A Microfluidic Platform for Stimulating Chondrocytes with Dynamic Compression

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 microenvironments^{1,2,3,4,5,6,7,8}. 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 s....

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 ....

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 diameters are 1.......

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
(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