A Highly Uniform Synchronized Culture Method for Caenorhabditis elegans Based on Inverted Starvation Arrest

Abstract

This invention discloses a highly uniform and synchronized culture method for *C. elegans* based on inverted starvation arrest. By limiting key process parameters such as NGM agar thickness, bacterial spread range, and inverted culture temperature, humidity, and time, gravity is used to induce natural egg detachment and hatching. Relying on the nematode's inherent L1 stage starvation arrest mechanism, newly formed larvae are stably arrested at the L1 developmental stage, completely eliminating the problem of premature molting and development. This method significantly improves the survival rate of nematode larvae, greatly increases the synchronization rate of population development, effectively eliminates physiological interference from chemical reagents, and ensures uniform growth and development rates of nematodes with minimal differences in parallel sample data.

Description

Technical Field

[0001] This invention belongs to the field of model organism nematode culture and experimental sample pretreatment technology, specifically involving a standardized process for the synchronized culture of Caenorhabditis elegans that is chemically non-destructive, high-throughput, and highly consistent, and its application in biological research and pharmacological experiments. Background Technology

[0002] *Caenorhabditis elegans* is a core model organism in life sciences, anti-aging research, exploration of oxidative stress mechanisms, and pharmacological validation of natural drugs. It possesses advantages such as a short lifespan, clear genetic background, and ease of experimental manipulation, and is widely used in various basic research and drug screening experiments. The core prerequisite for nematode experiments is obtaining synchronized nematode samples with highly uniform developmental stages, stable physiological states, and free from external damage or interference. The synchronization and physiological activity of these samples directly determine the authenticity and reproducibility of subsequent experimental data.

[0003] Currently, the mainstream method for nematode synchronization in laboratories is the sodium hypochlorite-sodium hydroxide bleaching and lysis method. This method relies on a mixture of strong alkali and strong oxidant to break down adult nematodes and separate and collect eggs to achieve sample synchronization. However, this technique has several inherent drawbacks: First, the strong alkali and oxidant can cause irreversible oxidative stress damage to eggs and newly hatched larvae, altering the nematode's baseline physiological state and severely interfering with the results of subsequent precise experiments such as lifespan detection, antioxidant evaluation, and drug pharmacology and efficacy, leading to biased and unreliable experimental data. Second, this method is cumbersome, requiring multiple centrifugation, washing, and lysis processes, which is time-consuming and prone to human error, making it unsuitable for the simultaneous preparation of large batches of samples. Third, the concentration and reaction time of the lysis reagent are difficult to standardize and control, resulting in significant differences in the consistency of nematode development between different batches of experiments and extremely poor experimental repeatability.

[0004] While the conventional upright natural hatching synchronization method avoids damage from chemical reagents, it relies entirely on the spontaneous development of the worms, making it impossible to artificially lock in a uniform developmental period. This results in inconsistent egg hatching and larval molting times, easily leading to mixed developmental stages and making it difficult to obtain highly homogeneous experimental samples. It only meets the needs of rudimentary experiments. Existing simple inverted culture procedures are merely rudimentary laboratory explorations, lacking standardized and quantifiable process parameters. These procedures commonly suffer from incomplete egg shedding, unstable hatching rates, some larvae prematurely molting into the L2 stage, and low sample synchronization. A standardized, replicable, and scalable technical solution has yet to be developed, severely hindering the development of precise scientific research experiments related to *C. elegans*.

[0005] In summary, current nematode synchronization technologies generally suffer from shortcomings such as chemical damage, poor synchronization effect, complex operation, low batch stability, and inability to produce high-throughput nematodes. There is an urgent need to develop a nematode synchronization culture method that is free from physiological damage, has standardized parameters, high synchronization, and can be produced in batches. Summary of the Invention

[0006] This invention aims to overcome the shortcomings of existing nematode synchronization culture techniques, such as chemical toxicity damage, low synchronization of nematode development, poor experimental reproducibility, cumbersome operation, and inability to prepare samples in large quantities. It provides a highly uniform synchronized culture method for *C. elegans* based on inverted starvation arrest. This invention requires no chemical lysis reagents, completely preserves the original physiological state of the nematodes, and stably obtains highly active and homogeneous nematode populations arrested in the L1 phase through a precise and controllable physical culture process. It simplifies experimental procedures, lowers the experimental threshold, increases sample preparation throughput, and is suitable for various high-precision nematode research scenarios, addressing the core pain points of existing technologies.

