A simple single nematode culture model and its sleep monitoring method

Abstract

The application belongs to the technical field of nematode culture and sleep detection, and particularly relates to a simple single nematode culture model and a sleep monitoring method thereof. The model fixes a single nematode in a culture chamber for culture, and the culture chamber is divided into two layers, the upper layer is a micro cavity for accommodating the nematode, and the lower layer is a culture medium (dry base). A nematode dry bacteria solution is arranged in the culture medium for providing growth of the nematode. The application discards the problem that the sleep behavior of the nematode cannot be monitored due to the use of liquid or droplet delivery in microfluidics. Meanwhile, the problem that the nematode cannot be fixed and a single nematode cannot be analyzed in the sleep monitoring model of Caenorhabditis elegans is improved, the throughput and accuracy are improved, the sleep behavior of the nematode can be directly analyzed by using wormlab, naked eye observation is not needed, the labor and material costs are reduced, and the scientificity and effectiveness of the experiment are improved.

Description

Technical Field

[0001] This invention belongs to the field of nematode culture and sleep detection technology, specifically relating to a simple single nematode culture model and its sleep monitoring method. Background Technology

[0002] *C. elegans*, a model organism, is characterized by its small size, short growth cycle, and ease of observation. It shares 60%-80% gene homology with humans, exhibits high conservation in sleep-related genes, and key gene mutants are readily available. *C. elegans* shares a common evolutionary history of sleep with humans and exhibits several common characteristics with mammalian sleep, including inactivity (i.e., reduced movement and cessation of pharyngeal pumping or feeding), specific postures, reduced response to aversive stimuli, and rapid reversibility. Therefore, *C. elegans* is an efficient and accurate experimental model for studying sleep mechanisms and has the potential to serve as a sleep monitoring model.

[0003] This model has been used in current research for the evaluation and screening of active ingredients. For example, in 2006, a large-scale drug screening study using *Caenorhabditis elegans* was first reported (Kwok et al., 2006). Kwok et al. used the fully automated Complex Object Parametric Analyzer and Sorter (COPAS). TM BIOSORT (Union Biometrica) and semi-automated image acquisition were used to screen 14,100 small molecules. Lehner et al. developed a fully liquid workflow based on 96-well plates (Lehner et al., 2006), which eliminated agar in the workflow and used automated liquid processors and integrated automated imaging platforms, greatly reducing the manual labor required for experiments. Moy et al. used a *C. elegans* *Enterococcus faecalis* infection model to conduct the first liquid-culture-based screening of antimicrobial compounds, screening 6,000 synthetic compounds and 1,136 natural product extracts, identifying 16 compounds and 9 extracts that extended the lifespan of nematodes (Moy et al., 2006). Although the above high-throughput drug screening workflows have incorporated some automation improvements, they still require significant manual labor for observing, recording, and evaluating characterization indicators.

[0004] In our preliminary research, we established a method for monitoring the sleep of *C. elegans*. First, the nematodes were cultured. After a 58-hour growth period, nematodes preparing to enter sleep were selected and placed on a 15cm diameter plate, with 15 nematodes selected from each plate. Video recordings were performed using the Wormlab nematode behavior monitoring platform. The videos were then visually observed and evaluated to track the nematodes entering sleep. The time from entering to exiting sleep was manually confirmed, and the total sleep duration of the target nematodes was calculated. The sleep-inducing efficacy of the active ingredient was evaluated based on the difference in total sleep duration. While this method allows for sleep monitoring of *C. elegans*, some problems exist, such as (e.g.) Figure 5 As shown in the figure: nematodes are placed on an open, large culture medium surface, which can easily cause entanglement during video recording, leading to misidentification during data analysis; nematodes move freely on the open plate and often crawl out of the recording range, resulting in data loss; effective data recovery is low, generally 15 nematodes are selected, with an average of only 3 data points recovered, resulting in low throughput of the entire model.

[0005] Two improvements are needed to address these issues;

[0006] (1) It is necessary to fix individual Caenorhabditis elegans within a certain range so that it can move freely without escaping or becoming entangled.

[0007] (2) Optimize sleep monitoring methods using a device that fixes Caenorhabditis elegans.

[0008] Common models for screening nematode bioactivity include lifespan models, high uric acid models, high lipid models, and alcohol intoxication models. Sleep monitoring models, as a relatively new method, require further refinement and improvement. Considering the extremely small size of adult nematodes, only on the micrometer scale, microfluidic technology offers valuable insights. Microfluidic chips are typically made of polydimethylsiloxane (PDMS), which has excellent permeability and can provide oxygen to the nematodes. Researchers have designed CD-shaped microfluidic chips that can culture nematodes on-chip for several days. Food enters the culture chamber via centrifugal force, while waste is ejected, thus facilitating material exchange. Although early microfluidic chips could culture nematodes for three generations without affecting their growth and motility, their simple structure made it impossible to distinguish between different generations of nematodes and to track and image individual nematodes.

