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

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

Simply put, “cells in 3D more closely mimic the phenotype of real tissues and organs than those in 2D,” informs Jeffrey Morgan, Ph.D., a professor of medical science and engineering at Brown University and founder of Microtissues.

Built upon knowledge gleaned over several decades, “what we are seeing now—the big picture—is the emergence of a variety of novel 3D cell culture technologies,” he declares.

Anticipation is also in the air at Emulate. “We are undergoing a major evolution from 3D cell culture to organs-on-chips,” exclaims the company’s president and CSO, Geraldine Hamilton, Ph.D. The Emulate evolution is incorporating a host of microfluidic approaches and microfabrication technologies.

Yet another change-is-coming statement comes from Mamunur Rahman, Ph.D., principle investigator and laboratory director, Scivax Life Sciences, a subsidiary of JSR based in Japan. “Just two years ago, we compared 3D versus 2D,” recalls Dr. Rahman. “Now we compare 3D versus 3D. People understand that 3D is more important than 2D.”

Drs. Morgan, Hamilton, and Rahman were among the presenters at CHI’s 3D Cellular Models conference recently held in Boston. The conference showcased microengineered biomimetic systems, outstanding examples of which are discussed in this article. Multiple presenters emphasized that these systems could be used in pharmaceutical applications.

“The pharmaceutical industry is very interested,” Dr. Morgan elaborates. “It wants better models to replace animals in research and to provide more predictive information on toxicity as well as efficacy. So, it is shifting away from screens of 2D cells and starting to do 3D screens for phenotypic drug discovery.”

The human blood-brain barrier growing in the OrganoPlate™, an organ-on-chip technology provided by Mimetas. Structural components include astrocytes, shown in green, and blood vessel endothelium cells, shown in red. Cell nuclei are shown in blue. The blood vessel has the width of a human hair.

Catch the Rhythm, Feel the Beat

Pharmaceutical companies would definitely be interested in 3D models that could help them evaluate drugs for cardiac toxicity. “Cardiac toxicity accounts for almost one-third of drugs failing during preclinical or clinical stages,” says Tetsuro Wakatsuki, Ph.D., co-founder and CSO at InvivoSciences.

“The heart muscle contracts, but it is not easy to measure the force of contraction using cells grown in 2D culture,” Dr. Wakatsuki continues. “Physiology is based on 3D tissue.”

InvivoSciences mass produces 3D contractile engineered heart tissues (EHTs). The company differentiates human induced pluripotent stem cells (iPSCs) into cardiac lineages and fabricates them into tissue. “We grow EHTs in 96-well plates and perform the same physiological assays in miniature format that have been done in animals or isolated heart tissue,” explains Dr. Wakatsuki. “Many drugs are potentially toxic because they may induce cardiac arrhythmia by disrupting functions of the ion-channels on heart muscle cells.”

EHTs can be employed in drug screening assays for identifying compound-induced cardiac arrhythmia risks. While EHTs make a “heartbeat,” electrophysiological parameters, such as real-time cardiac action potentials, can be measured and applied to the analysis of channel function.

Robotic systems automate production processes, bringing practical applications to personalized medicine. For example, muscular dystrophy is associated with cardiac risk factors.

“To understand underlying mechanisms and drug responses, you cannot just rely on one cell line generated from a given patient,” Dr. Wakatsuki advises. “Triplicates may be needed to ensure that the phenotype observed in vitro is reproducible. For genetic diseases, you also need the right control group, which typically consists of cell lines from the patients parents or siblings.” Automation is key for handling this workload.

The proof is in the pudding as results indicate EHTs from muscular dystrophy donors with heart failure are dysfunctional, whereas EHTs from muscular dystrophy donors without heart failure behave similarly to control groups. “This is an example,” Dr. Wakatsuki concludes, “of recreating the patient phenotype in 3D tissue.”

InvivoSciences has developed an automated engineered heart tissue production and compound screening system. Patient-specific cells can be reprogrammed into induced pluripotent stem cells, which can produce a large number of erythrocytes by means of a 96-well-plate automated cell culture system. Tissue fabrication and phenotype assays are automated using a robotic liquid handler and phenotype assay devices. The system can enable precision medicine applications such as modeling patient-specific disease phenotypes, including muscular dystrophy and genetically determined congenital heart defects.

Atmospheric Feng Shui

The utility of biomimetic 3D models facilitates the growing adoption of 3D cell spheroid technologies for drug discovery. The off-the-shelf and ready-to-use NanoCulturePlate (NCP) is a core product of Scivax’ 3D cell culture system for spheroid growth.

