1Department of Mechanical and Industrial Engineering, University of Toronto, 2Institute of Biomaterials and Biomedical Engineering, University of Toronto, 3Faculty of Dentistry, University of Toronto
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Moraes, C., Sun, Y., Simmons, C. A. Microfabricated Platforms for Mechanically Dynamic Cell Culture. J. Vis. Exp. (46), e2224, doi:10.3791/2224 (2010).
The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.
A. Device description and operation
Devices are fabricated using multilayer soft lithography1 in polydimethylsiloxane (PDMS), and are capable of simultaneously generating a range of mechanical conditions in individual cell culture locations across the microfabricated array. In this protocol, the steps to fabricate an array of pneumatically-actuated microposts are first described, followed by steps to modify the device to enable mechanically dynamic culture in both two-dimensional (2D) and three-dimensional (3D) culture paradigms. The outlined microfabricated approach increases throughput over existing macroscale systems, and is best suited for screening for the effects of a variety of mechanical conditions.
The operational principle for the device is based on an array of vertically actuated microposts. Microposts are fabricated on a freely-suspended diaphragm, and are raised and lowered by applying positive and negative pressures beneath the actuation diaphragm (Figure 1). A key feature of the array is that by varying the size of the actuation diaphragm, a single pressure source can be used to obtain a range of vertical displacements across the array. This principle is used to rapidly screen cellular response to a large number of mechanical stimulatory conditions across a single device.
Based on an analogous macroscale design by Schaffer et al.2, our design includes a second suspended and lubricated cell culture film over the post, allowing cells to experience 2D substrate deformation as the culture film slips over the raised loading post. Alternatively, photopatterning an array of cell-laden hydrogels over the loading posts allows for compressive stimulation of cells in 3D culture. Detailed instructions in setting up these systems follow.
B. Fabrication of the pneumatic microactuator array
Fabricating the pneumatic microactuator array requires stringent alignment in multilayer PDMS structures. This is challenging, due to shrinkage-induced alignment registration errors. To address this, we use a fabrication process termed 'sandwich mold fabrication'3, shown to effectively eliminate this issue4.
Sandwich Mold Fabrication
Constructing the multilayer device
The schematic for fabrication of the microactuator array is provided in Figure 2A, and operation of the actuators is shown in Figures 2B and C. To fabricate the device:
C. Mechanically active 2D culture substrates
The array of actuated microposts can be used to create various strain profiles in a suspended polymer film, on which cells are cultured. Raising the post into a lubricated film causes the film to slip and deform around the post. Using a circular loading post results in an equibiaxial strain distribution, but the design is versatile in that different post shapes can be used to create a variety of strain fields. To modify the actuator array for 2D culture experiments (schematic in Figure 3):
Detailed characterization of microdevice operation and biological experiments conducted on this platform have been published elsewhere7.
D. Mechanically active 3D hydrogels
Modifications to the device can also be used to enable compression of photopatterned. cell-laden, three-dimensional hydrogels in a high-throughput manner. In this example, we create 350 μm diameter polyethylene glycol (PEG) hydrogel cylinders, encapsulating C3H10T1/2 mouse mesenchymal stem cells, and apply compressive strains ranging from 5 to 25% across the array. This protocol can be used with more advanced hydrogel photopolymerization chemistries. To use the platform for experiments in three-dimensional mechanobiology (schematic in Figure 4):
In situ polymerization:
Cell-laden PEG hydrogels were then photolithographically patterned into the devices8,9 as follows:
Detailed characterization of microdevice operation and biological experiments conducted on this platform have been published elsewhere10.
E. Peripheral equipment
Modified petri dishes are used to maintain sterility of cell cultures during actuation of the devices. A blunted and stripped 18G needle is epoxied into a hole drilled in the side of the petri dish. Tubing (Clay Adams Intramedic PE190; VWR International, Arlington Heights, IL, USA) is press-fitted to these connectors and connects to the controlled solenoid valves and pressure source.
Pressures to the devices are provided by external micropumps. For the 2D experiments, positive pressure values from 30-55 kPa were generated using a voltage-controlled eccentric diaphragm pump (SP 500 EC-LC 4.5VDC; Schwarzer Precision, Germany). A pulse-width modulation based voltage controller is used to vary the applied voltage to control pressure output. Solenoid valves and manifolds (S10MM-30-12-3 and MSV10-1; Pneumadyne, Plymouth, MN, USA) are used to create a cyclic pressure waveform. The valves are actuated with a 12 V signal, controlled by a square waveform function generator.
F. Device characterization and use
Imaging on the device
A common issue with imaging cells or particles on the device is poor optical resolution due to the condensation of droplets beneath the PDMS film supporting the loading post. This condensation arises from temperature and humidity differences inside the incubator and after fixation or on a microscope stage. To resolve this problem:
For live cell imaging, this procedure can be done before seeding the cells on the devices. However, channels must be designed large enough to prevent any viscous losses of pressure between units on the device.