[0009] Meanwhile, current technology struggles to precisely deliver individual nematodes to each culture chamber without disrupting system operation. This is because nematodes are small, and in microfluidics, where liquid or droplet delivery is typically used, they often become entangled and cannot be separated. In the previously established sleep monitoring model, the selected nematodes were those with a growth time of 54 hours prior and a translucent crescent-shaped mark on their vulva. The monitored nematodes have certain physiological requirements, making it more difficult to utilize simple microfluidic technology, thus increasing both the complexity and cost.

[0010] Furthermore, after sorting specific nematodes, these nematodes will be videotaped using the Wormlab nematode behavior imaging device to record their behavior. Simple microfluidic technology requires constant liquid flow within the culture chamber, making video recording difficult. Moreover, microfluidic culture chambers cannot monitor nematode sleep behavior. There are two reasons for this: first, the nematodes are not fixed in a specific location, causing them to become entangled or escape the camera's field of view, making it impossible for automated instruments (such as Wormlab) to analyze nematode speed data, resulting in less effective data; second, the released nematodes move too far, making it impossible to determine sleep behavior using Wormlab, requiring human visual identification of sleeping nematodes; third, the microfluidic culture chamber is filled with liquid, forcing nematodes to swim rather than crawl, making it impossible for the machine to recognize sleep behavior characteristics, and current microfluidic technology cannot achieve precise single-nematode sampling.

[0011] Finally, the current sleep monitoring model for *C. elegans* involves releasing worms onto large plates, visually observing them to identify those entering sleep, and then using WormLab to determine sleep characteristics and analyze the speed to obtain relevant data. However, the current model is inefficient, has insufficient throughput, and yields limited effective data, requiring significant human and material resources. Summary of the Invention

[0012] To overcome the aforementioned problems in the prior art, the primary objective of this invention is to provide a simple single nematode culture model.

[0013] Another object of the present invention is to provide a sleep monitoring method based on the above model.

[0014] This application designs a reusable, simple single-needle micro-culture chamber to fix nematodes in small holes for monitoring their sleep physiological activities.

[0015] The present invention achieves the above objectives through the following technical solutions:

[0016] A method for monitoring nematode sleep based on a simple single nematode culture model, wherein a single nematode is fixed in a culture chamber for cultivation. The culture chamber is divided into upper and lower layers, with the upper layer being a microcavity for containing the nematode and the lower layer being a culture medium. The culture medium contains a nematode-derived bacterial solution to provide growth for the nematode.

[0017] Preferably, the culture chamber is square and has multiple small holes.

[0018] Preferably, each small hole has a diameter of 3-5 mm, a spacing of 2-2.5 mm, and a thickness of 200 μm.

[0019] This application uses microfluidics technology to design a single nematode microculture chamber. Compared with the existing Caenorhabditis elegans sleep monitoring model, the single nematode microculture chamber preferably has 25 small holes, and the shape is a 4cm×4cm square grid plate. Each small hole has a diameter of 5mm, a spacing of 2.5mm, and a thickness of 200μm. This invention changes the traditional method of culturing nematodes on large plates to culturing them in small 5mm culture chambers. The upper layer is a PDMS perforated plate, and the lower layer is nematode agar medium. The combination of these two forms a simple and reusable single nematode micro-culture system. The single nematode micro-culture chamber of this invention can simultaneously culture and observe 25 nematodes individually for a long period of time. The Wormlab nematode behavior video recorder performs sleep analysis on each nematode. Compared with the previous method of culturing nematodes on plates, the advantages of fixing nematodes on agar are obvious. Each small culture chamber contains one nematode, which solves the problem of data loss caused by nematodes entanglement. At the same time, the nematodes cannot escape the range of the Wormlab camera, which greatly improves the efficiency of nematode sleep monitoring experiments, reduces experimental errors caused by subjective human judgment, improves the scientific nature of the experiment, reduces labor costs, improves the effectiveness of the Caenorhabditis elegans sleep monitoring model, and reduces the difficulty of operation.

[0020] Preferably, the method for preparing a single nematode microculture chamber includes the following steps:

[0021] Step 1: Set the incubation chamber parameters and prepare the corresponding mask;

[0022] Step 2: Apply photoresist to the mask in Step 1, take a 10cm*10cm single crystal silicon wafer, absorb the photoresist, and spread it evenly according to the height until the photoresist is evenly spread all over the single crystal silicon wafer.

[0023] Step 3: Use ultraviolet light to etch and obtain the culture chamber model;

[0024] Step 4: Mix the polydimethylsiloxane adhesive with the curing agent, stir until uniform, spread evenly on the photolithographic single crystal silicon mold, place it in a vacuum chamber to degas until the air bubbles completely disappear, and then dry.

