The Atlantic KET Med Project (AKM), with the aim of facilitating a transnational advanced pilot manufacturing ecosystem for producing next generation medical devices has provided direct support to companies to improve innovation management, value chain analysis, technology-specific education, access to research, and enable greater integration of KETs into education, production and innovation in the Atlantic Area.
One of the key aspects targeted by Atlantic KET Med is the provision of better and more open access to high-tech pilot production facilities. To this end, a network of KETs experts, which forms the core of the project consortium, has worked directly with companies and other stakeholders to develop both the large-scale development strategy for the Atlantic Area regions and specific product development strategies on a case-by-case basis for new biomedical products, offering companies technical support for conducting case studies for a novel product, thus facilitating, with companies using applied KETs, the development of new medical products and devices.
From the Fundación Instituto de Investigación Sanitaria de Santiago de Compostela (FIDIS) we have collaborated with the company 3DBIOLUX, proposing a case study focused on its product called FABRILUX (TRL 8-9), a 3D Bioprinter, which applies KETs – photonics, laser, and advanced manufacturing techniques. FABRILUX is a tool intended for use in both research and manufacturing environments, so it is necessary to validate the product for use in both environments.
The approach for this case study is as follows: in a first phase, FIDIS – 3DBIOLUX, for evaluation in a laboratory environment, analyzing those components of the 3D bioprinter that are, or are not, more or less prone to contamination. 3D bioprinters need to be proven to work properly in a sterile environment. This means that the designs of new printers must be compatible with the sterilization protocols used in cell culture chambers (chemicals and UV radiation). In addition, to avoid any contamination, these printer designs must not disturb the laminar flow of sterile air in the biological safety chambers.
Validation was carried out in the facilities and research laboratories of the Instituto de Investigación Sanitaria de Santiago de Compostela (IDIS).
In a later phase, NUI Galway – 3DBIOLUX, testing 3D bioprinting with cells in the combined additive and subtractive pilot manufacturing test bed for future biomaterials and printed electronics applications for the validation of the 3D bioprinter in an pre-industrial manufacturing environment.
Bioprinter (Fabrilux II)
Fabrilux II (figure 2) is a bioprinter system for the generation of complex and multimaterial artificial tissues. The innovation of 3D Bioprinting systems developed by 3DBiolux is based on the application of photonics concepts in combination with microfluidic systems and “customized” heads for the biofabrication requirements of each particular tissue.
Figure 2. Fabrilux II bioprinter.
Bone tumor model generation
Organ-on-a-chip (OoC) systems garnered enormous interest from researchers for their ability to monitor real-time physical parameters by mimicking the in vivo microenvironment. OoC systems can also provide a precise response of xenobiotics, i.e., drug efficacy and toxicity over conventional two-dimensional (2D) and three-dimensional (3D) cell cultures, as well as animal models.
Recent advancements of OoC systems evidenced the fabrication of ‘multi-organ-on-chip’ (MOC) models, which connect separated organ chambers together for monitoring the complex interactions between multiple organs (tumor niches) and the resultant dynamic responses of multiple organs to pharmaceutical compounds. The use of tumor / metastasis-on-a-chip is not limited to the studies described so far, but the potential of this device will allow the application of personalized medicine thanks to ex vivo models of patients.
3D bioprinting allows the generation of tissues that incorporate a variety of cell types in a complex and defined spatial architecture whose main aim is to better mimic human physiology and functions at multiple scales, from the molecular to the cellular, tissue, and organ level. Monocultures 2D assays are cost-efficient and simple to use, but have limited predictive competence, since they fail to mimic human physiology.