[0007] As a first aspect of the present invention, the present invention provides a method for highly uniform synchronous culture of Caenorhabditis elegans based on inverted starvation arrest, comprising the following steps: S1: Prepare sterile NGM culture plates free of contaminants; S2: Adult *C. elegans* were inoculated into the mycelium-covered area of ​​a plate and pre-cultured for oviposition. S3: After oviposition, the plate is inverted and kept in the dark and constant temperature. Gravity causes the eggs to fall, and the starvation environment on the cover allows the eggs to hatch and remain stably in the L1 stage. S4: Collect and purify diapause L1 stage larvae; S5: The obtained synchronized larvae are transferred and revived for culture to achieve synchronous development of the nematode population.

[0008] Preferably, in S1, the agar plate solidification thickness is 3.5~4.5mm, and OP50 Escherichia coli bacterial moss is only coated in the central area at the bottom of the plate, with a moss diameter of 3~5cm. The inner surface of the petri dish lid is kept free of sterile residue and contamination by other bacteria.

[0009] Preferably, the pre-culture oviposition conditions described in S2 are as follows: select adult *C. elegans* that are vigorous in reproduction, physiologically stable, and free from bacterial contamination, inoculate them in the central mycelial area of ​​a plate, and pre-culture them at room temperature in the dark for 10-12 hours to allow the adults to lay enough eggs and obtain a sufficient number of mature free eggs.

[0010] Preferably, the process parameters for the inverted, light-protected, and constant-temperature culture in S3 are: culture temperature of 20~25℃, culture environment humidity of 55%~65%, inverted culture time of 14~18h, and culture in a completely dark and sealed environment.

[0011] Preferably, the step of collecting and purifying diapause L1 larvae in S4 is as follows: the inner surface of the petri dish lid is rinsed multiple times with sterile M9 buffer, all detached and hatched L1 diapause larvae are collected, and the larvae are washed 2-3 times by low-speed centrifugation to remove egg residue and metabolic impurities, thereby obtaining a high-purity nematode sample with high developmental uniformity.

[0012] Preferably, the larval resuscitation culture step in S5 is as follows: the collected and purified L1 stage diapause nematodes are transferred to a standard NGM plate containing fresh activated OP50 Escherichia coli bacterial culture, and cultured in the dark at a constant temperature of 20-25°C and 55%-65% humidity to simultaneously restart the growth and development of the nematodes.

[0013] As a second aspect of the present invention, the present invention provides the application of the above-mentioned highly uniform synchronous culture method of Caenorhabditis elegans based on inverted starvation arrest in the study of the anti-aging mechanism of Caenorhabditis elegans, evaluation of antioxidant stress, verification of the pharmacological efficacy of natural drugs, detection of nematode life cycle, and molecular biology experiments of nematodes.

[0014] The beneficial effects of this invention are: 1) No chemical damage throughout the process, and the physiological state of the sample is real: This invention abandons the traditional strong alkali and strong oxidant lysis process, and realizes the synchronization of nematodes through physical culture only. There is no chemical toxicity stress, the original physiological state of nematodes is completely preserved, and there is no additional oxidative stress damage. This ensures the authenticity and accuracy of subsequent experimental data from the source and is suitable for the needs of high-precision scientific research experiments.

[0015] 2) Excellent synchronization effect and extremely high sample consistency: This invention utilizes the inherent L1 stage starvation diapause mechanism of nematodes and, with precise control of culture parameters, can stably lock all newborn larvae in the L1 development stage, completely eliminating the problems of premature molting and mixed development. The synchronization of population development is significantly better than that of traditional processes, solving the core pain point of poor sample uniformity in existing technologies.

