Anhypnia zebrafish model and modeling method thereof

Abstract

The invention relates to the technical field of animal evaluation models, and discloses an insomnia zebrafish model and a modeling method thereof.The insomnia zebrafish model is prepared from zebrafish and a culture solution, the culture solution comprises one of water and dilution water, the culture solution is vortex, the vortex flow rate is 0.07-0.1 m / s, and the vortex flow rate is 0.07-0.1 m / s; comprising the following steps: adding a culture solution into a culture dish to form eddy current, and then putting zebra fish into the culture dish for modeling culture; according to the method, vortex stimulation is adopted for modeling the insomnia zebra fish, the vortex stimulation can prevent water flow from damaging the zebra fish, the interference effect of pain factors on the insomnia zebra fish model can be avoided, and the non-painful insomnia can be accurately evaluated.

Description

Technical Field

[0001] This invention relates to the field of animal evaluation model technology, and in particular to a zebrafish model of insomnia and its modeling method. Background Technology

[0002] Insomnia is a common sleep disorder characterized by difficulty falling asleep, frequent awakenings, early awakenings, and poor sleep quality. It is often accompanied by daytime functional impairments such as fatigue, memory loss, poor concentration, and mood swings. Causes of insomnia include psychological and emotional factors, irregular lifestyle habits, environmental factors, illnesses, and dietary factors.

[0003] Animal evaluation models of insomnia are an important tool for studying the mechanisms and treatments of insomnia. Currently, mice and zebrafish are the most common animal models for insomnia evaluation. Existing technologies indicate that the hypothalamic-pituitary-adrenal (HPA) axis in mammals and the hypothalamic-pituitary-interrenal (HPI) axis in zebrafish are evolutionarily homologous systems, with highly conserved core hormonal regulation and stress regulation functions, making zebrafish a popular research model for stress-related issues. Currently disclosed stimulation methods for zebrafish insomnia evaluation models include continuous light stimulation, environmental fluctuation stimulation, drug stimulation, and physical stress stimulation, with drug stimulation being the most common. For example, CN120615793A discloses a method for constructing and applying a zebrafish model of insomnia. This method uses a combination of light and pentylenetetrazol to induce insomnia in zebrafish, specifically targeting drug-induced insomnia.

[0004] Currently, circadian rhythm disorder-related insomnia is on the rise due to factors such as staying up late, shift work, and living across time zones. Therefore, to study the formation mechanism and treatment methods of this type of insomnia, existing technologies disclose a zebrafish model that prevents zebrafish from entering a resting sleep state through continuous water flow stimulation. After in-depth research on this technical solution, this invention found that the aforementioned technical solution, by continuously impacting the zebrafish with water, easily damages them, leading to painful insomnia. This means that pain factors are incorporated into the influencing factors of zebrafish insomnia, making it impossible to accurately evaluate non-painful insomnia. Summary of the Invention

[0005] The purpose of this invention is to construct a zebrafish model of insomnia by treating zebrafish with eddy current stimulation. Eddy current stimulation can avoid injury to zebrafish, avoid the interference of pain factors on the evaluation of non-painful insomnia, and improve the evaluation accuracy of non-painful insomnia.

[0006] The specific technical solution of this invention is as follows: A zebrafish model of insomnia, comprising zebrafish and a culture medium, wherein the culture medium consists of either water or diluted water, and the culture medium is a vortex with a flow velocity of 0.07~0.1 m / s.

[0007] A method for establishing the above-mentioned zebrafish model of insomnia includes the following steps: adding culture medium into a culture dish to form a vortex, and then placing the zebrafish in the culture dish for modeling culture. The culture conditions include: stimulation time of 10-15 h.

[0008] As the preferred choice, the zebrafish is the wild AB type zebrafish.

[0009] As a preferred choice, zebrafish are zebrafish that are 4-7 dpf after fertilization.

[0010] As a preferred choice, zebrafish are zebrafish that have undergone fertilization for 4 dpf.

[0011] Preferably, the standard dilution water consists of calcium chloride, magnesium chloride, sodium bicarbonate, and potassium chloride.

[0012] Preferably, the concentration ratio of calcium chloride, magnesium chloride, sodium bicarbonate and potassium chloride is 27~30:6~7:10~12:1.

