The advent of labs- and organs-on-chips, thanks to joint advances in design techniques, materials science, micro-manufacturing technologies, and cell-culture processes, is providing researchers with new ways of modeling and studying disease, while helping to develop related therapies in a non-invasive, sustainable, and quite straightforward approach [1
]. Within these labs-and organs-on-chips cells grow, move, communicate, and interact with their companions in much more physiological and biomimetic conditions than when cultured upon conventional Petri dishes.
Even if some key aspects, such as obtaining a real three-dimensional representation of the tissues and organs being studied or promoting in vitro vascularization, are still unsolved, these microsystems are already having a relevant impact in biomedical research. Foundational experiences in this field resorted to the use of UV-lithography and etching upon silicon wafers, following procedures from the microelectronics industry [5
], although the biochemical and biomechanical interactions between cells and silicon does not always provide the desired level of biomimicry. The development and application of soft-lithographic procedures, to achieve polymeric (mainly polydimethylsiloxane (PDMS) but sometimes even paper) parts, provided a new set of resources for researchers and for the rapid manufacture of labs- and organs-on-chips [6
More recently, progresses in additive manufacturing technologies and developments linked to the materials abilities or characteristics for being additively processed have also made an impact in the biomedical engineering arena and, especially, in the development of labs- and organs-on-chips [8
]. These additive manufacturing technologies provide designers with an increased level of geometrical complexity and flexibility, which is necessary for devices trying to imitate the intricate shapes of the human body, organs, and tissues. In addition, additive manufacturing technologies also promote the level of integration and even monolithic labs- and organs-on-chips have been recently obtained, which constitutes and advance, in terms of ergonomics, for researchers usually having to deal with several parts and devices in their labs.
However, in many cases, the required level of precision for interacting at single cellular level is difficult to achieve; hence alternative techniques or multi-scale approaches are required. Despite the aforementioned advances, the success of labs- and organs-on-chips as transformative technologies in biomedical engineering still depends on our ability of solving some current challenges related to their design, modeling, manufacturability, and usability. Among present needs for the industrial scalability and impact promotion of these bio-devices, their sustainable mass production constitutes a breakthrough for reaching the desired level of repeatability in systematic testing procedures based on labs- and organs-on-chips. The use of adequate biomaterials for cell-culture processes and the achievement of the multi-scale features required, for in vitro modeling the physiological interactions among cells, tissues, and organoids, prove to be demanding requirements in terms of production.
This study presents an innovative synergistic combination of technologies, including: laser stereolithography, laser materials processing on micrometer scale, electroforming, and micro-injection molding, which enables the rapid creation of 3D multi-scale mold cavities for the industrialized mass production of labs- and organs-on-chips using thermoplastics apt for in vitro testing. The procedure is validated by the design, rapid prototyping, mass production, and preliminary testing with human mesenchymal stem cells (hMSCs) of a conceptual multi-organ-on-chip platform, which is conceived for future studies linked to modeling cell-to-cell communication, cell-material interactions, and metastatic processes. Current research works upon initial trials developed by our team [11
], but achieves here more precision, a larger final size of the mold inserts and micro-injected replicas and an increased degree of complexity, including multiple organ chambers and biomimetic features for an improved performance. At the same time, a more complete validation of the developed multi-organ-on-chip, by testing with hMSCs, is presented and analyzed. The biological testing stands out by the employment of a human mesenchymal stem cell (h-MSC) conditioned medium (CM), which is a solution of growth factors generated progenitor stem cells themselves, that helps to enhance the microsystem’s response by promoting cell adhesion, proliferation, and final long-term viability in the cell-culture trials.
The relevant advantages of the employed synergistic combination of technologies, especially in terms of multi-scale geometrical freedom, structure accuracy, use of industry-capable methods and productivity, towards mass-manufacture of polymeric labs- and organs-on-chips for increased industrial and social impact are finally discussed, considering also the preliminary assessment of material’s viability achieved thanks to the cell-culture tests performed.
3. Results and Discussion
Once the computer-aided designs are improved taking account of the desired network of chambers and channels and once the master models additively generated by using laser stereolithography, the procedures previously described in the “Materials and Methods” section are applied to generate a metallic mold for micro-injection molding and to successfully produce a series of multi-scale organ-on-chip platforms using a transparent thermoplastic adequate for cell-culture studies, which are also performed. Relevant results, in connection with the validation of the different manufacturing steps, have been already presented in the “Materials and Methods” section for illustrating the different design and manufacturing processes. This section also discusses the results of the different processes for illustrating how the synergistic combination of technologies presented here finally works and how cells interact with the manufactured substrates. The potential of these synergistic technologies for the field of biomedical microdevices and microfluidic systems is also discussed.
