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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Results
  • Discussion
  • Disclosures
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This article provides detailed methods for fabricating and characterizing a pneumatically actuating microfluidic device for chondrocyte compression.

Abstract

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.

Introduction

Micro-engineered platforms are valuable tools for studying the molecular, cellular, and tissue level biology because they enable dynamic control of both the physical and chemical microenvironments1,2,3,4,5,6,7,8. Thus, multiple hypotheses can be simultaneously tested in a tightly controlled manner. In the case of growth plate cartilage, there are increasing evidences of an important role of compressive stress in modulating bone growth through action on the growth plate cartilage9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25. However, the mechanism of action of compressive stress – in particular, how stress guides the formation of chondrocyte columns in the growth plate – is poorly understood.

The goal of this protocol is to create a pneumatically actuating microfluidic chondrocyte compression device26 to elucidate mechanisms of mechanobiology in growth plate chondrocytes (Figure 1a-c). The device consists of two parts: the pneumatic actuation unit and the alginate gel construct. The microfluidic pneumatic actuation unit is fabricated using polydimethylsiloxane (PDMS) based on the photo- and soft-lithography. This unit contains a 5 x 5 array of thin PDMS membrane balloons which can be inflated differently based on their diameters. The alginate gel construct consists of the chondrocytes embedded in a 5 x 5 array of alginate gel cylinders, and the entire alginate-chondrocyte constructs are assembled with the actuation unit. The alginate gel constructs are compressed by the pneumatically inflated PDMS balloons (Figure 1b). The microfluidic device can generate five different levels of compressive stress simultaneously in a single platform based on differences in the PDMS balloon diameter. Thus, a high-throughput test of chondrocyte mechanobiology under multiple compression conditions is possible.

The microfluidic device described in this protocol has many advantages over the conventional compression device such as external fixators14,21,23 and macroscopic compression devices16,19,27,28 for studying chondrocyte mechanobiology: 1) The microfluidic device is cost effective because it consumes smaller volume of samples than the macroscopic compression device, 2) The microfluidic device is time effective because it can test multiple compression conditions simultaneously, 3) The microfluidic device can combine mechanical and chemical stimuli by forming a concentration gradient of chemicals based on the limited mixing in microchannels, and 4) Various microscopy techniques (time-lapse microscopy and fluorescence confocal microscopy) can be applied with the microfluidic device made of transparent PDMS.

We adopted and modified the method of Moraes et al.7,29 to create different compressive stress levels in a single device to enable high-throughput mechanobiology studies of chondrocyte compression. Our approach is appropriate for cells (e.g., chondrocytes) which need three-dimensional (3D) culture environment and for biological assays after compressing cells. Although some microfluidic cell compression devices can compress cells cultured on two-dimensional (2D) substrates30,31,32, they cannot be used for chondrocytes because 2D cultured chondrocytes dedifferentiate. There are microfluidic platforms for compressing 3D cultured cells in photopolymerized hydrogels7,33, but they are limited in isolating cells after compression experiments because isolating cells from photopolymerized hydrogel is not easy. Additionally, the effects of ultraviolet (UV) exposure and photo crosslinking initiators on cells may need to be evaluated. In contrast, our method allows rapid isolation of cells after compression experiments for post biological assays because alginate hydrogels can be depolymerized quickly by calcium chelators. The detailed device fabrication and characterization methods are described in this protocol. A brief procedure for fabricating the microfluidic chondrocyte compression device is shown in Figure 2.

Protocol

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.

