Immobilization of Live Caenorhabditis elegans Individuals Using an Ultra-thin Polydimethylsiloxane Microfluidic Chip with Water Retention

Summary

A series of immobilization methods has been established to allow the targeted irradiation of live Caenorhabditis elegans individuals using a recently developed ultra-thin polydimethylsiloxane microfluidic chip with water retention. This novel on-chip immobilization is also adequate for imaging observations. The detailed treatment and application examples of the chip are explained.

Abstract

Radiation is widely used for biological applications and for ion-beam breeding, and among these methods, microbeam irradiation represents a powerful means of identifying radiosensitive sites in living organisms. This paper describes a series of on-chip immobilization methods developed for the targeted microbeam irradiation of live individuals of Caenorhabditis elegans. Notably, the treatment of the polydimethylsiloxane (PDMS) microfluidic chips that we previously developed to immobilize C. elegans individuals without the need for anesthesia is explained in detail. This chip, referred to as a worm sheet, is resilient to allow the microfluidic channels to be expanded, and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals. Furthermore, the worm sheet shows water retention and allows C. elegans individuals to be subjected to microscopic observation for long periods under live conditions. In addition, the sheet is only 300 µm thick, allowing heavy ions such as carbon ions to pass through the sheet enclosing the animals, thus allowing the ion particles to be detected and the applied radiation dose to be measured accurately. Because selection of the cover films used for enclosing the animals is very important for successful long-term immobilization, we conducted the selection of the suitable cover films and showed a recommended one among some films. As an application example of the chip, we introduced imaging observation of muscular activities of animals enclosing the microfluidic channel of the worm sheet, as well as the microbeam irradiation. These examples indicate that the worm sheets have greatly expanded the possibilities for biological experiments.

Introduction

Radiation, including X-rays, gamma rays, and heavy-ion beam, is widely used for biological applications such as in cancer diagnosis and treatment, and for ion-beam breeding. Numerous studies and technical developments are currently focusing on the effects of radiation^1,2,3. Microbeam irradiation is a powerful means of identifying radiosensitive sites in living organisms⁴. The Takasaki Advanced Radiation Research Institute of National Institutes for Quantum and Radiological Science and Technology (QST-Takasaki) has been developing a technology to irradiate individual cells under microscopic observation using heavy-ion microbeams⁵, and has established methods to enable targeted microbeam irradiation of several model animals, such as the nematode Caenorhabditis elegans^4,6, silkworms⁷, and Oryzias latipes (Japanese medaka)⁸. Targeted microbeam irradiation of the nematode C. elegans allows the effective knockdown of specific regions, such as the nerve ring in the head region, thus helping to identify the roles of these systems in processes such as locomotion.

A method for on-chip immobilization of C. elegans individuals without the need for anesthesia has been developed to allow for microbeam irradiation⁴. In addition, to improve microfluidic chips used in the previous study⁴, we have recently developed wettable, ion-penetrable, polydimethylsiloxane (PDMS) microfluidic chips, referred to as worm sheets (see Table of Materials), for immobilizing C. elegans individuals⁹. These comprise of ultra-thin soft sheets (thickness = 300 µm; width = 15 mm; length = 15 mm) with multiple (20 or 25) straight microfluidic channels (depth = 70 µm; width = 60 µm or 50 µm; length = 8 mm) at the surface (Figure 1A-D). The microfluidic channels are open and allow multiple animals to be enclosed in them simultaneously (Figure 1E). The sheets are resilient to allow the microfluidic channels to be expanded (by ~10%, Figure 1F), and the elasticity allows animals to be enveloped gently. Also, owing to the self-adsorption capacity of the PDMS, animals can be sealed in the channels by covering the surface of the worm sheet with a thin cover film, in which animals are not pushed into the channels for enclosure. By turning the cover film over, we can easily collect the animals.

The channels do not hurt the worms when they are being enclosed or when they are collected. Furthermore, the sheets are made from PDMS, which is essentially hydrophobic, but water retention can be achieved by imparting hydrophilicity to the material. The water retention and thickness are favorable characteristics of the worm sheets. The water-retention capacity prevents dehydration of the animals after prolonged immobilization and enables long-term observations to be carried out.

