Tissue engineering is bringing together advances from stem cell biology, microfluidics, robotics, and 3-D cell culture to develop novel products for the drug development and toxicity testing sectors. The ability to create miniature tissue or even human organs-on-a-chip is valuable for a number of reasons.
First, it has been shown that cells growing in more physiological 3-D cultures behave differently to the same cells when grown in the type of 2-D cultures currently being used in the drug development sector. Second, when analyzing the potential toxic effects that drug metabolites may have on other cell types, it would be useful to have an in vitro system that links the human liver to a chamber containing the test cell type.
Third, current analysis of potential metabolite toxicity involves the use of a large number of experimental animals (mostly rodents). This is not ideal as animal models are both costly to run and less likely to give an accurate representation of metabolism and toxicity in the human organ. Ongoing model development also seeks to reduce the number of animals used in such work for ethical reasons.
Researchers at Edinburgh’s Heriot-Watt University have developed valve-based cell-printing processes that are able to deliver cells in specific patterns in volumes as low as 2 nL or less than 5 cells per droplet. Using cells derived by Roslin Cells, sister company Roslin Cellab has demonstrated the first example of printing human embryonic stem cells using this valve-based printing approach. The printed cells are to be subjected to a directed differentiation protocol to produce human hepatocyte-like cells. In order to analyze the printed and differentiated cells, a development product from Reinnervate Limited will be used to enable us to maintain printed 3-D stem cells on the upper surface.
The Cell Printer
In recent years, the use of a simple inkjet technology for cell printing has triggered tremendous interest and established the field of biofabrication. In laboratories, we have seen exciting demonstrations of printing 2-D tissue like skins and 3-D structures such as artery and kidney; however, there are still many challenges to overcome before the technology can be used clinically to generate transplantable organs.
One of the key challenges has been the development of printing nozzles that are more controllable and gentle on the cells to preserve cell and tissue viability. We recently developed a valve-based dual-nozzle printer (Figure 1) that has been validated to print highly viable cells including the first example of printing human embryonic stem cells for tissue regeneration.
Human embryonic stem cells (hESC) are pluripotent and can thus be used to generate many cell types present in the human body. In addition, the cells are highly expandable, which enables large numbers to be produced prior to differentiation into the cell type required.
A panel of 5 of the 20 hESC lines derived by Roslin Cells were used to make embryoid bodies, which are cell aggregates that are allowed to differentiate in an undirected manner. Our special interest is in the production of hepatocytes, so we selected the hESC line that showed the strongest natural tendency to make these cells (RC10).
After 17 days of directed differentiation, key markers of mature hepatocytes were expressed by RC10 derived hepatocyte-like cells (Figure 2).
In addition, the hepatocyte-like cells were shown to be metabolically active, demonstrating basal CYP3A activity, albumin secretion, urea genesis, and testosterone metabolism.
A feature of hESC lines that has made them more difficult to work with is that, when dissociated into a single-cell suspension and re-seeded into fresh plates, they have a tendency to differentiate spontaneously. For this reason, many laboratories prefer to passage the cells physically without using trypsin; however, this introduces great variance in cell number between wells after re-plating and should be avoided.
Interestingly, the RC10 hESC line is also more resistant to the deleterious effects of single-cell passaging, making it a good choice for producing hepatocytes for cell-based assays. This feature also means that RC10 can be used effectively in cell printing processes to establish artificial stem cell colonies of specific sizes and shapes and to create cell spheroids.
For some applications it is useful to deliver cell aggregates rather than a cell suspension. For example, when differentiating stem cells into blood cells such as macrophages, it is necessary to proceed via an embryoid body (EB) intermediate. The size of these EBs partly determines the efficiency with which the macrophages can be produced and is controlled by the number of cells used to make the initial pre-EB spheroids.
Printing of pre-formed stem cell spheroids may also enable 3-D tissue to be built up more quickly. Figure 3 shows how stem cell spheroids of varying size can be created reproducibly using a dual printing head that delivers cells in medium or medium alone. After printing, the well plate is inverted to allow cells to gravity aggregate and begin dividing.
Once spheroid growth has reached the required level, the spheroids can be transferred to the surface upon which the new tissue is to be created. Establishing spheroids with between 5 and 140 dissociated cells resulted in spheroids of 0.25–0.6 mm diameter.
Discussion and Perspectives
This work demonstrates that the valve-based printing process is gentle enough to maintain stem cell viability, accurate enough to produce spheroids of uniform size, and that printed cells maintain their pluripotency.
In subsequent experiments, we will print and differentiate cells into hepatocytes and test for metabolic activity and other markers of mature hepatocyte phenotype. Following on from this we will look to establish 3-D stem cell cultures that will be differentiated and analyzed for an expected improvement in metabolic activity and functional life span. These will be made either by delivering the cells to a surface in a hydrogel/medium mixture, or printing them on top of a matrix and building up cell layers with incorporation of additional matrix compounds.
We see these products as being potentially valuable to the in vitro drug development and toxicity-testing sectors, though clearly by demonstrating improved hepatocyte function and longevity in 3-D cultures we will also be paving the way for cells to be incorporated into clinical protocols either for patient implantation or inclusion in a bio-artificial liver device.
Will Wenmiao Shu, Ph.D. (W.Shu@hw.ac.uk), is a lecturer at Heriot-Watt University, Jason King, Ph.D. (Jason.King@RoslinCellab.com), is business development manager at Roslin Cellab. Stem cell printing and analyses were carried out by Seb Greenhough and Alan Faulkner.