An automated microfluidic platform for toxicity testing based on Caenorhabditis elegans
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Abstract
Humans are frequently exposed to a multitude of chemicals daily, necessitating efficient methods for rapidly assessing toxicity and potential health risks. Microfluidics has shown promise as an intelligent tool for rapid compound testing, owing to its flexibility in integrating with automated devices. The article introduces an automated microfluidic platform, based on Caenorhabditis elegans (C. elegans) , designed for chemical toxicity testing. This platform consists of three modules – worm culture, monitoring, and image analysis – which enable automated worm culturing, drug delivery, periodic monitoring, and automated phenotypic analysis. Researchers have designed a bridged microfluidic chip that permits worms to move freely during experiments and established an economical monitoring module for long-term tracking and periodic imaging. Furthermore, they have developed an automated image analysis algorithm to automatically determine worm bending frequency. The platform was subsequently utilized for long-term toxicological assessments of the organophosphate pesticide and environmental pollutants. Results indicated that the platform can effectively evaluate the general and developmental impacts of chemicals. The automated microfluidic worm analysis platform holds significant potential for applications in drug safety assessmentand drug screening research, contributing to human health and industry advancement.
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Firstly, synchronized worm suspension in the reservoir was loaded into microfluidic chip. During the experiment, culturing medium can be replenished and consistently loaded into the chip at a low flow rate, to provide sufficient nutrients for worms.
How labor intensive is this loading process? Are you able to just open the channel briefly and load a set number of animals into the chip or do you have to carefully monitor the flow of the liquid until you see the correct amount of worms and then turn it off? I'm wondering about this as in regards to making this more high-throughput. If the loading can be standardized, do you think it would be possible to keep the worms in a 96 cell plate, with an automatic sampler and bring worms into the chamber one well at a time for imaging, and then either dispose of them or return them to their well …
Firstly, synchronized worm suspension in the reservoir was loaded into microfluidic chip. During the experiment, culturing medium can be replenished and consistently loaded into the chip at a low flow rate, to provide sufficient nutrients for worms.
How labor intensive is this loading process? Are you able to just open the channel briefly and load a set number of animals into the chip or do you have to carefully monitor the flow of the liquid until you see the correct amount of worms and then turn it off? I'm wondering about this as in regards to making this more high-throughput. If the loading can be standardized, do you think it would be possible to keep the worms in a 96 cell plate, with an automatic sampler and bring worms into the chamber one well at a time for imaging, and then either dispose of them or return them to their well for later imaging? e.g. if you were going through a lot of mutants or drugs or doing a mutagenesis screen.
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The worm cultivation module includes a microfluidic chip for worm culturing and observation, equipped with pressure-based delivery system. Based on this module, diets and test substances can be automatically added at regular intervals, and worms can be cultured in microfluidic chip until death. During chronic toxicity testing assays, the phenotypes of worms in bright filed can be monitored and collected regularly with the monitoring module, which includes a motion stage and a Pi camera. After capturing images or videos of worms, the body length and bend frequency can be calculated automatically with the image analysis module. Fig.1B shows the physical photograph of the entire platform, which is controlled through the control center.
I think this is a very powerful device with a lot of applications. This is a small issue, but the …
The worm cultivation module includes a microfluidic chip for worm culturing and observation, equipped with pressure-based delivery system. Based on this module, diets and test substances can be automatically added at regular intervals, and worms can be cultured in microfluidic chip until death. During chronic toxicity testing assays, the phenotypes of worms in bright filed can be monitored and collected regularly with the monitoring module, which includes a motion stage and a Pi camera. After capturing images or videos of worms, the body length and bend frequency can be calculated automatically with the image analysis module. Fig.1B shows the physical photograph of the entire platform, which is controlled through the control center.
I think this is a very powerful device with a lot of applications. This is a small issue, but the diagram is a bit confusing the first time you look at it. You show the chip between the waste and reagent, and then an image of the chip on the right side of the red monitoring module box. Is that second box what the camera is seeing from the chip and sending to the control center? It's a bit confusing as it looks like there are two different chips in the system.
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In addition, fluorescence images also showed that the body length of nematodes exposed to L511A in the high-dose group was shorter than that in the control group, suggesting that L511A may have growth and developmental toxicity.
It would help to show this data in figure 6 to give more detail on how much body length is impacted.