In addition, as described previously⁹, the sheets are only 300 µm thick, allowing heavy ions such as carbon ions (with a range of about 1 mm in water) to pass through the sheet enclosing the animals. This allows the ion particles to be detected and the applied radiation dose to be measured accurately. Moreover, the worm sheets can be reused and are thus economical. With the conventional injection method, the animals enclosed are sometimes dead and they cannot be taken out of the channel; their eggs can also clog the channels. This makes the chip unusable. Chips are, therefore, basically disposable and the cost-benefit ratio is poor.

In the present paper, we describe in detail a series of methods for on-chip immobilization of live C. elegans individuals using worm sheets. Through locomotion assays of animals 3 h after on-chip immobilization, we evaluated the suitable cover film. In addition, we showed the examples of on-chip immobilization for both imaging observations and microbeam irradiation.

Protocol

1. Strains and maintenance

1. Select a suitable strain of C. elegans and Escherichia coli (food) depending on the purpose of the experiment.
   NOTE: In the present paper, wild-type N2¹⁰C. elegans (Figure 2A) is generally used, and HBR4:goeIs3[pmyo-3::GCamP3.35::unc-54 - 3'utr, unc-119(+)]V¹¹ is only employed for imaging assay. E. coli OP50 was used as food for C. elegans. Some mutants with abnormal body shape, such as the unc-119(e2498) III mutant with a coiled shape (Figure 2B), also can be enclosed in the straight microfluidic channels (Figure 2C).
2. Maintain the C. elegans at 20 °C on 6 cm Petri dishes containing 10 mL of nematode growth medium (NGM) spread with overnight-incubated (37 °C) E. coli as described previously¹⁰. If possible, synchronize the developmental stages of C. elegans from the embryo stage.
3. Use well-fed adult animals, approximately 3-4 days after hatching with a width of about 50-60 µm, which are optimally suited to the size of the microfluidic channels in the worm sheets.

2. Selection of buffer solution for on-chip immobilization

1. For wettable worm sheets, use any buffer solution depending on the purpose of the experiments.
   NOTE: Suitable buffer solutions for on-chip immobilization on PDMS microfluidic chips have been defined previously⁹ and the following buffer solutions were shown to have no effect on the motility of the animals after immobilization: S basal buffer solution (5.85 g of NaCl, 1 mL of cholesterol (5 mg/mL in ethanol), 50 mL of 1 M pH 6.0 potassium phosphate, H₂O to 1 L; sterilized by autoclaving)¹⁰ containing a large amount of NaCl, M9 phosphate buffer solution (5 g of NaCl, 3 g of KH₂PO₄, 6 g of Na₂HPO₄, 1 mL of 1 M MgSO₄, H₂O to 1 L; sterilized by autoclaving)¹⁰, wash buffer solution (5 mL of 1 M pH 6.0 potassium phosphate, 1 mL of 1 M CaCl₂, 1 mL of 1 M MgSO₄, 0.5 g pf gelatin, H₂O to 1 L; sterilized by autoclaving)¹² containing gelatin, and ultrapure water⁹. In the present paper, wash buffer solution is typically used, and S basal buffer solution is only employed for evaluating on-chip immobilization using different cover films in the section 3.
2. For conventional microfluidic chips without wettability, use a wash buffer solution, which provides the most effective means of maintaining moisture in the microfluidic channels of the chip and thus prevents drying of C. elegans individuals (regardless of the wettability of the microfluidic chips).