[0013] Preferably, the apparatus used for cultivation includes a horizontal shaking table.

[0014] Preferably, the rotation speed of the horizontal shaking table is 90~100 r / min.

[0015] Preferably, the cultivation temperature is 28~30 ℃.

[0016] This invention provides a method for constructing a zebrafish model of insomnia. The method uses eddy current stimulation to treat zebrafish to construct the insomnia zebrafish model. Using eddy current stimulation can avoid the damaging effect of water flow on zebrafish, significantly reduce the interference of pain factors on the non-painful zebrafish insomnia model, and improve the evaluation accuracy of the zebrafish model.

[0017] In practical applications, this invention has found that traditional water flow stimulation of zebrafish, which uses a continuous water flow to impact the zebrafish and prevent them from entering a resting, sleep-like state, causes damage to the zebrafish before the insomnia model is established. This damage leads to pain in the zebrafish, interfering with the formation of insomnia and making it impossible to accurately evaluate non-painful zebrafish. Further research in this invention has revealed that using eddy currents to stimulate zebrafish can also induce insomnia, while simultaneously preventing damage and significantly minimizing the interference of pain factors.

[0018] Compared with the prior art, this application has the following technical effects: This method uses vortex stimulation to model insomnia in zebrafish. Vortex stimulation can significantly reduce the damage of water flow to zebrafish and significantly reduce the interference of pain factors on the insomnia zebrafish model, and can accurately evaluate non-painful insomnia. In this method, zebrafish can effectively form an insomnia zebrafish model when the vortex velocity is 0.07~0.1 m / s and the stimulation time is 10~15 h. Detailed Implementation

[0019] The present invention will be further described below with reference to embodiments.

[0020] To better understand the content of this invention, further explanation is provided below with reference to specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of this invention.

[0021] Example 1: A method for creating a model of insomnia in zebrafish includes the following steps: Wild-type AB zebrafish, 4 dpf post-fertilization, were randomly selected and placed in plastic culture dishes (Nest Biotech, 100 mm, 50 mL). Each dish contained 30 zebrafish, and 30 mL of standard dilution water was added to each dish. The standard dilution water consisted of 294.0 mg / L calcium chloride dihydrate, 123.3 mg / L magnesium chloride heptahydrate, 63.0 mg / L sodium bicarbonate, and 5.5 mg / L potassium chloride. The culture dishes were placed on a horizontal shaker (OS-20F) for modeling culture. The modeling culture conditions were: dark, temperature 28℃, shaker speed 90 rpm (vortex flow rate 0.09 m / s), and stimulation time 15 h. After the modeling culture was completed, modeled zebrafish were obtained and placed in a zebrafish behavior analyzer (ZebraLab3.22.3.31) to test the zebrafish's arousal activity.

[0022] Example 2: A method for creating a model of insomnia in zebrafish includes the following steps: Wild-type AB zebrafish, 4 days post-fertilization (dpf), were randomly selected and placed in plastic culture dishes (Nest Biotech, 100 mm, 50 mL). Each dish contained 30 zebrafish, and 30 mL of standard dilution water was added to each dish. The standard dilution water consisted of 294.0 mg / L calcium chloride dihydrate, 123.3 mg / L magnesium chloride heptahydrate, 63.0 mg / L sodium bicarbonate, and 5.5 mg / L potassium chloride. The culture dishes were placed on a horizontal shaker (OS-20F) for modeling culture. The modeling culture conditions were: dark environment, temperature 28℃, shaker speed 70 rpm (vortex flow rate 0.07 m / s), and stimulation time 15 h. After the modeling culture was completed, modeled zebrafish were obtained and placed in a zebrafish behavior analyzer (ZebraLab3.22.3.31) to test the zebrafish's arousal activity.

[0023] Example 3: A method for creating a model of insomnia in zebrafish includes the following steps: Wild-type AB zebrafish, 4 days post-fertilization (dpf), were randomly selected and placed in plastic culture dishes (Nest Biotech, 100 mm, 50 mL). Each dish contained 30 zebrafish, and 30 mL of standard dilution water was added to each dish. The standard dilution water consisted of 294.0 mg / L calcium chloride dihydrate, 123.3 mg / L magnesium chloride heptahydrate, 63.0 mg / L sodium bicarbonate, and 5.5 mg / L potassium chloride. The culture dishes were placed on a horizontal shaker (OS-20F) for model culture. The model culture conditions were: dark, temperature 28℃, shaker speed 80 rpm (vortex flow rate 0.08 m / s), and stimulation time 15 h. After the model culture was completed, model zebrafish were obtained and placed in a zebrafish behavior analyzer (ZebraLab3.22.3.31) to test the zebrafish's arousal activity.

