A Simple Microfluidic Chip for Long-Term Growth and Imaging of Caenorhabditis elegans

Summary

The protocol describes a simple microfluidic chip design and microfabrication methodology used to grow C. elegans in presence of a continuous food supply for up to 36 h. The growth and imaging device also enables intermittent long-term high-resolution imaging of cellular and sub-cellular processes during development for several days.

Abstract

Caenorhabditis elegans (C. elegans) have proved to be a valuable model system for studying developmental and cell biological processes. Understanding these biological processes often requires long-term and repeated imaging of the same animal. Long recovery times associated with conventional immobilization methods done on agar pads have detrimental effects on animal health making it inappropriate to repeatedly image the same animal over long periods of time. This paper describes a microfluidic chip design, fabrication method, on-chip C. elegans culturing protocol, and three examples of long-term imaging to study developmental processes in individual animals. The chip, fabricated with polydimethylsiloxane and bonded on a cover glass, immobilizes animals on a glass substrate using an elastomeric membrane that is deflected using nitrogen gas. Complete immobilization of C. elegans enables robust time-lapse imaging of cellular and sub-cellular events in an anesthetic-free manner. A channel geometry with a large cross-section allows the animal to move freely within two partially sealed isolation membranes permitting growth in the channel with a continuous food supply. Using this simple chip, imaging of developmental phenomena such as neuronal process growth, vulval development, and dendritic arborization in the PVD sensory neurons, as the animal grows inside the channel, can be performed. The long-term growth and imaging chip operates with a single pressure line, no external valves, inexpensive fluidic consumables, and utilizes standard worm handling protocols that can easily be adapted by other laboratories using C. elegans.

Introduction

Caenorhabditis elegans has proved to be a powerful model organism to study cell biology, aging, development biology, and neurobiology. Advantages such as its transparent body, short life cycle, easy maintenance, a defined number of cells, homology with several human genes, and well-studied genetics have led to C. elegans becoming a popular model both for fundamental biology discoveries and applied research^1,2. Understanding cell's biological and developmental processes from repeated long-term observation of individual animals can prove to be beneficial. Conventionally, C. elegans is anesthetized on agar pads and imaged under the microscope. Adverse effects of anesthetics on the health of animals limit the use of anesthetized animals for long-term and repeated intermittent imaging of the same animal^3,4. Recent advances in microfluidic technologies and their adaptation for anesthetic-free trapping of C. elegans with negligible health hazards enable high-resolution imaging of the same animal over a short and long period of time.

Microfluidic chips have been designed for C. elegans'⁵ high throughput screening^6,7,8, trapping and dispensing⁹, drug screening^10,11, neuron stimulation with high-resolution imaging¹², and high-resolution imaging of the animal^12,13,14. Ultra-thin microfluidic sheets for immobilization on slides have also been developed¹⁵. Long-term studies of C. elegans have been performed using low-resolution images of animals growing in liquid culture to observe growth, calcium dynamics, drug effects on their behavior^16,17,18,19, their longevity, and aging²⁰. Long-term studies using high-resolution microscopy have been carried out to assess synaptic development²¹, neuronal regeneration²², and mitochondrial addition²³. Long-term high-resolution imaging and tracing of cell fate and differentiation have been done in multichannel devices^24,25. Several cellular and sub-cellular events occur over the time scales of several hours and require trapping the same individual at different time points during their development to characterize all intermediate steps in the process to understand cellular dynamics in vivo. To image biological process such as organogenesis, neuronal development, and cell migration, the animal needs to be immobilized in the same orientation at multiple time points. We have previously published a protocol for high-resolution imaging of C. elegans for over 36 h to determine where mitochondria are added along the touch receptor neurons (TRNs)²³.

This paper provides a protocol for establishing a microfluidics-based methodology for repeated high-resolution imaging. This device, with a single flow channel, is best suited for repeated imaging of a single animal per device. To improve throughput and image many animals at once, multiple devices could be connected to the same pressure line but with separate three-way connectors controlling a single animal in each device. The design is useful for studies that demand high-resolution time-lapse images such as post-embryonic developmental processes, cell migration, organelle transport, gene expression studies, etc. The technology could be limiting for some applications such as lifespan and aging studies that require parallel growth and imaging of many late-stage animals. Polydimethylsiloxane (PDMS) elastomer was used for fabricating this device due to its biostability²⁶, biocompatibility^27,28, gas permeablility^29,30, and tunable elastic modulus³¹. This two-layer device allows the growth of animals with continuous food supply in a microfluidic channel and the trapping of individual C. elegans via PDMS membrane compression using nitrogen gas. This device is an extension of the previously published device with the advantage of growing and imaging the same animal in the microchannel under a continuous food supply³. The additional isolation membrane network and a 2 mm wide trapping membrane enable efficient immobilization of developing animals. The device has been used to observe neuronal development, vulval development, and dendritic arborization in sensory PVD neurons. The animals grow without adverse health effects in the device and can be repeatedly immobilized to facilitate imaging sub-cellular events in the same animal during its development.

