WormSpace μ-TAS enabling automated on-chip multi-strain culturing and multi-function imaging of Caenorhabditis elegans at the single-worm level on the China Space Station

Abstract

As a model organism for space biology experiments, Caenorhabditis elegans (C. elegans) has low demand for life support and strong resistance to unfavorable environments, making experimentation with C. elegans relatively easy and cost-effective. Previously, C. elegans has been flown in several spaceflight investigations, but there is still an urgent need for analytical platforms enabling on-orbit automated monitoring of multiple phenotypes of worms, such as growth and development, movement, changes of biomarkers, etc. To solve this problem, we presented a fully integrated microfluidic system (WormSpace μ-TAS) with an arrayed microfluidic chip (WormChip-4.8.1) and a replaceable microfluidic module (WormChip cartridge), which was compatible with the experimental facility on the China Space Station (CSS). By adopting technologies of programmed fluid control based on liquid medium CeMM as well as multi-function imaging with a camera mounted on a three-dimensional (3D) transportation stage, automated and long-term experimentation can be performed for on-chip multi-strain culturing and bright-field and fluorescence imaging of C. elegans at the single-worm level. The presented WormSpace μ-TAS enabled its successful application on the CSS, achieving flight launch of the sample unit (WormChip cartridge) at low temperature (controlled by a passive thermal case at 12 °C), automated 30-day cultivation of 4 strains of C. elegans, on-orbit monitoring of multiple phenotypes (growth and development, movement, and changes of fluorescent protein expression) at the single worm-level, on-chip fixation of animals at the end of the experiment and returning the fixed samples to earth. In summary, this study presented a verified microfluidic system and experimental protocols for automated on-chip multi-strain culturing and multi-function imaging of C. elegans at the single-worm level on the CSS. The WormSpace μ-TAS will provide a novel experimental platform for the study of biological effects of space radiation and microgravity, and for the development of protective drugs.

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Introduction

Spaceflight for humans is accompanied by a wide range of physiological changes such as fluid redistribution, bone demineralization, muscle atrophy, decreased erythrocyte lifetime and immunosuppression.¹ With new horizons for long-duration and deep space exploration, it becomes more and more important to fully understand and provide countermeasures to the effects of the space environment on the human body. In addition, space provides a unique laboratory to study how life and physiologic functions adapt from the cellular level to that of the entire organism.² Due to the high cost and unavailability of launch platforms, only a few biology experiments have been carried out in space and no conclusive evidence has been found. Animal models are recognized as cost-effective solutions to some problems intrinsic with studying humans (e.g. high cost, low throughput, slow discovery rate and endangerment of human health).³ As a popular model organism, C. elegans is small and transparent in body, short in life- and reproduction-span, known and highly homologous to humans in genome, and available with a large library of genetic mutants and fluorescently reporting strains, enabling wide applications in biological experiments both on earth and in low earth orbit.⁴C. elegans has low demand for life support and strong resistance to unfavorable environments, making experimentation with C. elegans relatively easy and cost-effective. At the same time, the results obtained with flown C. elegans appear to have strong similarities in human beings.⁵ Together, C. elegans has been recognized as an excellent model system for space biology research.⁶

From 1992 to 2021, C. elegans was flown in several spaceflight investigations through the space transportation system (STS),^3,7–11 DELTA MISSION (ICE-FIRST)^2,5,12 and SpaceX mission^4,13,14 to the International Space Station (ISS), or from China's Shenzhou-8 spacecraft^15,16 and Shijian-10 recoverable scientific satellite.¹⁷ These studies mainly focused on the biological effects of space radiation and microgravity, and most of them used genomics or proteomics to analyze C. elegans populations with multiple generations and different developmental stages. The results show that C. elegans reproduction and development, cell apoptosis and genomic instability were not affected by the space environment, but expressions of the whole genome and space radiation-induced mutation ratios were altered significantly. Microgravity can affect C. elegans expressions of muscle-related genes and proteins, structures of muscle fiber and ability of movement.

The spaceflight hardware used in these C. elegans experiments is mainly to supply nutrients (with solid or liquid medium in large-volume culture chambers or bags) and control the culturing temperature. The samples launched were mostly L1 larvae, and the worms returning to the ground were often in mixed states with multiple-generations and different developmental stages,^3,7–10 which could have a great impact on the stability and reproducibility of the results. To solve this problem, several spaceflight missions carried C. elegans experiments with Dauer larvae induced by axenic solid medium,^2,15–17 which can maintain the development of the recovered samples at the same stage and avoid interference between multiple generations, but it is difficult for this method to achieve automatic experimentation as well as in-orbit C. elegans growth and development. Meanwhile, the test results of these studies were all obtained by returning the samples to the ground after space-flight and then performing growth observation and omics analysis, without in-orbit monitoring the growth and development of C. elegans at the single-worm level. Under this circumstance, it is difficult to obtain the results of on-orbit phenotype verification and accurate statistical data, which could result in inconsistency of the phenotypic changes observed on the ground with the corresponding molecular regulation mechanisms.

Microfluidics, also known as lab-on-a-chip,^18–20 is a science and technology for manipulating fluids at the micrometer scale, which holds features of miniaturization, integration and automation, with benefits of high throughput, low consumption and improved data quality/reproducibility. The micro total analysis system (also known as μ-TAS)^21–23 integrates required functional units, such as sample preparation, reaction, separation and detection, on a single microfluidic system, enabling on-site rapid analysis of biomarkers and/or microscale organisms (such as C. elegans).²⁴ There are already lots of microfluidic-based technologies and assays for research on C. elegans,^24–26 which holds great potential for construction of μ-TAS for C. elegans-based research on space biology. Culture automation is feasible using chemically defined axenic liquid medium, C. elegans maintenance medium (CeMM),^27,28 and automated analysis of liquid-culture animals is possible using either video recordings or fluorescence sensors.^2,3,9 Recently, microfluidic-based C. elegans analysis has been successfully performed on the ISS,⁶ which achieved detection of nematode muscle strength with multiple-generations at the single-worm level by designing a microfluidic device and a worm-loading apparatus for single worm sorting and sampling, and establishing a fluid handling protocol as well as the optimized procedures for spaceflight experiments. This study preliminarily verified the feasibility of in-orbit nematode analysis on the microfluidic device. However, integration of the needed functional units was not achieved in this microfluidics-integrated hardware, reducing the degree of automation for on-orbit operation, therefore astronauts were required to periodically replace culture bags, connect tubing and operate microscopes during the experiment. Moreover, this method cannot realize on-orbit multi-strain C. elegans culturing and multi-phenotype (such as growth and development, movement, changes of biomarkers, etc.) monitoring, which is very important for C. elegans space experiments.^11,13,29 It remains an area of active research for analytical platforms that are capable of testing multiple strains of C. elegans at the same time (to study biological effects of different space environments), and on-orbit multi-functional control of fluids and detection of worms (such as automated renewal of culture medium, immobilization and release of worms, bright-field and fluorescence imaging of animals, on-line fixation of worms, etc.) for spaceflight experiments.