Strain characterization by bead tracking
On the 2D culture systems:
A similar procedure can be followed to characterize strains in 3D systems. Mix a quantity of fluorescent beads into the hydrogel precursor solution. In the case of the PEG hydrogel system, the pore sizes are much smaller than those of the 1 μm diameter beads. Beads are encapsulated in the hydrogel, and the system can be imaged using a confocal microscope. Bead displacements can then be tracked, analyzed and fitted to a deformation model.
G. Representative Results
Representative results for device fabrication, characterization and operation are provided in Figures 5 and 6.
Figure 1. Sandwich mold fabrication process. (A) An overhead transparency is carefully lowered onto uncured PDMS on an SU-8 master. (B) The sandwich is placed in a foam, glass and metal stack, and cured in an oven under compression. (C) The stack is disassembled and the transparency peeled away, retaining the patterned PDMS layer3. Reproduced by permission of the Institute of Physics.
Figure 2. Schematic of vertically actuated micropost at (A) rest and (B) when actuated. (C) Schematic of multilayer fabrication process required to make an array of microposts3. Adapted by permission of the Institute of Physics.
Figure 3. Process to modify microactuator array to conduct experiments for cells cultured on a deforming two-dimensional substrate7. Reproduced by permission of the Royal Society of Chemistry http://dx.doi.org/10.1039/B914460A.
Figure 4. Process to modify microactuator array to conduct experiments for cells cultured in three-dimensional photopatterned hydrogel constructs10. Reproduced by permission of Elsevier.
Figure 5. (A) Sample device for 2D mechanostimulatory culture. Red dye used to mark the actuating pressure delivery channels, and blue dye used to mark the lubrication channels. (B) Sample image of a strain characterization experiment. Red spots represent the undeformed location of the beads, while green spots represent the deformed locations7. Reproduced by permission of the Royal Society of Chemistry http://dx.doi.org/10.1039/B914460A.
Figure 6. (A) Sample device for 3D compressive experiments. Green dye used to mark actuation pressure channels. (B) Top-down view of micropost array with (C) a hydrogel construct patterned on top of the post. (D, E) Side-reconstructed images of fluorescent beads in the hydrogel construct under (D) rest and (E) 55 kPa actuation pressures, demonstrating the strain characterization process in a 3D culture system10. Reproduced by permission of Elsevier.
Though conceptually simple, device fabrication does take some experimental skill and practice. Particularly in the case of 2D cell culture, alignment of the multiple layers in the device can be challenging, especially over a large-area array. Practically speaking, we can reliably achieve a 100% alignment success rate using devices with 50 μm of tolerance in spacing between adjacent features in multiple layers. We have also successfully demonstrated alignment with tolerances as low as 15 μm, but the alignment time required is substantially greater and is achieved at a lower success rate. Alignment of the mask to photopattern biomaterials onto the array is similarly challenging, but the posts can be designed to be quite large in comparison to the hydrogel cylinders, alleviating this constraint.
Other challenges involve cell culture on the materials described in this protocol. In the 2D stretching system, using PDMS as a culture film limits biological investigation to short-term experiments, as cell adhesion to the substrate begins to be substantially affected after 6 hours of stimulation. This can be rectified by covalently binding matrix proteins to the PDMS surface11, or by replacing the PDMS cell culture substrate with a more clinically relevant polyurethane material, to improve cell adhesion and provide greater flexibility in conducted biological experiments12. In the described 3D system, the hydrogel chemistry is intended as an illustrative example only. Long-term culture of adherent cells requires the use of alternative natural or synthetic hydrogels, or the inclusion of adhesive ligands into the PEG matrix (which can be easily achieved using standard PEGylation chemistries)13.
No conflicts of interest declared.
We acknowledge financial support from the Natural Sciences and Engineering Research Council of Canada and the Canadian Institutes of Health Research (CHRPJ 323533-06), the Ontario Graduate Scholarship program to CM, and the Canada Research Chairs in Micro and Nano Engineering Systems to YS, and in Mechanobiology to CAS.
|Sylgard 184 PDMS Monomer and Crosslinker Kit||Dow Corning|
|Silanization agent: (tridecafluoro-1, 1, 2, 2-tetrahydrooctyl)-1-trichlorosilane||United Chemical Technologies|
|Foam pads||Craft supply stores, 1-2 mm thick|
|Overhead inkjet transparencies||Grand & Toy|
|Micromanipulator system||Siskiyou, Inc.|
|Custom-made vaccum mount|
|Vision system, Navitar 12x zoom||Navitar|
|Connecting tubes||VWR international||Clay Adam Intramedic PE190|
|Blunt 18G needles||Small Parts (www.smallparts.com)|
|Eccentric diaphragm micropump||Schwarzer Precision||SP 500 EC-LC4.5V DC|
|3-(trimethoxysilyl) propyl methacrylate||Sigma-Aldrich|
|Polyethylene glycol diacrylate 3.4 kDa||Laysan Bio Inc.|
|Polyethylene glycol 8 kDa||Sigma-Aldrich|
|Irgacure 2959||Ciba Specialty Chemicals|
|Standard cell culture reagents|