September 15, 2014 (Vol. 34, No. 16)

3D tissue engineering is advancing rapidly, enabling researchers to mimic the complexity of in vivo conditions with 3D in vitro assays.

According to Jonathan Garlick, D.D.S, Ph.D., a professor of oral pathology at Tufts University and one of a growing cadre of scientists pushing beyond the limits of traditional 2D cell-based assays, 3D tissue assays are driving a transition from “disease in a dish” to “disease in a tissue” systems.

This transition is happening now thanks to sophisticated microfluidics, innovative scaffold building, and reliable techniques for deriving many cell types from human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs).

One result of this transition is that 3D tissue assay platforms are being developed to test the efficacy and safety of drugs against cancer, heart disease, and many other diseases. By mirroring complex tissue organization and myriad microenvironment signals, these platforms are expected to be far more predictive than 2D cell-based assays of what happens in vivo. Work is also ongoing on transplantable tissue.

“The idea is to provide what I call the missing link between valuable observation in 2D cell-based assays and valuable observations made in animal models. There needs to be this human predictive correlate, and that’s the role that 3D tissue biology plays, says Dr. Garlick. “It serves as a preclinical setting that can accelerate the drug [and potentially transplant tissue] development.”

One of the most ambitious programs is the Wyss Institute’s Organs-on-Chips effort. It seeks to create microfabricated versions of human organs on chips, interconnect them, and create a platform for drug research that could potentially eliminate or drastically reduce animal testing.

Dr. Garlick’s lab has several 3D tissue initiatives. “Our goal is to work toward the development of disease-specific and pathway-specific tissue models. If we can adapt these 3D models further to have a patient-specific basis, we’ll be able to screen drugs and study disease pathways in a human-like disease context that has direct personal relevance for the patients,” explains Dr. Garlick, who is scheduled to speak at Cambridge Healthtech Institute’s Functional Analysis and Screening Technologies (FAST) Congress in November.

One example of Dr. Garlick’s work is the study of epigenetic changes that complicate wound healing in diabetes. “If you take cells from diabetic wounds that have been altered because of aberrant repair conditions and reprogram them back to iPSCs, you can manipulate them to differentiate into many cell types,” discusses Garlick. “You then modulate their disease properties in the hope you can direct the phenotype to a reparative phenotype. But how do you test whether those cells would be effective therapy for the patient? One way is to incorporate those cells into a 3D tissue model to determine if we have enhanced cell behavior.”

A number of researchers believe that the use of stem cells along with 3D bioprinting eventually will provide replacements for human tissues and organs. [crystaleyemedia/]

Tackling Cardiac Tissue

Engineering 3D cardiac tissue, despite significant progress, remains a formidable challenge. Consider this brief description of the heart’s dynamism and complexity excerpted from a recent overview (Coulombe et al. Annu. Rev. Biomed. Eng., July 11, 2014):

“The healthy adult human heart weighs 200–350 g, is approximately the size of your fist, and contains 2–4 billion cardiomyocytes. The average cardiac output is 5 L/min at rest with a 60% ejection fraction, which increases with exercise to 15 L/min with up to 85% ejection fraction. The architecture of the heart muscle enables efficient pumping of blood, exemplified by the fiber angle and orientation of cardiomyocytes within the extracellular matrix (ECM) that enable torsional squeezing to maximize ejection fraction.

“Each cardiomyocyte is surrounded by 3–4 capillaries, which have a single layer of endothelial cells (ECs) stabilized by pericytes that share a common basement membrane. Cardiac fibroblasts lie between cardiomyocytes, and larger coronary vessels provide blood flow to the CVU [cardiovascular unit] and are surrounded by vascular smooth muscle cells (VSMCs) and other perivascular cells.”

2D cell-based assays (drug efficacy, toxicity, etc.) have been used in heart research for decades, but emulating in vivo conditions with 2D platforms is less effective. The full panoply of in vivo constituents and crosstalk is required for optimum results.

“Cardiomyocytes in 2D culture show things like excitation and contraction, but it’s very different than in a 3D form,” points out Kareen Coulombe, Ph.D., assistant professor of engineering at Brown University and the lead author of the paper excerpted above. “There we find things like faster conduction velocities and more physiological interactions between the cardiomyocytes within the tissue.”

Among the many challenges in engineering 3D cardiac tissue are: producing sufficient numbers of primary cardiomyocytes (a billion or so cardiomyocytes die in a major heart attack); developing effective 3D environments; and ensuring adequate electrical integration and vascularization.

Substantial progress has been made deriving cardiomyocytes from hESCs and hiPSCs. “These cells definitely mimic the human heart much better than any current standard the FDA has for testing drugs before they go into a clinical trial,” notes Dr. Coulombe, who emphasizes that proven protocols for deriving cardiomyocytes in quantity, as well as for deriving smooth muscle cells (SMCs) and endothelial cells (ECs), are already available.