This work aims to culture multiple strains and monitor multiple phenotypes of C. elegans at the single-worm level in the space environment by using a microfluidic system as the culture platform and adopting technologies of automated fluid control based on liquid medium CeMM as well as periodic imaging with a camera mounted on a 3D transportation stage. Based on the experimental facility (Ecology Science Experiment Rack, ESER) and the small universal biological culture module with an electronic control box (the SUBCM drawer) provided by the China Space Station (CSS), we designed a replaceable microfluidic module (WormChip cartridge) and a 3D-movable imaging module, and then constructed the experimental device containing the 2 modules. Upon inserting the experimental device into the SUBCM drawer, a fully integrated microfluidic system (WormSpace μ-TAS) was presented for automated on-chip multi-strain culturing and multi-function imaging of C. elegans at the single-worm level. Wild-type and multiple mutant strains of C. elegans can be investigated simultaneously. Multiple phenotypes can be monitored through the protocols of multi-functional control of fluids (automated renewal of culture medium/collection of progenies, immobilization and release of worms, on-line fixation of worms) and imaging of worms (video recording, bright-field and fluorescence imaging). The WormSpace μ-TAS and the experimental protocols presented here have demonstrated their successful application on the CSS from flight launch of the sample unit (WormChip cartridge), on-orbit operation and automated 30-day experiment, to returning to earth of the fixed samples, enabling a novel experimental platform for the study of biological effects of space radiation and microgravity, and for the development of protective drugs.

Results and discussion

Design of the microfluidic chip (WormChip-4.8.1)

We aim to develop a micro-total analysis system (μ-TAS) suitable for automated C. elegans space experiments, realizing on-chip multi-strain culturing, on-orbit monitoring of multiple phenotypes, single-worm level tracking, flight launch and recovery of C. elegans samples, etc. The key is to design the structure of the microfluidic chip that meets these experimental requirements. Previous efforts have been made in studying the phenotype of C. elegans in longitudinal chips,^30–32 and worms were loaded into these chips for periodic observation. However, these microfluidic chips do not meet the needs of multiple strains of C. elegans parallel space-flight studies. Moreover, the chambers were not connected to worm reversible immobilization structures, not allowing monitoring of worms with labelled fluorescent proteins. Additionally, these earth-based studies mainly used bacteria-based liquid culture media, which possibly results in the clogging of microfluidic channels and brings the risks of biosafety to the crew and workstation on the space station. Recently, Soni et al.⁶ verified the feasibility of microfluidic-based C. elegans spaceflight experiment. However, in this work, nematodes were cultured in liquid bags and periodically loaded into the chambers of the chip for observation by astronauts, resulting a low degree of automation and deficiency of validating on-chip long-term cultivation in orbit. In addition, the current method could not achieve testing of multiple strains of C. elegans at the same time and performing multi-functional control of fluids and monitoring of multiple phenotypes.

The design of the microfluidic chip for in-orbit automated C. elegans culturing and monitoring needs to meet the following requirements: (i) the chip should accommodate multi-strain C. elegans samples to investigate the effects of different space environment factors (space radiation, microgravity, synergistic effects of radiation and microgravity, etc.) on individual nematodes at the same time, (ii) allow simultaneous loading and parallel manipulation of multi-strain nematodes, (iii) maximize the number of chambers and nematodes on one chip to obtain more data of in-orbit nematode monitoring, (iv) minimize the number of fluid control channels of the chip to reduce the difficulty of fluid control, (v) the culture chambers and their connecting channels should be designed to accommodate CeMM-grown animals (Table S1†), and (vi) minimize the influence of debris, impurities and bubbles on the chip channels during the in-orbit long-term experiments of nematodes.

The schematic illustration of the WormChip-4.8.1 is shown in Fig. 1(A) and consists of 4 arrayed units for simultaneous analysis of wild-type and multiple mutant strains of C. elegans to study biological effects of different space environments, such as radiation, microgravity, and synergy of radiation and microgravity. Each unit contains an independent inlet, bifurcated network of delivery channels, and 8 parallel analytical channels including culture chambers and worm clamps for analysis of separated nematode worms, through another bifurcated network of distribution channels, and then converges to a common channel connected to the only 1 outlet of the chip. This design makes it possible to introduce a fluid, such as a suspension of one strain of C. elegans or culture medium (CeMM), into parallel chambers of the specific unit at the corresponding inlet, enabling simultaneous loading and parallel manipulation of 4 strains of C. elegans worms. The spacing between the adjacent chambers was designed to be 3.4 mm to maximize the number of chambers in one chip and to facilitate imaging of each chamber. To minimize the number of fluid control channels thus lowering the level of the difficulty of fluid control, only one outlet of the chip was designed to provide a point of exit from the 4 units for waste that has passed through all of the analytical channels.

Fig. 1 Design of the WormChip-4.8.1 for multi-strain culturing and monitoring of C. elegans at the single-worm level. (A) Schematic diagram of the WormChip-4.8.1 with 4 arrayed units, each with 8 parallel analytical channels, converged to a common channel connected to the only 1 outlet of the chip. (B) Schematic illustration of one of the analytical channels, including 3 regions (i) of loading filters with 2 worm trappers, culture chamber and worm-clamp array with characteristic dimensions. Actual images of the culture chamber with the loading filters (ii) on the left and the worm-clamp array (iii) on the right, respectively. Scale bar, 300 μm. (C) Picture of the WormChip-4.8.1 filled with red food dye for better visualization of the structure. Scale bar, 10 mm.

Fig. 1(B) shows the details of one of the analytical channels, including 3 regions (i) of the loading filters with 2 worm trappers having wide openings and narrow ends, an oval-shaped culture chamber (ii) for 1–4 worms to swim, and a microfluidic array of worm clamps (iii). The oval-shaped chamber has a length of the major and minor axes of 1400 μm and 1300 μm, respectively; this size provides sufficient room for the 1 mm long adults to move freely within the chamber, but is small enough to confine the worms to an area that fits within the field of view of video recording (∼1300 × 1300 μm²) for the imaging module. The cross-sectional area of the channels connected to the chamber was based on the body diameter of CeMM-grown worms and the minimum size needed to confine the worms within the chamber. The body diameters of the middle stage of L4 and adult worms grown in CeMM are about 23–27 μm and 40–45 μm, respectively. Therefore, a minimum channel width of 20 μm was designed for the loading channel, so that worms in the middle stage of L4 could be loaded into the chamber and stay at a higher flow rate. To increase the number of worms loaded into the chamber and to decrease clogging of loading channels due to debris, impurities or bubbles, parallel channels (width of 20 μm) with 2 worm trappers (openings at 100 μm and ends at 20 μm with a length of 800 μm) were used as loading filters, serving the dual purpose of loading worms or fresh CeMM into the chamber and allowing a path for the washout of progenies and waste. To enable periodic, temporary immobilization of the worms in one chamber individually for bright-field and fluorescence microscopic imaging, we introduced an array of tapered channels (width narrowing from 100 μm to 15 μm) as worm clamps adjacent to the chamber into the design. The ends of the worm-clamp array were designed to be narrow channels (width of 15 μm and length of 500 μm) to prevent loss of animals during loading and immobilization. The channel height of 35 μm was implemented, so that the movement of adult worms would be restricted in the vertical direction within the clamps. To facilitate the ability of the worms to swim freely within the chamber, we increased the height of the chambers to approximately 200 μm (see the Experimental section). Fig. 1(C) shows the picture of the WormChip-4.8.1 (130 × 50 × 6 mm³) with 5 ports (inlet 1–4 and the outlet) for insertion of cannulas.