  1. Glass treatment
    NOTE: Wear a face shield, gloves, and a lab coat for step 1.1.
    1. Make Piranha solution (60 mL) by mixing sulfuric acid (H2SO4) and hydrogen peroxide (H2O2) with a volume ratio of 3:1.
      CAUTION: Do not use Piranha solution and acetone in the same fume hood due to the explosion hazard.
    2. Place a glass plate (50.8 mm × 76.2 mm × 1.2 mm) in Piranha solution for 30 min at 40 °C.
    3. Rinse the glass plate with deionized water (diH2O).
    4. Place the glass plate in acetone at room temperature for 10 min.
    5. Rinse the glass plate with isopropanol and dry it with nitrogen (N2) gas.
    6. Bake the glass plate at 200 °C for 20 min on a hot plate. All subsequent baking steps use a hot plate.
  2. SU-8 seed layer formation on the glass plate
    1. Spread SU-8 5 on the glass plate with a disposable pipette to cover around 2/3 of the plate’s surface area.
    2. Spin the glass plate with SU-8 5 photoresists at 500 rpm for 35 s (initial spinning cycle) and then 2,500 rpm for 40 s (final spinning cycle) using a spin coater. All subsequent spin coating steps include the same initial spinning cycle.
    3. Bake the SU-8 5 coated glass plate at 65 °C for 2 min and then at 95 °C for 5 min.
    4. Expose the SU-8 5 coated glass plate to UV light (60 mW/cm2, distance between UV lamp and photomask is 20 cm, total amount of UV light energy = 60 mJ/cm2) for 1 s.
      NOTE: The UV exposure time should be adjusted according to the power of a used UV light.
    5. Bake the SU-8 5 coated glass plate at 65 °C for 2 min and at 95 °C for 5 min.
    6. Place the baked glass plate in the SU-8 developer for 2 min.
    7. Bake the SU-8 5 coated glass at 180 °C for 20 min.
  3. SU-8 channel pattern fabrication using photolithography (Figure 2a Step 1-3)
    1. Pour SU-8 100 on the SU-8 5 seeded glass plate to cover around 2/3 of the plate’s surface area.
    2. Spin the glass plate with SU-8 100 at 3,000 rpm for 38 s (≈ 90 μm thick).
    3. Bake the SU-8 100 coated glass plate at 65 °C for 10 min and then at 95 °C for 30 min. If SU-8 100 is still sticky after this procedure, bake the glass plate for a longer time at 95 °C until SU-8 100 becomes non-sticky.
    4. Place a high-resolution microchannel photomask (25,400 dpi, see Supplementary Figure 1) on the SU-8 100 coated glass plate and expose the photomask covered glass plate to UV light for 4 s (total amount of UV light energy = 240 mJ/cm2).
      NOTE: The UV exposure time should be adjusted according to the power of a used UV light.
    5. Remove the photomask from the glass plate and bake the glass plate at 65 °C for 2 min and then 95 °C for 20 min.
    6. Keep the baked glass plate in a container, which is wrapped with aluminum foil to block any light, for overnight curing.
    7. Develop SU-8 100 channel patterns on the glass plate in the SU-8 developer for 15 min.
    8. Wash the SU-8 patterned glass plate (master mold) with isopropyl alcohol and dry it with N2 gas. If white particles remain during this process, repeat step 7 for 5 min.

2. Pneumatic actuation unit

  1. Microchannel layer (Layer 1)
    NOTE: Perform step 2.1.1 - 2.1.2 in a fume hood.
    1. Drop 200 μL of (Tridecafluoro-1,1,2,2-Tetrahydrooctyl)-1-Trichlorosilane on a coverslip and place it in a vacuum chamber with the master mold.
    2. Apply vacuum for 2 min in the chamber and wait for 6 h (or overnight) for silanization of the mold.
    3. Mix PDMS with a weight ratio of 10:1 (prepolymer:curing agent) for 5 min.
      NOTE: All subsequent PDMS casting step contains the same prepolymer and curing agent weight ratio (10:1).
    4. Pour PDMS on the master mold and degas PDMS in a vacuum chamber for 30 min.
    5. Sandwich PDMS with a piece of transparency film.
    6. Clamp the above sandwiched assembly with a glass plate, foam pads and plexiglass plates (Figure 2a Step 4).
    7. Cure PDMS in an oven at 80 °C for 6 h.
    8. Isolate the PDMS layer (Layer 1) with the transparency film from the sandwiched structure (Figure 2a Step 5).
    9. Activate surfaces of Layer 1 and a clean glass plate (Glass plate 1; 50.8 mm × 76.2 mm × 1.2 mm) using a plasma cleaner for 1 min.
      NOTE: Plasma cleaning time may vary based on the power of a used plasma cleaner.
    10. Bond Layer 1 onto Glass plate 1 and place them in the oven at 80 °C for 30 min.
    11. Remove the transparency film from Layer 1.
  2. Thin PDMS membrane (Layer 2)
    1. Spin coat uncured PDMS on a transparency film at 1,000 rpm for 1 min to obtain a 60 μm thick PDMS layer.
    2. Partially cure spin coated PDMS (Layer 2) in the oven at 80 °C for 20-30 min.
    3. Activate Layer 1 on Glass plate 1 and Layer 2 using the plasma cleaner for 1 min.
      NOTE: Plasma cleaning time may vary based on the power of a used plasma cleaner.
    4. Bond the Layer 2 onto Layer 1 and place them in the oven at 80 °C overnight.
  3. Tubing block
    1. Place metal tubes vertically on a petri dish.
    2. Gently pour PDMS in the dish to submerge about 3/4 of the metal tubes.
    3. Cure PDMS in the oven at 60 °C for 6 h (or overnight).
    4. Cut a piece of PDMS block containing one metal tube.
    5. Punch a hole on Layer 2 for the inlet.
    6. Activate the PDMS block and Layer 2 using the plasma cleaner for 1 min.
    7. Attach the PDMS block onto the inlet part of Layer 2 and place the entire actuation unit in the oven at 80 °C for overnight.