[0016] 3) High larval survival rate and lower experimental cost: It avoids the killing and damage of chemical reagents to nematode eggs and larvae, greatly improves the survival rate of nematode samples, reduces experimental sample loss, eliminates the need for repeated sample preparation, and effectively reduces scientific research experimental costs and labor costs.

[0017] 4) Extremely simple operation and high-throughput preparation: The process steps of this invention are simple, the parameters are controllable, and the error tolerance is high. There is no need for multiple centrifugation, lysis and washing operations. The human operation error is small. Multiple sets of plate samples can be processed simultaneously in batches, realizing high-throughput and standardized preparation of nematode samples, which is suitable for large-scale drug screening and batch experimental research.

[0018] 5) Wide range of applicable scenarios and strong versatility: The method of this invention can be perfectly adapted to various scientific research scenarios such as anti-aging research of Caenorhabditis elegans, evaluation of oxidative stress, pharmacological verification of natural drugs, life cycle detection, and molecular biological mechanism analysis. The technology has strong versatility and has extremely high scientific research and promotion value.

[0019] 6) Standardized process and strong reproducibility: This invention clearly defines all core parameters such as plate thickness, mycelial size, temperature and humidity, and culture time, forming a standardized and finalized process. There is no reliance on experience, and different operators and different experimental batches can replicate stable experimental results, completely solving the problem of poor repeatability of traditional technology. Attached Figure Description

[0020] Figure 1 This is a schematic diagram comparing the survival rate and developmental synchronization rate of nematodes obtained in Example 1 of the present invention with those obtained in the comparative example. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. It should be noted that, unless otherwise specified, the following embodiments and features can be combined with each other. It should also be understood that the terminology used in the embodiments of this invention is for describing specific implementation schemes and not for limiting the scope of protection of this invention.

[0022] When a range of values ​​is given, it should be understood that, unless otherwise stated in this invention, the two endpoints of each range and any value between the two endpoints may be selected.

[0023] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly used in the field to which this invention pertains. The terminology used to describe this invention is intended only to describe a particular implementation and is not intended to limit the scope of the teachings. This invention can be implemented using any prior art methods, apparatus, and materials similar to or equivalent to those described, used, or made in the embodiments of this invention.

[0024] The term “and / or” as used herein should be understood to mean any one of the options or any combination of two or more of the options.

[0025] As used herein, the term "about" indicates a range of ±20% of the following value. In some embodiments, the term "about" indicates a range of ±10% of the following value. In some embodiments, the term "about" indicates a range of ±5% of the following value.

[0026] This invention provides a highly uniform synchronous culture method for *C. elegans* based on inverted starvation arrest. The core principle is as follows: NGM culture plates containing oviposition-completed nematodes are inverted for culture. Gravity causes free eggs to naturally detach from the plate and fall onto the inner surface of the petri dish lid. Through differentiated bacterial distribution design, mycelium is retained at the bottom of the plate, while the inner surface of the lid is free of any nutrient source. After the eggs hatch on the lid, the newborn larvae trigger the conservative L1 starvation arrest mechanism of *C. elegans*, stably arresting at the L1 developmental stage and preventing subsequent molting and development, resulting in a highly uniform nematode sample. Subsequent transfer to a plate containing fresh mycelium allows for synchronous recovery and development.