“NCP is the only commercially available plate that is a gel-free, scaffold-type nano-patterned plate,” asserts Dr. Rahman. “The 3D nanostructure design of the NCP plate mimics the scaffold without the need for gel, pretreatment, or coating of any kind.”

The evenly patterned nanoscale structure is on a special plastic plate bottom film. Seeded cells grip the nanostructure (while lightly attaching to the plate bottom); migrate; adhere to neighboring cells; and form spheroids.

“Although one can certainly obtain nice spheroids with common gel scaffolds such as Matrigel,” remarks Dr. Rahman, “NCP technology avoids lot-to-lot variations of pre-coated plates.” NCP, which performs as well as Matrigel in drug-sensitivity studies, is designed for routine high-throughput screening of 3D cell culture using automated image analyzers.

Scivax’ 3D primary cancer cell culture kit enables drug-sensitivity testing from small tumor specimens. The kit includes dissociation solution, medium, and NCP, and “the tumor cells are cultured altogether on the NCP,” notes Dr. Rahman. “The beauty of our plate is you don’t need to separate out the fibroblasts because they do not overgrow the culture.” The fibroblasts actually establish co-culture with the cancer cells, and the resulting 3D spheroid system mimics in vivo tumor environments.

By obviating fibroblast separation, the 3D system demonstrates its big advantage over 2D systems. In 2D systems, fibroblasts adhere tightly to plates and overgrow. Although the mechanism remains to be fully elucidated, the patterned nanoscale structures of NCPs limit attachment. This feature is essential for keeping fibroblast growth in check.

NanoCulture Plate (NCP) and NanoCulture Dish (NCD) technologies from Scivax are engineered with micropatterned surfaces that minimize cell attachment and enhance cell migration and proliferation. Upper image: Minimum attachment of cell spheroid to the nanostructure was observed by SEM. Lower right: Cells migrate and form spheroids. Lower left: Various systems—including three NCPs (24-, 96-, 384-well plates) and one NCD (35-mm dish)—are available.

Do-It-Yourself Mold

Dr. Morgan develops easy-to-use micromold spheroid technology for drug discovery and toxicity testing. Cells descend by gravity from seeding chambers of casted agarose gels and settle into microwells, “where they contact each other, aggregate, and self-assemble into tens to hundreds of spheroids,” says Dr. Morgan.

Agarose gel is nonadhesive, so cells can’t attach to agarose. Small cell–cell adhesion forces take over, and cells form spheroids. Essentially, 2D tissue culture dishes are designed for cells to stick to surfaces, whereas casted 3D Petri Dishes® are designed for cells to stick to each other.

Dr. Morgan investigates drug uptake and the role of efflux pump transporters to control drug uptake. Efflux pumps use ATP to pump a drug out of the cell against a concentration gradient; pumps can be upregulated during multidrug resistance.

“We made spheroids by mixing two different cell types, high- and low-pump-expression cells, and they formed three different architectures,” Dr. Morgan details. “The location of the pump cells had a big influence on how much drug could be taken up by that spheroid. When the high-pump-expression cells coated the outside of the spheroid, they protected the entire spheroid from drug exposure.”

Dr. Morgan indicates that he is aware that “pumps are very important in the drug discovery testing space,” and he asserts that Microtissues’ technology allows investigation in a 3D setting mimicking dynamic in vivo microenvironments.

“The gel is transparent, so we can use time-lapsed images to investigate uptake of a fluorescent molecule that mimics the behavior of a drug,” Dr. Morgan continues. “The pumps will pump this fluorescent molecule out of the cell or prevent it from entering the cell. We quantify ‘drug’ uptake, which is measured by the amount of fluorescence in the spheroid within the radial dimension of the spheroid.”

To train students in 3D culture, Dr. Morgan runs boot camps at a center for predictive biology that he co-founded with his colleagues. This center, which is located at Brown University, focuses on developing next-generation toxicity testing based on the 3D platform.

Dr. Morgan anticipates that the micromold will become a mainstay technology. He says that his first publication about micromolds stimulated so many requests that he “licensed 3D culture technology from Brown University, started Microtissues, and began distributing Microtissues’ signature green micromolds through Sigma-Aldrich.”

To predict drug response or toxicity, pharma is increasingly performing smaller-scale validation studies of experimental drug compounds in novel 3D cell culture models intended to mimic more closely the structure, activity, and extracellular environment of tissues in vivo. [IStock/Hakat]

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

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.

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]

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