3. Selection of suitable cover film for on-chip immobilization

1. Prepare different cover films as follows. Based on the ease of handling, cut the following transparent, biocompatible cover films to a suitable size (i.e., width of 10-15 mm and length of 30-50 mm): 130-170 µm thick cover glass, 125 µm thick polyester (PET) film, and ~130 µm thick polystyrene (PS) film.
2. Perform 3 h on-chip immobilization using different cover films as follows. Based on steps 4.1-4.6, enclose ≥10 washed adult animals (3.5 days after hatching) in the worm sheet using each cover film, respectively, and leave for 3 h.
3. For comparison purposes, allow 3 h of free movement as follows. Place ≥10 washed adult animals on a 3.5 cm plate containing 3 mL fresh NGM, cover with a lid, and leave for 3 h.
4. Collect animals immediately after 3 h of on-chip immobilization according to steps 5.1-5.3. Remove the cover film from the worm sheet and add a drop of buffer solution to each animal. Pick up animals swimming in the droplet using a platina picker and transfer them to a 3.5 cm plate containing 3 mL fresh NGM without food (assay plate).
5. Collect animals after 3 h of free movement. Add a drop of buffer solution to each animal. Pick up animals swimming in the droplet on the NGM plate and transfer them to an assay plate using a platina picker.
6. Evaluate the effects of cover film on motility (locomotion assay).
   1. At least 5 min after transfer to the assay plate, count 'body bends' (defined as the number of bends in the anterior body region at 20-s intervals) manually under a microscope¹³.
   2. Carry out locomotion assays five times independently for each cover film.
      NOTE: In the experiments shown in the representative results, 10 animals were evaluated for each experiment in which multiple animals were enclosed simultaneously, compared with only five animals counted in the previous study¹³.
   3. Calculate the average number of body bends for the 10 animals in each group for each experiment. Then average the values from five independent experiments to evaluate the effects of on-chip immobilization. Analyze the data statistically using a one-way analysis of variance (ANOVA) test in a spreadsheet software at significance levels of 0.01 and 0.05.
      NOTE: Based on these experiments, a PS cover film with high oxygen permeability was deemed suitable (see the representative results). These PS films are included with the worm sheets. PET film, which has low oxygen permeability, was not deemed suitable because the animals tended to suffocate if covered with it during long-term immobilization on the worm sheets. It is also important that the cover film does not break during a series of procedures; glass covers were therefore also unsuitable (see the representative results).

4. On-chip immobilization

NOTE: Disposable, sterile gloves should be worn to avoid contaminating the worm sheets.

1. Place a thin transparent sheet such as a PS or glass cover film which has no autofluorescence, for use as a bottom cover film, onto the experimental desk or the microscopic stage. Place a worm sheet gently onto the bottom cover film using flat tweezers.
   NOTE: In addition to the worm sheets with 60 µm-wide microfluidic channels (suitable for adults 3-4 days after hatching), sheets with 50 µm-wide channels (suitable for young adults 3 days after hatching and mutants with small body in adult stage) can also be employed. Select a suitable sheet based on the size of the animals to be used.
2. To collect animals, pick up an individual adult C. elegans from the culture plate under a stereomicroscope using a platina picker. Repeat the process if multiple animals are needed.
3. Wash animals to remove food (bacteria).
   1. Place at least three droplets (5 µL drops) of buffer solution on the surface of a 6 cm non-treated (water-repellent) Petri dish.
   2. Transfer the animals to a droplet using a platina picker and allow them to remove any food by swimming. Wash them twice in two separate droplets, and then rinse the animals in another droplet.
      NOTE: It is necessary to practice this procedure to be able to perform it quickly before carrying out any experiments.
4. Drop 2-3 µL of buffer solution onto the surface of a worm sheet. Pick up the washed animals from the droplet on the Petri dish and transfer them to the droplet on the worm sheet.
5. Enclose animals in microfluidic channels as follow. Place a PS cover film over the worm sheet using flat tweezers and press gently over the channels from one end of the sheet to the other to maintain humidity (as described previously^4,9).
   NOTE: Animals are randomly enclosed in the channels. Droplets containing animals are spread across the worm sheet by covering the sheet, resulting in droplets being widely extended over several channels. Each animal can be enclosed in any one of these channels. The important point regarding the use of the worm sheet for on-chip immobilization is that multiple channels are available over a wide area.
6. Immediately after enclosing the animals in the microfluidic channels, confirm that they are alive by checking for movement of the head under a microscope (e.g., 1x or 2x magnification).
7. Record animal's position, at the same time as carrying out step 4.6 and note the following on a dedicated sheet of paper (Supplementary File 1): number of the channel in which the animal is enclosed; position of each animal in the channel (left/center/right); and direction of the head.
8. Draw an arrow to mark the position of each animal in the channel, with the direction of the arrow corresponding to the animal's head.
   NOTE: The channel numbers (e.g., 1, 5, 10, 15, 20, 25) are engraved on the surface of the worm sheet near the left and right edges of the microfluidic channels (Figure 1B). This information on the dedicated sheet allows animals to be located quickly, making the process more efficient, and also helps to prevent radiation leakage during subsequent steps.

