Emulating Human Tissues via 3D Bioprinting

Cell-Based Models for Drug Screening

The pharmaceutical industry has been relying on the use of planar and static cell cultures in well plates for a century now (Figure 1a). These monolayer cell-based models are instrumental to drug screening as they provide extremely high throughput allowing for simultaneously testing of hundreds to millions of compounds through established automation in handling and analyses, which are further downsized for subsequent animal experimentation before moving to the costly and time-consuming multi-phase clinical trials. Nevertheless, the use of the two-dimensional (2D) models to screen drug molecules presents significant problems such as the low success rate, largely due to the inabilities of these overly simplified systems to reproduce the complex human tissue structural-functional relationships outside the human body in the dishes. In particular, safety (>50%) and efficacy (>10%) issues are the primary causes of drug development failure, more than other contributing factors such as those that are strategic, commercial, and operational altogether.^1,2 Indeed, almost no tissues in the human body are flat or static – they are unanimously finely structured, multi-layered, and/or complex in composition; furthermore, a variety of dynamics exist, ranging from the blood flows that cause shear stresses on vascular cells and pressure-driven interstitial flows to the mechanical movements of certain tissues that are either spontaneous or passive. Therefore, incorporating all these volumetric structural aspects and dynamic cues into the engineered in vitro tissue models is critical, and will improve their biological and (patho)physiological relevancy allowing us to predict the drug efficacy/toxicity at much higher accuracy.

Bioprinting as an Enabling Technology for Tissue Model Fabrication

There are numerous strategies to generate three-dimensional (3D) models of human tissues. The simplest one lies in the use of self-aggregating behaviors of most adherent cells to form spheroids usually of several hundred micrometers in size (Figure 1b), giving the volumetric tissue matter but still lacking the microscale tissue architecture. Hydrogel matrices or polymeric scaffolds produced using conventional molding methods are an alternative (Figure 1c), whose ability to form defined patterns in 3D is limited, besides the added complexity of the fabrication procedures. To this end, the recently emerging 3D bioprinting technology seems to be an enabling solution to addressing the challenges associated with the existing bio-fabrication approaches, by providing unprecedented ease to generate volumetric tissue constructs containing precisely defined internal architecture using optimized bioinks (i.e., biomaterials mixed with cells). The computer- programmed robotic control further allows for potential of large-scale microtissue bioprinting at high reproducibility.

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Common 3D bioprinting modalities can be divided into a few categories based on their different operational modes.⁴ Inkjet bioprinting that ejects microdroplets of the bioink generated by thermal or piezoelectric actuations was the first bioprinting system available (Figure 2a).⁵ Alternatively, extrusion bioprinting is perhaps currently the most popular bioprinting modality due to its simple operations and wide availability, which relies on extrusion of the bioink from the nozzle using pneumatic or mechanical pressure (Figure 2b). Other bioprinting modalities include laser-assisted bioprinting that deposits bioink droplets with localized laser heating (Figure 2c), multi-photon lithography that polymerizes the bioink locally (Figure 2d), and stereolithography that layer-by-layer crosslinks varying patterns of the bioink (Figure 2e).

Each bioprinting modality has its unique advantages and associated limitations, which can be meticulously chosen according to the desired tissue model to be fabricated. For example, inkjet bioprinting is cost-effective but requires low viscosities of the bioinks to facilitate droplet formation at good resolution, alongside with the limited ability to build 3D objects; extrusion bioprinting is low in cost and permits convenient 3D deposition of the bioinks of high viscosities (unless in situ crosslinking can be done) and of medium resolution at lower speeds; laser-induced forward transfer is usually fast allowing for bioprinting of medium-to-high densities of cells at relatively high costs; multi-photo lithography gives extremely high resolution of the bioprinted structures but is also posed at very high costs with the use of dedicated lasers; stereolithography on the other hand, enables high-resolution, fast bioprinting of complex tissue constructs at reasonable costs, which however, has a strong requirement on the properties of the bioinks.⁶

