Method for improving success rate of single cell culture under external stimulation

Abstract

The invention provides a method for improving the success rate of single cell culture under external stimulation, belongs to the field of cell culture, and can solve the problem that the contradiction between a Raman spectrum signal and the survival rate can be formed by improving laser energy in the existing single cell culture. The method comprises the following steps: preparing a working solution containing an allogenic material; pretreating an experimental strain by using a culture medium containing the allogenic material to obtain a cell suspension; introducing the cell suspension into chambers of a micro-fluidic chip, and adjusting the cell suspension until each chamber contains at most one cell; in the presence of the allogenic material, culturing the single cell; wherein the allogenic substance is selected from one or more of an antioxidant substance, an anti-radiation substance, an anti-apoptosis substance and a repair promoting substance. According to the method, the exogenous material is added into the culture medium, and the micro-fluidic chip with a plurality of chambers is adopted for single cell culture, so that the survival rate and the clone formation rate of the single cells after Raman sorting can be remarkably improved, meanwhile, the advantage of low background interference is kept, and the survival rate of the single cells is improved.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of cell culture, and particularly relates to a method for improving the success rate of single cell culture under external stimulation. BACKGROUND

[0002] Traditional microbiology research generally takes a cell population as the research object and studies the cells of the same type at the population level. Studies have found that even microorganism cells in the same population can have significant differences at multiple levels, such as gene transcription and translation, protein activity, and metabolite abundance, indicating that there is heterogeneity among microorganism cells at multiple levels. At the same time, traditional microbiology research is generally carried out at the population level and often relies on the separation and pure culture of a certain type of cell. However, more and more studies have found that microorganisms generally exist in interdependent forms in nature, and separation and pure culture techniques are not necessarily suitable for all microorganisms. In addition, only a small part of microorganisms in nature can be cultured in the laboratory, and a large number of microorganisms cannot be explored and studied by traditional methods. Therefore, the traditional research method may not reflect the true state of microorganisms in nature, and it is also difficult to study target microorganisms that cannot achieve a pure culture system. Although techniques such as metagenomics and metatranscriptomics do not require pure culture enrichment, they still have certain difficulties in analyzing the heterogeneity among microorganism cells in complex populations and the deep-level analysis of community structure.

[0003] Single-cell Raman sorting-culturing technology has become an important means to obtain rare functional cells and establish pure cultures due to its advantages of label-free, in-situ functional recognition, and non-destructive sorting. However, this method generally has the bottleneck of low survival rate and insufficient clonal formation rate of single cells after sorting: first, during the sorting process, even if a low-power near-infrared laser is used, cells still experience transient increases in reactive oxygen species (ROS) and energy metabolism imbalance, leading to some cells entering a sub-lethal state; second, traditional culture media are designed only for population cells and lack specialized nutrients and signal support for single-cell isolation, making it difficult for cells to initiate the first division after sorting; third, the volume in microdroplets or micro-well plates is extremely small, usually tens to hundreds of picoliters, and the diffusion of exogenous antioxidant buffers and growth-promoting signal substances is limited, further exacerbating the problems of oxidative stress and growth arrest.

[0004] Currently, existing technologies mainly focus on improving laser power, sorting chip structure, or droplet generation methods, but have not systematically addressed the key issue of physiological recovery of cells after sorting. However, simply increasing laser power can improve Raman signal quality, but it also increases the level of reactive oxygen species (ROS), creating a conflict between Raman signal quality and survival rate.

[0005] Therefore, there is an urgent need in the art for a method for improving the success rate of single cell culture under external stimulation. SUMMARY

[0006] The present application aims at the technical problem that the existing single cell culture forms a contradiction between the Raman spectrum signal and the survival rate by increasing the laser energy, and provides a method for improving the success rate of single cell culture under external stimulation, which can significantly improve the survival rate and the clonal formation rate of single cell Raman sorting by adding exogenous substances in the culture medium and using a microfluidic chip with multiple chambers, while maintaining the advantages of low background interference and improving the survival rate of single cells.

[0007] In order to achieve the above-mentioned purpose, the technical scheme adopted by the present application is as follows: a method for improving the success rate of single cell culture under external stimulation, comprising the following steps: preparing a working solution containing exogenous substances; using the culture medium containing the exogenous substances to pretreat the experimental strain to obtain a cell suspension; introducing the cell suspension into the chambers of the microfluidic chip, and adjusting to contain at most one cell in each chamber; culturing the single cells in the presence of the exogenous substances; wherein the exogenous substances are selected from one or more of antioxidant substances, anti-radiation substances, anti-apoptotic substances, and repair-promoting substances, and the final concentration of the exogenous substances in the culture medium is preferably 5 µg / mL-1 mg / mL.