Design of the microfluidic module (WormChip cartridge)

The WormChip-4.8.1 loaded with C. elegans and the fluidic components need to be integrated in a portable and fully-closed manner to allow automated fluid control during on-orbit culturing and monitoring of 4 strains of C. elegans grown in CeMM. This chip-to-module integration was achieved using a WormChip cartridge (microfluidic module) to prevent manual operation and contamination that presents additional crew time and a biohazard on the CSS. The design of the WormChip cartridge addressed several requirements for the on-orbit C. elegans experiment in an automated and safe manner. These requirements include (i) integration of various components needed for fluid control to automate fluid-control operation during the experiment, (ii) complete closure of the entire fluid lines to ensure sterile operation throughout the experimental process, (iii) a fluid line design that achieves the 3 fluid-control modes (periodic renewal of culture medium for long-term culturing, reversible immobilization of worms for bright-field and fluorescence microscopic imaging, and fixative injection after the experiment to fix samples for further study) involved in the nematode experiment on orbit, (iv) a volume and weight that can meet the requirements of the microfluidic module during flight launch, and (v) the procedures of installation, disassembly and decomposition of the microfluidic module according with the requirements of in-orbit operations for astronauts.

The schematic of the fluidic components and their connection pattern for the WormChip cartridge shown in Fig. 2(A) was designed for leak-proof and automated fluid control during C. elegans experiments. The WormChip cartridge has 5 key hardware components: (i) a chip cassette (Fig. 2(B)); (ii) pumping/valving components, containing 6 miniature peristaltic pumps (PP, RP-Q1-S-P45A-DC3V, TAKASAGO ELECTRIC, INC) and 6 pinch valves (PV, PCK-1015 W, TAKASAGO ELECTRIC, INC); (iii) 11 liquid-storage bags (PL 07/30/70-2G, OriGen Biomedical GmbH) made of fluorinated ethylene propylene (FEP), including 5 CeMM-storage bags (30 mL), 4 worm-collection bags (70 mL, infusing ∼10 mL CeMM in advance), one waste bag (70 mL) and one RNALater bag (7 mL, RNA Keeper Tissue Stabilizer used as fixative solution); (iv) specially designed fluid lines containing silicone flexible tubing and FEP tubing, connectors, 5 check valves (CV), one bubble trapper, and 18 hose clamps (HC); and (v) a custom-made cartridge to mount hardware components and fluid lines, showing a chip holder, 6 valves and 6 pumps, a box for collection of liquid storage bags and an electrical connector (Fig. 2(C)).

Fig. 2 Design of the portable and fully-closed WormChip cartridge for automated fluid control during culturing and monitoring of C. elegans grown in CeMM. (A) Design of the fluid lines of the WormChip cartridge, which contains the WormChip-4.8.1, 6 miniature peristaltic pumps (PP) and 6 pinch valves (PV), 11 fluorinated ethylene propylene (FEP) bags, silicone flexible tubing and FEP tubing, connectors, 5 check valves (CV), one bubble trapper, and 18 hose clamps (HC). (B) Schematic illustration of the chip cassette, including an aluminum manifold with 5 connectors and FEP tubes, a polycarbonate (PC) cover plate, an aluminum outer frame and a PC bottom plate, which are secured together with screws on the 4 sides of the outer frame. (C) Structure diagram of the WormChip cartridge showing PV 1–6, PP 1–6, the chip holder, the box for collection of liquid-storage bags and the electrical connector.

The chip cassette schematic shown in Fig. 2(B) housed the polydimethylsiloxane (PDMS)/glass microfluidic chip with a polycarbonate (PC) cover plate, a bottom plate and an aluminum outer frame. A PDMS replica of the WormChip-4.8.1 of 4.0 ± 0.3 mm thickness can be integrated into the cassette. The inlet and outlet cannulas are connected to 5 pinch valves (PV 1–4 for inlet 1–4, PV-5 for the outlet) through silicone tubing and connectors with 2 hose clamps (HC) attached on each hose between the inlet/outlet port and the PV. The 2 fluid lines of the PV (1–4) are connected to the PP (1–4) ended with the CeMM-storage bag (1–4) and to a CV (1–4) ended with the worm-collection bag (1–4), respectively. Two HCs are attached on the hose between the CV and the worm-collection bag. The 2 fluid lines of PV-5 are connected to a CV (CV-5) ended with a waste bag and to another PV (PV-6), respectively, with a bubble trapper between PV-5 and PV-6. The 2 fluid lines of PV-6 are connected to 2 PPs (PP-5 and PP-6), which ended with the CeMM-storage bag 5 and the RNALater bag, respectively. The chip cassette was mounted on the chip holder using a fastener. All of the 11 liquid-storage bags were stored in the box covered with a lid. The actual image of the WormChip cartridge is shown in Fig. S1.†

The miniature peristaltic pumps deliver CeMM or fixative solution (RNALater) through the WormChip-4.8.1 with flow rates of 20–60 μL min⁻¹ adjusted by a duty ratio control technology. The pinch valves are used for selecting flow path. The collected worm progenies were stored in biocompatible FEP bags, which are highly permeable to oxygen and carbon dioxide while remaining impermeable to water, facilitating the culture of progenies during the experiment and on-orbit storage. Volumes of the CeMM-storage bags, worm-collection bags and waste bag are large enough for a long-term (30 days) experiment. A bubble trapper prevents air bubbles in tubing or CeMM-storage bag 5 and RNALater bag from entering the microfluidic chip. The check valves are connected to the fluid lines to prevent liquids in the 4 worm-collection bags and the waste bag from flowing back into the chip. The hose clamps attached on the silicon tubing facilitate the clamping and closing of the flexible tubing by buckling the hose clamps before cutting the marked tubes between 2 adjacent clamps for detachment of the chip cassette and the 4 worm-collection bags after the experiment to recover C. elegans samples.