[0024] Example 4: A method for creating a model of insomnia in zebrafish includes the following steps: Wild-type AB zebrafish, 4 days post-fertilization (dpf), were randomly selected and placed in plastic culture dishes (Nest Biotech, 100 mm, 50 mL). Each dish contained 30 zebrafish, and 30 mL of standard dilution water was added to each dish. The standard dilution water consisted of 294.0 mg / L calcium chloride dihydrate, 123.3 mg / L magnesium chloride heptahydrate, 63.0 mg / L sodium bicarbonate, and 5.5 mg / L potassium chloride. The culture dishes were placed on a horizontal shaker (OS-20F) for model culture. The model culture conditions were: dark, temperature 28℃, shaker speed 100 rpm (vortex flow rate 0.1 m / s), and stimulation time 15 h. After the model culture was completed, model zebrafish were obtained and placed in a zebrafish behavior analyzer (ZebraLab3.22.3.31) to test the zebrafish's arousal activity.

[0025] Example 5: A method for creating a model of insomnia in zebrafish includes the following steps: Wild-type AB zebrafish, 4 days post-fertilization (dpf), were randomly selected and placed in plastic culture dishes (Nest Biotech, 100 mm, 50 mL). Each dish contained 30 zebrafish, and 30 mL of standard dilution water was added to each dish. The standard dilution water consisted of 294.0 mg / L calcium chloride dihydrate, 123.3 mg / L magnesium chloride heptahydrate, 63.0 mg / L sodium bicarbonate, and 5.5 mg / L potassium chloride. The culture dishes were placed on a horizontal shaker (OS-20F) for modeling culture. The modeling culture conditions were: dark, temperature 28℃, shaker speed 90 rpm (vortex flow rate 0.09 m / s), and stimulation time 10 h. After the modeling culture was completed, modeled zebrafish were obtained and placed in a zebrafish behavior analyzer (ZebraLab3.22.3.31) to test the zebrafish's arousal activity.

[0026] Example 6: A method for creating a model of insomnia in zebrafish includes the following steps: Wild-type AB zebrafish, 4 days post-fertilization (dpf), were randomly selected and placed in plastic culture dishes (Nest Biotech, 100 mm, 50 mL). Each dish contained 30 zebrafish, and 30 mL of standard dilution water was added to each dish. The standard dilution water consisted of 294.0 mg / L calcium chloride dihydrate, 123.3 mg / L magnesium chloride heptahydrate, 63.0 mg / L sodium bicarbonate, and 5.5 mg / L potassium chloride. The culture dishes were placed on a horizontal shaker (OS-20F) for modeling culture. The modeling culture conditions were: dark, temperature 28℃, shaker speed 90 rpm (vortex velocity 0.09 m / s), and stimulation time 12 h. After the modeling culture was completed, modeled zebrafish were obtained and placed in a zebrafish behavior analyzer (ZebraLab3.22.3.31) to test the zebrafish's arousal activity.

[0027] Example 7: A method for creating a model of insomnia in zebrafish includes the following steps: Wild-type AB zebrafish, 4 days post-fertilization (dpf), were randomly selected and placed in plastic culture dishes (Nest Biotech, 100 mm, 50 mL). Each dish contained 30 zebrafish, and 30 mL of standard dilution water was added to each dish. The standard dilution water consisted of 294.0 mg / L calcium chloride dihydrate, 123.3 mg / L magnesium chloride heptahydrate, 63.0 mg / L sodium bicarbonate, and 5.5 mg / L potassium chloride. The culture dishes were placed on a horizontal shaker (OS-20F) for model culture. The model culture conditions were: dark, temperature 28℃, shaker speed 90 rpm (vortex flow rate 0.09 m / s), and stimulation time 18 h. After the model culture was completed, model zebrafish were obtained and placed in a zebrafish behavior analyzer (ZebraLab3.22.3.31) to test the zebrafish's arousal activity.