[0027] As one specific implementation method, the method includes the following steps: S1 NGM Plate Precision Preparation: Prepare NGM solid culture medium, control the agar solidification thickness to be 3.5~4.5mm, spread OP50 Escherichia coli bacterial growth only in the center area of ​​the bottom of the plate, limit the bacterial growth diameter to 3~5cm, strictly ensure that the inner surface of the petri dish lid is free of bacteria and contaminants, and set aside for use; S1 Adult worm loading and inoculation: Adult Caenorhabditis elegans with vigorous reproduction and stable physiological state are enriched and inoculated into the central mycelial area of ​​the plate, and pre-cultured in the dark as usual until a large number of mature worm eggs are produced in the plate. S3 Inverted starvation arrest culture: The plate after oviposition is inverted and cultured in the dark at a temperature of 20~25℃ and an ambient humidity of 55%~65% for 14~18 hours to allow the eggs to fall off, hatch and be stably arrested in the L1 stage. S4 Collection of diapause larvae: The inner surface of the petri dish lid was rinsed multiple times with sterile M9 buffer to collect all L1 stage diapause larvae. The larvae were then washed 2-3 times by low-speed centrifugation to remove impurities and residual metabolites, resulting in a high-purity synchronized nematode sample. S5 Nematode Resuscitation Culture: The collected L1 stage synchronized nematodes are transferred to a standard NGM plate containing fresh OP50 Escherichia coli bacterial culture, and normal culture will synchronously restart the growth and development of the nematodes.

[0028] The following embodiments and accompanying drawings are provided to aid in understanding the present invention. However, it should be understood that these embodiments are for illustrative purposes only and do not constitute any limitation. The actual scope of protection of the present invention is set forth in the claims. It should be understood that any modifications and changes can be made without departing from the spirit of the present invention. Unless otherwise specified, the raw materials used in the embodiments are all commercially available products in the art, their specifications are conventional specifications in the art, and the methods used are conventional methods in the art. The instruments and equipment used in the embodiments are conventional instruments and equipment in the art.

[0029] Example 1 This embodiment provides a standardized and batch-reproducible method for the synchronized culture of Caenorhabditis elegans, the specific steps of which are as follows: 1) Prepare NGM solid plates, strictly control the agar solidification thickness to 4mm, spread OP50 Escherichia coli bacterial growth with a diameter of 4cm in the center of the bottom of the plate, wipe and clean the inner surface of the petri dish lid to ensure that there are no bacterial residues, and let it stand aseptically for later use. 2) Select vigorous, uncontaminated adult *C. elegans* and inoculate them evenly onto the mycelial area of ​​the plate. Pre-culture at room temperature in the dark for 12 hours to ensure that the plate produces a large number of mature eggs. 3) Invert the plate and place it in a constant temperature incubator at 22℃ and 60% humidity for 16 hours in the dark. 4) Remove the culture plate, rinse the inner surface of the plate lid three times with sterile M9 buffer, collect the eluent, centrifuge twice at low speed to remove impurities, and obtain pure L1 stage diapause nematodes. 5) The collected nematodes were transferred to NGM plates containing fresh OP50 bacterial culture. The nematodes were able to resume growth and development simultaneously. The resulting samples showed uniform development and excellent activity, and could be directly used for subsequent pharmacological experiments and mechanism detection.

[0030] Example 2 The only difference between this embodiment and Example 1 is that the thickness of the NGM agar plate is controlled at 3.5 mm, the diameter of the bacterial growth is 3 cm, and the plate is inverted and cultured for 18 h at 20°C and 60% humidity.

[0031] This embodiment ensures complete egg detachment and hatching, and stable larval diapause. No chemical intervention is involved throughout the process, maintaining the nematode's baseline physiological state without deviation. Synchronized nematodes exhibit highly consistent growth after recovery, completely eliminating the interference of chemical damage and developmental differences on lifespan experimental data. This significantly improves the accuracy and repeatability of experimental data, making it suitable for high-precision nematode lifespan detection and anti-aging mechanism research.

[0032] Example 3 The only difference between this embodiment and Example 1 is that the NGM agar plate thickness is 4.5 mm, the bacterial growth diameter is 5 cm, and the culture is carried out upside down for 14 hours at 25°C and 65% humidity to quickly complete the hatching of insect eggs and the arrest of L1 stage.

[0033] The nematodes obtained in this embodiment showed no abnormal oxidative damage, avoiding the stress interference of traditional chemical synchronization methods. It can accurately reflect the effects of drugs and environment on the antioxidant capacity of the nematodes and is suitable for various antioxidant pharmacological screening experiments.

