3D-printed biological organs: medical potential and patenting opportunity

Abstract

Three-dimensional (3D) bioprinting has emerged as a new disruptive technology that may address the ever-increasing demand for organ transplants. 3D bioprinting offers many technical features that allow for building functional biological tissue constructs by dispensing the individual or group of cells into specific locations along with various types of bio-scaffold materials and extracellular matrices, and thus, may provide flexibility needed for on-demand individualized construction of biological organs. Several key classes of 3D bioprinting techniques are reviewed, including potential medical and industrial applications. Several unanswered engineering components for the ultimate creation of printed biological organs are also discussed. The complicated nature of the human organs, in addition to the legal and ethical requirements for safe implantation into the human body, would require significant research and development to produce marketable bioprinted organs. This also suggests the possibility for further patenting and licensing opportunities from different sectors of the economy.

Keywords:

• additive manufacturing
• bioprinting
• organs
• regenerative medicine
• tissue engineering

Advancement of surgical techniques and post-op immunosuppressive therapies, combined with better immunological matching between donor and recipient, has enabled the organ transplantation to become mainstream practice in modern medicine [1]. To substantially reduce concerns for chronic shortage of transplantable organs, scientists and doctors have sought after on-demand production and cultivation of replaceable biological organs. Unfortunately, the production of artificial organs that have full functionality, not to mention their commercialization, has not been realized.

The ranges for the types and function of biological tissue, including the highly organized cell-extracellular matrices (ECM) structure, are immensely diverse. Therefore, the on-demand reproduction of human organs would certainly be a daunting task, considering that our current knowledge on the method of composing functional cells along with the appropriate epicellular environment is still in its infancy. The development of microelectromechanical systems (MEMS) and microfluidic techniques have enabled the micro/meso-scale construction of a cell–hydrogel composite that mimics the structure-of-interest to some degree; however, multilayered, three-dimensional (3D) production of biological tissue at a large-scale remains challenging.

The advent of the 3D rapid prototyping (RP) technology, represented mostly by solid-state 3D printing, is changing the landscape of commercial manufacturing. Recently, open-source software algorithms and shared control hardware platforms have rendered the technology more affordable. Biological 3D printing technologies, based on the similar principle as the 3D RP, place the desired types of cells into the strategic locations with allocation of ECMs around the cells that support various cellular functions (the reference [2] provides excellent technical review), and are anticipated to pave the way for the on-demand production of implantable biological organs.

The 3D bioprinting, however, requires a different set of production requirements than conventional 3D RP, and also casts a set of unique challenges in terms of manufacturing/fabrication processes that were previously unseen. Most of the human organs originate from soft tissue structures (e.g., even the compact bone stems from the malleable osseous tissue), and therefore, hydrogel/biocompatible scaffold materials that mimic the soft tissue ECM and the micro tissue architecture should be printed along with the cells. The printing process should also ensure excellent cell viability and spatial accuracy while having the ability to replicate the myriad and complexity of the human organs.

There are several ways to manufacture 3D biological tissue (outlined in Table 1). One is done through the extrusion of high-viscosity, novel hydrogel precursor biomaterials that can be stacked on to each other in a layer-by-layer fashion (via gelation) along with concurrent embedment of cells during the printing of 3D mesh frames made of solid polymers [3]. In this case, highly viscous hydrogel precursors make the printing resolution marginal while the solid supporting frames may cause mechanical incompatibility. To overcome these limitations, there has been an effort to micro-assemble spherical ‘pellets’ of cell–hydrogel aggregates (which contains thousands of cells per each pellet) into a 3D structure using high-precision robots [4]. The cell–hydrogel aggregates, each approximately half a millimeter, can be strategically placed to form different spatial patterns; however, its rather large dimension prohibits the integration of small vasculatures and fluidic channels that are necessary to replicate structures in the endocrine and renal systems. Therefore, perhaps the most versatile technique could be found from the ‘true’ 3D bioprinting method, in which the phase-changing hydrogel precursors are printed in a liquid phase as nanoliter-sized liquid droplets, and immediately gelated to maintain the structures in 3D [5]. The method has been used to print > 15 layers of cell-containing hydrogel in an on-demand fashion [6], and has been applied to embed fluidic channels [7] as well as to create vascular networks [8]. The technique is also applied in printing a structure that time-releases soluble cell growth factors to support neural cell differentiation and migration [9]. These techniques are still undergoing further refinements, and anticipated to provide further patenting opportunities.