[0028] Example 8: A method for creating a model of insomnia in zebrafish includes the following steps: Wild-type AB zebrafish, 4 dpf post-fertilization, were randomly selected and placed in plastic culture dishes (Nest Biotech, 100 mm, 50 mL). Each dish contained 30 zebrafish and 30 mL of aquaculture water. The dishes were placed on a horizontal shaker (OS-20F) for modeling culture. The modeling culture conditions were: dark, temperature 28℃, shaker speed 90 rpm (vortex velocity 0.09 m / s), and stimulation time 15 h. After the modeling culture was completed, modeled zebrafish were obtained and placed in a zebrafish behavior analyzer (ZebraLab3.22.3.31) to test the zebrafish's arousal activity.

[0029] Comparative Example 1: Comparative Example 1 is zebrafish that did not undergo modeling experiments.

[0030] Comparative Example 2: The difference between Comparative Example 2 and Example 1 is that continuous water flow stimulation was used, with a water flow rate of 0.07 m / s and a stimulation time of 10 h.

[0031] Test example: The modeled zebrafish obtained using the modeling methods of Examples 1-7 and Comparative Examples 1-2 were tested. The test items included: the percentage of damaged area of ​​the zebrafish, the level of arousal and activity, and the mortality rate of the zebrafish. The test results are shown in Table 1: Table 1 Test Results As shown in Table 1, the results indicate that an eddy flow velocity of 0.07 m / s did not effectively establish the insomnia zebrafish model, while an eddy flow velocity above 0.08 m / s was effective. Furthermore, the level of arousal activity in the zebrafish significantly increased with increasing eddy flow velocity. A stimulation time of 10 h was sufficient to effectively establish the insomnia zebrafish model, and the level of arousal activity also significantly increased with increasing stimulation time. Zebrafish mortality occurred after 18 h of stimulation, therefore the stimulation time should be less than 18 h. The composition of the culture medium also affected the insomnia zebrafish model; compared to standard dilution water, standard dilution water significantly increased the level of arousal activity in zebrafish. Furthermore, a comparison of the effects of continuous water flow and eddies revealed that continuous water flow caused damage to zebrafish before the insomnia model was established, and the damaged area of ​​zebrafish reached 30% after the model was established, which is significant for the insomnia model. The eddy flow velocity also affected zebrafish, with the damaged area reaching 5% when the eddy flow velocity was 0.1 m / s, indicating the presence of zebrafish injury. Therefore, to avoid interference from pain factors, the flow velocity of eddy stimulation should be set below 0.1 m / s.

[0032] Verification Example 1: The method for evaluating the efficacy of melatonin (batch number F1804064, Aladdin) using a zebrafish model of insomnia includes the following steps: Normal control group, model control group, sample A group, sample B group and sample C group were set up respectively.

[0033] Normal control group: zebrafish that did not undergo modeling experiments.

[0034] Model control group: Wild-type AB zebrafish, 4 dpf post-fertilization, were randomly selected and placed in plastic culture dishes (NestBiotech, 100 mm, 50 mL). Each dish contained 30 zebrafish. 30 mL of standard dilution water was added to each dish. The standard dilution water consisted of 294.0 mg / L calcium chloride dihydrate, 123.3 mg / L magnesium chloride heptahydrate, 63.0 mg / L sodium bicarbonate, and 5.5 mg / L potassium chloride. The culture dishes were placed on a horizontal shaker (OS-20F) for model culture. The model culture conditions were: dark, temperature 28℃, shaker speed 90 rpm (vortex velocity 0.09 m / s), and stimulation time 15 h. Model zebrafish were obtained after the model culture was completed.

[0035] Sample A: Wild-type AB strain zebrafish, 4 dpf post-fertilization, were randomly selected and placed in plastic culture dishes (NestBiotech, 100 mm, 50 mL). Each dish contained 30 zebrafish. 30 mL of standard dilution water and 62.5 μg / mL of melatonin were added to each dish. The standard dilution water consisted of 294.0 mg / L calcium chloride dihydrate, 123.3 mg / L magnesium chloride heptahydrate, 63.0 mg / L sodium bicarbonate, and 5.5 mg / L potassium chloride. The culture dishes were placed on a horizontal shaker (OS-20F) for model culture. The model culture conditions were: dark, temperature 28℃, shaker speed 90 rpm (vortex flow rate 0.09 m / s), and stimulation time 15 h. Model zebrafish were obtained after the model culture was completed.

