Microfluidics-integrated spaceflight hardware for measuring muscle strength of Caenorhabditis elegans on the International Space Station

Abstract

Caenorhabditis elegans is a low-cost genetic model that has been flown to the International Space Station to investigate the influence of microgravity on changes in the expression of genes involved in muscle maintenance. These studies showed that genes that encode muscle attachment complexes have decreased expression under microgravity. However, it remains to be answered whether the decreased expression leads to concomitant changes in animal muscle strength, specifically across multiple generations. We recently reported the NemaFlex microfluidic device for the measurement of muscle strength of C. elegans (Rahman et al., Lab Chip, 2018). In this study, we redesign our original NemaFlex device and integrate it with flow control hardware for spaceflight investigations considering mixed animal culture, constraints on astronaut time, crew safety, and on-orbit operations. The technical advances we have made include (i) a microfluidic device design that allows animals of a given size to be sorted from unsynchronized cultures and housed in individual chambers, (ii) a fluid handling protocol for injecting the suspension of animals into the microfluidic device that prevents channel clogging, introduction of bubbles, and crowding of animals in the chambers, and (iii) a custom-built worm-loading apparatus interfaced with the microfluidic device that allows easy manipulation of the worm suspension and prevents fluid leakage into the surrounding environment. Collectively, these technical advances enabled the development of new microfluidics-integrated hardware for spaceflight studies in C. elegans. Finally, we report Earth-based validation studies to test this new hardware, which has led to it being flown to the International Space Station.

Fig. 1. C. elegans culture in CeMM and the multigenerational culture protocol.

a An FEP bag containing wildtype C. elegans culture in CeMM. Image used in this figure was captured at TTU. b A schematic protocol for culturing C. elegans for multigenerational studies over an 8-week period. Here, d0 represents day 0 (start of the culture), and w2, w4, w6, and w8 represent week 2, week 4, week 6, and week 8, respectively.

Fig. 2. Characterization of culture growth and health across the eight-week multi-generational experiment.

a Characterization of the culture density including adults, larvae, eggs, and dead animals. b Swim-induced thrashing frequency of gravid adults as a measure of their locomotory health (Supplementary Table 1). Error bars represent standard deviation. All the data pass the normality test. There is no significant difference between thrashing frequency as calculated by one-way ANOVA, P ≥ 0.7.

Fig. 3. Design of the NemaFlex-S device for strength measurement of C. elegans.

a An actual image of the NemaFlex-S device with two identical sections NF-A and NF-B, the device is filled with red food dye for better visualization of the salient features. b Image of a gravid adult crawling in the pilar chamber. The zoomed inset image shows the eggs inside the worm. Scale bar, 100 µm. c Design of the individual pillar chamber. The chamber is connected to the flow channel (highlighted with blue arrows) with a tapered neck for trapping the worms and with sieve channels for removing the progenies. d Scanning electron microscopy (SEM) image of deformable micropillars. Scale bar, 50 µm. e SEM image of sieve channels. Scale bar, 100 µm. f Image of an adult worm trapped in the neck Scale bar, 500 µm. See Supplementary Video for trapping and loading of an adult into the chamber. Images used in this figure were captured at TTU.

Fig. 4. Microfluidics-integrated worm loading apparatus.

a Schematic diagram of the worm loading apparatus (b) and (c) shows the schematic top and bottom view of the WLA board and its key component, respectively. d A leak proof imaging cassette for loading the worms into NemaFlex-S Cassette. The highlighted parts are: (1) inlet lines connected to the distribution valve (2) clips holding the inlet and outlet cannulas (3) L-shaped cannulas connected to the inlet of NemaFlex-S chip (4) cannula connected to air-vent purge port (5) outlet cannula opening into the waste collection chamber (6) porous membrane for releasing the air. e Image of the actual worm loading apparatus showing the three major hardware components – Imaging cassette, distribution valve, and the syringe pump. The worm culture bag and 10 mL waste collection syringe are connected to the distribution valve; the worm suspension loop carries either the CeMM from the media syringe or worm solution from the culture bag. The distribution valve has a feeding port (shown in fig. c) underneath the mounting board. Image used in this figure was captured at TTU.

Fig. 5. Schematic diagram of protocol of loading worms into NemaFlex-S device using the Worm Loading Apparatus.

a Shows the schematic of the worm loading apparatus. (b) Step I: priming of the valve with CeMM, flow will be from CeMM media syringe to waste collection syringe. (c) Step II: aspirating a specified worm aliquot from worm culture bag to worm suspension loop. d Step III: priming of the valve with worm suspension, flow is from suspension loop to waste collection syringe. (e) Step IV: taking a specified worm aliquot (400 µL) from worm bag to suspension loop. (f) Step V: load the worms into A side of the NemaFlex-S device. Repeat step IV and step V to load the worms into the B side of the NemaFlex-S device.

Fig. 6. Optimization of parameters used for efficient loading of worms into NemaFlex-S device.

a Effect of flow rate used for worm trapping in the tapered neck (Supplementary Table 3). b Effect of worm suspension volume (Supplementary Table 4). The density of animals in the culture bag was 1060 ± 160 adults/mL. Error bars represent standard deviation.

Fig. 7. Measurement of muscle strength for wild-type C. elegans during multigenerational experiment.

a Worm diameter over multiple generations. b Muscle strength over multiple generations. n = 32 for week 2, n = 36 for week 4, n = 38 for week 6, and n = 41 for week 8 (Supplementary Table 5). Error bars represent standard deviation. All the data pass the normality test. There is no significant difference between force values using one-way ANOVA with P = 0.8.