[0008] In one embodiment, the exogenous substances are selected from one or more of the following categories: antioxidant substances, including vitamin C, glutathione, ferrous salt, coenzyme Q10, catalase, superoxide dismutase, reduced coenzyme II, astaxanthin, N-acetylcysteine; anti-radiation substances, including tetrahydropyrimidine, melanin, microbial polysaccharide, chitooligosaccharide; anti-apoptotic substances, including ferroptosis inhibitors; repair-promoting substances, including growth factors, cytokines, heat shock protein inducers, and DNA repair enzyme cofactors; and derivatives, precursors or combinations thereof.

[0009] In one embodiment, the pretreatment step comprises inoculating single colonies of the experimental strain in the culture medium for activation to obtain a cell suspension, and then diluting the cell suspension with the culture medium containing the exogenous substances to make the sample concentration reach a predetermined range. In order to prevent cell adhesion in the chip, buffer and culture medium containing exogenous substances are added at the same time when the cell suspension is diluted.

[0010] In one embodiment, the step of adjusting each chamber to contain at most one cell comprises: using fluid driving to inject the pretreated cell suspension into the multiple chambers of the microfluidic chip; and then removing the excess cells so that at most one cell remains in each chamber. In actual operation, the excess cells in each chamber of the chip can be dragged into the channel using optical tweezers to adjust at most one cell in each chamber; and the excess cells in the channel of the chip can be flushed out of the chip using the corresponding medium.

[0011] In one embodiment, after adjusting each chamber to contain at most one cell, the occupancy rate of the chambers containing single cells is detected; when the occupancy rate reaches or exceeds a preset target value, the subsequent culture step is performed; if not, the cell suspension injection and adjustment steps are repeated until the requirement is met. Preferably, the preset target value of the occupancy rate of the chambers containing single cells is 2 / 3.

[0012] In one embodiment, the step of culturing the single cells comprises: isolating each chamber containing single cells from the channel of the chip to form independent micro-culture units; and then culturing under suitable cell growth conditions. Preferably, air is introduced into the channel of the chip to expel the medium in the channel of the chip, so as to divide each chamber into an independent culture unit, and then the chip is sealed at both ends and placed in a culture box for 16-20 hours of culture. More preferably, the culture box has a temperature of 37°C. In the technical solution of the present application, air is used to isolate the chambers, so that each chamber is divided into an independent micro-culture unit, which can avoid the influence of the connection between adjacent chambers on the result judgment during cell growth.

[0013] In one embodiment, the method improves the survival rate of cells after being subjected to external stimulation to 48.15%.

[0014] In one embodiment, the external stimulation is selected from one or more of physical stimulation, chemical stimulation or biological stimulation. Preferably, the physical stimulation uses laser irradiation, the wavelength of the laser is adjusted to 532 nm, the laser intensity is 25-50 mW, and the irradiation time of the single cells in the chamber is 0.5-2 s.

[0015] Compared with the prior art, the present application has the advantages and positive effects that: by adding a specific concentration of exogenous substances to the culture medium and using a microfluidic chip with multiple chambers for single cell culture, the present application can significantly improve the survival rate and cloning efficiency of single cells after Raman sorting, and improve the success rate of single cell culture; the exogenous substances added in the present application will not affect the Raman signal of the cells, and at the same time will help the cells to resist oxidation, protect the cells in a radiation environment, promote cell growth, etc. to ensure the survival of single cells, thereby improving the success rate of single cell culture. When the laser energy is 25 mJ, the survival rate of single cells can reach 48.15%. BRIEF DESCRIPTION OF DRAWINGS

[0016] Figure 1 Structure diagram of one embodiment of the chip used for single cell culture in the embodiment of the present application; Figure 2 Comparison chart of single cell survival rates of the embodiment 1 and the comparative example 1 of the present application; Figure 3 Comparison chart of single cell survival rates of the embodiment 2 and the comparative example 1 of the present application; Figure 4 Comparison chart of single cell survival rates of the embodiment 3 and the comparative example 2 of the present application; Figure 5 Comparison chart of single cell survival rates of the embodiment 4 and the comparative example 2 of the present application; Wherein, 1, channel; 2, chamber. DETAILED DESCRIPTION

