3D printing – a key technology for tailored biomedical cell culture lab ware
-
Florian Schmieder
,
Joachim Ströbel
Abstract
Today’s 3D printing technologies offer great possibilities for biomedical researchers to create their own specific laboratory equipment. With respect to the generation of ex vivo vascular perfusion systems this will enable new types of products that will embed complex 3D structures possibly coupled with cell loaded scaffolds closely reflecting the in-vivo environment. Moreover this could lead to microfluidic devices that should be available in small numbers of pieces at moderate prices. Here, we will present first results of such 3D printed cell culture systems made from plastics and show their use for scaffold based applications.
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 [1], cell growth [2], cell migration [3] 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 [4], [5]. The advantage of these technologies is the opportunity to create scaffolds from different cell types and to combine different, growth factor coupled hydrogels [6]. 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.
Schematic view of a multilayer based microfluidic system (basic chip; 1–4) with different on-top components to integrate scaffolds into a microfluidic system. (5) compartment to integrate 3D printed scaffolds, (6) compartment to integrate membrane based models and (7) compartment to integrate molded hydrogel scaffolds or pre-formed customized scaffolds.
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 [7] 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 [8] or silicone (e.g. PDMS) sheets was developed [9]. 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 [10]. The basic principle of the integrated micro pump is shown in Figure 2.
Schematic working principle of the integrated membrane pump. In the first phase the entrance valve (8) is opened to enable the fluid flow into the pump chamber. In the second phase the membrane of the pump chamber (9) is moved upwards drawing the fluid into the pumping chamber. In the third phase the inlet valve gets closed and the outlet valve (10) opens up. This enables the fluid flow out of the pump chamber within the forth phase by deflecting the membrane downwards.
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).
Sectional view of a 3D printed compartment to house membrane inserts that should be perfused. Functional features like internal channels (11), threads (12), guide notches (13) and undercuts for sealing rings (14) could be easily produced by 3D printing.
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).
Microfluidic system with integrated pump (15), 3D printed compartment for the integration of multi well based scaffolds (16), reservoir with Luer-Lock fittings (17) and membrane insert (18).
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.
HUVEC cells covering the inner surface of the microfluidic system. The nuclei were stained using DAPI (blue), while the endothelial cell marker von-Willebrand factor was stained in red.
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.
Comparison of different cell culture compartments regarding to applications, major benefits and limitations.
| Cell culture compartment | 3D printed scaffolds | Multi well based scaffolds | Pre-formed or molded scaffolds |
|---|---|---|---|
 |  |  | |
| Application | Bone like constructs; tubular structures; artificial blood vessels | Barrier models like blood brain barrier; skin; mucosa; gut; kidney; placenta; endothelium | Pre-defined customized bone cement models; molded pre-formed liver models |
| Benefits | Realistic 3D models possible; easy to apply different materials | Models for substance transport; well established; easy to use | Easy to apply for customized models; robust cell culture models |
| Limitations | 3D printer needed | Just suitable for barrier models | Pre-defined model needed |
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 [11], [12], [13] evaluating the possibilities and problems of printed microfluidics for biological applications.
Author’s Statement
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.
References
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©2016 Florian Schmieder et al., licensee De Gruyter.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 License.
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- Usability evaluation of a locomotor therapy device considering different strategies
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- Fabrication of MEMS-based 3D-μECoG-MEAs
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- Biocompatibility of photopolymers for additive manufacturing
- Biochemical piezoresistive sensors based on pH- and glucose-sensitive hydrogels for medical applications
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- Deep brain stimulation: increasing efficiency by alternative waveforms
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- Determining cardiac vagal threshold from short term heart rate complexity
- Classification of cardiac excitation patterns during atrial fibrillation
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- Deriving respiration from high resolution 12-channel-ECG during cycling exercise
- Reducing of gradient induced artifacts on the ECG signal during MRI examinations using Wilcoxon filter
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- Postprocessing algorithm for automated analysis of pelvic intraoperative neuromonitoring signals
- Best practice: surgeon driven application in pelvic operations
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- Classification of morphologic changes in photoplethysmographic waveforms
- Novel computation of pulse transit time from multi-channel PPG signals by wavelet transform
- Efficient design of FIR filter based low-pass differentiators for biomedical signal processing
- Nonlinear causal influences assessed by mutual compression entropy
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- Evaluation of open-source software for the lung segmentation
- Automatic determination of lung features of CF patients in CT scans
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- Effect of key parameters on synthesis of superparamagnetic nanoparticles (SPIONs)
- Radiopacity assessment of neurovascular implants
- Development of a desiccant based dielectric for monitoring humidity conditions in miniaturized hermetic implantable packages
- Development of an artifact-free aneurysm clip
- Enhancing the regeneration of bone defects by alkalizing the peri-implant zone – an in vitro approach
- Rapid prototyping of replica knee implants for in vitro testing
- Protecting ultra- and hyperhydrophilic implant surfaces in dry state from loss of wettability
- Advanced wettability analysis of implant surfaces
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- Plasma treatment on novel carbon fiber reinforced PEEK cages to enhance bioactivity
- Wear of a total intervertebral disc prosthesis
- Digital health and digital biomarkers – enabling value chains on health data
- Usability in the lifecycle of medical software development
- Influence of different test gases in a non-destructive 100% quality control system for medical devices
- Device development guided by user satisfaction survey on auricular vagus nerve stimulation
- Empirical assessment of the time course of innovation in biomedical engineering: first results of a comparative approach
- Effect of left atrial hypertrophy on P-wave morphology in a computational model
- Simulation of intracardiac electrograms around acute ablation lesions
- Parametrization of activation based cardiac electrophysiology models using bidomain model simulations
- Assessment of nasal resistance using computational fluid dynamics
- Resistance in a non-linear autoregressive model of pulmonary mechanics
- Inspiratory and expiratory elastance in a non-linear autoregressive model of pulmonary mechanics
- Determination of regional lung function in cystic fibrosis using electrical impedance tomography
- Development of parietal bone surrogates for parietal graft lift training
- Numerical simulation of mechanically stimulated bone remodelling
- Conversion of engineering stresses to Cauchy stresses in tensile and compression tests of thermoplastic polymers
- Numerical examinations of simplified spondylodesis models concerning energy absorption in magnetic resonance imaging
- Principle study on the signal connection at transabdominal fetal pulse oximetry
- Influence of Siluron® insertion on model drug distribution in the simulated vitreous body
- Evaluating different approaches to identify a three parameter gas exchange model
- Effects of fibrosis on the extracellular potential based on 3D reconstructions from histological sections of heart tissue
- From imaging to hemodynamics – how reconstruction kernels influence the blood flow predictions in intracranial aneurysms
- Flow optimised design of a novel point-of-care diagnostic device for the detection of disease specific biomarkers
- Improved FPGA controlled artificial vascular system for plethysmographic measurements
- Minimally spaced electrode positions for multi-functional chest sensors: ECG and respiratory signal estimation
- Automated detection of alveolar arches for nasoalveolar molding in cleft lip and palate treatment
- Control scheme selection in human-machine- interfaces by analysis of activity signals
- Event-based sampling for reducing communication load in realtime human motion analysis by wireless inertial sensor networks
- Automatic pairing of inertial sensors to lower limb segments – a plug-and-play approach
- Contactless respiratory monitoring system for magnetic resonance imaging applications using a laser range sensor
- Interactive monitoring system for visual respiratory biofeedback
- Development of a low-cost senor based aid for visually impaired people
- Patient assistive system for the shoulder joint
- A passive beating heart setup for interventional cardiology training