[0034] Comparative Example 1 This comparative example uses the conventional upright natural hatching synchronization method to culture nematodes. The specific steps are as follows: 1. Prepare standard NGM agar plates with an agar thickness of 3.5-4.5 mm. Spread OP50 Escherichia coli bacterial growth with a diameter of 3-5 cm in the center of the plate. Let the plates stand at 20-25℃ in the dark for later use. 2. Select vigorous adult *C. elegans* that are free from contamination by other bacteria, inoculate them into the mycelium area of ​​agar plates, and pre-culture them at 20-25°C in the dark for 10-12 hours to allow the adults to lay eggs fully; 3. Keep the plate upright and incubate at 20-25℃ and 55%-65% humidity in the dark for 14-18 hours. The insect eggs will hatch naturally on the surface of the mycelium on the plate. 4. Rinse the agar plate surface with sterile M9 buffer, collect all larvae at different developmental stages, and wash 2-3 times by low-speed centrifugation at 300-500 r / min to remove egg residue and metabolic impurities; 5. The collected mixed-age nematodes were directly transferred to NGM plates containing fresh OP50 mycelium and cultured at 20-25℃ in the dark. The nematodes developed spontaneously and disorderly, and the population contained multiple stages of larvae, including L1, L2, and L3.

[0035] Comparative Example 2 This comparative example uses the classic sodium hypochlorite-sodium hydroxide bleaching-lysis synchronization method to culture nematodes. The specific steps are as follows: 1. Take an NGM plate covered with adult Caenorhabditis elegans and eggs, wash the plate surface with sterile M9 buffer, collect the suspension of worms and eggs, centrifuge at 400 r / min for 2 min, discard the supernatant, and collect the worm pellet; 2. Preparation of pyrolysis working solution: Mix 5% sodium hypochlorite solution and 1 mol / L NaOH solution at a volume ratio of 1:1, and use immediately after preparation; 3. Add lysis working solution to the precipitate of insect bodies, with a volume ratio of lysis solution to precipitate of insect bodies of 5:1. Shake vigorously at room temperature for 6-8 minutes, inverting and mixing once every 2 minutes, until the adult insect bodies are completely broken down and only intact eggs remain. 4. Immediately after lysis, add 3 volumes of sterile M9 buffer to terminate the lysis, centrifuge at 400 r / min for 2 min, and discard the supernatant; repeat the M9 buffer centrifugation and washing 3 times to thoroughly remove residual strong base and oxidizing agent; 5. Resuspend the purified eggs in sterile M9 buffer, drop them onto the surface of a standard NGM plate, and incubate them upright in the dark at 20-25°C and 55%-65% humidity for 14-18 hours. The eggs will hatch to obtain L1 stage larvae. 6. Collect the hatched larvae from the plates and transfer them to fresh OP50 NGM plates for resuscitation and culture.

[0036] Experimental Example This experiment tested two core indicators: nematode larval survival rate and population development synchronization rate in Example 1, Comparative Example 1, and Comparative Example 2. The differences and stability of data from each group were statistically analyzed, and the results are as follows: Figure 1 As shown, the experimental group used nematodes cultured in Example 1, while control group 1 and control group 2 were nematodes cultured in Comparative Example 1 and Comparative Example 2, respectively.

[0037] The experimental results showed that the experimental group was not subjected to any chemical reagent stress or physical damage throughout the process, and the larval survival rate remained stable at over 98%, which was significantly higher than that of the control group 2 bleaching lysis method (survival rate 72.5%). This completely avoided problems such as egg death, larval damage, and decreased activity caused by strong alkali and oxidants. The larval survival rate of the control group 1 natural hatching method could reach over 94%, but the larval development synchronization was extremely poor, which could not meet the requirements of high-precision experiments.

[0038] The experimental group, through precise parameter control and the inherent starvation diapause mechanism of nematodes, could stably lock the larvae at the L1 stage 100% without premature molting, mixed development, or individual differences, and the population development synchronization rate was 100%. The control group 2 had 20% to 30% of the larvae with disordered development, uneven physiological state, and delayed or premature development, with a synchronization rate of only about 70%. The control group 1 had disordered larval development stages, with multiple stages of larvae including L1, L2, and L3, and a synchronization rate of less than 60%.