[0017] The technical solutions in the embodiments of the present application will be described clearly and completely below. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0018] The embodiment of the present application provides a method for improving the success rate of single cell culture under external stimulation. By adding exogenous substances in the culture medium and using a microfluidic chip with multiple chambers for single cell culture, a higher quality Raman signal can be obtained, and the success rate of single cell culture is promoted. The exogenous substances added in the present application will not affect the Raman signal of the cells, and at the same time will help the cells to resist oxidation, protect the cells in the radiation environment, promote the growth of the cells, etc. to ensure the survival of the single cells, thereby improving the success rate of single cell culture. When the laser energy is 25mJ, the single cell survival rate is 31.2%-48.15%; when the laser energy is 50mJ, the single cell survival rate is 25.6%-28.4%.

[0019] In order to more clearly and specifically introduce the method for improving the success rate of single cell culture under external stimulation provided by the embodiments of the present application, the following will be described in combination with specific embodiments.

[0020] Embodiment 1 The method for improving the success rate of single cell culture under external stimulation in the present embodiment specifically comprises the following steps: S1, preparation of vitamin C working solution: 10 mg of vitamin C powder was weighed and dissolved in 1 mL of sterile water, and then filtered and sterilized with a 0.22 μm filter to prepare a 10 mg / mL vitamin C working solution; part of the vitamin C working solution was added to the LB medium to make the final concentration of vitamin C 5 μg / mL, and the remaining vitamin C working solution was stored at -20°C after being divided into small portions to prevent loss of efficacy due to repeated freeze-thawing; S2, activation and quantification of experimental strain: E. coli ATCC 35218 was used as the experimental strain in this example, a single colony was inoculated into LB medium and incubated at 37°C overnight to activate the sample; the sample was diluted with a buffer containing 1% PF127 and a vitamin C working solution, and the concentration was adjusted to 10^6 CFU / mL by counting with a hemocytometer; S3, sample injection and adjustment of cell number in the chamber: S3.1, using a negative pressure injection device, 95 kPa negative pressure for 1 min, the sample with adjusted concentration was injected into the 60-hole culture chip, and the structure of the chip is shown in Figure 1 ; S3.2, after the sample entered the chamber 2, the number of cells in each chamber 2 was observed under a microscope to ensure that at least 2 / 3 of the chambers 2 had cells, otherwise the sample was re-injected; S3.3, the excess cells in the chamber 2 were dragged into the channel 1 using a light tweezer, and the chamber 2 without cells was ignored to ensure that there was only one cell in the chamber 2 with cells; S3.4, the excess cells in the channel 1 of the chip were flushed out of the chip using the corresponding medium to prevent cells from entering the chamber 2; S4, single cell culture: S4.1, adjust the laser intensity to 532 nm, irradiate the single cell in the chamber 2 for 1 s in the range of 25 mW; air was introduced into the channel 1 of the chip to completely drain the medium in the channel 1, and each chamber 2 was divided into an independent culture unit; S4.2, the chip was sealed and placed in a culture dish with water to prevent the chamber 2 from drying out, and then cultured in a 37°C incubator for 18 h, and the single cell growth rate was calculated according to the following formula, .

[0021] Example 2 The method of this example is used to improve the success rate of single cell culture under external stimulation, which specifically includes the following steps: S1, preparation of Fe 2+ working solution: 10 mg of ferrous sulfate heptahydrate powder was weighed and dissolved in 1 mL of sterile water, and then filtered and sterilized with a 0.22 μm filter to prepare a 10 mg / mL Fe2+ Working solution; take part Fe 2+ Working solution is added to LB medium to make Fe 2+ The final concentration is 27.8 μg / mL, and the remaining Fe 2+ The working solution is divided and stored at -20℃. S2, Activation of experimental strain and quantification of bacterial solution concentration: In this example, Escherichia coli ATCC 35218 is used as the experimental strain. A single colony is inoculated into LB medium and activated overnight at 37℃. The sample is diluted with a buffer containing 1% PF127, Fe 2+ Working solution, and the sample concentration is adjusted to 10^6 CFU / mL by counting with a hemocytometer. S3, Sample injection and adjustment of cell number in the chamber of the microfluidic chip: S3.1, The sample with adjusted concentration is injected into the 60-hole culture chip using a negative pressure injection device at 95 kPa for 1 min. The structure of the chip is shown in Figure 1 . S3.2, After the sample enters the chamber 2 of the chip, the number of cells in each chamber 2 is observed under a microscope to ensure that at least 2 / 3 of the chambers 2 have cells. Otherwise, the sample is re-injected. S3.3, The excess cells in the chamber 2 are dragged into the channel 1 using a light tweezer. The chambers 2 without cells are ignored, and only one cell is ensured in the chambers 2 with cells. S3.4, The excess cells in the channel 1 of the chip are flushed out of the chip using the corresponding medium to prevent the cells from entering the chamber 2. S4, Single cell culture: S4.1, The laser intensity is adjusted to 532 nm, and the single cell in the chamber 2 is irradiated for 1 s in the range of 25 mW. Air is introduced into the channel 1 of the chip to completely drain the medium in the channel 1, and each chamber 2 is divided into an independent culture unit. S4.2, The chip is sealed and placed in a culture dish with water to prevent the chamber 2 from drying. After 18 h of culture in a 37℃ incubator, the single cell growth rate is calculated according to the following formula, .

