3D Bioprinting – Enabling Drug Discovery and Development and Cell Based Therapies

Yes. I believe acoustic dispensing will be a very big player in this industry, as it allows for much higher resolution than any syringe-based model. It is also very fast and accurate, and I believe it is gentle on cells so their viability remains high. It is a noncontact method, too!

Another technology that could be interesting uses some sort of structured illumination (like a hologram or a projection) to create the 3D shapes out of photopolymerizable biomaterials. Resolution would be much better than the syringe dispensing, and it would be a matter of finding the appropriate biomaterials and applications.

Yes, of course there are, as with any new technology. Though dispensing methods might not be new, the applications we are seeing haven’t been done until now. Printing with live cells and biological reagents definitely requires not only a lot of caution on the part of the researcher, but it opens doors to regulatory questions (is 3D printed tissue going to be considered a drug? a medical device? a Cell Therapy application? does the FDA need to be involved in particular applications?) and potential ethical questions as well (some people have referred to these as “playing God” type questions: should we even be printing organs at all? should we enhance the function of an organ if we figure out how? will this lead to us engineering better humans, and if so, at what cost? where will we ever stop?).

Even in pharmaceutical development, there will be looming questions. 3D cell culture models are still being researched and tweaked, and there is not one standard model being used. Comparing the data from a 3D assay to a 2D assay could prove difficult, even though I’m thoroughly convinced 3D-derived data is far superior. One big hurdle is that, as the industry has been standardized around a 2D system thus far, validating the results around a 3D model will not be the most straightforward task. However, I believe it is an imperative that companies jump on board, as those that do will ultimately be bringing large efficiency improvements to their processes. This will pay off in due time, and allow more of a streamlined process.

That is a great question! We will start small. Many companies have “innovation” groups, or automation teams. These are the people that are currently looking at bioprinting and bringing 3D cell culture into the drug discovery and development workflows. I am working with them to get the dispensing technology that works for their particular material and their particular specs. Once we know which type of dispenser will work, we can begin to augment their current automation framework and integrate our components. We’ve done this in both bioprinting R&D labs as well as highly automated production environments.

Without information on the equipment and materials you’re using (What is the medium that provides your cells with an extracellular matrix-like environment? How are the cells being dispensed, etc?), it is hard to say how bioprinting would compare with what you are currently doing. Bioprinting encompasses a wide range of technologies for depositing cells and other biological molecules in particular patterns so as to allow the cells to interact and form into tissue constructs, so perhaps you are already actually “bioprinting”. One bioprinting technique might work for your particular cells, while another one might not. It would likely come down to running some trials using your cells and media and determining which variables yield the most viable tissues for the drug testing.

In my experience, bioprinting has played a big part in the drug screening process in these ways: automating the creation of 3D microenvironment by dispensing high viscosity materials that could give the construct some structure (a Matrigel-cell mixture, for example) and layering in 3D, being able to deposit materials in a noncontact application wherein using a syringe/pipette isn’t ideal, raising throughput, and meanwhile producing more physiologically relevant data.

So in summary, there are so many parameters to consider, that it is hard to make a generalization about “bioprinting” vs. your current culture methods without having more details.

Moving beyond the adherent 2D culture, building cells up in any 3D geometry means you must keep the cells alive. As the volumes we are attempting to construct get thicker than about 200um, we will have to somehow address the innermost cells and keep them healthy. If you have ever done cell culture, you might not think about this, but it is pretty simple because the cells are pretty flat and you can address a large surface area-- simple steps of aspirating old media and changing to fresh media will usually do the trick to keep your culture happy. But in considering the structure of thicker tissues, it is quite amazing to study the vascular design that nature uses to deliver nutrients (and remove wastes) to our tissues and to every cell. In order to print out thicker viable tissues and organs, how can we get a flow of oxygen and nutrients into thicker tissues? Can we somehow print out blood vessels along with the layers of cells and growth factors? ArtiVasc 3D is just one example of a group dedicated to solving this very problem, and researchers all over the world are working on this. In addition to vascular constructs, there are alternative approaches that could be considered, such as: Can we exploit diffusion properties, and perhaps develop materials that create structure and allow for diffusion deep into the structures?

Another challenge to building any type of tissue is in getting the resolution needed to deposit the various cell types into the patterns needed to mimic natural tissue. While it’s true that biology takes the reins and cells can self-organize, they do need to first be placed in proximity to one another. To this end, researchers have developed laser assisted bioprinting: a method in which people can actually control individual cells and deposit them very precisely. Another method uses acoustic energy to generate very small droplets and can also be used to get the resolution needed.

