NOTE: Wear personal protective equipment (PPE) such as gloves and lab coat for every step in this protocol.
1. Master mold fabrication
NOTE: Perform step 1.1 – 1.3 in a fume hood.
2. Pneumatic actuation unit
3. Alginate-chondrocyte (or bead) constructs
4. Device assembly (Figure 2d)
NOTE: PDMS spacers and 3D printed clamps need to be prepared separately.
5. Actuation of the device
6. Imaging of chondrocytes in the device
NOTE: To obtain a good image quality, image chondrocytes (or fluorescent beads) in alginate gel through Glass plate 2 because expanded PDMS balloons and air chambers can distort optical images. If an inverted microscope is used for imaging, the device needs to be setup so that Glass plate 2 faces downward.
This article shows detailed steps of the microfluidic chondrocyte compression device fabrication (Figure 2). The device contains a 5 x 5 arrays of cylindrical alginate-chondrocyte constructs, and these constructs can be compressed with five different magnitudes of compression (Figure 1, Figure 3 and Figure 4). The height of the pneumatic microchannel is around 90 μm, and the PDMS balloon diameters are 1.2, 1.4, 1.6, 1.8 and 2.0 mm, respectively. The performance of the device was quantified with confocal microscopy with static compression conditions and image processing. Static compression was employed for the microscopic imaging because the z-stack imaging with the confocal microscopy takes a few minutes, so it is too slow for quantitative imaging during the dynamic compression.
Figure 3a shows the 5 × 5 arrays of alginate hydrogel columns (diameter: ~0.8 mm, height: ~1 mm) cast on Glass plate 2. These gel constructs were imaged by adding fluorescent beads in the gel. Figure 3b shows an example case that the gel column was compressed by 33.8% in height by the largest PDMS balloon. The resultant compressive strain of the gel constructs increased by approximately 5% per 0.2 mm increment in the PDMS balloon diameter as shown in Figure 3c.
Compressive deformation of chondrocytes was determined by imaging the cells in a 613 μm × 613 μm × 40–55 μm (x × y × z) volume near the gel construct center as shown in Figure 4a. Figure 1d shows an example image of a chondrocyte that was compressed by 16% by the largest PDMS balloon. Figure 4b shows the distribution of the measured cell compression strain values, and overall cells were compressed more by larger PDMS balloons. Therefore, the amount of alginate gel and chondrocyte compression were controlled by the diameter of PDMS balloons (Figure 3 and Figure 4) with a constant pressure of 14 kPa.
Figure 1. Microfluidic chondrocyte compression device.
(a) Schematic of the assembled device. A 5 × 5 array of alginate–chondrocyte constructs are aligned on PDMS balloons with 5 different diameters (D = 1.2, 1.4, 1.6, 1.8 and 2.0 mm), where D is the diameter of PDMS balloon (or air chamber). (b) Schematic of the device operation. The device is actuated by pneumatic pressure which expands PDMS balloons. (c) Image of an actual device (coin diameter = 19 mm). (d) Vertical cross-sections of a chondrocyte before (left) and under (right) compression on the largest PDMS balloon (D = 2.0 mm) (cell compressive strain, εcell = |cell height change/initial cell height| x 100 = 16%). This figure is reproduced from 26.
Figure 2. Detailed steps of microfluidic chondrocyte compression device fabrication.
(a) Photolithography for generating a SU-8 master mold and following soft lithography for creating PDMS layer with pneumatic microchannels (Layer 1). (b) Thin PDMS membrane (Layer 2) on a transparency film generated by spin coating. (c) Cylindrical alginate gel casting method on glass (Glass plate 2). (d) Assembly of the microfluidic chondrocyte compression device. This figure is reproduced from 26. Please click here to download a larger version of this figure.
Figure 3. Measurement of alginate gel deformation under static compression.
(a) 5 x 5 arrays of cylindrical alginate gel constructs (diameter: ~800 μm, height: ~1 mm). (b) Alginate gel compressed by the largest PDMS balloon (D = 2.0 mm). The compressive strain of the alginate gel is 33.8%. (c) Compressive strain of alginate gel (εgel) increases around 5% per 0.2 mm increment of PDMS balloon diameter (D). Error bar: standard deviation. Red line: linear fitting line This figure is reproduced from 26.