3. Alginate-chondrocyte (or bead) constructs

  1. Amino-silanized glass plate
    1. Cut a glass plate (50.8 mm × 76.2 mm × 1.2 mm) into two half-sized glass plates (50.8 mm × 38.1 mm × 1.2 mm) using a diamond scriber.
    2. Place the glass plates in 0.2 M hydrogen chloride (HCl) and gently shake (e.g., 55 rpm) them overnight.
    3. Rinse the glass plates with diH2O.
    4. Shake the glass plates in 0.1 M sodium hydroxide (NaOH) for 1 h at 55 rpm and rinse them with diH2O.
    5. Shake the glass plates in 1% (v/v) 3-aminopropyltrimethoxysilane (APTES) for 1 h at 55 rpm and rinse them with diH2O.
    6. Dry the amino-silanized glass plates in a fume hood overnight.
  2. Agarose gel mold for alginate gel constructs
    1. Mix 5% (w/v) agarose and 200 mM calcium chloride (CaCl2) in diH2O.
    2. Boil the agarose gel solution with a microwave oven (or a hot plate). Boiling time varies based on the volume of the agarose gel solution and the power of the microwave oven (or the temperature of the hot plate).
    3. Pour the boiled agarose gel solution onto an aluminum mold (see Supplementary Figure S2), and sandwich it with a glass plate.
    4. Wait for 5 min and unmold the solidified agarose gel from the aluminum mold.
  3. Growth plate chondrocyte harvest
    1. Isolate growth plates from the hind limbs of neonatal mice.
    2. Place the growth plates in 1 mL of 0.25% collagenase for 3 h in an incubator at 37 °C and 8% carbon dioxide (CO2), to remove extracellular matrix (ECM).
    3. Centrifuge the digested sample for 5 min at 125 x g to make a chondrocyte pellet and remove supernatant from the sample. Here, 1 x g is the acceleration of gravity.
    4. Resuspend the chondrocyte pellet in 1 mL of Dulbecco's modified Eagle's medium (DMEM).
    5. Count the number of chondrocytes in DMEM using a cell counter.
    6. Centrifuge the chondrocytes in DMEM for 5 min at 125 x g to make a chondrocyte pellet again and remove supernatant from the sample.
    7. Optionally, resuspend the chondrocyte pellet in the chondrocyte culture media (CCM)34 containing 2 μM calcein AM, and incubate the sample at 37 °C for 30 min. Repeat step 3.3.6 and move to step 3.3.8.
    8. Resuspend the chondrocyte pellet in a desired volume of CCM and keep them in the incubator at 37 °C and 8% CO2 before use.
  4. Alginate-chondrocyte (or bead) constructs (Figure 2c)
    1. Mix 1.5% (w/v) alginate in phosphate-buffered saline (PBS) with 5 mg/mL of sulfo-NHS, 10 mg/mL of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC).
    2. Add 8 x 106 chondrocytes into the 1 mL of alginate gel solution [or Add 3 μL of 1 μm-diameter fluorescent beads (542/612 nm) in 1 mL of alginate gel solution (0.3% (v/v))].
    3. Place 150 μL of the alginate-chondrocyte (or bead) solution on the amino-silanized glass plate fabricated in Step 3.1.
    4. Cover the alginate gel solution with the agarose gel mold for 3 min.
    5. Remove excessive alginate gel solution overflowing from the agarose gel mold with a razor blade and remove the agarose gel mold. Then, cylindrical alginate-chondrocyte (or bead) constructs are obtained on the amino-silanized glass plate.
    6. Place the alginate-chondrocyte constructs in cross-linking solution (50 mM CaCl2 / 140 mM NaCl in diH2O) for 1 min for further polymerization.