Starting with the laser processing of the master prototypes, Figure 2
shows the results from the laser material processing performed upon the master epoxy resin prototypes for generating the micro-pillars or connecting gates between vascular channels and organ chambers. Figure 2
a provides a dimensional overview of chambers, channels and connecting gates, while Figure 2
c provides details of the surface finish after laser material processing. The layer-by-layer generation of the master models leaves some slight horizontal marks in the walls of the channels and chambers, which can be appreciated in Figure 2
a. However, the lateral walls of the opening gates, obtained by laser processing are extremely vertical(without the previously mentioned horizontal walls), which helps to highlight the viability of processing the epoxy resin of stereolithographic masters and the possibility of obtaining very precise cuts, in order to achieve the desired multi-scale geometries, as finally presented also in Figure 2
The repeatability of the process is also noteworthy, as all the opening gates are 260 ± 5 µm and the remaining micro-pillars are 340 ± 5 µm. Such level of definition can be obtained with extremely high-precision additive manufacturing techniques, mainly resorting to two-photon polymerization (i.e., Photonic Professional system by NanoScribe GmbH), but the working field is normally limited to less than 1 mm2
. Consequently, for organ-on-chip devices, in which relevant portions of tissues must be cultured and studied, the multi-scale combination of large field additive manufacturing techniques, for generating the overall chip structure, and the use of laser material processing, for defining the smaller details, proves more adequate with the current state of technology. Alternative approaches relying on the combination of multi-scale processes, such as mask-based lithography and high-precision direct laser writing (by two-photon polymerization), also allow for the generation of multi-scale organs-on-chips [25
], although their productivity (parts manufactured per time unit) is still far from matching the performance of mass-produced systems by micro-injection molding, as presented here. Nevertheless, in terms of geometrical complexity and integration level [26
], additive procedures remain unrivalled, so further synergies between processes may prove highly beneficial.
Once the master model is processed, the key challenge is linked to creating the metallic mold insert by nickel electroforming without damaging the master model and without losing the previously created details, which provide the desired functionality. Figure 3
shows the different steps for the metallization of master prototypes and the results of this process, which stands out for the surface quality achieved (see SEM images presented in Figure 4
). The generated metallic insert, after dissolving the epoxy resin of the master model and final mechanical treatment using a milling process, is adjusted to create a mold cavity, which is machined and polished for improved flatness and mirror-like surface finish, as shown in Figure 5
. The set of replicas is created by micro-injection molding (Figure 6
), as previously detailed. Final productivity can be approximated to around 100–200 parts per hour, once the micro-injection process is adjusted and set to automated production. Multi-part molds are also possible, which would increase production, but the productivity achieved in present study is more than enough for our validation purposes and for illustrating the potentials of this synergistic combination of technologies.
Improvements in terms of multi-scalability and precision may further be achieved by synergic nano-replication techniques, including coatings upon patterns obtained by nano-imprint lithography [27
] and mold tools obtained by synergic combination of electron beam lithography, nano-imprint lithography, and ion beam etching [28
Regarding cell-culture tests, the available results from hMSCs interacting within the multi-organ-on-chip biomedical microdevice are presented further on in the images of Figure 7
To this end one of the micro-injected replicas was exposed to CM produced by hMSC, as reported in early work, then seeded with cells and incubated with Dulbecco’s Modified Eagle’s Medium (DMEM) low glucose and 10% FBS during 48 h. The process is detailed in the “Materials and Methods” section. Figure 7
a provides a global view of the biomedical microdevice and highlights the zones magnified in Figure 7
b,c. Figure 7
b shows cell adhesion to an inlet well and across a vascular channel entering or colonizing one of the organ chambers. Figure 7
c shows cells colonizing an organ chamber after reaching it through the vascular channel and in between the micro-pillars although in other experiments the organ chamber may well be seeded independently (and even using different cell types for a true multi-organ-on-chip study).