Design of the fluid lines of the WormChip cartridge (Fig. 2(A)) enables 3 fluid-control modes, shown in Fig. 3 for schematic and in Table S2† for details. After assembly of the WormChip cartridge, the initial state of the valves/pumps is shown in Fig. 2(A). To renew the culture medium in the WormChip-4.8.1 (Fig. 3(A)) for long-term culturing, after PV 1–5 switched on, PP-5 drives the culture medium in CeMM-storage bag 5 from the outlet, through chambers and channels, to inlet 1–4 and finally into worm-collection bag 1–4, with the worms retained in the chambers. Also in this mode, the worm progenies in the 4 units of the chip can be collected into the corresponding worm-collection bags, and the animals immobilized in the clamp array can be released to culture chambers after photographing. To immobilize worms for bright-field and fluorescence imaging (Fig. 3(B)), PP 1–4 drive liquids in CeMM-storage bag 1–4 from inlet 1–4, through chambers and channels, to the outlet and finally into the waste bag, pushing the worms immobilized in the clamp array. At the end of the experiment, fixative solution should be injected into the chambers to fix animals and protect cellular RNA. After PV 1–6 switched on, PP-6 drives the fixative in the RNALater bag into the chip (Fig. 3(C)), during which we monitor one of the culture chambers with worms. After observing that the worms in the chamber were fixed (changing from normal swimming to stiffness), fixative injection mode was stopped by sending an immediate command code to shut off PP-6 and then switch off PV 1–6. This prevents the fixative solution from flowing through the tubing and entering into the worm-collection bags. To test the feasibility of the 3 fluid-control modes, a WormChip-4.8.1 was loaded with worms and the WormChip cartridge was assembled according to Fig. 2(A). Valves and pumps were controlled by a ground-based valve/pump controller on the basis of Table S2† and the analytical channels were imaged using a microscope. The efficacy of the fluid control protocols was demonstrated by the results shown in Fig. 3(D).

Fig. 3 Design and verification of the 3 fluid-control modes of the WormChip cartridge. (A) Mode-1: culture-medium renewal/progeny collection/immobilized worm release, flow will be from CeMM-storage bag 5 to worm-collection bag 1–4 with PV 1–5 switched on and PP-5 in operation. (B) Mode-2: worm immobilization, flow is from CeMM-storage bag 1–4 to the waste bag with PP 1–4 in operation. (C) Mode-3: fixative injection, flow will be from the RNALater bag to the tubes between inlet 1–4 and PV 1–4 with PV 1–6 switched on and PP-6 in operation. (D) Validation testing of the 3 fluid-control modes by imaging the analytical channels of the WormChip-4.8.1 using a microscope. (i) CeMM renewal/worm release. (ii) Worm immobilization. (iii) Fixative injection. Red arrow shows the flow direction of the fluid.

The developed WormChip cartridge (Fig. 2(A)) integrates required components for fluid control, making it possible for fully automated control of valves and pumps during the entire process of C. elegans experiment by uploading commands through the electrical connector. At the same time, the whole fluid lines are closed with the help of an aluminum manifold (Fig. 2(B)) to hold the inlet/outlet cannulas and by using self-locking nylon cable ties to fasten all of the connectors in fluid lines, ensuring sterility throughout the experiment. Three fluid-control modes of the WormChip cartridge shown in Fig. 3 enable C. elegans culturing and manipulation on the CSS. The assembled WormChip cartridge is portable with a weight of less than 2.8 kg and dimensions of ∼224 × 224 × 119 mm³, facilitating flight launch and on-orbit operation (installation and disassembly). Operation of recovery of the worm chip and bags after the experiment is easy and friendly for astronauts, simply involving buckling the hose clamps and cutting the tubes between 2 adjacent clamps (cleaning the tips with disinfectant wipes) to detach the chip cassette and the 4 worm-collection bags in the on-orbit Science glove box.

C. elegans loading and WormChip cartridge assembly

Unless otherwise stated, all operations were performed in the clean bench at 20 °C. According to the chip design (shown in Fig. 1) and experimental requirements, nematode samples suitable for on-chip loading were 23–27 μm in body width and no less than 2000 in quantity. We synchronised the worms growing in CeMM and then cultured them to the appropriate body width. Different strains of nematodes show different rates of growth and development. To obtain 4 strains of C. elegans samples of a specific body width (23–27 μm) for loading into the WormChip-4.8.1 simultaneously, we need to measure the growth curves of different worms in CeMM and then determine the synchronization-to-loading time for each sample. After that, we can choose the starting point of synchronization for different animals and get the fit samples at the time of loading. Before the loading process, we adjusted the densities of the 4 worm suspensions to ∼6000 worms per mL and 8 μL of each suspension was aspirated into the tip of an FEP tubing connected to a 1 mL syringe with CeMM. After inserting the 4 tips of the FEP tubing into inlet 1–4 of the chip, the connected 1 mL syringes were mounted on a 4-channel syringe pump. A loading filter-based strategy for size-based loading of the middle stage of L4 worms was implemented by using 2 particular geometric features to the culture chambers – loading filters with 2 worm trappers and worm-clamp array, as can be seen in Fig. 1(B). The principle underlying this loading strategy is that an animal of a given size is arrested in the worm trapper during the loading and distributing process (∼3 μL min⁻¹), while smaller animals are driven into the chambers or washed out from the worm-clamp arrays (minimum width of 15 μm). The worm trapper was designed to be 0.8 mm long with the minimum width being 20 μm such that the middle stage of L4 worm remains trapped until the flow rate is increased to push the trapped animal inside the chamber. Once most of the worm trappers are occupied, the trapped worms can be pushed into the chambers and stay when the pump is set at higher flow rates. Meanwhile, the smaller animals in the chambers would be flushed out from the worm-clamp arrays. After 6–10 hours, the worms in the chambers become too large to pass through the loading filters. A syringe pump was used to drive CeMM from the outlet, through the chambers, to inlet 1–4 to remove the larger animals in the distribution channels that could not enter into the chambers, retaining the loaded worms in the chambers. On average, more than 80% of the chambers could be loaded with 1–4 worms within about 30 min based on ground-based loading tests. Fig. S3† shows representative images of the culture chambers of the chip unit 1–4 after sample loading, and Table S3† shows ratios of chambers with worms and with single worm after the C. elegans loading process of three chips.

After on-chip worm loading, the WormChip cartridge was assembled according to Fig. 2. Before assembly, the liquid-storage bags were injected with CeMM or RNALater and then degassed to prevent release of the gas dissolved in liquid during the experiment. The assembly process consists of 4 steps: (1) connection of the flexible silicone tubing, the silicone tubing is connected to the liquid storage bags, peristaltic pumps, check valves and the bubble trapper following the fluid line design shown in Fig. 2(A). The CeMM-storage bag 1–5 is connected to the inlet of pump 1–5, respectively, and the RNALater bag is connected to the inlet of pump 6. Attach hose clamps in the specified position of the silicone tubing, and insert the specific silicone tube to the corresponding pinching head of the pinch valve, then start the valves and pumps to fill the fluid lines with CeMM; (2) assembly of the chip cassette according to Fig. 2(B), the FEP tubes inserted into the chip inlets/outlet after loading are fixed through the 5 mounting holes and connectors on the manifold, then the chip cassette is secured by the screws on the outer frame; (3) connection of the chip and the fluid lines, the FEP tubes fixed on the chip and the corresponding silicone tubing are connected through the connectors to form closed flow lines. Make the 2 ends of the fluid line overflow liquid before connecting them to prevent introduction of bubbles at the joint; (4) connector reinforcement and fluid line arrangement, the connectors in fluid lines are fastened using self-locking nylon cable ties to prevent liquid leakage; liquid storage bags should be placed in the box in order (the 4 worm-collection bags should be placed on the top for easy recovery after the experiment) and covered with a lid.