4. Device assembly (Figure 2d)

NOTE: PDMS spacers and 3D printed clamps need to be prepared separately.

  1. Locate four 1 mm thick PDMS spacers on the four corners of the Layer 2 of the actuation unit.
  2. Place 700 μL of CCM to cover the air chambers of Layer 2.
  3. Place the alginate-chondrocyte (or bead) constructs on Layer 2 while carefully aligning the constructs with the air chambers.
  4. Clamp the device with 3D printed clamps (Figure 1c).

5. Actuation of the device

  1. Connect the outlet of an air pump (see Table of Materials) with the inlet of a solenoid valve with a silicon tubing.
  2. Connect the outlet of the solenoid valve with the inlet of the assembled device with a silicon tubing.
  3. Connect the solenoid valve with a function generator.
  4. Manipulate the solenoid valve with a square wave (e.g., 1 Hz) generated by the function generator.
  5. Turn on the air pump to actuate the device pneumatically.

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.

  1. Prepare the device with chondrocytes (or fluorescent beads) as shown in previous sections.
  2. Take z-stack images of chondrocytes (or fluorescent beads) with a confocal microscope before and under compression, respectively. Choose a z-step size based on the depth of field of a used confocal imaging system.
  3. The height of chondrocytes (or an alginate-bead construct) can be measured with automatic image processing method shown in previous literatures26,35.

Results

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 diame...

Discussion

To test the effects of compressive stress on growth plate chondrocytes, we developed the microfluidic chondrocyte compression device (Figure 1) to apply various levels of compressive stress to the chondrocytes in the alginate hydrogel scaffold for 3D culture in high throughput ways. To assist other researchers to adopt our device or to develop similar devices, we provided details of the device fabrication steps in this protocol article.

The crucial steps in this p...

Disclosures

The authors have nothing to disclose.

Acknowledgements

We thank Drs. Christopher Moraes and Stephen A. Morin for their support for device design and fabrication. This study was supported by Bioengineering for Human Health grant from the University of Nebraska-Lincoln (UNL) and the University of Nebraska Medical Center (UNMC), and grant AR070242 from the NIH/NIAMS. We thank Janice A. Taylor and James R. Talaska of the Advanced Microscopy Core Facility at the University of Nebraska Medical Center for providing assistance with confocal microscopy.

Materials

NameCompanyCatalog NumberComments
(3-Aminopropyl)triethoxysilane (ATPES)Sigma-Aldrich741442-100ML
(Tridecafluoro-1, 1, 2, 2-Tetrahydrooctyl)-1-TrichlorosilaneUnited Chemical TechnologiesT2492-KG
Acrylic sheetMcMaster-Carr8560K354
Air pumpSchwarzer PrecisionSP 500 EC-LC4.5V DCWe 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 powderFMC CorporationPronova UP MVG
Barb Straight Connectors (Metal tube)PneumadyneEB40-250
Calcein AMInvitrogenC3100MP
Dulbecco's Modified Eagle Medium (DMEM)Gibco11960-044
Dyed red aqueous fluorescent particlesThermo Fisher ScientificR0100
EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride)Thermo Fisher Scientific22980
Foam padGRAINGERItem # 5GCE8
Function / Arbitrary Waveform GeneratorKeysight Technologies33210A
Hydrochloric acidFisher ChemicalA144-500
Hydrogen peroxideFisher BioReagentsBP2633500
Isopropyl alcoholBDH1174-4LPVWR
Microscope slidesThermo Fisher Scientific22-267-013
Plasma cleanerHarrick PlasmaPDC-001
Polydimethylsiloxane (PDMS)Dow Corning184 SIL ELAST KIT 0.5KG
Power supplyKeysight TechnologiesE3630A
SeaKem LE AgaroseLonza50004
Sodium hydroxideFisher ChemicalS318-1
Solenoid manifoldPneumadyneMSV10-1
Solenoid valvePneumadyneS10MM-30-12-3
Spin coaterLaurell TechnologiesWS-650Mz-23NPPB
SU8 DeveloperMicroChem Corp.Y020100 4000L1PE
SU8-100MicroChem Corp.Y131273 0500L1GL
SU8-5MicroChem Corp.Y131252 0500L1GL
Sulfo-NHS (N-hydroxysulfosuccinimide)Thermo Fisher Scientific24510
Sulfuric acidEMD MilliporeMSX12445

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