The cells are stained with crystal violet and, when analyzed with the microscope, show an expanded and healthy morphology. By careful visual inspection we can note that more than 100 cells/mm2
are present in such healthy conditions in several zones of the microsystem. Besides, it is important to highlight that hMSCs are adherent cells and that when they do not have enough metabolic energy to adhere they become spheroid, apoptotic and die in just a couple of hours. When hMSCs die, they appear floating in the cell-culture assays and disturb microscope imaging. In our tests we did not find a relevant number of dead and floating cells within the presented organ-on-chip platform and we could not appreciate any spheroidal cells, all of which helps to validate the adequate biological performance of the technologies, materials and methods used. As an example of the aforementioned unhealthy behavior of hMSCs, recent studies have demonstrated how the number of cells adhered is reduced if mitochondria have diminished electron transport and how this is directly linked to the progressive spheroidal configurations, apoptotic behavior, and final detachment [29
]. Taking into consideration the biofunctionalization post-process performed by using the hMSCs-CM, we would like to mention that the coating improves hMSCs adhesion up to a 30–40%, when compared to the use of uncoated microsystems, as previous studies by our team have shown [12
]. However, the manufactured layers can be also used for culturing other cell types and even hMSCs, just after micro-injection and polishing, without the need for a final biofunctionalization step.
Even if we have worked with a single micro-structured device and cultured the cells within such platform just closing it with a transparent coverslip, which cannot be considered a completely functional organ-on-chip device, in which pumped fluids would support cells during their growth, proliferation and phenotype expression, we would like to mention that in many cases these preliminary cell-culture tests provide interesting information regarding cell-cell and cell-material interactions. In fact, the performed cell cultures have helped us to validate the viability of the micro-injected material for the industrial production of organ-on-chip platforms, or of their functional layers, and the possibility of working with these multi-chamber and multi-channel platforms, whose potential applications are varied. Future studies with these or similar micro-injected organ-on-chip platforms and cell-culture platforms will be linked to exploring the co-culture of different cell types and their communication, in accordance with recent progresses in cell-culture microsystems [1
]. Synergies with other recent approaches to multi-scale mold insert fabrication, with which very special contact phenomena can be promoted, will be studied [31
Finally, considering the issue of potential industrialization of the proposed approach, we envision that these set of technologies, with some additional supporting facilities, such as bonding and welding line, could be implemented in the form of a pilot line for the rapid engineering of lab- and organ-on-chip devices. The line would consist of: (1) a rapid prototyping technique working through additive photopolymerization, typically laser stereolithography or digital light processing, either in their industrial or low-cost modalities; (2) a laser micro-machining or ablation system for manufacturing the finer details and for performing surface modification and texturing for tuning the master models; (3) a electro-deposition system by galvanic bath for transforming the polymeric master models into metallic mold inserts and (4) an industrial micro-injection system with mold structures prepared for rapidly changing the inserts with the desired configurations of channels and chambers. In addition, supporting bonding and welding lines, together with fluid pumping systems, for the mounting and testing of complete microfluidic systems would be necessary.
For simple lab- and organ-on-chip devices, in which the functional layer includes just micro-channels and chambers, the set of operations performed via laser stereolithography and subsequent laser ablation and metallization could be possibly replaced by a micro-milling machine working upon a metallic surface for directly generating the mold inserts. However, if complex geometries are involved, if aspect ratios above 1.5 are needed, or if details below 20 µm are needed, the combination of laser stereolithography and subsequent laser ablation performs better than micro-milling, especially when working with thermoset polymeric masters, whose machining by conventional subtractive methods is not so straightforward due to the higher heat-affected zones and debris generated. In fact, laser ablation reaches aspect ratios up to 50 for drilling and 10 for cutting and enables structuring materials with resolutions down to 1–10 µm, which result extremely demanding for micro-milling. In addition, laser material processing may be applied to obtain special surface textures, which can be applied to defining hydrophobic/hydrophilic transitions for controlling cell aggregation upon the surfaces of these types of microsystems.
The main challenge for the proposed industrialization may be connected to training a community of researchers and technicians capable of managing this populated set of technologies, which requires vision, time, and resources. In any case, interesting alternatives may include: (a) the cooperation among delocalized research communities, as in fact we have shown in present study, or (b) the opening of large research infrastructures to external researchers through competitive calls, a possibility for which the KNMF-KIT constitutes a paradigmatic example. We expect these types of pilot lines and collaborative communities to impact at the bio-application level in this field of microfluidic devices by helping researchers to develop, in a straightforward way, sets of lab- and organ-on-chip devices, probably with a common framework or outer structure but with interchangeable functional layers, in which different patterns of channels, chambers and surface textures may help to mimic different organs and configurations of vascular networks and parenchymal tissues, towards the human-on-chip concept by integration of organ-on-chip microdevices.