3D bioprinting can be used for reproducing the micro physiological systems that reconstitute tissue–tissue interfaces and the TME, thus expanding the capabilities of OoC models. The 3D bioprinting technology used for bioprinting will affect the quality shape maintenance and cell viability of the final tissue structure. Digital light projection (DLP) bioprinting is based on the photopolymerization of biomaterials. DLP usually uses ultraviolet (UV or Visible) light to solidify a photosensitive bioink in a layer-by-layer fashion to generate a 3D structure. This bioprinting process presents a high bioprinting speed with high resolution especially working with hydrogels, which also allows a certain control over matrix properties, and so facilitates more realistic 3D modelling of tumour models. Based on such premises we have demonstrated the capabilities of Fabrilux II bioprinter for generation bone tumours models on a chip.
Evaluation of contamination risk
To evaluate the presence of any potential contaminating organism (bacteria, fungi, etc.) the cell cultures were monitored with an optical microscope before and after (48h) the exposition to the bioprinter. As shown in figure 2, it was not identified any contaminating organism, cell death, proliferation alteration, or morphological change associated to the exposition to the bioprinter. Nitrite determination in the cell culture media. To confirm the absence of any potential contaminating organism in the culture, nitrite accumulation was determined in the cell culture supernatant of ATDC5 cells 48h after the exposition to the bioprinter (fig 4a).
Data obtained revealed that nitrite accumulation, a NO metabolite, was not induced due to the exposition to the bioprinter. In contrast to this, the supernatant of LPS-(bacterial wall component) treated cells accumulated a great amount of nitrite (fig 4b).
Figure 4. Optical images of cells: a) after printing and b) at 48 H. c) graphics showing no nitrite accumulation on bioprinted samples.
Microfluidic chip
The microfluidic chip of the bioprinting platform consists of a multi-layer configuration. The material used was to build the chip was Polydimethylsiloxane (PDMS) due to its properties: chemically inert, autoclavable, optically transparent, flexible, and allows gas exchange. Firstly, the chip mold was designed using CAD inventor and imported to a 3D printer.
The mold include 1mm wide inlet, one printing region that will form the chip’s culture chamber (The upper ring has 10 mm diameter and the lower, 8 mm) and 1 mm outlet. Those rings provide the support for attaching the 8 µm porous membrane, which is fixed by the 10 mm diameter and 1 mm high plastic ring (Figure 5), on which the model will be bioprinted.
Figure 5. Schematic representation of the circulatory system and culture chamber inside the chip.
The resulting microfluidics device consists of a perforated PDMS brick (75 mm x 25 mm x 8 mm) which has a culture chamber where the 3D study model is bioprinted. Stainless steel adaptors (20G) were connected the inlets/outlets. The porous membrane is used as a bed for bioprinting while allowing the interchange between the up and down parts of the culture chamber.
Osteosarcoma tumor model bioprinting
We have used the bioprinter for generating osteosarcoma tissue. A pre-defined 3D pattern of bone tissue was bioprinted using mesenchymal stem cells (MSCs) and osteoblasts. Both cell lines were stained using two distinct cell-trackers, HUVECs were stained in green (Cell Tracker Green (CTG)) and MG-63 spheres in red (Cell Tracker Red (CTR)) to monitor the state of the CSC migration assay on days 1, 3 and 7 by confocal images.
The MG-63 spheres present within the construct have a uniform shape and a diameter between 100-150 µm. Moreover, the X-axis view images clearly show how the spheres migrate from the bottom of the hydrogel to the HUVECs zone since on day 1 only a few independent cells (represented by red dots) are visible close to the HUVECs zone. However, on day 7, a large number of migrating individual cells from the spheres are visible close to the HUVECs monolayer (Figure 6).
Figure 6: X axis view HUVECs in green and MG-63 detached cells or spheres in red.
The data obtained indicate that the 3D bioprinter does not increase the infection rate of the cultures. Therefore, it can safely be used for bioprinting purposes in the sterile environment of biological safety hoods. To validate the result on a bioprinting process, we have developed a bone tumour model on a-chip which reveals as a potential strategy through the generation of 3D in vitro models for recapitulating the tumor process. Such models could be used for pharmacological screening, for clinical application in precision medicine and decision making through the generation of personalized models with patient samples, which will improve patient’s survival and outcome.