There are rich nutrients in CeMM, so it is easy to cause bacterial contamination. Moreover, the entire procedure of loading and assembly involves a variety of equipment and fluid line fittings, and the operation takes a relatively long time (>6 h), therefore sterile control during the operation is crucial to the success of the experiment. A four-step process is used to achieve sterile operation: (1) control of the experimental environment, the laboratory is treated with disinfectant and irradiated by using a UV lamp in advance, and all operations are carried out in the clean bench with flame sterilization using an alcohol lamp; (2) sterilization of tubing, connectors, accessories, etc., perform high-temperature autoclave treatment (121 °C for 20 min) and then dry; (3) cleaning of the peristaltic pump flexible tubes and the WormChip-4.8.1, rinse with 75% alcohol for at least 30 min, and then irradiate with the UV lamp for at least 30 min; (4) treatment of CeMM, antibiotics (100 U mL⁻¹ of penicillin and 100 μg mL⁻¹ of streptomycin) are added to the CeMM to prevent bacterial contamination during operation,²⁸ and a syringe filter (filter membrane with a pore size of 0.22 μm) is utilized to remove possible bacteria and impurities from the liquid before use. Ground testing demonstrated that the ratio of worms retained within the chambers after a 15-day experiment was more than 85% (Table S4†), and a properly assembled WormChip cartridge went well for more than 100 days without leakage and contamination.

Design and experimentation of the WormSpace μ-TAS

To realize automated multi-strain cultivation and multi-function detection of nematodes on orbit, the experimental device should contain an imaging module, and be fully compatible with the WormChip cartridge, so as to build an integrated WormSpace μ-TAS. The design of the WormSpace μ-TAS should consider the following factors: (i) compatibility of the interfaces (installation, optical detection, control circuit, etc.) between the experimental device and the WormChip cartridge; (ii) an imaging module that enables videotaping of the parallel chambers and bright field and fluorescence microscopic imaging of the worm-clamp arrays in the WormChip-4.8.1; (iii) automation of the on-orbit nematode experiment to reduce the crew time and dependence on astronauts; (iv) environmental conditions (temperature, atmospheric pressure, etc.) compatible with C. elegans experiments.

Fig. 4(A) and (B) show the structural design and composition of the integrated microfluidic system (WormSpace μ-TAS) for automated culturing and monitoring of C. elegans. The WormSpace μ-TAS consists of 3 parts: a 3D-movable imaging module, a microfluidic module (WormChip cartridge) and an electronic control box. The 3D-movable imaging module includes a bright-field detection unit and a fluorescence detection unit, sharing a camera mounted on a 3D transportation stage (Fig. 4(A) and (B)) for videotaping and photographing of C. elegans in the WormChip-4.8.1. The design of the optical system was based on a transmission-type bright-field lighting unit and a coaxial excitation lighting unit. In bright-field videotaping mode, the field of view is ∼1.3 × 1.3 mm², the resolution is 1024 × 1024 pixels and the frame rate is >15 FPS (frame per second), which is feasible for video recording of worms within a chamber. In fluorescence photographing mode, the field of view is ∼3.3 × 2.5 mm², the resolution is 2592 × 1944 pixels and the wavelengths for excitation and emission are 488 ± 5 nm and 507 ± 5 nm, respectively, which is compatible for fluorescence imaging of GFP and YFP strains of worms immobilized in the worm-clamp array. By using a 3D transportation stage, the camera could be positioned within the range of the WormChip-4.8.1 and focusing could be achieved. Given that a transmission-type bright-field lighting unit was designed for the optical system, the chip cassette was mounted on the chip holder in the vertical direction (Fig. 2(C) and 4(B)), and the WormChip cartridge was installed on the baseplate of the experimental device from the bottom (Fig. 4(C)). The electronic control box was pre-fixed in the back of the small universal biological culture module (SUBCM drawer, Fig. 4(D)), enabling remote control of the WormSpace μ-TAS and automatic collection and periodic transmission of experimental data obtained on the CSS (Fig. 4(A)). Upon insertion of the experimental device into the SUBCM drawer, the complete WormSpace μ-TAS was formed. Fig. S2† shows the actual images of the WormSpace μ-TAS.

Fig. 4 Design of the WormSpace μ-TAS for automated culturing and monitoring of multi-strain C. elegans on the CSS. Block diagram of structural design (A) and schematic of structural composition (B) of the WormSpace μ-TAS. Schematic diagram shows (C) the experimental device with a pre-fixed 3D-movable imaging module and installed microfluidic module on the baseplate from the bottom, and (D) the WormSpace μ-TAS in the SUBCM drawer, insertion of the experimental device into the small universal biological culture module pre-fixed with an electronic control box (the SUBCM drawer).

To image C. elegans worms inside the WormChip-4.8.1 using the WormSpace μ-TAS, Fig. 5(A) shows the schematic of the motion path of the 3D-movable camera. With regard to the 3D transportation stage, the distance that can be moved horizontally is 124 mm, allowing successive detection of parallel 32 chambers and worm-clamp arrays; the distance of vertical movement is 6 mm, making it possible for moving the camera from the culture chamber to the worm-clamp array; the movable distance for focusing of 2 mm enables high-resolution imaging with the microscopy camera. To validate the capability of the multi-function imaging protocols, we loaded a WormChip-4.8.1 as the procedures mentioned above, assembled the WormChip cartridge (Fig. 2(A)) and installed it on a ground-based experimental device (Fig. 4(C)), which was then inserted into the SUBCM drawer to form the WormSpace μ-TAS (Fig. 4(D)). By using a one-by-one imaging strategy, the 3D-movable imaging module can achieve videotaping of the 32 parallel chambers with swimming worms (Fig. 5(B)) and photographing of the worm-clamp array 3.1–3.8 with immobilized YFP strain AM141 in bright-field and fluorescence mode (Fig. 5(B)). Fig. S4† shows the representative images of the chip unit-3 with C. elegans strain AM141 before and after worm immobilization during ground-based testing, and the ratio of immobilized worms during the process of fluorescence imaging is more than 90% (Table S5†).

Fig. 5 Detection of the C. elegans worms inside the WormChip-4.8.1 by one-by-one imaging of the chambers and worm-clamp arrays using the WormSpace μ-TAS. (A) Schematic diagram of the motion path of the camera mounted on a 3D transportation stage. Ground-based validation testing of the multi-function imaging protocols: (B) sequentially videotaping of the 32 culture chambers with swimming worms from chamber-4.8 to chamber-1.1 (pictures extracted from the corresponding videos of each chamber with a scale bar of 200 μm); (C) bright-field and fluorescence photographing of worm-clamp array 3.1–3.8 with immobilized YFP-strain of C. elegans AM141 that were loaded into chip unit-3. Scale bar, 500 μm.