Table 1. The classes of bioprinting techniques and their advantages and disadvantages.

Classes of methods (and cited example)	Technology highlights	Advantages	Disadvantages
Extrusion of highly viscous hydrogel (with or without the cells) along with plastic polymers that make a solid-state framework [3]	–High-viscosity hydrogel materials, with or without the cells, are mechanically extruded. –Cells are embedded between the spaces of extruded hydrogel. –Solid-state polymers are also printed.	–High viscosity of the hydrogel can partially support the printed three-dimensional (3D) structure. –Solid polymer-based framework can provide structural rigidity.	–Hydrogel alone cannot entirely support the 3D morphology. –Viscous material and its continuous extrusion limit the printing resolution and ability to create complicated morphology. –The mechanical characteristics of the solid framework may limit the scope of applications.
3D micro-assembly/printing [4]	–Hydrogel–cell aggregates/pellets are prepared and assembled with hydrogel rods in 3D. –Combinatory placement of spherical cell aggregates and the linear rods produces tissue topology, including large tubular structures.	–Cells are prepared in large numbers as an aggregate, and it expedites cell integration/fusion during culture. –The manufacturing process can be also expedited due to the use of preassembled cell–hydrogel units.	–The micro assembly technique and (relatively) large cell-aggregates limit the printing resolution and the ability to create complicated on-demand morphology, including capillary-level vascular systems. –Printing multiple cell lines on to substrates with high-spatial resolution is difficult due to the use of large, pre-made cell aggregates. –The material characteristics of hydrogel-rods may limit the scope of applications. –The 3D cellular hydrogel construct may require additional mechanical support.
3D bioprinting with phase-changing hydrogel [5-9,11,12]	–Liquid state, phase-changing hydrogel precursors are printed at high-spatial resolution. –Various types of cells, prepared in a liquid-state suspension, are printed separately and embedded into the hydrogel. –Novel layer-by-layer gelation process enables the formation of the desired 3D structures.	–Versatile printing features allow for the demonstration of various tissue topologies and functions in 3D. –Capillary-level angiogenesis/vascularization models have been demonstrated, along with on-demand construction of fluidic channels and embedment of growth factors.	–The choice of hydrogel precursor materials may limit the scope of applications. –The 3D cellular hydrogel construct may require additional mechanical support.

Important aspect of 3D bioprinting is the ability to print/dispense cells (in suspension) as well as the scaffold materials and ECMs on to a substrate. Ink-jet printing, based on either a bubble jet or piezoelectric dispensing mechanism, has been explored in this context [10,11]. A laser printing technique has been reported, whereby the cells can be ejected from a cell-laden hydrogel surface (as a ‘cell ribbon’) by heating the hydrogel (and concurrent bubble-formation) using high-intensity focal laser [12]. The laser-based approaches are orifice-free, and are therefore has greater flexibility in ability to print viscous materials. A printing technique based on using micro-valve dispensing has also been used [5-9], whereby the mild (< 10 psi) pneumatic pressure is given to liquid cell-suspension or liquid-phase hydrogel precursors while the ultra-fast mechanical gating (on the order of hundreds of microseconds) of the flow path is used to dispense nanoliter-sized liquid droplets. By adjusting the density of cell suspension, applied pneumatic pressure and gating duration, a predetermined number of cells can be reliably printed onto the target substrates. A single-cell dispensing, although not demonstrated in the context of 3D bioprinting, will also be possible through the use of MEMS-based single cell sorting and loading [13].

Expert opinion

Many technical hurdles and elements in manufacturing the bioprinted organs/organoids implicate new opportunities for further development and subsequent formations of new patents (summarized in Table 2).

Table 2. Potential patenting areas in three-dimensional (3D) bioprinting.

