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September 15, 2015 (Vol. 35, No. 16)

Feng Shui Basics for 3D Cell Culture

Tissue Engineering and Advanced Robotic Systems Come Together To Improve Success Rates in Phenotypic Drug Discovery

  • Play Legos with Tissues

    A microfluidics-based organ-on-chip technology platform called OrganoPlates™ is available from Mimetas. “There’s quite a few game-changing features associated with OrganoPlates,” says Jos Joore, Ph.D., Mimetas’ co-founder and managing director. “Basically, the platform lets us build any tissue complexity in a 3D device.

    “Many devices are restricted to just one chamber with a spheroid or maybe one or two layers in a chamber. In contrast, we basically play Legos with tissues. We combine multicolor blocks into different tissue types, using a variety of cell types to build functional tissues.”

    OrganoPlates employ PhaseGuides™, a patented liquid-handling technology enabling precise 3D constructions of structures in a gel-culture matrix. A typical OrganoPlate, according to Dr. Joore, consists of an epithelial layer, the underlying fibroblast layer, and a layer with immune cells—finished with an endothelial tubule that actually perfuses the entire system.

    “This level of complexity is, as far as I know, unprecedented in the organ-on-chip world,” Dr. Joore declares. “The practical advantages are second to none.” The system runs autonomously without any pumps or tubes and is in a 384-well plate format. “It’s like coming home for a biologist,” he exclaims. “All the equipment for pipetting, imaging, and reading the plates is basically compatible with the traditional well-plate format.”

    Dr. Joore brings us to the crux of the matter, the customers and their needs: “Most of our customers are actually pharma customers, and they work with small hydrophobic compounds that tend to bind strongly to some of the materials currently being used in microfluidics such as polydimethylsiloxane.”

    “We strive to stay away from them and just use low-absorbent materials to make it possible for the pharma industry to do screens on these plates, Dr. Joore explains. “They don’t have to be afraid that their compounds will actually stick to a device or even be totally absorbed by the device.”

  • Organs-on-Chips for All Comers

    Emulate spun out of Harvard’s Wyss Institute to focus on commercializing the Organs-on-Chips system. The company hopes to advance product development in pharma as well as cosmetic, agriculture, and chemical-based consumer products. The biologically inspired organs-on-chips system integrates chips, instrumentation, software, and big data to investigate human physiology in an organ-specific context and enable novel in vitro disease models.

    “We are providing input to the $37 million DARPA grant awarded to our collaborators at Harvard to develop instrumentation for connecting multiple organs-on-chips to create a virtual human-on-chips,” says Dr. Hamilton.

    Emulate’s system affords scientific capabilities not found in other organs-on-chips, such as the ability to recreate tissue–tissue interfaces, exert and control mechanical forces, introduce immune system components, and establish air–liquid interfaces.

    “Each chip has three microfluidic channels,” details Dr. Hamilton. “The central channel has a porous, flexible membrane that can be coated with extracellular matrix protein, providing a scaffold to anchor cells in the organ. We then seed the cells in the chip.”

    Endothelial cells lining the exterior of the blood vessel are on one side of the membrane. Epithelial cells, which constitute the major cell type in an organ such as alveolar cells in the lung, are on the other side. “And these two cell types form the basic unit, the tissue–tissue interface,” she explains. “We have shown time and time again the endothelial component is critical to organ functionality.”

    “We use engineering techniques to apply mechanical forces to the cells,” she continues. Alveolar cells experience mechanical forces when the lung stretches, expands, and contracts from breathing. Emulate recreates those mechanical forces within the chips.

  • Modeling Renal Toxcity

    Renal toxicity is a major cause of drug attrition at the clinical trial stage, and the primary site of this toxicity is within the proximal tubule. Conventional renal cell culture models lack the complexity of native tissue and thus have a limited capacity for predicting tissue-level responses, according to Deb Nguyen, Ph.D., director of R&D at Organovo. Moreover, as Dr. Nguyen has noted, the predictive potential of preclinical animal trials is limited due to species-specific differences between human and animal renal functions, including differential sensitivity to insults.

    3D Tissue Model of Proximal Tubule
    Dr. Nguyen, along with colleagues Shelby King, Olivia Creasey, and Sharon Presnell, designed and fabricated a human 3D tissue model of the tubulointerstitial interface in which human renal interstitial tissue supports proximal tubule epithelial cells to facilitate their optimal morphology and function. Histological characterization demonstrated that the interstitial layer is viable and well organized, containing well-developed CD31+ endothelial cell networks, said Dr. Nguyen.

    Method Optimization
    Method optimization resulted in the formation of a polarized layer of renal epithelium on top of the interstitial layer, and formation of a basement membrane between the layers. Gene-expression analysis showed that the renal tissues expressed key enzymes involved in metabolism and protein processing (CYPs, renin-angiotensin system), suggesting that physiologic function is retained.

    “These bioprinted human tissues may provide an opportunity to accurately study how compounds affect the renal proximal tubule,” explained Dr. Nguyen. “They may also advance the modeling of pathogenic processes that involve tubular transport, cell–cell interactions, and the development of tubulointerstitial fibrosis,” said Dr. Nguyen.

  • Studying Keratinocytes with Ker-CT Cells

    Click Image To Enlarge +
    Micrograph (10x magnification ) of primary keratinocytes at 11 days post airlift stained with DAPI (blue) as well as antibodies directed against keratin 14 (green) and filaggrin (red). Keratin 14 is translated in the basal and spinous layers and persists through the stratum granulosum; filaggrin is expressed in late-differentiated keratinocytes and can be observed in the stratum granulosum and the stratum corneum. The yellow staining reflects the overlap of keratin 14 and filaggrin in the stratum granulosum. [ATCC]

    Earlier this year, scientists from ATCC presented a poster (“Characterization of a Three-Dimensional Organotypic Skin Model Using Keratinocytes and Mesenchymal Stem Cells Immortalized by hTERT”) at the Annual Society of Toxicology conference in San Diego. In this study, the researchers compared the differentiation capacity of primary keratinocytes with human telomerase (hTERT)-immortalized keratinocytes (Ker-CT cells), co-cultured with a variety of stromal cells.

    Stromal cells included primary fibroblasts, primary adipose-derived mesenchymal stem cells (primary MSCs), hTERT-immortalized fibroblasts (BJ-5ta cells), or hTERT-immortalized mesenchymal stem cells (hTERT-MSCs). All the cell lines were obtained from the ATCC collection.

    The scientists confirmed that both primary keratinocytes and Ker-CT cells are able to fully differentiate into skin equivalents in a 3D air–liquid interface (ALI) culture model when co-cultured with primary fibroblasts, primary MSCs, BJ-5ta cells, or hTERT-MSCs.

    To verify the functionality of the co-culture models, both the primary keratinocyte and the Ker-CT ALI co-cultures were subjected to a scratch assay. Healing, monitored by re-epithelialization, occurred in both Ker-CT cells and primary keratinocytes.

    The researchers also tested rapid penetration using 1% Triton X-100 added to the fully differentiated skin model. For both cell types, the IC50 was 5–10 hours. The ATCC team concluded that the differentiation capacity and continuous nature of the Ker-CT cell line make it an invaluable model for the research of keratinocyte biology. Ker-CT cells yield physiological data without the short lifespan and donor variation seen with primary cells.

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