No doubt many of us have watched videos on social media of a “human ear” being 3D printed in a lab, indeed, many of us will be the ones posting such clips, where we use the complex structure of the ear to demonstrate the printability and performance of a bio-ink material. Besides the visually engaging results, it is worth to consider how such 3D bioprinting technology can offer real hope for patients with conditions such as microtia.
Microtia is a birth defect where the external ear is underdeveloped. Although there are several different options for auricular reconstruction, today the ‘Rib Cartilage Graft Reconstruction’ is the most effective. This treatment is based on sculpting the patient’s own rib cartilage into the form of an ear. Because the cartilage is the patient’s own living tissue, the reconstructed ear continues to grow as the child does. This is a very detailed and complicated surgical procedure with associated high-risk as well as chest-wall deformities.
3D bioprinting technology aims to create transplantable organs by applying cell-supporting bio-inks. One of the leading research areas of this technology focuses on the development of cartilage tissues for use in reconstructive surgery. This process aims to utilize the natural self-organizing properties of cells in order to produce a functional tissue. Although bioprinting technology is a promising method in itself, the properties of the bio-inks required are also crucial for the development of functional living tissues such as cartilage.
Synthetic biocompatible polymers and peptides have been utilized 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.
In the last few years, there has been a huge variety of research in cartilage bioprinting techniques, cell types, and bio-inks which have demonstrated the promising potential of such advanced bio-manufacturing process. Today this technology still has many challenges. However, it is hoped that in the future 3D bioprinting as a process to create transplantable cartilage can be translated into clinical practice and can provide a low-risk surgical solution for diseases such as Microtia.
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.
3D bioprinted in vitro models have received increasing attention as a potential means to improve in vitro cancer research by bridging the gap between 2D in vitro and animal in vivo platforms and mimicking more accurately human cancer biology. In vivo cell behavior is modulated through different interactions, including mechanical and chemical signals between cells themselves and their extracellular microenvironment (extracellular matrix and stromal cells). These signals are responsible for cellular growth, self-organization and cell-dependent deposit of matrix proteins, and are impaired in tumors. Indeed, tumor microenvironment determines different cancer phenotypes for a same tissue, and subsequent therapeutic response or resistance. In line with in vivoevidence and in contrast to 2D monoculture, a number of studies have shown that 3D microenvironment substantially alters the tumor response to anti-cancer drugs; perhaps, due to the absence of a well-established microenvironment and defined tissue architecture in 2D models. Therefore, relevant 3D in vitro platforms that enable the formation of pertinent interactions between cancer cells and their microenvironment are required for the assessment of different anti-tumorigenic strategies. Furthermore, modeling the complicated tumor architecture in a very precise and controlled way is currently possible with innovative bioprinting techniques, which has opened new venues for the creation of more realistic 3D cancer in vitro platforms.
Recently, Langer et al. (2019) demonstrated that 3D bioprinting might be used to generate multicellular architecturally defined tissue models of human tumors capable of effectively mimicking in vivo tissue biology. In particular, they modeled different 3D tumor phenotypes with a variety of cancer cell types, including multiple human breast cancer and pancreatic cell lines, and, importantly, primary tissue from pancreatic cancer patients. The generated platforms were used to interrogate both intrinsic and extrinsic signals from the diverse cell types, including their corresponding responses to different microenvironments and a range of anti-cancer drugs.
The models were initially created as bio-printed structures comprising the corresponding cancer cell type, different stromal cell types and alginate-containing hydrogel as bio-ink, which was removed during subsequent culture. The resulting tumor tissue showed similarities to in vivo solid tumor architecture, involving a tumor cell core surrounded by normal stromal cells. These cells were able to survive, self-organize, proliferate, migrate and, importantly, interact with one another to form tissue-like structures once the bio-ink was removed. For instance, an estrogen receptor-positive model of breast cancer was created with a mix of the MCF-7 cancer cell line, stromal fibroblasts, and human umbilical vein endothelial cells. In this model, close interaction between epithelial cancer cells and stromal fibroblasts, and progressive formation of endothelial networks was evidenced several days after the bio-ink was removed.
Additionally, the study compared the proliferation rate of different models of breast cancer containing the same stromal cell mix. The proliferation rate was highest in the platform containing the claudin-low MDA-MB-231 breast cancer phenotype, which has been widely recognized as highly invasive in vivo. Conversely, no significant differences were reported when the equivalent 2D platforms were compared.
The authors also demonstrated that distinct microenvironments might be modelled in their architecturally defined breast cancer models, by incorporating additional cell types known to be present in human breast tumors to the initial stromal cell mix. These included mesenchymal stem cells, which have been shown to affect tumor progression, growth, and migration.
Importantly, Langer et al. evidenced that their bio-printed 3D tissues can be used to assess therapeutic efficacy, better recapitulating in vivo phenotypes than 2D monocultures. As an example, an equivalent 2D monoculture to one of their breast cancer models was treated with the targeted PI3K/mTOR inhibitor BEZ235, which led to a substantial decrease in cell proliferation. In contrast, the same experiment didn’t lead to any significant changes in the bio-printed 3D tissue.
