Bioprinting is becoming widely used not only in regenerative medicine and tissue engineering applications but also in many other biomedical fields such as cancer research, as recently discussed on the Biogelx blog (see article). Thus, it is not surprising that scientists are now focusing on using this tool for novel approaches in stem cell research as well.
A recent article by Stephanie Willerth from University of Victoria in Canada describes advances in printing stem cells (particularly hiPSCs) by microfluidic extrusion and discusses the opportunities these advances open for high-throughput production of hiPSC-derived neural tissues. Willerth explains how these tissues can then be utilized for screening drug candidates for Alzheimer’s disease; which is yet another example of how bioprinting is becoming important in drug development.
Another innovative application of bioprinting as an enabling tool in stem cell research is the development of novel treatments for cardiac injury. Michael Davis from Emory University and Georgia Institute of Technology has worked extensively with stem cells and focuses his research in solving pediatric congenital heart defects using bioprinting technology combined with the reparative properties of stem cells. Scientists at Davis’ lab are working on a 3D printable patch which contains stem cells that will repair the surrounding damaged cardiac tissue when inserted into place. The group is also using bioprinting to create heart valves using skin cells from patients, thus minimizing the risk of organ rejection. Importantly for pediatric patients, this also allows the organ to grow with the patient, which means that a replacement will not be needed in the future.
In summary, the bioprinting of stem cells is being increasingly recognized as an innovative tool with extensive applications in biomedicine. Whilst the promise of such techniques is undeniable, it is important to remember that the bioinks utilized and several other factors in the printing process (as well as the culture period post printing) will be key to their success. Such factors can influence stem cells viability, differentiation, and function, so they should be carefully considered for future applications.
You can read a 2018 review of stem cell bioprinting technology here.
The future of drug screening, disease modeling, and precision medicine.
In the last few decades, both high-throughput screening and three-dimensional (3D) bioprinting technologies have emerged and have been proven to be instrumental for the advancement of drug screening, drug development, and tissue modelling.
High-throughput screening (HTS) is well established within the pharmaceutical industry for compound and drug discovery. It is, for the most part, the primary method of testing prior to compound validation and preclinical animal model studies prior to clinical trials. Today, the industry standard is still the use of two-dimensional (2D) cell culture for screening, as it has a long record of use and thus well-characterised cell lines and methods. This is despite the fact that the use of 2D culture is limiting due to its inability to capture in vivo-like cell-cell and cell-matrix interactions, and the fact many cell types display different phenotypes and varying genomic profiles in 2D versus 3D. Whilst the advantages of 3D cell culture are well understood, the reality is that many 3d culture systems can be challenging to reproduce to the levels required for HTS.
This is where 3D bioprintingis becoming recognised as a promising technology for HTS. It has the potential to produce more realistic, highly reproducible 3D cell cultures and support the bio-fabrication of 3D tissues and organoids.
The future of bioprinting in pharmaceutical applications
Bioprinting technology has advanced significantly in recent years, although it is still limited by the speed of printing and resolution. Therefore depending on the complexity of the printed construct, it may currently be a better candidate for use in lower throughput, larger scale models in 96-well or fewer plates. Nonetheless, in the future, with the advances being made in the precision, accuracy, and scale of the bioprinters, this technology might offer solutions for many current challenges of the drug development such as disease modelling, drug optimisation, toxicity screening and precision medicine.
Disease modelling and drug optimization
Better disease models are crucial for better understanding the behaviour, development, treatment, prevention, and cure of disease. Bioprinting of 3D cell-hydrogel constructs, when combine with microfluidic systems, has allowed the creation of complex, reproducible organ-on-a-chip models which can replicate body wide diseases by incorporating many external features such as fluid or air flow and a combination of tissue types into a single 3d model. Such technology enables researchers to conduct disease progression studies for better understanding and ultimately treating the disease.
Drug toxicity screening
When developing new drug treatments, toxic effects must be well characterised and monitored in vivo in animal models. The opportunity to more effectively model such effects prior to animal studies by utilising 3D cell culture models generated using bioprinting technology may allow for the drug to be modified or withdrawn prior to costly to in-vivo modelling.