WormSpace μ-TAS has the characteristics of integration, modularization and compatibility (Fig. 4(C) and (D)), which not only ensures the convenience of astronauts' on-orbit operation, but also maximizes the adaptation to the needs of different C. elegans space experiments. The WormChip cartridge is an independently installable and replaceable module that is installed on the baseplate of the experimental device from the bottom (Fig. 4(C)). Upon installation, the WormChip-4.8.1 can be adapted to the optical system and the 3D transportation stage, and the electrical connector on the WormChip cartridge can be reliably connected to that on the experimental device. The imaging module is equipped with a bright-field and fluorescence detection unit and a 3D transportation stage, which can record videos of worms in parallel chambers and take pictures of animals immobilized in worm-clamp arrays of different chip units, completely meeting the requirements of in-orbit multi-function imaging of nematodes. During the in-orbit experiment, valve/pump-based fluid control and nematode imaging were operated automatically through uploading commands from the ground, without participation of astronauts. The SUBCM drawer where the WormSpace μ-TAS is located has good compatibility with the experimental facility (Ecology Science Experiment Rack, ESER) on the CSS, and can make full use of the existing life support conditions (liquid-cooling module, normal air pressure) of the ESER to provide suitable environmental conditions (20 ± 1 °C, one atmosphere) for in-orbit nematode experiments.

Procedures for C. elegans spaceflight experiment

The above mentioned ground testing demonstrated that the experimental procedures developed for the WormSpace μ-TAS make the technology amenable to automated C. elegans culturing and monitoring on the CSS. Fig. 6 illustrates the procedures for the C. elegans spaceflight experiment. C. elegans samples were prepared in the lab of Jiuquan Satellite Launch Center, Gansu, China. Four strains of C. elegans (N2, LS292, AM141, TJ356) were selected as the samples for flight because of their different biological effects under space environment (microgravity and radiation), shown in Table S6.† According to the synchronization-to-loading time of these worms (5–7 d), synchronization was arranged to obtain the 4 samples (Fig. 6(Ai)) applicable for loading 2 days before launch (T-2 d). Wild-type (N2) and mutant (LS292, AM141, TJ356) strains of C. elegans samples were loaded into inlet 1–4 of the WormChip-4.8.1 (Fig. 6(Aii)) using the loading protocol mentioned above. As a result, ∼84.4% of the 32 chambers were loaded with 1–4 worms, and more than 8 worms in total existed in the chambers of each unit of the chip, according with the design and experimental requirements. The loaded chip was then assembled into a closed WormChip cartridge (Fig. 6(Aiii)) with lithium fluoride thermoluminescent detector chips (TLDs) and CR-39 plastic nuclear track detectors (PNTDs) accompanied the worm-collection bags to provide radiation dosimetry.⁷ After fluid control testing, the WormChip cartridge was incubated at 12 °C to slow down nematode development before the experiment until flight turnover.

Fig. 6 Illustration of the procedures for the C. elegans experiment on the CSS. (A) Ground preparations: (i) preparation of samples of 4 strains of C. elegans, (ii) on-chip sample loading, (iii) assembly of the WormChip cartridge and (iv) installation of the protective cover. (B) Flight launch: installation of the WormChip cartridge in a passive thermal case (12 °C) and flying to the CSS. (C) In-orbit experiment: (i) installation of the WormChip cartridge on the experimental device; (ii) inserting the experimental device into the SUBCM drawer; (iii) installation of the SUBCM drawer on the experimental facility (ESER) in Lab module I of the CSS for the 30-day experiment through remote operation. (D) Sample storage: after experiment, detachment and storage of the chip cassette and the 4 worm-collection bags in the −20 °C and 4 °C containers, respectively. (E) Returning to earth: putting the chip cassette and the worm-collection bags in low temperature boxes and returning to earth for further investigations.

Before turnover, the WormChip cartridge was inspected for any possible contamination and a protective cover was then installed to protect the components (Fig. 6(Aiv)). After that, the WormChip cartridge was packed into a passive thermal case (12 °C, Fig. 6(B)), which consists of a phase-change material (phase-transition temperature at 10–12 °C) unit and a valve and pump control unit powered by a battery. The passive thermal case enables maintaining the WormChip cartridge at 12 ± 3 °C for 16 h and automatically renewing CeMM in the WormChip-4.8.1 at an 8-hour interval. The WormChip cartridge was fixed vertically in the passive thermal case with one side of the protective cover facing upward (Fig. 6(B)) to keep the WormChip-4.8.1 in a horizontal state during the launch. The assembled passive thermal case was flown to the CSS aboard the Shenzhou-15 manned spacecraft on Nov. 29th, 2022 (T 0 d).

The SUBCM drawer and the experimental device, except for the WormChip cartridge, were already onboard the CSS before the Shenzhou-15 spacecraft arrived. After receiving the passive thermal case at the CSS, the astronauts firstly took out the WormChip cartridge, removed the protective cover, then installed the WormChip cartridge on the baseplate of the experimental device from the bottom (Fig. 6(Ci)), mounted the experimental device in the SUBCM drawer (Fig. 6(Cii)), and finally installed the drawer on the experimental facility (ESER) (Fig. 6(Ciii)) in Lab module I of the CSS. The crew successfully installed the experimental device with guidance provided by the authors of this study. The on-orbit C. elegans experiment was performed for 30 days by remote uplink commands using the 48-hour periodic protocols shown in Fig. S5,† including renewal of culture medium at a 12 h interval, videotaping of the 32 chambers at a 24 h interval, and immobilization/photographing in bright-field and fluorescence/release of worms in chip unit 3–4 at a 48 h interval. The experiment went well, and sufficient movies (Fig. S6†) and pictures (Fig. S7†) were obtained and downlinked for analysis of C. elegans phenotypes. At the end of the experiment (T + 33 d), command codes were sent to execute the fixative injection protocol until observing that the animals in chambers were fixed. After that, the WormChip cartridge was disassembled from the experimental device and then transferred to the on-orbit Science glove box, where the chip cassette and the 4 worm-collection bags were detached to store in the −20 °C and 4 °C containers (Fig. 6(D)), respectively. Upon returning to earth (T + 6 months), the chip cassette and the worm-collection bags were in low temperature boxes (Fig. 6(E)) and were handed over to scientists for further studies, enabling us to correlate phenotype changes to alterations in gene expressions.

