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Sep 15, 2013 (Vol. 33, No. 16)

Novel 3D Cell Culture Systems

  • Click Image To Enlarge +
    96-well microtissue assay plate (InSphero’s GravityTRAP™) loaded with tumor microtissues composed of the colon cancer cell line HCT116 (left) expressing a green fluorescent protein and stromal mouse fibroblasts NIH3T3 expressing a red fluorescent protein. Visualization of the individual cell populations using the PerkinElmer Opera QEHS equipped with a 10x Air objective. The single z-stack through the microtissue was processed with the Acapella High Content Imaging and Analysis Software to generate a maximum projection image (right). [Image provided by PerkinElmer, Karin Boettcher and Stefan Letzsch]

    In the latter case, the ability to simulate the microtumor environment, communication between tumor cells, the effects of the tumor stroma environment, and the interaction of tumor cells with other surrounding cell types such as epithelial cells or fibroblasts, can enhance the ability to study mechanisms of tumorigenesis, cell migration and invasion, and metastasis.

    One of the main and ongoing challenges has been to develop in vitro 3D cell culture systems that are compatible with industrial-scale applications and are readily automatable for high-throughput screening assays.

    Jens Kelm, Ph.D., CSO and co-founder of InSphero, described three 3D cell culture methods that are currently most broadly accepted in the industry: cell constructs embedded in hydrogels; cells grown in scaffolds; and cellular self-assembly leading to spheroid formation. InSphero’s approach leads to the formation of scaffold-free 3D multicellular spheroids in a 96-well format. The company’s GravityPLUS™ technology automates the classic hanging drop methodology, in which cells in hanging drops of culture media descend and assemble into microtissue spheroids without the need for any support matrices or contact with any surfaces.

    “We have exploited the versatility of hanging drop production by uncoupling the generation of microtissues from downstream applications,” said Dr. Kelm. “This was the creation of our platform, producing very uniform spheroids or microtissues while also allowing for compound treatments, microscopic analysis and assays, to get the best of both worlds!”

    In his presentation at the SMi meeting, Dr. Kelm described the availability of an increasing variety of model systems available to industry and the research community for testing the safety, toxicity, and efficacy of drug compounds in in vitro systems and minimizing the need for animal testing. The demand for good in vitro model systems for screening extends beyond the pharmaceutical industry, with safety assessment of chemicals and other compounds important for the chemicals and cosmetics industries as well.

    “We are using reporter systems that are allowing us to perform target validation in 3D model systems,” said Dr. Kelm. This has revealed sometimes substantial differences in the effects of drugs on microtissues versus cells grown in monolayer systems, and in particular in models of tumor growth and proliferation.

    One goal going forward is standardization. It would be convenient, for example, to have standardized 3D liver and cardiac model systems available for safety testing. “It is important to generate comparable results, to test on the same model and be able to compare results over years and across drugs,” added Dr. Kelm.

    Dr. Kelm foresees continued progress in mimicking interconnected, complex tissue constructs in functional living tissue models—so-called body-on-a-chip concepts. Two consortia, one in the United States and one in Europe, are working on this concept. InSphero is participating in the European project, in which the different tissue types are produced externally as 3D spherical constructs and then loaded onto a microchip, which is composed of interconnected compartments.

    “There is consensus in the pharmaceutical industry that 3D model systems can provide a higher quality of information in vitro,” concluded Dr. Kelm.

  • Nanofibers

    Peptisyntha, a member of the Solvay group, applied its expertise in peptide synthesis chemistry to develop a family of short peptides that are able to self-assemble and form nanofibers. These nanofibers exist in a hydrogel capable of supporting 3D cell cultures.

    “The major benefit achieved with 3D or pseudo-3D culture is the higher cell density that can be obtained when compared to 2D culture,” said Marc Fouassier, business development manager. “This is an extremely important aspect for the manufacture of certain cell lines, and certain targeted cell applications for instance in cell therapy.”

    Using Peptisyntha’s hydrogel system, cells grow on a soft coating prepared from the self-assembling peptides, which mimics the extracellular matrix. The peptide coatings can be prepared on various plastic surfaces traditionally used for cell culture, including polystyrene, polyethylene terephthalate, and polycarbonate.

    According to Fouassier, advantages include the fully synthetic, animal-free, GMP origin of these hydrogel peptides, which are particularly useful for culturing cells that require adhesion.

  • Human Brain Tissue Grown in Test Tubes

    Researchers from the Institute of Molecular Biotechnology (IMBA) of the Austrian Academy of Sciences report the development of human brain tissue in a 3D cell-culture system. Their technique, which is discussed in an article last month in Nature (“Cerebral organoids model human brain development and microcephaly”), permits pluripotent stem cells to develop into cerebral organoids, or “mini brains,” that consist of several discrete brain regions.

    “Our goal was to create a model system of the human brain,” said Jürgen Knoblich, Ph.D., IMBA’s deputy scientific director during a press conference.

    Intrinsic cues from the stem cells guided the development toward different interdependent brain tissues. Using the mini brains, the scientists were able to model the development of a human neuronal disorder (i.e., microcephaly) and identify its origin.

    “A normal developing brain has a stem cell population that undergoes rounds of division at specific times to make more stem cells and eventually neurons,” noted Dr. Lancaster. “But the microcephalic patient-derived stem cells made neurons too early in the process. This led to a depletion of the stem cell population, which resulted in fewer neurons being made.”

    Putting it another way, Dr. Knoblich explained that this finding led to the hypothesis that, during brain development of patients with microcephaly, the neural differentiation happens prematurely at the expense of stem and progenitor cells, which would otherwise contribute to a more pronounced growth in brain size. “Further experiments also revealed that a change in the direction in which the stem cells divide might be causal for the disorder,” he continued.

    The new method offers great potential for establishing model systems for human brain disorders, according to the researchers. Such models are urgently needed, as the commonly used animal models are of considerably lower complexity and often do not adequately recapitulate the human disease.

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