The present 3D bio-printing methodology was applied to additional tumor types (i.e. pancreatic ductal adenocarcinoma) both in the human pancreatic cell line HPAFIIA and primary tissue from patients. In order to demonstrate that these models respond to microenvironmental signals, they treated the HPAFIIA 3D platform with transforming growth factor beta (TGFbeta), a well-studied cytokine that activates pancreatic stellate cells thereby inducing their proliferation and tumor cell intrinsic migratory capacity in vivo. In line with this, pancreatic stellate cell proliferation was increased in the developed 3D platforms treated with TGF-beta, as well as the percentage of HPAFFII cancer cells that migrated into the surrounding stroma. Finally, primary tumor tissues from pancreatic cancer patients that were able to recapitulate in vivo morphology were generated. The morphology, connectivity between cells and proliferative capacity of these innovative tissue platforms were indeed similar to structures found in primary tissue and xenografts of the corresponding patients.
All in all, Langer and colleagues have generated physiologically realistic 3D bio-printed tumor tissues from distinct subtypes of breast and pancreatic cancer in relevant microenvironments and demonstrated that the same technique can model patient-specific tumors using primary cells. Moreover, the models can be used to mimic different tumorigenic reactions to multiple stimuli, including drug resistance or response in distinct cancer phenotypes. In the future, bio-printed tissues containing cells from patient tumors could be used for translational studies aiming at generating personalized therapies for the treatment of cancer.
Source: Langer et al. Modeling Tumor Phenotypes In Vitro with Three-Dimensional Bioprinting. Cell Reports, 2019.
Extrusion-based bioprinting methods have been employed in recent years to provide researchers with cost-effective alternative methods for scaffold fabrication. The popularity of these methods relies on clear-cut processing methods leading to a technique which offers simplicity, diversity, and predictability. The wide range of printable biomaterials and inexpensive equipment are also among the advantages of this printing technique.
The extrusion-based bioprinting method is categorized into pneumatic, piston-driven, and screw-driven dispensing. In pneumatic dispensing, air pressure provides the required driving force, while in piston and screw-driven dispensing, vertical and rotational mechanical forces initiate printing, respectively. All of these methods required a bioink which needs to be in the liquid phase to avoid nozzle clogging, but viscous enough that it holds the printed shape, protects cells during extrusion and provides the cells with an in vivo-like environment.
Researchers prefer to use biomaterials previously proven to be compatible with cells to decrease the variables in their projects. However, many biomaterials are not printable without making extensive changes to their mechanical and potentially, therefore, their biological properties. Thus, it is very important to choose a material for your research which does not only provide you with a 3D cell culture matrix but is also printable.
At Biogelx, we manufacture peptide-based hydrogels which offer you both benefits. We apply extrusion-based methods to print with our novel synthetic bioinks. See how perfect the shape is by using standard BiogelxTM-Ink.
BiogelxTM-Ink S is commercially available from April 2019!
In many respects, 3D biofabrication remains a young area of research, facing many of the same barriers to entry as any other emerging technology. The cross-disciplinary requirements for success in 3D bioprinting involve expertise in both engineering and biology, and many of the bioprinters on the market today have resulted from successful collaborative projects which satisfy key requirements from each of these disciplines. Many of the early commercially-available printers were bespoke machines with fittingly high price tags. However, over recent years the presence of more affordable printers on the market has resulted in a lower cost of entry to the field, driving research in this area forward at an ever-increasing rate.
A recently accepted paper in the journal Bioprinting is now available online, and details the design and performance of a new low-cost, open-source printer which is capable of printing using both inkjet and extrusion printheads. The potential for this technology is to make both high-resolution and high-throughput printing affordable for many more research labs, while allowing those researchers with more complex bioprinting needs to adapt the model to their specific requirements. The researchers from the University of Connecticut USA, acknowledge the biological constraints placed on the mechanical design of the printer, including limitations on heating and applied forces which would limit the compatibility with cells in a cell-laden bioink. The parts and instructions to build this dual function 3D printer are clearly presented in the paper, and its performance is then tested using GelMA (with UV photo-crosslinking) and alginate (with chemical cross-linking) bioinks. Using mouse embryonic fibroblast cells for cell-laden bioprinting, cell viability was then assessed using live/dead staining, and while cell viability was similar for both inkjet and extrusion printing, the inkjet printed constructs had a slightly higher cell viability %, while the concentration of alginate material used for extrusion printing had to be carefully optimised to give comparable results.
Having shown the competitive performance of this new bioprinter, the researchers also compared the cost of sourcing the components used. The cost analysis totalled just $1,370, or six times less than the most competitive quote received from a commercial bioprinter vendor.
Using open-source principles, this work should help place affordable and adaptable 3D bioprinters into many more research labs at a fraction of the initial cost. There is clearly high demand for new and improved hardware,bioinks and protocols for 3D printing, as evidenced by the growing body of literature. By lowering a key barrier to entry, projects such as this may help to accelerate progress in the growing and widely applicable area of 3D bioprinting.