1 Introduction – limitations of today’s cell culture lab ware
Plastic based vascular cell culture systems have proven to be suitable tools in biomedical research investigating single aspects of cellular mechanisms like cell physiology , cell growth , cell migration  and cell interactions. Nevertheless, there is an unmet need in science and industries for more powerful lab ware to close the gap between poorly predictive in-vitro models and real 3D in-vivo models without the use of laboratory animals or animal derived perfused organ models or cells. Different cell and material sources were used to create such 3D models. While biopsies of e.g. the foreskin are used since a long time to create in-vitro tissue cultures new artificial tissue models like hydrogel scaffolds are widely used as an alternative for creating almost in-vivo like tissue constructs by different shaping technologies like molding or 3D printing of cell loaded hydrogels , . The advantage of these technologies is the opportunity to create scaffolds from different cell types and to combine different, growth factor coupled hydrogels . By applying complex cultivation protocols to these scaffolds maintenance of cell culture conditions is possible. Nevertheless, some issues are still remaining. The major problem of these tissue models is the difficulty to apply well defined biologically relevant (in-vivo like) conditions such as shear stress in printed tubules or on-demand supply with nutrients or oxygen in hydrogel blocks. To overcome these issues, we present a microfluidic system that comprises the ability to combine well defined microfluidic conditions with customized 3D printed cell culture compartments.
2 Setup of the microfluidic system
The microfluidic system that is shown in Figure 1 comprises a basic microfluidic system (1), reservoirs for media storage (2), channels and valves to direct and switch the current conditions (3) and an integrated micro pump (4) to create well defined fluidic conditions within the scaffold. The basic microfluidic system consists of several layers of laser micro-structured foils. The reservoirs are created by 3D printing and contain Luer ports for fluidic connection of the microfluidic system to external media reservoirs.
3 Manufacturing of the microfluidic system
3.1 Layer laminate manufacturing
The basic chip was produced by layer laminate manufacturing. It is based on geometrically defined sheets that are pre-structured by laser based micro-cutting, blade based micro-cutting  or micro punching. Afterward these sheets are stacked and fused together by gluing, chemical bonding with plasma processes, laser welding or thermal diffusion bonding. A closed technology chain to manufacture such devices from different substrates including materials like pressure sensitive and thermal activated adhesive films, thermal bonded plastic sheets like polycarbonate (PC) or cyclic olefin copolymer  or silicone (e.g. PDMS) sheets was developed . The presented system contains a stack of PC and PDMS to build the basic part allowing distribution and control of fluidic flow within a microfluidic network by the use of integrated pneumatic actuated elements like pumps and valves . The basic principle of the integrated micro pump is shown in Figure 2.
3.2 3D printing
To produce the cell culture compartments we used stereolithography. All parts were constructed in CAD and exported as STL files. The production process took place at an industrial production 3D printer with a scanning laser. Afterwards they were washed with ethanol and distilled water. The printing of these compartments is needed to integrate functional features that could hardly be realized with conventional production technologies. Figure 3 shows the CAD model of a 3D printed compartment that integrates features like internal channels to perfuse cells or tissues inside of the compartment (11), threads (12), guiding notches to place membrane inserts in the right position (13) and undercuts to integrate sealings (14).
4 Combination of the microfluidic system and 3D printed components
To couple scaffold based tissue models with this basic vascular microfluidic system a standard interface was established. Depending on the requirements of the tissue model, several compartments were designed to integrate either 3D printed scaffolds (5), multi well plate inserts (6; compare Figure 4) or customized pre-formed scaffolds (7) (compare Figure 1).
The connection of tailored cell culture compartments and the basic microfluidic system allows combing well-defined fluidic conditions with high cell densities and accurate matrix properties of engineered scaffolds in an easy way. Furthermore, by seeding endothelial cells like human umbilical vein endothelial cells (HUVEC) into the microfluidic channels (Figure 5) it is possible to interconnect different 3D printed vascular systems creating an artificial vascular network. This enables on the one hand an in-vivo like interconnection of the scaffold or membrane based tissue models that are integrated into the system and on the other hand features the possibility to introduce blood cells like erythrocytes or peripheral blood monocyte cells (PBMCs) to the system.
5 Comparison of different cell culture compartments
Different cell culture models are necessary to mimic and reflect the main tissue specific functionality in vitro. For instance, to design barrier models well plate inserts with standardized integrated technical membranes will be most suitable. Table 1 shows an overview of major benefits and limitations of the presented cell culture compartments as well as applications of the shown technologies.
6 Conclusion and outlook
Additive manufacturing technologies open a new spectrum for the fabrication of microfluidic cell culture devices that will help researchers to overcome the major issues of conventional production technologies. Today’s additive manufacturing processes include well defined but 2.5 D limited processes like layer laminate manufacturing as well as 3D printing processes such as stereolithography. By combining a layer laminate manufactured basic microfluidic system with 3D printed cell culture compartments it is possible to house scaffold constructs and to realize fluidic interfaces. Seeding endothelial cells into the channels of the microfluidic system results in an artificial vascularization of the walls and thus providing the applicability of the chip system to investigate for instance endothelial cell-blood cell interactions within this system. Nevertheless new developments and process innovations will be necessary to overcome the major optical and spatial issues of today’s 3D printing processes. This fits well to the findings of other researchers , ,  evaluating the possibilities and problems of printed microfluidics for biological applications.
Research funding: The authors want to express great appreciation to the Free State of Saxony and the European Union (SAB project “UNILOC”) as well as to the BMWi and the AiF (IGF project “Vascularized bioreactor”) for the financial support. Conflict of interest: Authors state no conflict of interest. Material and Methods: Informed consent: Informed consent is not applicable. Ethical approval: The conducted research is not related to either human or animal use.
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About the article
Published Online: 2016-09-30
Published in Print: 2016-09-01
Citation Information: Current Directions in Biomedical Engineering, Volume 2, Issue 1, Pages 105–108, ISSN (Online) 2364-5504, DOI: https://doi.org/10.1515/cdbme-2016-0026.
©2016 Florian Schmieder et al., licensee De Gruyter.. This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License. BY-NC-ND 4.0