One of the most heavily investigated side effects in drug development is the liver toxicity since it can result from over-exposure to toxins, leading to toxic hepatitis, inflammation of the liver, and eventual cirrhosis if exposed over a long period of time. These side effects further lead to what is known as drug-induced liver injury which is life-threatening. The complexity of the liver combined with the substantial role it plays in drug metabolism has created a demand for physiologically improved liver models which can be addressed by novel bioprinting approaches and improved liver models.
Precision medicine is the tailoring of therapy based on a patient’s genetic information. The use of a patient’s own cells allows for an amount of data to be collected for long term studies of disease progression and drug treatment response.
The use of 3D cell culture models in precision medicine allows for cells from the patient to be cultured and expanded to study the disease ex vivo while still maintaining in vivo genotype. Using the patient’s own cells, 3D bioprinting can be leveraged to create a large quantity of patient-specific disease models for use in parallel with clinical trials or independently through precision medicine initiatives for targeting patient or disease-specific genetic, proteomic, and phenotypic characteristics.
In summary, the potential of 3d bioprinting applications in high-throughput screening is vast, with drug screening and toxicity studies, disease modelling, and most recently precision medicine applications having a greater potential in the future due to the increased complexity, physiological relevance and reproducibility bioprinting can offer. By using bioprinting as a tool in high-throughput screening, physiologically relevant models can be realized for the improvement of healthcare.
Printing techniques and materials which have the potential in this research area
Chondral and osteochondral lesions represent one of the most challenging scenarios for the orthopaedic surgeon and for the patient, as the current therapeutic strategies are not providing satisfactory results in restoring function or slowing the progression of osteoarthritis. Based on research outcomes in the recent years, tissue engineering seems to have the potential to address the issue of osteoarticular loss and provide a viable alternative to current treatments by applying 3d bioprinting techniques in the clinical environment.
It is known that cartilage injuries cannot heal spontaneously, and that any type of repair will be characterised by fibrocartilage i.e. scar tissue. This tissue lacks properties such as resistance to shear and compression which make hyaline cartilage so distinctive, and in turn leads to degenerative changes and arthritis.
Which bioprinting techniques work for osteoarticular tissues?
With the boom of 3D bioprinting, there has been widespread development of printers and additive manufacturing techniques, which allow the biofabrication of complex structures. These technologies include fused deposition modelling (FDM), stereolithography, extrusion printing, inkjet printing and laser-based techniques. With regards to cartilage regeneration, the hydrogel-based scaffolds are the main materials used due to their inherent compatibility with chondral tissue. Therefore, inkjet and extrusion printers are the most commonly used machines in cartilage tissue engineering. Having said this, it has been extrusion-based methods that have been most widely used in recent years. The popularity of this printing technique is most likely due to its simplicity, diversity, and predictability. Whilst in comparison to inkjet, for the moment extrusion-based bioprinting has lower speed and resolution, it offsets this by being able to offer advantages such as higher cell densities, a wider range of printable biomaterials (viscosity in the range of 30-6 x 107 mPa/s) and relatively inexpensive equipment.
Featured materials for cartilage bioprinting
Properties of bio-inks are also crucial for the development of functional living tissues such as cartilage by 3D bioprinting. Bio-inks based on the combination of scaffold and cells should satisfy biological features, biodegradability, and printability. Generally, available scaffold options for bio-inks are hydrogels, decellularized ECM (dECM) and microcarriers.
Hydrogels are water-swellable, yet water-insoluble, cross-linked networks. Often these are naturally-derived polymers, in which the 3D environment provided is able to maintain a high-water content, which resembles biological tissues and facilitates cell proliferation. The only limitation of such hydrogels for tissue engineering might be their inability to maintain a uniform 3D structure. However, researchers have been able to overcome this problem by applying synthetic hydrogels or natural-synthetic hydrogels hybrids.
Synthetic biocompatible polymers and peptides have been utilised to develop hydrogels for cartilage tissue engineering. Their properties can be controlled and custom-designed to match the requirements of a given cell type. This is especially true for peptide-based hydrogels., in which the physical and the biomimetic properties can be tailored.
It has been proven that chondrocytes change their function and morphology based on the ECM. Therefore, being able to provide the appropriate ECM structure is considered paramount in cartilage tissue engineering.