As The Cell Culture Dish has reported in the past, bioprinting isn’t just about the act of printing out a structure. A successful bioprinting process means that the cells will form higher order structures when given the time and the proper biochemical/environmental cues. As such, it isn’t the printing process that takes the most time. The time it takes also depends on the complexity of the structure, and the size. There is not really a hard answer for this, as so many variables affect this time.

Material properties that allow for ease of dispensing (general printing logistics) should be considered, as well as properties allowing for the tissue to remain viable. That is, the material should be “printable” yet also provide structural integrity in a 3D form to maintain the physiological environment.

As such, many medical implant companies are having to tweak their formulations to allow for printing, rather than their traditional applications using molds to create implant shapes. Additionally, different printing technologies can handle different viscosities, so that plays a factor as well and should be considered in the overall design or choice of materials used. Having biocompatibility and providing tissue-specific cues is of course important, so finding ECM-like materials will give cells the most physiologically similar environment. Furthermore, the application might dictate some of these requirements; for example, if this is to be implanted in a body for regenerative therapies, the bioink should enable cell viability for some time until remodeling begins takes place and the biomaterial can be resorbed /replaced with the body’s tissue. FDA approvals will also be a consideration for these applications.

It should be noted that some bioinks must processed after printing (curing, controlling temperature, applying an additional agent) to allow for the kind of structural stability needed; some are based on photopolymers that use a UV (or other type) light to crosslink the polymers after they are printed. Special care must be taken that any post-processing does not damage any cells that are present, so a potentially easier alternative is to use biomaterial formulas that do not require this step. One bioink that I think shows promise is made by Xanofi; they are able to create nanofibers out of different starting materials (for a wide range of functional properties, depending on the application) to mimic the ECM of tissue. This has been shown to increase cell proliferation rates, while allowing the user control of the viscosity for the printing technique used (by varying the concentrations of the starting fibers and carrier gels).

There are many potential ways to injure cells during any bioprinting process, and each dispensing technology exposes the cells to varying degrees of these stresses. Some of these stresses include: Acceleration of the cells, pressure, thermal effects, shear forces, impact stresses, etc.

With pneumatic dispensing, the driving pressure and nozzle size can have an effect on the pressure (experienced by the cells) within the chamber, so using a lower pressure or a larger orifice could help lessen the damage.

Thermal effects should be minimized; avoiding biomaterials that require extreme temperature control or minimizing/localizing the thermal damage from the actual printing process (as in laser and inkjet systems).

Ensuring cells are provided nutrition/oxygen is also important, so if there are any long processes or delays, this could impact viability (for example, when cells are encapsulated within hydrogel layers that might require crosslinking, it could take some time).

Shear forces induced by the walls of the needles and impact forces should be reduced, and the surrounding matrix should provide enough structural support for cells yet still allow mobility.

These are just a few examples that will help increase viability in bioprinting methods.

While you could organize this information in different ways, I think of it as the use of contact methods vs. noncontact methods of dispensing.

Contact Methods (also referred to as “extrusion”, as one would extrude the material out from the tip of a pipette/syringe; the tip comes close enough to the substrate for the material to make contact)
Air pulse (pneumatics push the fluid down the syringe barrel)
Displacement syringe (piston pushes fluid down the barrel)
Screw (screw motion pulls fluid down the barrel)

Noncontact Methods (produces droplets, which shoot at a substrate from a specified distance)
Jet - there are different types like piezo, thermal, mechanical
Laser - uses pulsed laser beam to generate droplets
Acoustic - uses acoustic energy to generate droplets
Photopolymerization - (stereolithography and related techniques)

Note: some people are erroneously calling the technique of magnetic levitation (used for creating 3D cultures) a type of bioprinting, when I would argue that it is not. It does not depend on a particular kind of printing, but rather on interactions between magnetic nanoparticles incorporated into the cells and an external magnet.

As long as you keep all variables consistent, this shouldn’t be a problem. It is very important to maintain proper environmental control. Also, you should use a high quality dispensing system with repeatability specs that meet your needs (repeatability both in the xyz motion as well as in the dispensing itself). For example, if the dispenser is not giving you the same exact droplet size every time (or the same pressure and flow rate if we are dealing with a pneumatic syringe/needle system), then you can’t guarantee that your assays will be valid. I have seen systems that do not in fact give consistent dispensing results, so the researchers cannot get consistent data.

Yes, you do, and each printing technology and the printing parameters specified have varying effects. One way to visualize this is using DNA or membrane stains, as shown here.

For a pressure-driven system (syringe + needle), there is data that shows that varying the pressure generally has more effect on morphology change and viability than does varying the needle size, and there are even empirical models to predict the number of live/injured/dead cells according to these dispensing parameters.