Areas of potential IP	Examples of possible IP contents
Hydrogel/extracellular matrices (ECM) materials	–New formulation of ECM/hydrogel precursor materials with tissue-like functional properties with inert immunological responses. –Novel methods on how to derive/synthesize such materials.
Isolation and growth/differentiation of cells in the context of bioprinting	–Novel methods on how to derive cells, either primary or pluripotent, that can be used as sources for bioprinting. –Methods to prepare the cells for printing, with or without the combination of scaffold materials.
Bioreactor/method to grow printed organ precursors	–Methods of integration and coupling of printed organ precursors with the external bioreactors. –Development of modular bioreactor/bioreactor components that sub-serve different culture conditions. –Novel methods that enable automated organ growth after printing.
Methods of dispensing/printing techniques	–Single-cell precision loading and dispensing of various types of cells –Method to improve speed of printing, for example, deployment of multiple dispensing arrays. –Novel methods to dispense liquid materials with higher viscosities, in form of droplets, which may broaden the choices of printable materials.
Methods of novel 3D fabrication techniques	–Hybrid approaches to constructively enhance the current 3D printing principles.

IP: Intellectual property.

First, the choice of hydrogel materials that provides the structural support and epicellular environment is surprisingly limited. The materials that have excellent immune-compatibility and functional support of the printed cells have been grossly unexplored, not to mention the mechanical characteristics after printing (such as contractility or mechanical rigidity). In similar regard, methods for harvesting appropriate types and amount of cells for bioprinting should be carefully addressed. Unless the cells are autologous in nature, the immunocompatibility would ultimately pose serious issues for the implantation, therefore, the method of securing appropriate cell lines, either primary or nonprimary, is crucial. In addition, for the amelioration of immune-compromised organ functions (e.g., aberrant beta islet cell function implicated in the type-1 diabetes), autologous cells would be inevitably subjected to immune-related damages, and appropriate immunological isolation/modification is needed. Ongoing advancement in stem cell technology, such as induced pluripotent stem cells [14] or even embryonic stem cells lines acquired by somatic cell nuclear transfer [15], is anticipated to diversify the sources and types of cell lines used in bioprinting for better immune-compatibility.

Other important aspects for the bioprinted organs include its integration of vascular and lymphatic systems, and its coupling to existing networks during transplantation. Progress has been made in terms of the generation of (micro and macroscopic) vascular models as well as support of angiogenesis using bioprinting [7,8] to facilitate the integration of the printed organs into the body. It is also important to note that the cells, right after the printing, are subject to additional growth and morphological/functional changes, dictated by cell-to-cell and cell-to-ECM interactions. Therefore, bioprinting warrants the development and conjunctional use of a ‘bioreactor,’ which supports the functional and structural maturation of the printed organs/tissue prior to its implantation. This bioreactor should also support the proper gas-exchange and provision/removal of nutrients/wastes. The further development of a bioreactor, dependent or independent from the usage of bioprinting, may cast additional patenting opportunities.

Based on the current standings of technology, it would take at least a few years of research and development, perhaps even decades, to have marketable bioprinted organs with high-order functionality that fulfill the ethical and legal requirements for ultimate use in humans. However, it is important to realize that 3D bioprinting can be used to exploit a few niche applications, with less stringent FDA-related regulations, which can be readily put to practical use. These include, but not limited to, applications to create: i) the artificial skin constructs as testing beds for cosmetic industries (3D bioprinting can readily be adapted to create skin layers with different phenotypes, along with skin blemishes and colors); ii) the 3D tissue structures/mini-organs, replicating major key components of liver/kidney tissues that have relevance to systemic toxicity/efficacy screening of pharmacological drugs in high-throughput fashion; and iii) the in vitro tumor tissue models that enable the examination of tumor infiltration, growth and metastasis.

Due to untapped potentials for fulfilling many technical aspects in 3D bioprinting, it would be hardly feasible for one or few identities to dominate the intellectual properties in the field of organ printing. Multifaceted and diverse applications of printed organs/tissue would definitely have a profound impact on human health and associated industries, and the number of patent applications in this field is expected to increase steadily. Advancement in biotechnology will eventually make the production of human organs an achievable concept.

Declaration of interest

The author has no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.