Peptide-based Hydrogel Inks
Synthetic ECM mimics are increasingly being used as they can provide solutions to some of the issues that naturally-derived materials pose. The main benefit of synthetic hydrogels is consistency. There should be no batch to batch variations as they are produced using known quantities, optimised procedure, and generally the chemistry of the polymer does not change unless required. As they are synthetic, there are no components from animal-derived sources which is an important factor for cell culture and tissue engineering research with the ultimate aim of clinical applicability.
As the field of 3D bioprinting matures, increasingly challenging applications of biofabrication are being undertaken. One such challenge is the construction of vascular networks, which can support the growth of larger tissue constructs by transporting vital nutrients and growth factors to the construct, while removing metabolic waste. This area of tissue engineering was recently reviewed by authors from the University of Saskatchewan in the Journal of Pharmaceutical Analysis, with a particular focus on recent examples of 3D printing directed to recreating vascular networks for tissue regeneration.
In addition to the complex three-dimensional architecture which must be recreated when printing a vascular structure, a truly representative construct must also allow incorporation of the relevant progenitor cells (such as endothelial progenitor cells). In this sense, a successful effort to fabricate a functional vascular construct will require high print fidelity and optimal methods, while providing sufficient biocompatibility to allow proliferation of embedded cells within the structure.
In terms of printing methodology, extrusion printing and inkjet deposition were highlighted as common methods used for the construction of vascular structures. Often these approaches require the use of sacrificial or fugitive materials which can be used in a print, encapsulate, dissolve workflow to form a hollow channel akin to a vascular structure. Commonly applied materials for this purpose include carbohydrate glass filaments and Pluronic F127. In addition, through emerging co-axial printing methodologies it is possible to print hollow fibres in real time.
The biopolymers used in the examples highlighted in this review vary from naturally-derived (alginate, collagen, fibrinogen, silk fibroin, laminin, Matrigel), synthetically-modified (GelMA, modified ceramics, alginate-PVA) to fully synthetic (PEG, Pluronic F127, PEUU). Each example describes the specific hardware, methodology, and materials to address a specific application, and it is clear that judicious choice of each variable is necessary for success in this fledgling area.
The generation of relevant 3D in vitro tumor models presents many challenges, but they are increasingly recognized as one of the best preclinical drug-screening platforms and an improved method to study cancer in controlled conditions in the laboratory, due to their enormous potential for recapitulating the appropriate three-dimensional and physiological features of human tumor tissues.
Traditional methods in which cancer cells are grown in a monolayer in two dimensions result in flat cells where there is no opportunity for cellular contact on all sides. This modifies cellular function due to loss of these interactions, altered cell polarity, and changes in cell shape resulting in a deficient model for understanding cancer biology or establishing appropriate antitumoral therapies. A high number of drugs have been shown to be effective in killing cancer cell monolayers, only to go on to fail in demonstrating any relevance when reaching the clinical stage. However, even though 2D culture models lack realistic complexity, the alternative animal models are very expensive and time consuming and often fail to replicate in vivo human tumor biology. Furthermore, in animal xenografts human cancer cells are usually transplanted to sites in the mouse that are convenient for experimental reasons but unfortunately do not necessarily reflect the original microenvironment of the parent tumor. Thus, 3D in vitro models can be found to be much more realistic than such animal models.
The main challenge and a priority aspect for relevant in vitro 3D models is the ability to mimic the complexity of the tumor microenvironment appropriately. In order to reproduce the complex interactions between tumor cells, stromal cells and ECM, and replicate the typical tumor compartmentalization in a precise manner, cancer cells would need to be grown in a sphere-shaped organoid, and would have to be combined with biomaterials that allow tunability of both the setup and experimental handling.
Whilst there are several 3D cell culture techniques available for the generation of tumor spheroids including hanging-drop and non-adherent surface technologies, bioprinting techniques for the generation of tumor spheroids are receiving increasing attention due to their ability to incorporate appropriate tumor architecture in a precise and controlled manner. 3D printing multiple cell types into specific scaffolds can help the generation of improved tumor organoids in which cancer cells are able to self-organize, grow, secrete their extracellular matrix and behave as they would in vivo, thus accurately representing the tumor microenvironment.
Three-dimensional bioprinting of live human cells has shown that effective in vitro replication of tumor biology is achievable. Several recent articles outline current developments in the use of bioprinted models used in cancer research, opening up a new frontier for the understanding of tumor biology and advancement of cancer therapies.
Image source: Charbe N. et al. 3D bio-printing in oncology research. WJCO, 2017