[0025] Step 5: After demolding, punch holes to obtain the culture chamber;

[0026] Step 6: Combine the dried polydimethylsiloxane from Step 5 with agar medium containing dried bacterial solution to obtain a micro-culture chamber.

[0027] This invention, based on the single-nematode microculture chamber, further constructs a sleep monitoring model for *C. elegans*, defining the entire nematode culture process, identifying characteristic indicators of nematodes before sleep onset, and establishing overall indicators for the *C. elegans* sleep monitoring model. This model is determined by combining nematode sleep behavior indicators and physiological indicators during sleep. The sleep behavior indicators of nematodes are monitored using Wormlab, including total sleep duration, sleep frequency, number of periods of stillness, and duration of stillness. The sleep physiological indicators of nematodes are measured by determining changes in sleep-related γ-aminobutyric acid (GABA) levels. When Wormlab monitors a nematode transitioning from a state of activity to stillness and then back to activity over a period of time, combined with changes in neurotransmitter levels during this state, it indicates that the nematode has undergone a developmental sleep cycle. Compared to previous models, the improved sleep monitoring model of this invention provides a more comprehensive evaluation of nematode sleep and is more scientifically sound in its sleep data acquisition.

[0028] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0029] The model and sleep detection method provided in this application improve upon the limitations of the *C. elegans* sleep monitoring model, which cannot immobilize nematodes and analyze individual nematodes. This increases the model's throughput, allowing direct analysis of nematode sleep behavior using Wormlab without visual observation, reducing manpower and material costs, and enhancing the scientific validity and effectiveness of the experiment. Attached Figure Description

[0030] Figure 1 The preparation process for a single-nematode microculture chamber;

[0031] Figure 2 This is a top-view schematic diagram of the culture chamber;

[0032] Figure 3 The process of implementing the photomask;

[0033] Figure 4 To utilize this model for sleep detection, an imaging state map from wormlab was used;

[0034] Figure 5 This is an imaging state diagram obtained in the previous research of this application;

[0035] Figure 6 To observe the characteristic images of the vulva of nematodes before sleep using the model in this application;

[0036] Figure 7 The figures show the sleep characteristic curves of nematodes obtained using the model and detection method of this application; the upper figure shows the sleep characteristic curves of nematodes, and the lower figure shows the comparison of the content of sleep-related neurotransmitters in nematodes at the same time.

[0037] Figure 8 For comparison of GABA in the body during nematode sleep obtained using the model and detection method of this application;

[0038] Figure 9 This is a top view of the culture chamber (where 1 is dry bacterial culture; 2 is agar; 3 is PDMS culture chamber; 4 is micropores). Detailed Implementation

[0039] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0040] Unless otherwise specified, the experimental methods used in the embodiments of this invention are conventional methods; unless otherwise specified, the materials and reagents used are commercially available.

[0041] Example 1: A method for preparing a single nematode microculture chamber, comprising the following steps:

[0042] 1) The structure of the micro-culture chamber was designed using AutoCAD and specific parameters were set to obtain the structural diagram of the single nematode micro-culture chamber. After a series of operations including graphic creation and conversion, exposure, development, etching, demolding, cleaning, defect inspection, defect repair, shipping cleaning, and film application, a quartz mask was obtained.

[0043] 2) Coat the photoresist onto the photomask and spin coat it in a spin coater. Take a 10cm*10cm single-crystal silicon wafer, take 10g of SU-8 photoresist, and set the spin coater parameters to 500rpm / 5s according to the height of the micro-culture medium of 200μm, until the photoresist is evenly spread over the single-crystal silicon wafer;

[0044] 3) The single-crystal silicon coated with photoresist was placed on a Cchip-0019 high aspect ratio ultraviolet lithography machine. The ultraviolet light intensity was 86.2 mJ / cm*cm. Before each experiment, measurements were taken according to the type of photoresist and the desired etching height to calculate the exposure ultraviolet intensity and exposure time. A model of a single-needle worm micro-culture chamber was obtained through photolithography.

[0045] 4) Mix polydimethylsiloxane (PDMS) adhesive and curing agent in a ratio of 10:1, stir quickly with a glass rod until uniform, spread evenly on the single crystal silicon mold after photolithography, put it in a vacuum hood to degas until the air bubbles disappear completely, and finally put it in an oven with the temperature set at 80℃-120℃ for more than 6 hours.

[0046] 5) Remove the material from the oven and demold it in a clean bench. After demolding, cut out the shape with scissors and use a punch to make a 5mm hole in the culture chamber to obtain the culture chamber. Soak it in 2% SDS for 30 minutes, take it out and wipe it clean with lens paper for later use.