5. Collection of animals from a worm sheet

1. Immediately after immobilization, remove the cover film from the worm sheet using flat tweezers.
2. Drop 10-15 µL buffer solution onto the microfluidic channels enclosing the animals and observe the animals starting to swim in the droplets under a stereomicroscope.
   NOTE: The animals start swimming in the new droplet on their own without any additional pressure, help, or push. Just dropping some buffer on top of them is enough to make them swim out of the channel. An animal may be unable to swim in a droplet if it has been vitally damaged by irradiation in step 7.9 or by the immobilization process in steps 4.2-4.5, though this is rare. In addition, if the animal remains attached to the cover film when it is removed, add the droplets onto the cover film instead of into the channels.
3. Pick up the animals swimming in the droplet using a platina picker and place them on an assay plate.

6. Application of worm sheets for imaging observations

NOTE: Worm sheets can be widely used in microscopic observations. The chip can retain water and does not affect the motility of C. elegans individuals after 3 h of on-chip immobilization⁹. In addition, the chip itself has no autofluorescence, making it suitable for use in fluorescence imaging assays. A sample application for fluorescence imaging assay is given below.

1. Select a fluorescence microscope and mount a digital camera or digital video camera on the microscope to capture images or videos, respectively (Figure 3A).
   NOTE: The working distance (WD) of microscope is more than 0.2 mm depending on objective lens specification (see Figure 3B,C). The specification of the fluorescence microscope system used in this paper is shown in the Table of Materials. However, the specification is not limited to our example because it depends on the purpose of observation or/and users.
2. Select any fluorescent strain of C. elegans and maintain as described in section 1.
   NOTE: If it is necessary to observe the body-wall muscular contraction (see representative results), use young adult HBR4¹¹C. elegans, in which a reporter gene is used to express the calcium indicator GCaMP3.35 in all body-wall muscle cells. It is important to use young adults (≤3 days after hatching) that are thinner than the width of the microfluidic channels (50 or 60 µm) of the worm sheet. The small degree of clearance means that the animals can bend slightly, making it possible to observe the muscular contraction and extension during crawling, using a calcium-ion indicator.
3. Carry out enclosure of animals according to the immobilization procedure in section 4.
4. Observe fluorescent spots of animals (e.g., green fluorescent protein-labeled strains) using a fluorescence microscope, and capture images using a digital camera mounted on the microscope.
   NOTE: Follow the previously established methods^14,15,16, since the microscope observation methods (including fluorescence observation) and the specification of the microscope system depends on the purpose of the observation.
5. Image calcium-ion wave propagation using video acquisition to observe dynamic activities, such as the body-wall muscular contraction and extension in the HBR4 worms (Video 1).

7. Application of worm sheets for microbeam irradiation

NOTE: The collimating microbeam irradiation system⁵ can use several heavy-ion particles accelerated from the azimuthally varying field cyclotron installed at the Takasaki Ion Accelerators for Advanced Radiation Application (TIARA) facility of QST-Takasaki (Figure 4A). There is an automatic stage for irradiation under the beam exit (Figure 4B). The procedure for heavy-ion microbeam irradiation of C. elegans using this system is as follows.