Longitudinal phenotype detection of C. elegans on the CSS

As can be seen from Fig. S5,† the in-orbit C. elegans monitoring protocols involved in video recording of the chambers with swimming worms and fluorescence imaging of worm-clamp arrays containing immobilized animals. From these videos (Fig. S6†), we can observe the status of the animals in real time and obtain the data of multiple phenotypes, such as body length, body width, body bending frequency, etc., along days to analyze the changes of worm growth and development, and movement on the CSS; from the fluorescence images (Fig. S7†), we can obtain the data of number and intensity of the fluorescent aggregates to monitor the changes of the fluorescent protein markers expressed in transgenic strains under space environment. To evaluate the efficacy of the on-chip multi-strain culturing protocol on the CSS and the feasibility of on-orbit multi-function imaging at the single-worm level, we measured the changes of body length, body bending frequency, and fluorescence of the C. elegans samples as a measure of the phenotypes of worm growth and development, movement, and fluorescent protein expression, respectively. These measurements were conducted during the 30-day experiment at a 48 h interval. At the early stage of the in-orbit experiment, it was found that the number of worms in chip unit-1 (N2), unit-2 (LS292) and unit-4 (TJ356) decreased significantly (n < 8), which may be due to the relatively small body width of N2, LS292 and TJ356 worms during the on-chip loading process, resulting in some of the animals being washed out of the chambers during the flight stage and the early stage of the experiment. This makes it difficult to perform data processing and statistical analysis, so the results were represented by a scatter plot.

Fig. 7 shows the curves of body length and body bending frequency of the 4 strains of C. elegans samples. As illustrated in Fig. 7(A), the average body length of the strain AM141 (iii, n ≥ 12) increased gradually from ∼780 μm on day 3 to ∼850 μm on day 11 after the experiment began, and then remained relatively stable, fluctuating within a small range. The curves of the body length of the strain N2 (i), LS292 (ii), and TJ356 (iv) are similar to that of the strain AM141 (iii), but more on-orbit data is required to perform statistical analysis. With regard to movement (Fig. 7(B)), we find that the average body bending frequency of the strain AM141 (iii) tended to decrease from more than 120 bends per minute (day 3) to about 20 bends per minute (day 11), and then fluctuated within a range along the day of on-orbit culture. These results are similar to those of preliminary experiments on the ground. As for the strain N2 (i), LS292 (ii) and TJ356 (iv), the tendencies of the body bending frequency were not obvious because of the small amount of data.

Fig. 7 Characterization of on-chip culture growth and development, and movement across the 30-day multi-strain experiment on the CSS. Changes of body length (A) and body bending frequency (B) obtained from the videos of the 4 strains of C. elegans grown in CeMM within the 32 chambers of the WormChip-4.8.1. (i) N2 (n = 1–2), (ii) LS292 (n ≥ 5), (iii) AM141 (n ≥ 12) and (iv) TJ356 (n = 2–4). Error bars represent standard deviation.

Fig. 8 demonstrates the changes of number and intensity of the fluorescent aggregates expressed in the transgenic strain AM141 and TJ356 during the in-orbit experiment. The fluorescence images were obtained by photographing of the 8 worm-clamp arrays of chip unit-3 and unit-4 with immobilized AM141 and TJ356 worms, respectively. After hatching, AM141 exhibits Q40::YFP in muscle cells of the body wall and the fluorescence becomes more and more obvious with the increase of age. In adults, Q40::YFP exhibits a complete aggregation phenotype.^27,33,34 Muscular dystrophy of astronauts is one of the significant impediments of space exploration. Research indicates that expression of muscle proteins and the number of Q40::YFP aggregates in AM141 decreased under microgravity.³⁵ The sensitivity of AM141 to microgravity is chosen as the phenotype of muscle-specific expression of polyQ::YFP fusion proteins, which can be easily observed in its body wall to quantify the expression of relevant genes.¹² As shown in Fig. 8(A), the number of Q40::YFP aggregates in AM141 varied between ∼30 and ∼65, and the mean values increased from less than 45 (day 3) to more than 50 (day 17), then fluctuated around 50. The average fluorescence intensity of AM141 (Fig. 8(B)) increased gradually from ∼80 (day 3) to more than 90 (day 17), and then decreased slowly. The strain TJ356 expresses fluorescent protein DAF-16::GFP. The daf-16 (ref. 36 and 37) is a stress receptor gene, which can be used as a marker gene of radiation biodosimeter to characterize the radiation damage. Fig. 8(C) shows that the tendency of the average fluorescence intensity of TJ356 is similar to that of the strain AM141 (iii), but the change is not statistically significant because of the low n number. As expected, the fluorescence intensity of AM141 (∼60–100) was significantly higher than that of TJ356 (∼31–42).

Fig. 8 Characterization of on-orbit single-worm level imaging of C. elegans with fluorescent protein markers across the 30-day multi-strain experiment on the CSS. Changes of the number (A) and intensity (B) of the Q40::YFP aggregates expressed in AM141 (n ≥ 12), and (C) intensity of the DAF-16::GFP aggregates expressed in TJ356 (n = 2–4). The data came from the fluorescence images of AM141 and TJ356 worms immobilized in the 8 worm-clamp arrays of chip unit-3 and unit-4, respectively. Error bars represent standard deviation.

As mentioned above, we have achieved microfluidic chip-based 4-strain C. elegans cultivation for 30 days on the CSS and successfully obtained the videos and bright-field and fluorescence images of C. elegans samples along days. With these on-orbit data, multiple phenotypes can be monitored and the changes can be used to compare with the results of ground control tests in the future to investigate the biological effects of the space environment on C. elegans. Fig. S8† shows comparison of the results of growth and fluorescence obtained on the CSS to those obtained on earth during ground-based testing, indicating differences between them. No drying, leakage or glass cracks was observed in the WormChip-4.8.1 and the fluid lines over the 30-day experiment. Although a bubble trapper was connected in the fluid lines, we saw an obvious increase in air bubbles in the chambers and channels of the chip at the CSS, which could be due to permeability of liquid-storage bags and silicone tubing, release of gas dissolved in liquids under microgravity condition, or maybe a combination of both that need to be ascertained. These bubbles can be effectively removed by performing the fluid-control mode of worm immobilization (Fig. 3(B)) at lower flow rates.

Conclusions

To fulfill the needs of automated multi-strain C. elegans culturing and monitoring on the CSS, we have developed an arrayed nematode microfluidic chip (WormChip-4.8.1), a closed microfluidic module (WormChip cartridge) and an integrated microfluidic system (WormSpace μ-TAS). By programmed control, automated experimentation can be performed for periodic renewal of culture medium, video recording of moving worms, bright-field and fluorescence imaging of fluorescent strains of worms when immobilized and then released, and fixation of animals at the end of the experiment. Furthermore, we have validated the capability to implement the on-chip 4-strain culture on the CSS, on-orbit 48 h-interval videotaping and photographing at the single-worm level for 30 days, on-line automated fixation, and analysis of the videos and fluorescence pictures. Thus, the WormSpace μ-TAS for multi-strain C. elegans culturing and multi-function imaging has been successfully launched and operated onboard at the CSS while maintaining sterile culture over the 30-day experiment. Videos and pictures were collected and can be used to analyse changes of multiple phenotypes of C. elegans samples. Meanwhile, fixed culture samples have been returned to earth for further gene expression analysis and the results will be reported in the future. The presented WormSpace μ-TAS enables automated tracking and monitoring of model animals at the individual level, providing a new experimental platform for studying the biological effects of space radiation and microgravity.