[0047] 6) Prepare a 1cm thick agar solid culture medium for nematodes. Air-dry the medium in a clean bench with a level 2 ventilation system for 36 hours. In the clean bench, slowly attach the PDMS micro-culture chamber to the solid culture medium, gently pressing it down with a smear ring to ensure complete adhesion between the agar and PDMS, thus obtaining a single nematode micro-culture chamber (e.g., ...). Figure 9 (as shown);

[0048] 7) Culturing *Caenorhabditis elegans* to the L4 pre-stage, select crescent-shaped, translucent nematodes from the vulva and place them in a micro-culture chamber. Record the process on Wormlab, taking a photo every 10 seconds for 12 hours. After recording the video, use Wormlab's built-in analysis software to analyze the nematodes' total sleep duration, sleep frequency, number of pauses, and pause duration to obtain a marker sleep curve.

[0049] 8) Collect nematodes exhibiting sleep characteristics in the L4 pre-stage and determine their GABA content. Wash the nematodes from the culture medium with 1 mL of M9 buffer and transfer to a 2.0 mL centrifuge tube. After the nematodes settle naturally, discard the supernatant. Repeat the washing process three times with sterile M9 buffer, discarding the supernatant each time. Add 9 times the volume of nematode lysis buffer (methanol:acetonitrile:0.2% formic acid water = 1:1:1) for homogenization. Use a bead mill with the following parameters: 60 Hz, 30 s run, 10 s interval, 10 min continuous operation. Centrifuge at 12000 rpm for 20 min, collect the supernatant, filter through a membrane, determine the BCA concentration, and analyze by LC-MS / MS.

[0050] in, Figure 5 This is an imaging state map obtained in the previous study of this application; from Figure 5 It is evident that the field of view is not clear enough. This is because nematodes tend to become entangled during video recording, leading to misidentification during data analysis.

[0051] also, Figure 1 The preparation process of the single nematode microculture chamber provided in this application; Figure 2 This is a top-view schematic diagram of the culture chamber; Figure 3 The process of implementing a photomask (conventional); Figure 4 To utilize this model for sleep detection, imaging state maps from WormLab were used. Combined with the culture chamber prepared above and subsequent routine testing procedures, WormLab can be used to directly analyze the sleep behavior of nematodes. For example, Figure 6 To observe the characteristic images of the vulva of nematodes before sleep using the model in this application; Figure 7 The sleep characteristic curve of nematodes obtained using the model and detection method of this application; Figure 8 This study compares the levels of GABA in nematodes during their dormancy period using the model and detection method described in this application. The device and detection method provided in this application enable precise single-worm culture, and the analysis of dormancy characteristics using Wormlab provides rapid and accurate data with high throughput and a large amount of effective data, thus improving monitoring efficiency.

[0052] Obviously, the specific implementation schemes described above are merely a further detailed explanation of the purpose, technical solution and beneficial effects of the present invention. It should be understood that the above descriptions are only specific examples of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for monitoring sleep in nematodes based on a simple single nematode culture model, characterized by, The model fixes single nematodes in a culture chamber for culture, and the culture chamber is divided into upper and lower layers, the upper layer is a micro cavity for accommodating nematodes, and the lower layer is a culture medium; the culture medium is provided with a dry nematode bacteria solution for providing growth of nematodes; The preparation method of the culture chamber for single nematodes comprises the following steps: Step 1: set the culture chamber parameters and prepare the corresponding mask plate; Step 2: coat photoresist on the mask plate of step 1, take a 10cm*10cm single crystal silicon wafer, suck the photoresist, and uniformly coat the photoresist on the single crystal silicon wafer according to the height until the photoresist uniformly covers the single crystal silicon wafer; Step 3: etch using ultraviolet light to obtain a culture chamber model; Step 4: mix the polydimethylsiloxane glue with the curing agent, stir and beat until uniform, uniformly spread on the single crystal silicon mold after photoetching, put into a vacuum cover to degas until the bubbles completely disappear, and then dry; Step 5: after demolding, punching is performed to obtain the culture chamber; Step 6: the polydimethylsiloxane after drying in step 5 is laminated with the agar culture medium with dry bacteria solution to obtain a micro culture chamber.

2. The method according to claim 1, wherein the sleep of the nematode is monitored on a simple nematode culture model. The culture chamber is square and is provided with a plurality of small holes.

3. The method according to claim 1, wherein the method is characterized by, Each small hole has a diameter of 3-5mm, an interval of 2-2.5mm, and a thickness of 200μm.

4. The method according to claim 3, wherein the sleep of the nematode is monitored on the basis of the movement of the nematode in the simple nematode culture model. Sleep characteristics are determined and analyzed using conventional wormlab instruments.

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