Various bioprinting strategies have been adopted for tissue model fabrication. Note that the bioprinting strategies discussed here are different from the above- mentioned bioprinting modalities, as a single modality can be adapted, or modified to allow for several specific bioprinting approaches. Take extrusion bioprinting for instance – while the natural way of using an extrusion bioprinter is to deposit the bioink line-by-line in a layer-by-layer manner to produce 3D patterns (Figure 2b),⁸ the fact that it lays down a single microfiber during each extrusion procedure makes it possible to use these bioprinted microfibers as the fugitive templates in building hollow microchannels, termed as sacrificial extrusion bioprinting (Figure 3a). In this strategy, the microfibers are first placed onto a substrate; then a secondary hydrogel matrix is filled in the surrounding space of the microfibers and crosslinked; finally, the initially bioprinted microfibers are selectively removed, leaving hollow microchannels that mimic the vascular or other tubular space within a bulk tissue. This way, vascularized tissue models,⁹ vascular disorder models (e.g., thrombosis, Figure 3b),¹⁰ kidney proximal tubule models,11 or mammary ductal carcinoma models¹² may be generated to emulate their native counterparts. Using the same extrusion bioprinting modality, the printhead can also be modified to host more than one single layer, thus facilitating concentric co-extrusion of multiple phases (i.e., microfluidic bioprinting), where the bioink is delivered from the exterior and the crosslinking agent flown from the interior, resulting in direct bioprinting of hollow, perfusable cannular tissues (Figure 3c).¹³ The extrusion-bioprinted microfibrous structures encapsulating one cell type (e.g., endothelial cells) can also be seeded with a secondary cell population to form a composite tissue construct of multiple components, where the behaviors of the surrounding cells (e.g., alignment) are controllable by the local patterns of the bioprinted structures (Figure 3d).¹⁴

Besides extrusion bioprinting, other modalities have similarly been used for generating 3D tissue models in vitro. Stereolithography has allowed for high-resolution deterministic patterning of biomimetic human hepatic model with two bioinks, one containing induced pluripotent stem cell-derived hepatocytes for creating the lobule-like structures, another encapsulating endothelial cells for mimicking the sinusoids.¹⁵ The hepatic model showed relevant metabolisms and drug responses. The capacity of generating multi-component microtissues using stereolithography was further enhanced through integration with a microfluidic chip device that allowed for automated injections of multipe bioinks, as recently demonstrated by us.¹⁶

Conclusions and Perspectives

In summary, the 3D bioprinting technology perfectly solves some of the long-lasting challenges relating to the conventional biofabrication methods, primarily their inability to produce precisely defined architecture in space. Indeed, the utilization of 3D bioprinting has provided us with unprecedented flexibility in generating structures and shapes in a highly reproducible manner, reaching the complexity that matches that of the native tissues in the human body and thus promoting the accuracy when screening pharmaceutical compounds. Nevertheless, problems persist, as improving the throughput of bio- printing is still not straightforward and the multiplication will require significant optimizations to simultaneously ensure the quality/ functionality of the bioprinted microtissues. Other challenges remain including proper characterizations of the bioprinted microtissues hampered by their 3D structures that do not typically allow for volumetric imaging at deep depths. Despite these limitations, 3D bioprinting when used in cohort with microfluidic bioreactors, will facilitate the recapitulation of both the structural mimicry of their human counterparts as well as the dynamics involved in the native tissues, which together, are anticipated to translate to better functional reproduction to eventually improve our success in screening the efficacy and safety of pharmaceutical compounds.

Acknowledgments

The author acknowledges supports from the National Institutes of Health (K99CA201603) and the New England Anti-Vivisection Society.

References

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Author Biography

Dr. Zhang received a B.Eng. in Biomedical Engineering from Southeast University, China (2008), a M.S. in Biomedical Engineering from Washington University in St. Louis (2011) and a Ph.D. in Biomedical Engineering at Georgia Institute of Technology and Emory University (2013). He is currently a Research Faculty at Harvard Medical School and Associate Bioengineer at Brigham and Women’s Hospital. His laboratory is focused on use of 3D bioprinting, microfluidics, and bioanalysis to create functional tissue models.