[0039] Experimental data show that the method of the present invention achieves 100% synchronous development of nematode populations while ensuring a high larval survival rate, and there are no significant differences in data between batches, demonstrating good repeatability and stability.

[0040] In summary, this invention overcomes the inherent technical bottlenecks of traditional synchronized culture techniques for *C. elegans*, innovatively constructing an integrated standardized culture process combining inverted gravity egg drop and starvation inhibition. Verified through multiple control experiments, it possesses numerous advantages, including no chemical damage, high synchronization, high survival rate, good reproducibility, high throughput, and ease of operation. The invention features precise technical parameters, a mature process, and can be replicated in batches. It can provide high-quality, highly stable standardized experimental samples for various nematode-related basic research, natural product pharmacological screening, anti-aging mechanism research, and oxidative stress experiments, effectively improving the accuracy and standardization of nematode-related scientific research experiments. It has broad application prospects and extremely high promotional value in life science research, pharmacological development, and natural active ingredient evaluation.

[0041] The above embodiments merely illustrate several implementation methods of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the patent application. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of the present invention should be determined by the appended claims.

Claims

1. A highly uniform synchronous culture method for *Caenorhabditis elegans* based on inverted starvation arrest, characterized in that... Includes the following steps: S1: Prepare sterile NGM culture plates free of contaminants; S2: Adult *C. elegans* were inoculated into the mycelium-covered area of ​​a plate and pre-cultured for oviposition. S3: After oviposition, the plate is inverted and kept in the dark and constant temperature. Gravity causes the eggs to fall, and the starvation environment on the cover allows the eggs to hatch and remain stably in the L1 stage. S4: Collect and purify diapause L1 stage larvae; S5: The obtained synchronized larvae are transferred and revived for culture to achieve synchronous development of the nematode population.

2. The method according to claim 1, characterized in that, In S1, the agar solidification thickness is 3.5~4.5mm. Only the OP50 Escherichia coli bacterial growth is spread in the center area at the bottom of the plate, with a bacterial growth diameter of 3~5cm. The inner surface of the petri dish lid is kept free of sterile residue and contamination by other microorganisms.

3. The method according to claim 1, characterized in that, The pre-culture oviposition conditions described in S2 are as follows: Select adult *C. elegans* that are vigorous in reproduction, physiologically stable, and free from bacterial contamination, inoculate them in the central mycelial area of ​​a plate, and pre-culture them at room temperature in the dark for 10-12 hours to allow the adults to lay eggs fully and obtain a sufficient number of mature free eggs.

4. The method according to claim 1, characterized in that, The process parameters for the inverted, light-protected, and constant-temperature culture described in S3 are as follows: culture temperature is 20~25℃, culture environment humidity is 55%~65%, inverted culture time is 14~18h, and the entire process is carried out in a light-protected and sealed environment.

5. The method according to claim 1, characterized in that, The steps for collecting and purifying diapause L1 larvae in S4 are as follows: Rinse the inner surface of the petri dish lid multiple times with sterile M9 buffer, collect all the detached and hatched L1 diapause larvae, and wash them 2-3 times by low-speed centrifugation to remove egg residue and metabolic impurities, thereby obtaining a high-purity nematode sample with high developmental uniformity.

6. The method according to claim 1, characterized in that, The steps for larval resuscitation culture in S5 are as follows: The collected and purified L1-stage diapause nematodes are transferred to a standard NGM plate containing fresh and activated OP50 Escherichia coli bacterial culture and cultured in the dark at a constant temperature of 20-25℃ and 55%-65% humidity to simultaneously restart the growth and development of the nematodes.

7. The application of the highly uniform synchronous culture method of Caenorhabditis elegans based on inverted starvation arrest as described in any one of claims 1-6 in the study of the anti-aging mechanism of Caenorhabditis elegans, evaluation of antioxidant stress, verification of the pharmacological efficacy of natural drugs, detection of nematode life cycle, and molecular biology experiments of nematodes.

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