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We treated worms with two commercially available flavor mixtures (codenamed L511A and X6145A), two cigarette brands of smoke Cambridge filter collections (codenamed Cig-1 and Cig-7), and a single tobacco component (solanone, codenamed Fla-1) to investigate their effects on bend frequency of worms. The experimental design covered four dose groups of 1/100000, 1/10000, 1/1000 and 1/200 of the stock solution, and 0.5% DMSO was set up as the solvent control.
It would be helpful to discuss how these stock concentrations relate to the concentrations humans are exposed to in their environment. Also, a great addition to the paper would be to do longer term studies of these chemicals' impact on worms. This device is a really powerful way to measure impacts on worm health and behavior over long periods and the introduction to the paper is …
We treated worms with two commercially available flavor mixtures (codenamed L511A and X6145A), two cigarette brands of smoke Cambridge filter collections (codenamed Cig-1 and Cig-7), and a single tobacco component (solanone, codenamed Fla-1) to investigate their effects on bend frequency of worms. The experimental design covered four dose groups of 1/100000, 1/10000, 1/1000 and 1/200 of the stock solution, and 0.5% DMSO was set up as the solvent control.
It would be helpful to discuss how these stock concentrations relate to the concentrations humans are exposed to in their environment. Also, a great addition to the paper would be to do longer term studies of these chemicals' impact on worms. This device is a really powerful way to measure impacts on worm health and behavior over long periods and the introduction to the paper is framed from the perspective of human cumulative exposure to toxins. I'd love to see longer time course assays on how these drugs affect the worms, especially at environmentally relevant concentrations.
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The average lifespan (± SD) of the worms in multi-well plates and microfluidic chips were 12 (± 4) days and 11 (± 3) days, respectively, which revealed no significant difference between the survival curves of two groups (P > 0.05) (Fig. 2D).
These lifespans seem shorter than the average in the literature. Do you think the constrained space in the PDMS chip and on the 96-well plate, is reducing their longevity? Also did you check development rates on the chips and do they go through the larval stages at normal rates?
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Supplementary InformationMicrofluidic device fabricationDetermination of the gap height of the microfluidic chips
The supplemental information is not available
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Through the analysis of these data, the neural effects of chemicals or drugs296can be evaluated and the scientific basis for the safety assessment can be provided.
Really fascinating method for culturing, screening and imaging all on one platform. Using pillars to 'cage' the c. elegens while allowing liquid to pass through is a great idea. I'd be curious if you have thought of any ways this design could be adapted to sort c. elegens into collection chambers based on their phenotype?
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the342total body relative fluorescence intensit
The phrase "total body" here implies the intensity is a sum, which would mean it depends on worm length. I think it'd be important to clarify this.
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. In addition, fluorescence images showed that the324body length of nematodes after exposure to L511A was significantly shorter than325that of the control group,
Does "significantly shorter" mean that the difference was statistically significant? If not statistically significant, I'm not sure it's worth mentioning. If it is, could this data be added to Figure 5?
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the overall fluorescence intensity of all GFP-labeled neurons
Is this the average intensity? (so that the intensity is independent of worm length)
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Combining the above results, we can draw the following conclusions:
If the differences were not statistically significant, I don't think these conclusions can be drawn.
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This suggests that these two flavoring substances and298tobacco component may produce neuroexcitatory effects at low doses in nematodes
Is an increase in bend frequency known to indicate neuroexcitation? It seems like it could also result from metabolic changes, perturbation of feeding behavior, or direct effects on muscle cells.
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For X6145A and Fla-1, the bend296frequency of worms also showed a trend of slightly increasing in low-dose group and297decreasing in high-dose group
If the trend wasn't statistically significant, I'm not sure this is worth commenting on.
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its bend frequency per minute was basically consistent with that of the291control grou
Is it possible to make a more more precise statement than "basically consistent"? This is a little confusing, especially since there was a statistically significant difference between the control and the higher dose.
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ig. 3A showed that treatment of MCP266and BDE-47 induced significant general toxicity in worms on this platform
How was worm death measured?
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ig. 3C&F showed270that the bend frequency of worms decreased significantly (p < 0.001) with the271increase in dose after 24 h treatment
For the 1mM MCP condition, it looks like most worms were still alive after 24 hours, but the bend frequency of all worms was zero. Does this make sense?
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Fig. 3. Toxicity evaluation of MCP and BDE-47 on the platform.
It looks like this figure is missing the number of worms analyzed for each condition. Also, the survival curves in Panel A are in discrete steps of the same size, suggesting a smaller number of worms than there are dots in Figures B and C.
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