1. Locate the worm sheet enclosing multiple animals on an aluminum frame custom-made for the microbeam-irradiation facility and set it on the automatic stage for irradiation (Figure 4C).
2. Raise the irradiation sample (i.e., animals enclosed in a worm sheet on a frame), to immediately under (~2 mm) the beam exit based on the monitor image from a microscope located under the automatic stage (Figure 4D).
3. After vertical positioning, locate each animal to target with the microbeam irradiation using the custom-made software for targeted irradiation of animals and cell cultures. To locate each animal enclosed in the microfluidic channel, refer to the dedicated sheet described in step 4.7-4.8 (see Supplementary File 1) and confirm the channel number near the left and right edges of the channels.
4. Control the automatic stage in the X and Y directions to position the animals just under the beam exit using a remote-control system and a laser mouse operated with the irradiation software.
5. Roughly tune the irradiation area by watching the projection from the microscope located under the automatic stage of the sample and moving the cursor to the irradiation position of the animal to be targeted.
6. After rough positioning, exit and close the irradiation room and move to the adjacent control room to avoid irradiating the operators.
7. Set the desired number of ion particles for one irradiation procedure, corresponding to irradiation of a specific region of the animal, using the console linked to an ion-counter system.
   NOTE: This consists of a plastic scintillator and a photoelectron multiplier in the collimating microbeam irradiation system.
8. From the irradiation-control room, fine-tune the position of the animals based on the monitor image from the microscope located under the automatic stage, which is the same image projected on the monitor in the irradiation room (Figure 4E).
9. Target the microbeam irradiation by click the irradiation button of the software for irradiation.
   NOTE: In the example shown in Figure 4F, we target the pharynx in the head region and irradiate with the appropriate number of microbeam carbon ions. Because the number of ion particles passing through the sample is counted using an ion-counter system linked to the console and the irradiation software, the irradiation is stopped after delivery of the desired number of ion particles.
10. Locate each animal recorded on the dedicated sheet and carry out targeted irradiation to each animal. Repeat steps 7.8 and 7.9 until all animals have been irradiated.
11. Immediately after irradiation, enter the irradiation room and lower the automatic irradiation stage. Remove the sample on the custom-made frame located on the automatic stage.
12. Collect the animals as described in steps 5.1-5.3.
13. Carry out behavioral and/or molecular analyses as required to evaluate the effects of the targeted irradiation, depending on the purpose of the study.
    NOTE: The locomotion assay described in step 3.6 is an effective method for evaluating the effects of irradiation on motility^4,9.

8. Treatment of worm sheets for repeated use

NOTE: Worm sheets can be used repeatedly at least 10 times⁹ with no adverse effects on the animals if cleaned and sterilized properly after use as follows.

1. Cleaning
   1. Place the used worm sheet on a 6 cm Petri dish and drop about 100 µL of sterilized ultrapure water onto the whole sheet.
   2. Paddle the water on the surface of the sheet using gloved fingers to wash off dirt such as dust, bacterial food, and any eggs laid in the channels.
   3. Wipe the moisture off the worm sheet thoroughly using disposable wipes.
2. Sterilization
   1. Inject about 5 mL of 70% ethanol into the Petri dish containing the worm sheet and paddle the surface of the sheet using gloved fingers to wash off the dirt.
3. Drying
   1. Remove the chips from the Petri dish filled with 70% ethanol and allow to dry naturally.
4. Storage
   1. After drying, place the worm sheet on a sterile Petri dish and cover it. Sheets can also be stored on a sterile Petri dish filled with 70% ethanol.
      NOTE: It is better to dispose of the cover films after use, but if they are to be re-used, clean them as done for the worm sheet. However, if folds develop on the cover film after use it will no longer adhere to the chip, resulting in dehydration of the animals, and it should therefore be replaced.

Results

Active C. elegans individuals could be immobilized successfully using an ultra-thin, wettable PDMS, microfluidic chip (worm sheet). We investigated the suitability of different cover films for sealing the worm sheet, as described in protocol section 3. To evaluate the sealing effects of the cover films, we determined the motility of animals 3 h after on-chip immobilization using cover glass (thickness: 130-170 µm), PET film (thickness: 125 µm), and PS film (thickness: ~130 µm), r...