Experimental

Fabrication of PDMS-glass microfluidic chip

We fabricated the WormChip-4.8.1 using a 2-step soft lithographic technique.^38,39 The mold was fabricated on a 6′′ silicon wafer as a substrate. First, the silicon wafer was deeply etched with the design shown in Fig. 1(A) with features that were 35 μm in height using BOSCH processing. On top of this layer, a second layer of 165-μm tall SU-82075 negative photoresist (Microchem Corp., Newton, MA) was fabricated with an array of oval cylinders that form the culture chambers. This two-layer approach provides a total chamber depth of approximately 200 μm and creates microchannels of height 35 μm.

We then treated the mold with chlorotrimethylsilane (ALDRICH, Inc., Sigma-Aldrich) to prevent the adhesion of poly (dimethyl siloxane) (PDMS, Dow Corning Sylgard 184, Corning, NY) to the mold during the replica molding process. A PDMS layer of thickness 4.0 ± 0.2 mm was cast using Sylgard 184 part A (base) and part B (curing agent) 10 : 1 by weight over the silicon mold by curing for ∼3 h at 65 ± 1 °C. Four inlets and one outlet were cored with a ∼1 mm diameter hole puncher. The PDMS layer was thoroughly cleaned by sonication with absolute ethanol before bonding. After drying, the PDMS replica was treated in an air-plasma cleaner (Harrick Plasma, Ithaca, NY) for 120 s along with a glass slide (50 mm × 130 mm, 2 mm thick, Corning) and the two layers were then bonded together. The bonded PDMS-glass chip was immediately kept in an oven for about 48 h at 65 ± 1 °C. The WormChip-4.8.1 was treated with 10 wt% Pluronic F127 (Sigma-Aldrich) for 10 min to help reduce bubble formation during worm loading. After incubation, the injected Pluronic was removed by washing the chip with DI water. The Pluronic-treated WormChip-4.8.1 was kept in an oven for about 30 min at 55 ± 1 °C and then remained in a vacuum desiccator until the day of the experiment.

Preparing liquid axenic media (CeMM)

Sterile liquid culture medium, CeMM (C. elegans maintenance medium), was prepared according to the method described in the literature,²⁷ which mainly included the water soluble part, the TEA soluble part, amino acids, nucleic acid substituents (thymine, etc.), other growth factors and energy (D-glucose). The prepared liquid medium was adjusted to a suitable final volume with distilled water, mixed thoroughly, and filtered using a cellulose acetate filter with a pore size of 0.22 μm.

Cultivation and synchronization of C. elegans worms

Wild-type (N2) and mutant (LS292 [dys-1(cx18)], AM141[rmIs133(unc-54p::Q40::YFP)], TJ356 zIs356 [daf-16p::daf-16a/b::GFP; rol-6(su1006)]) strains of C. elegans were obtained from the Caenorhabditis Genetics Center (CGC) at the University of Minnesota (Minneapolis, MN).^40,41 Worms were maintained in CeMM at 20 ± 1 °C for 14–21 days before synchronization.

We obtained a synchronous population of worms in the L4 stage of development using the following protocol.^{32,34,41–43} We transferred approximately 3.5 mL of CeMM-grown gravid worms in M9 buffer (combining 3 g KH₂PO₄, 6 g Na₂HPO₄, 5 g NaCl and 1 mL 1 M MgSO₄, and adding H₂O to 1 L) to a 15 mL centrifuge tube. We then added 1.5 mL of alkaline lysis buffer (0.5 mL 5 M NaOH and 1 mL NaOCl with available chlorine content ≥5%) into the tube for mixing and incubation until most of the worms were dissolved (about 6–12 min). After that, we centrifuged the tube at 3500 rpm for 1 min, removed the supernatant and added immediately 10 mL M9 buffer to terminate the reaction. The centrifugation/M9-buffer cleaning cycles were repeated 3 times to neutralize the solution to pH 7. We then removed the supernatant from the tube, leaving behind a population of eggs at approximately the same developmental stage for growth in CeMM. We defined the day of synchronization to be day 0. On day 5–7, the synchronized worms had reached the middle of the L4 larval stage, and were ready for loading into the WormChip-4.8.1.

Analyzing body length, bending frequency and fluorescence in C. elegans

The 4 strains of C. elegans worms were loaded into the 4 units of WormChip-4.8.1 using the procedure described in the main text. The animals were allowed to swim freely in the culture chambers for 45 s video recording and to be periodically immobilized in the worm-clamp arrays for bright-field and fluorescence photographing every other day with the 3D-movable imaging module. All videos and images were acquired at a temperature of 20 ± 1 °C.

The nematode body length was measured by the line drawn from the head to tail along the middle of the animal body by using the software ImageView and the screenshots obtained from the on-orbit videos. The value of the length was corrected according to the scale (100 μm long) below the number of the culture chamber. We defined one body bend as bending of the body of the worm in one direction. In order to measure the changes in the movement of nematodes in orbit, 10s were selected from the 45 s-videos of each culture chamber to count the number of bending of the body, and this value was multiplied by 6, which was the body bends of nematodes per minute. The number of fluorescent aggregates (AM141) and the average fluorescence intensity of fluorescent strains (AM141 and TJ356) were measured by using the ImageJ software and the fluorescence images obtained on orbit (Fig. S9†). The fluorescent region of each nematode worm in the images was selected and the average fluorescence density was calculated by the software.

Data availability

Data from the China Space Station.

Author contributions

Qianqian Yang contributed in investigation, methodology, writing – review & editing. Runtao Zhong is supervisor and contributed in writing – original draft and validation. Wenbo Chang contributed in formal analysis. Kexin Chen contributed in data curation. Mengyu Wang provided methodology of research. Shuqi Yuan, Zheng Liang and Wei Wang contributed in resources of preparing four strains of C. elegans samples. Chao Wang developed software and hardware of the passive thermal case. Guanghui Tong and Tao Zhang developed software and hardware of the SUBCM drawer and the WormChip cartridge. Yeqing Sun is supervisor and project administration.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

The authors thank Mr. Qi Liu and Mr. Shaohua Wang from Institute of Environmental System Biology, Dalian Maritime University, for their help in preliminary design, fabrication and test of the nematode microfluidic chip. We thank China Manned Space Engineering for providing space science and application data products of the China Space Station. This research was supported by space science experiment projects on the China Space Station (2020-228), scientific research project in key fields of China manned space application system (2018-28) and the Fundamental Research Funds for the Central Universities (no. 3132022162).

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Footnotes

† Electronic supplementary information (ESI) available. See DOI: https://doi.org/10.1039/d4lc00210e

‡ Co-first author: Qianqian Yang, Runtao Zhong.

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