Scaffold development techniques are diverse and making progress. Examples:

  • Molding: Dr. Coulombe’s lab uses laser etching in acrylic to create the negative template and then cast mold from polydimethylsiloxane (PDMS), or silicone. “We can draw any geometry we want. Laser etching is fast and cheap,” remarks Dr. Coulombe. “We can then study how the mechanics in the physical form of the tissue affects its function.” For the upcoming FAST event, Dr. Coulombe plans to discuss scaling tissue from a “few hundred microns up to centimeters” in the engineering of macroscale 3D human cardiac tissue from hiPSCs.
  • 3D Printing: It is now also possible to print the scaffold itself. “Researchers are also coming up with ways to have cells mixed into gels [that polymerize quickly],” says Dr. Coulombe. If polymerization is fast enough, the scaffold that “comes out of the printer head…will actually maintain a geometry.”

The ultimate goal of cardiac tissue engineering is to build a complete replacement heart, but current efforts are focused on three components of the heart: valves, vasculature, and cardiac patches.

Advances in both stem cell research and culturing techniques are helping to drive progress in the tissue engineering arena. [dra_schwartz /]

Building Bone

Multiple myeloma (MM) is the second most common hematological malignancy in the United States, and like many cancers, it is prone to developing drug resistance. Because MM exists inside bone’s specialized environment, a major challenge has been developing an effective cell-based platform for testing new drugs.

“Ironically it is difficult to kill the cells inside the body, but once the cells are outside, they die,” observes Jenny Zilberberg, Ph.D., assistant scientist, John Theurer Cancer Center, Hackensack University Medical Center. “When you want to develop a personalized approach to treat patients you need to preserve their original cells. You want to have the source of the problem in its natural state, comparable to its physiological environment.”

Dr. Zilberberg and colleagues adapted a microfluidics 3D platform developed by others (see Lee et al., Biomaterials, February 2012) to support MM cell proliferation. The device consists of eight culture chambers shaped as hexagonal prisms and mounted on glass. Osteoblasts are added to create the ossified scaffold, so that once the patient’s bone marrow is introduced, all the elements of bone’s natural environment are present.

“We also created a medium [that incorporates] the serum of the transplant of the patient,” says Dr. Zilberberg. “In that way we provided the liquid and the solid milieu in conjunction with the fluidics of the system that provides the refresh needed constantly.”

Such a 3D platform could be used for many purposes. For example, a patient’s response to a drug could be tested without having to actually administer the drug to that particular patient. “Most MM patients develop drug resistance. Based on the assay, you could choose some drugs for initial treatment and hold others for later use, when resistance develops,” continues Dr. Zilberberg. “It gives the patient more chances to use different drugs.”

Dr. Zilberberg has been able to keep the MM cells alive for roughly six weeks: “They feed on the ossified structure we created.”

MM, she emphasizes, is an osteolytic disease in which bone is consumed: “Eventually the MM cells and all the secreted cytokines introduced by this tumor (and provided through the microfluidic device) affect the viability of the osteocytes, and in turn the MM cells won’t survive. It is a very interconnected system.”

Better Biomaterials

Work developing effective biomaterials for 3D tissue engineering is ongoing, and one promising area is work on hydrogels. Nasim Annabi, Ph.D., an instructor at Brigham and Women’s Hospital and Harvard Medical School, is part of a group that has developed hydrogels for use in building cardiac tissue and in surgical glues and sealants.

Dr. Annabi and colleagues note that “hydrogels provide mechanical support for cardiac cells to deposit extracellular matrix (ECM) and form the newly synthesized tissue as they degrade.” After emphasizing that that physical and chemical properties of hydrogels can be easily tuned to enhance cell viability and function, these investigators conclude that “hydrogels are attractive materials for regeneration of the damaged myocardium.”

There are many hydrogels. Dr. Annabi has worked on developing one based on the human protein tropoelastin. This hydrogel is very tunable (porosity, tensile characteristics, degradation characteristics, etc.), cures quickly under UV light, and can have its conductive properties enhanced by adding conductive nanoparticles.

“Cells like the material and attach very strongly,” says Dr. Annabi. “We also had good contractile activity.”

Slight changes to the formulation enabled the tuning of its stickiness and stretchiness such that it can be used as a glue to seal surgical sutures. This use has very broad applications, and efforts to commercialize the technology are being undertaken by an Australian start-up, Elastagen.

No doubt obstacles remain to wider use of engineered 3D tissues. Scale-up and commercialization are two prominent challenges.

“We have an understanding of the design principles but still need several things,” comments Dr. Garlick. “It would be very helpful to have scaled-up, rapid, robotized screening facilities that use these tissues—and again they should be tissues not just 3D structure.” It would also be helpful to have out-the-box disease- and pathway-specific platforms. “Ultimately, once you sort these cells from individual patients, you could develop patient-specific tissues. That would be incredibly powerful, but we are not quite there yet.”

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