The entire protocol is divided into five parts. Part 1 describes device fabrication for the growth and imaging chip. Part 2 describes how to set up a pressure system for the PDMS membrane deflection to immobilize and isolate individual C. elegans. Part 3 describes how to synchronize C. elegans on a nematode growth medium (NGM) plate for device imaging. Part 4 describes how to load a single animal in the device and grow the animal inside the microfluidic device for several days. Part 5 describes how to immobilize an individual animal at multiple time points, capture high-resolution images using different objectives, and analyze the images using Fiji.

Protocol

1. Fabrication of growth and imaging device SU8 mold fabrication Design patterns 1 (flow layer) and 2 (control layer) using rectangular shapes in a word processing software (or a computer-aided design CAD software) and print the photomasks with the help of a laser plotter with a minimum feature size of 8 µm on polyester-based film (Figure 1). Cut silicon wafers in 2.5 cm × 2.5 cm pieces and clean them with 20% KOH for 1 min. Rinse…

Representative Results

Device characterization: The growth and imaging device consists of two PDMS layers bonded together (Figure 1) using irreversible plasma bonding. The flow layer (pattern 1) which is 10 mm in length and 40 µm or 80 µm in height allows us to grow the animal in liquid culture (Figure 1A). The trapping layer (pattern 2) has a 2 mm wide membrane (Figure 1B) for immobilizing the animal for high-resolution imaging…

Discussion

In this paper, a protocol for fabrication and use of a simple microfluidic device for growing C. elegans with constant food supply and high-resolution imaging of a single animal during its development has been described. This fabrication process is simple and can be done in a non-sterile environment. A dust-free environment is critical during fabrication steps. The presence of dust particles would lead to improper contact between the two bonding surfaces, resulting in poor bonding and leakage of the device durin…

Materials

18 G needles	Sigma-Aldrich, Bangalore, India	Gauge 18
3-way stopcock	Cole-Parmer	WW-30600-02	Masterflex fitting with luer lock
CCD camera	Andor Technology	EMCCD C9100-13no
Circuit board film	Fine Line Imaging, Colorado, USA		The designs are printed with 65,024 dots per inch (DPI)
Convection Oven	Meta-Lab Scientific Industries, India	MSI-5
Coverslips	Blue stat microscopic cover glass		22mm x 10Gms
Ethanol	Hi media
Harris uni-core puncher 1mm	Qiagen	Z708801
Hexamethyldisilazane	Sigma-Aldrich, Bangalore, India	440191
Hot plate	IKA	RCT B S 22
Isopropanol	Fisher Scientific	26895
KOH	Fisher Scientific
Laser Scanning Microscope	ZEISS	LSM 5 LIVE
Micropipette tips	Tarsons		0.5-10 µL micropipette tips are used for food supply
Negative Photoresist-1	Microchem	SU8-2025	http://www.microchem.com/Prod-SU82000.htm
Negative Photoresist-2	Microchem	SU8-2050	http://www.microchem.com/Prod-SU82000.htm
Nitrogen gas	Local Supplier	Commercial nitrogen gas	Cylinder volume of 7 cubic meter
PDMS (Curing solution)	Dow Corning Corporation, MI, USA	Sylgard curing solution	curing agent
Petri plates	Praveen Scientific Corporation
Plasma cleaner	Harrick Plasma, NY, USA	PDC-32G
Razor and blades	Lister surgical Blade
Silicon Elastomer (Base)	Dow Corning Corporation, MI, USA	Sylgard 184 base	elastomer base
Silicon tubes	Fisher Scientific		Plastic tubes with the inner diameter 1.59 mm and the outer diameter 3.18 mm
Silicon wafer	University Wafer, MA, USA	[100] orientation, 4-inch diameter	Small pieces (2 mm × 2 mm) were cut from 100 mm diameter wafer
Spin Coater	SPS-Europe B.V., The Netherlands	SPIN 150
Spinning Disk microscope	Perkin Elmer ultra-view VOX system	CSU-X1-A3 N	The system was equipped with four (405/488/561/640 nm) lasers and controlled with the Volocity software package.
SU8 developer	Microchem, MA, USA	SU8 Developer
Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane	Sigma-Aldrich, Bangalore, India	448931	Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane vapor is toxic
UV lamp	Oriel Instruments, Bangalore, India		200 Watt and collimated UV light source
Volocity software	Perkin-Elmer		Image analysis