[0036] Sample B: Wild-type AB strain zebrafish, 4 dpf post-fertilization, were randomly selected and placed in plastic culture dishes (NestBiotech, 100 mm, 50 mL). Each dish contained 30 zebrafish. 30 mL of standard dilution water and 125 μg / mL of melatonin were added to each dish. The standard dilution water consisted of 294.0 mg / L calcium chloride dihydrate, 123.3 mg / L magnesium chloride heptahydrate, 63.0 mg / L sodium bicarbonate, and 5.5 mg / L potassium chloride. The culture dishes were placed on a horizontal shaker (OS-20F) for model culture. The model culture conditions were: dark, 28℃, shaker speed of 90 rpm (vortex flow rate of 0.09 m / s), and stimulation time of 15 h. Model zebrafish were obtained after the model culture was completed.

[0037] Sample C: Wild-type AB strain zebrafish, 4 dpf post-fertilization, were randomly selected and placed in plastic culture dishes (NestBiotech, 100 mm, 50 mL). Each dish contained 30 zebrafish. 30 mL of standard dilution water and 250 μg / mL of melatonin were added to each dish. The standard dilution water consisted of 294.0 mg / L calcium chloride dihydrate, 123.3 mg / L magnesium chloride heptahydrate, 63.0 mg / L sodium bicarbonate, and 5.5 mg / L potassium chloride. The culture dishes were placed on a horizontal shaker (OS-20F) for model culture. The model culture conditions were: dark, 28℃, shaker speed of 90 rpm (vortex flow rate of 0.09 m / s), and stimulation time of 15 h. Model zebrafish were obtained after the model culture was completed.

[0038] Ten model zebrafish were randomly selected from each group and placed in a zebrafish behavior analyzer (ZebraLab3.22.3.31). The arousal activity level, total arousal time, number of arousal rounds, sleep latency, total rest time, number of rest rounds, and length of rest rounds were tested. The test results are shown in Table 2.

[0039] Table 2. Results of evaluating the efficacy of melatonin in a zebrafish model of insomnia. Table 2 shows that arousal activity is a core indicator for studying sleep disorders, specifically focusing on the "duration" or "depth" of sleep maintenance. The efficacy evaluation results indicate that arousal activity in the zebrafish model control group was significantly higher than that in the normal control group, indicating that the zebrafish model of insomnia was successfully established. After administration of melatonin at concentrations of 62.5, 125, and 250 μg / mL, arousal activity in zebrafish was significantly lower than that in the model control group, demonstrating that melatonin has a significant effect on improving insomnia in zebrafish.

[0040] Total wake time is a key indicator for studying sleep quality in cases of difficulty falling asleep, based on the "total" dimension of sleep maintenance efficiency. The efficacy evaluation results above showed that the total wake time of zebrafish in the model control group was significantly higher than that of the normal control group, indicating that the zebrafish model of insomnia was successfully established. After administration of melatonin at 62.5, 125, and 250 μg / mL, the total wake time of zebrafish was significantly lower than that of the model control group, indicating that melatonin has a significant effect on improving insomnia in zebrafish.

[0041] The number of awakening rounds is a key indicator for studying difficulty falling asleep and fragmented sleep from the perspective of the "frequency" or "fragmentation" of sleep maintenance. The above efficacy evaluation results show that the number of awakening rounds in the zebrafish model control group was significantly higher than that in the normal control group, about three times higher, indicating that the zebrafish model of insomnia was successfully established. After administering melatonin 250 μg / mL, the number of awakening rounds in zebrafish was significantly lower than that in the model control group, indicating that melatonin has a significant effect on improving insomnia in zebrafish.

[0042] Sleep latency is the most direct manifestation of difficulty falling asleep, studied from the perspective of the "speed" or "difficulty" of sleep initiation. The above efficacy evaluation results show that the sleep latency of zebrafish in the model control group was significantly higher than that of the normal control group, indicating that the zebrafish model of insomnia was successfully established. After administering melatonin at 125 and 250 μg / mL, the sleep latency of zebrafish was significantly lower than that of the model control group, and even better than that of the normal control group, indicating that melatonin has a significant effect on improving insomnia in zebrafish.

