and numerical modeling leading to these ideas are reviewed.

Structure and mechanical quality of the collagen–mineral nano-composite in bone

Bone is a complex highly structured mechanically active 3D tissue composed of cellular and matrix elements. The true biological environment of a bone cell is thus derived from a dynamic interaction between responsively active cells experiencing mechanical forces and a continuously changing 3D matrix architecture. To investigate this phenomenon in vitro, marrow stromal osteoblasts were cultured on 3D scaffolds under flow perfusion with different rates of flow for an extended period to permit osteoblast differentiation and significant matrix production and mineralization. With all flow conditions, mineralized matrix production was dramatically increased over statically cultured constructs with the total calcium content of the cultured scaffolds increasing with increasing flow rate. Flow perfusion induced de novo tissue modeling with the formation of pore-like structures in the scaffolds and enhanced the distribution of cells and matrix throughout the scaffolds. These results represent reporting of the long-term effects of fluid flow on primary differentiating osteoblasts and indicate that fluid flow has far-reaching effects on osteoblast differentiation and phenotypic expression in vitro. Flow perfusion culture permits the generation and study of a 3D, actively modeled, mineralized matrix and can therefore be a valuable tool for both bone biology and tissue engineering.

/pdf/fluid-flow-increases-mineralized-matrix-deposition-in-3d-4n0q0gxj3g.pdf

Fluid flow increases mineralized matrix deposition in 3D perfusion culture of marrow stromal osteoblasts in a dose-dependent manner

Tissue engineering of human bone is a complex process, as the functional development of bone cells requires that regulatory signals be temporally and spatially ordered The role of three-dimensional cellular interactions is well understood in embryonic osteogenesis, but in vitro correlates are lacking Here we report that in vitro serum-free transforming growth factor (TGF)-β1 stimulation of osteogenic cells immediately after passage results in the formation of three-dimensional cellular condensations (bone cell spheroids) within 24 to 48 hours In turn, bone cell spheroid formation results in the up-regulation of several bone-related proteins (eg, alkaline phosphatase, type I collagen, osteonectin) during days 3–7, and the concomitant formation of micro-crystalline bone This system of ex vivo bone formation should provide important information on the physiological, biological and molecular basis of osteogenesis

Three-dimensional cellular development is essential for ex vivo formation of human bone.

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Three-dimensional cellular development is essential for ex vivo formation of human bone.