[0022] Example 3 This example is used to improve the success rate of single cell culture under external stimulation. The method specifically includes the following steps: S1, Preparation of Fe 2+ Working solution: 10 mg of ferrous sulfate heptahydrate powder is weighed and dissolved in 1 mL of sterile water. Then, it is filtered and sterilized using a 0.22 μm filter membrane to prepare a Fe 2+ Working solution: 10 mg of ferrous sulfate heptahydrate powder is weighed and dissolved in 1 mL of sterile water. Then, it is filtered and sterilized using a 0.22 μm filter membrane to prepare a Fe2+ The working solution was added to LB medium to allow Fe 2+ The final concentration was 278 μg / mL, and the remaining Fe... 2+ The working solution should be aliquoted and stored at -20°C. S2. Activation of the experimental strain and quantification of bacterial concentration: In this example, *Escherichia coli* ATCC 35218 was used as the experimental strain. Single colonies were picked and inoculated into LB medium, activated overnight at 37°C, and used as samples. A buffer containing 1% PF127 and Fe2+ were used. 2+ The working solution was used to dilute the sample, and the sample concentration was adjusted to 10^6 CFU / mL by counting with a hemocytometer. S3. Microfluidic chip sample introduction and adjustment of cell count within the chamber: S3.1 Using a negative pressure injection device, apply a negative pressure of 95 kPa for 1 minute to inject the adjusted sample concentration into a 60-well culture chip. The chip structure is as follows: Figure 1 As shown; S3.2 After the sample enters the chip chamber 2, observe the number of cells in each chamber 2 under a microscope to ensure that at least 2 / 3 of the chambers 2 contain cells; otherwise, re-inject the sample. S3.3 Use optical tweezers to drag the excess cells in chamber 2 into channel 1. Ignore chamber 2 which has no cells, ensuring that there is only 1 cell in chamber 2 with cells. S3.4 Use the corresponding culture medium to flush out excess cells from chip channel 1 to prevent cells from entering chamber 2; S4, Single-cell culture: S4.1 Adjust the laser intensity to 532nm and irradiate the single cells in chamber 2 for 1s within a range of 50mW; introduce air into the chip channel to drain all the culture medium in chip channel 1, and divide each chamber 2 into an independent culture unit; S4.2 Seal the chip and place it in a water-filled culture dish to prevent chamber 2 from drying out. Incubate at 37°C for 18 hours, then calculate the single-cell growth rate using the following formula. .