[0043] The study focused on the total amount of sleep opportunities, specifically the total duration of sleep onset. The efficacy evaluation results indicated that the total rest time of zebrafish in the model control group was significantly lower than that of the normal control group, suggesting the successful establishment of the zebrafish model of insomnia. Furthermore, administration of melatonin at 250 μg / mL significantly increased the total rest time of zebrafish compared to the model control group, demonstrating that melatonin has a significant effect on improving insomnia in zebrafish.

[0044] The number of rest rounds is a dimension of "fragmentation" or "lack of persistence" in rest behavior, used to study the persistence and stability of sleep-inducing behavior. During insomnia, although individuals occasionally enter a resting state, the duration is very short, leading to an increase in the number of rest rounds throughout the night. This indicates that sleep is frequently interrupted, sleep quality is reduced, and this is a typical manifestation of sleep fragmentation. The above efficacy evaluation results show that the number of rest rounds in the model control group zebrafish was significantly higher than that in the normal control group, indicating that the zebrafish model of insomnia was successfully established. After administering melatonin at 62.5, 125, and 250 μg / mL, the number of rest rounds in zebrafish was significantly reduced compared to the model control group, indicating that melatonin has a significant effect on improving insomnia in zebrafish.

[0045] The length of rest rounds is the average duration of sleep-inducing behavior studied from the perspective of the "persistence" dimension of each continuous attempt to fall asleep. Complementing the "number of rest rounds," it measures how long one can persist in each attempt to fall asleep without giving up. A short average length indicates that patients quickly lose patience, are unable to sustain relaxation and sleep-inducing behaviors, and frequently switch back to a state of wakefulness. This is a typical manifestation of sleep difficulty and behavioral anxiety. A long average length indicates that even with difficulty falling asleep, patients can maintain continuous attempts in bed, and their behavioral patterns are more stable. In the insomnia model, due to increased activity of the central arousal system (hypothalamus-pituitary-adrenal axis, HPA axis), sleep maintenance ability is poor, rest is frequently interrupted, and the average length of each round is shortened. The above efficacy evaluation results show that the rest round length in the model control group zebrafish was significantly lower than that in the normal control group, indicating the successful establishment of the zebrafish insomnia model. After administration of melatonin 250 μg / mL, the rest round length in zebrafish was significantly higher than that in the model control group, indicating that melatonin has a significant effect on improving insomnia in zebrafish.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, alterations, and equivalent transformations made to the above embodiments based on the technical essence of the present invention shall still fall within the protection scope of the present invention.

Claims

1. A zebrafish model of insomnia, characterized in that the raw materials... include: Zebrafish and culture medium, which includes either water or diluted water, and the culture medium is a vortex with a flow velocity of 0.07~0.1 m / s.

2. The zebrafish model of insomnia according to claim 1, characterized in that, The zebrafish is a wild AB type zebrafish.

3. The zebrafish model of insomnia according to claim 1 or 2, characterized in that, Zebrafish are zebrafish that have undergone fertilization for 4-7 days (dpf).

4. The zebrafish model of insomnia according to claim 3, characterized in that, The zebrafish is a 4-dpf zebrafish after fertilization.

5. The zebrafish model of insomnia according to claim 1, characterized in that, The standard dilution water consists of calcium chloride, magnesium chloride, sodium bicarbonate, and potassium chloride.

6. The zebrafish model of insomnia according to claim 5, characterized in that, The concentration ratio of calcium chloride, magnesium chloride, sodium bicarbonate and potassium chloride is 27~30:6~7:10~12:

1.

7. The zebrafish model of insomnia according to claim 5 or 6, characterized in that, The calcium chloride used is calcium chloride dihydrate.

8. The zebrafish model of insomnia according to claim 5 or 6, characterized in that, Magnesium chloride used is magnesium chloride heptahydrate.

9. A method for establishing a zebrafish model of insomnia according to any one of claims 1 to 8, characterized in that, Includes the following steps: The culture medium was added to the petri dish to form a vortex, and then the zebrafish were placed in the petri dish for model culture. The culture conditions included a stimulation time of 10-15 h.

10. The method according to claim 9, characterized in that, The culture medium volume is 30-50 mL.

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