Figure 4. Measurement of chondrocyte deformation under static compression.
(a) A z-stack image [613 μm × 613 μm × 40–55 μm (x × y × z)] was obtained in the middle of the gel construct, 300–400 μm from the gel bottom. (b) Different magnitudes of chondrocyte compressive strain (εcell) resulted as a function of the PDMS balloon diameter (D). : mean values.
: each data points. Top (or bottom) and middle lines of the box are the standard deviation and median value, respectively. This figure is reproduced from 26.
Figure S1. Microchannel photomask design for step 1.3.4 (unit = mm). Please click here to download this figure.
Figure S2. Aluminum mold design for step 3.2.3 (unit = mm). Please click here to download this figure.
Figure S3. Permanent deformation of the alginate gel (1.5%, w/v) under 1 h-long dynamic (1 Hz) and static compression. This figure is reproduced from 26. Please click here to download this figure.
(3-Aminopropyl)triethoxysilane (ATPES) | Sigma-Aldrich | 741442-100ML | |
(Tridecafluoro-1, 1, 2, 2-Tetrahydrooctyl)-1-Trichlorosilane | United Chemical Technologies | T2492-KG | |
Acrylic sheet | McMaster-Carr | 8560K354 | |
Air pump | Schwarzer Precision | SP 500 EC-LC4.5V DC | We used the model purchased in 2015. The internal design and performance of air pump (SP 500 EC-LC) changed in early 2016. Also, air pump performance has changed in the course of time. Thus, air pressure generated by an SP 500 EC-LC air pump should be calibrated before use. |
Alginate powder | FMC Corporation | Pronova UP MVG | |
Barb Straight Connectors (Metal tube) | Pneumadyne | EB40-250 | |
Calcein AM | Invitrogen | C3100MP | |
Dulbecco's Modified Eagle Medium (DMEM) | Gibco | 11960-044 | |
Dyed red aqueous fluorescent particles | Thermo Fisher Scientific | R0100 | |
EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) | Thermo Fisher Scientific | 22980 | |
Foam pad | GRAINGER | Item # 5GCE8 | |
Function / Arbitrary Waveform Generator | Keysight Technologies | 33210A | |
Hydrochloric acid | Fisher Chemical | A144-500 | |
Hydrogen peroxide | Fisher BioReagents | BP2633500 | |
Isopropyl alcohol | BDH1174-4LP | VWR | |
Microscope slides | Thermo Fisher Scientific | 22-267-013 | |
Plasma cleaner | Harrick Plasma | PDC-001 | |
Polydimethylsiloxane (PDMS) | Dow Corning | 184 SIL ELAST KIT 0.5KG | |
Power supply | Keysight Technologies | E3630A | |
SeaKem LE Agarose | Lonza | 50004 | |
Sodium hydroxide | Fisher Chemical | S318-1 | |
Solenoid manifold | Pneumadyne | MSV10-1 | |
Solenoid valve | Pneumadyne | S10MM-30-12-3 | |
Spin coater | Laurell Technologies | WS-650Mz-23NPPB | |
SU8 Developer | MicroChem Corp. | Y020100 4000L1PE | |
SU8-100 | MicroChem Corp. | Y131273 0500L1GL | |
SU8-5 | MicroChem Corp. | Y131252 0500L1GL | |
Sulfo-NHS (N-hydroxysulfosuccinimide) | Thermo Fisher Scientific | 24510 | |
Sulfuric acid | EMD Millipore | MSX12445 |
Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.
Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.
Mechanical stimuli are known to modulate biological functions of cells and tissues. Recent studies have suggested that compressive stress alters growth plate cartilage architecture and results in growth modulation of long bones of children. To determine the role of compressive stress in bone growth, we created a microfluidic device actuated by pneumatic pressure, to dynamically (or statically) compress growth plate chondrocytes embedded in alginate hydrogel cylinders. In this article, we describe detailed methods for fabricating and characterizing this device. The advantages of our protocol are: 1) Five different magnitudes of compressive stress can be generated on five technical replicates in a single platform, 2) It is easy to visualize cell morphology via a conventional light microscope, 3) Cells can be rapidly isolated from the device after compression to facilitate downstream assays, and 4) The platform can be applied to study mechanobiology of any cell type that can grow in hydrogels.