Discussion

On-chip immobilization of C. elegans under live conditions using a wettable PDMS microfluidic chip enables the efficient targeted microbeam irradiation of multiple animals. The ease of handling and features to prevent drying make this system suitable for applications not only in microbeam irradiation, but also in several behavioral assays. These worm sheets have already been commercialized and can be easily obtained. Conventional microfluidic chips, such as olfactory chips, are associated with problems including...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
C. elegans wild-type strain	Caenorhabditis Genetics Center (CGC) , Minnesota, USA	N2	Wild-type C. elegans strain generally used in this study
C. elegans unc-119(e2498) III mutant strain	Caenorhabditis Genetics Center (CGC) , Minnesota, USA	CB4845	C. elegans strain only employed as an example of mutants with abnormal body shape
C. elegans transgenic strain HBR4	Caenorhabditis Genetics Center (CGC) , Minnesota, USA	HBR4	The genotype of this transgenic C. elegans strain is HBR4:goeIs3[pmyo-3::GCamP3.35:: unc-54–3’utr, unc-119(+)]V. This strain was only employed for imaging observation.
E. coli strain	Caenorhabditis Genetics Center (CGC) , Minnesota, USA	OP50	E. coli strain used as food for C. elegans
Worm Sheet IR (50/60)	Biocosm, Inc., Hyogo, Japan	BCM17-0001	Microfluidic chip with 25 straight 50/60-µm width channels used in all experiments and observation in this paper
Worm Sheet 60	Biocosm, Inc., Hyogo, Japan	BCM18-0001	Microfluidic chip with 20 straight 60 µm-width channels. This is sitable for adults 3-5 days after hatching at 20°C.
Worm Sheet 50	Biocosm, Inc., Hyogo, Japan	BCM18-0002	Microfluidic chip with 20 straight 50 µm-width channels. This is sitable for youg adults ~3 days after hatching at 20°C.
MICRO COVER GLASS	MATSUNAMI GLASS IND. LTD.	C030401	Cover glass (thickness: 130-170 µm) used in locomotion assays in Protocol 3
Polystyrene Film	Biocosm, Inc., Hyogo, Japan	BCM18-0001/ BCM18-0002	Bundled items of Worm Sheets. PS filim (thickness: ~130 µm) used in locomotion assays in Protocol 3.
Polyester Film Lumirror	TORAY INDUSTRIES, INC., Tokyo, Japan	Lumirror T60 (t 125 µm)	PET filim (thickness: 125 µm) used in locomotion assays in Protocol 3
IWAKI 60 mm/non-treated dish	AGC Techno Glass Co., Ltd., Shizuoka, Japan).	1010-060	Non-treated dish used in incuvation of C. elegans in Protocol 1
IWAKI 35 mm/non-treated dish	AGC Techno Glass Co., Ltd., Shizuoka, Japan).	1010-035	Non-treated dish used in locomotion assays in Protocol 3
Milli-Q	Merck, France		Ultrapure water
Kimwipe S-200	Nippon Paper Crecia Co., Ltd., Tokyo, Japan	62020	120 mm x 215 mm; 200 sheets/ box
WormStuff Worm Pick	Genesee Scientific Corporation, CA, USA)	59-AWP	Platina picker specilized for picking up C. elegans
Research Stereo Microscope System	OLYMPUS CORPORATION, Tokyo, Japan	SZX16	Micriscope used in all experiments and observation in this paper
Motorized Focus Stand for SZX16	OLYMPUS CORPORATION, Tokyo, Japan	SZX2-ILLB	This was used for bright field observation in Protocol 3-8.
Objective Lens (×1)	OLYMPUS CORPORATION, Tokyo, Japan	SDFPLAPO1×PF	NA: 0.15; W.D.: 60 mm. This lends was used for bright field observation in Protocol 3-8.
Objective Lens (×2)	OLYMPUS CORPORATION, Tokyo, Japan	SDFPLAPO2XPFC	NA: 0.3; W.D.: 20 mm. This  lends was used for imaging observations.
Mercury Light Source	OLYMPUS CORPORATION, Tokyo, Japan	U-LH100HG	The broad emission spectrum enables a range of fluorescence imaging experiments to be conducted using all common fluorophores.
SZX16 Fluorescent filter unit (High performance for YFP)	OLYMPUS CORPORATION, Tokyo, Japan	SZX2-FYFPHQ	Ex: 490-500 nm; Em: 510-560. This was used for imaging observation of HBR4 strain.
Digital Camera High Speed EXILIM	CASIO COMPUTER Co., Ltd, Tokyo, Japan		EX-F1 Figure 1B, 1E, 1F, Figure 2A-C, and Video 1 were obtained by using this digital camera.
Office 2013	Microsoft Co. Ltd, Redmond, WA, USA		EXCEL Software used for statistical analyses