[0023] Example 4 This embodiment describes a method for improving the success rate of single-cell culture under external stimuli, which specifically includes the following steps: S1. Preparation of tetrahydropyrimidine working solution: Weigh 10 mg of tetrahydropyrimidine powder, add it to 1 mL of sterile water to dissolve, and then filter it through a 0.22 μm filter membrane to sterilize it, thus preparing a 10 mg / mL tetrahydropyrimidine working solution; add a portion of the tetrahydropyrimidine working solution to LB medium to make the final concentration of tetrahydropyrimidine 1 mg / mL, and aliquot the remaining tetrahydropyrimidine working solution and store it at -20℃; S2. Activation of experimental strain and quantitative bacterial concentration: In this example, Escherichia coli ATCC 35218 was used as the experimental strain. A single colony was picked and inoculated into LB medium and activated overnight at 37°C as a sample. The sample was diluted with a medium containing 1% PF127 and tetrahydropyrimidine working solution. The sample concentration was adjusted to 10^6 CFU / mL by counting with a hemocytometer. S3. Microfluidic chip sample introduction and adjustment of cell count within the chamber: S3.1 Using a negative pressure injection device, apply a negative pressure of 95 kPa for 1 minute to inject the adjusted sample concentration into a 60-well culture chip. The chip structure is as follows: Figure 1 As shown; S3.2 After the sample enters the chip chamber 2, observe the number of cells in each chamber 2 under a microscope to ensure that at least 2 / 3 of the chambers 2 contain cells; otherwise, re-inject the sample. S3.3 Use optical tweezers to drag the excess cells in chamber 2 into channel 1. Ignore chamber 2 which has no cells, ensuring that there is only 1 cell in chamber 2 with cells. S3.4 Use the corresponding culture medium to flush out excess cells from the chip channels to prevent cells from entering the chamber; S4, Single-cell culture: S4.1 Adjust the laser intensity to 532nm and irradiate the single cells in chamber 2 for 1s within a range of 50mW; introduce air into chip channel 1 to drain all the culture medium in chip channel 1 and divide each chamber 2 into an independent culture unit; S4.2 Seal the chip and place it in a water-filled culture dish to prevent chamber 2 from drying out. Incubate at 37°C for 18 hours, then calculate the single-cell growth rate using the following formula. .

[0024] Comparative Example 1 The difference from Example 1 is that this comparative single-cell culture method does not add exogenous working solutions to the culture medium, but uses a culture medium containing only buffer for dilution, specifically including the following steps: S1. Activation of experimental strains and quantitative bacterial concentration: In this comparative example, Escherichia coli ATCC 35218 was used as the experimental strain. Single colonies were picked and inoculated into LB medium and activated overnight at 37°C to serve as samples. The samples were diluted with medium containing 1% PF127 and the concentration was adjusted to 10^6 CFU / mL by counting with a hemocytometer. S2. Microfluidic chip sample introduction and adjustment of cell count within the chamber: S2.1 Using a negative pressure injection device, apply a negative pressure of 95 kPa for 1 minute to inject the adjusted sample concentration into a 60-well culture chip. The chip structure is as follows: Figure 1 As shown; S2.2 After the sample enters the chip chamber 2, observe the number of cells in each chamber 2 under a microscope to ensure that at least 2 / 3 of the chambers 2 contain cells; otherwise, re-inject the sample. S2.3 Use optical tweezers to drag the excess cells in the chamber to channel 1. Ignore the cellless chamber 2 to ensure that there is only 1 cell in the cell-containing chamber 2. S2.4 Use culture medium to flush out excess cells from chip channel 1 to prevent cells from entering chamber 2; S3, Single-cell culture: S3.1 Adjust the laser intensity to 532nm and irradiate the single cells in chamber 2 for 1s within a range of 25mW; introduce air into chip channel 1 to drain all the culture medium in chip channel 1 and divide each chamber 2 into an independent culture unit; S3.2 Seal the chip and place it in a water-filled culture dish to prevent chamber 2 from drying out. Incubate at 37°C for 18 hours, then calculate the single-cell growth rate using the following formula. .

[0025] Comparative Example 2 The difference from Example 3 is that this comparative single-cell culture method does not add exogenous working solutions to the culture medium, but uses a culture medium containing only buffer for dilution, specifically including the following steps: S1. Activation of experimental strains and quantitative bacterial concentration: In this comparative example, Escherichia coli ATCC 35218 was used as the experimental strain. Single colonies were picked and inoculated into LB medium and activated overnight at 37°C to serve as samples. The samples were diluted with medium containing 1% PF127 and the concentration was adjusted to 10^6 CFU / mL by counting with a hemocytometer. S2. Microfluidic chip sample introduction and adjustment of cell count within the chamber: S2.1 Using a negative pressure injection device, apply a negative pressure of 95 kPa for 1 minute to inject the adjusted sample concentration into a 60-well culture chip. The chip structure is as follows: Figure 1 As shown; S2.2 After the sample enters the chip chamber 2, observe the number of cells in each chamber 2 under a microscope to ensure that at least 2 / 3 of the chambers 2 contain cells; otherwise, re-inject the sample. S2.3 Use optical tweezers to drag the excess cells in chamber 2 into channel 1. Ignore chamber 2 which has no cells, ensuring that there is only 1 cell in chamber 2 with cells. S2.4 Use culture medium to flush out excess cells from chip channel 1 to prevent cells from entering chamber 2; S3, Single-cell culture: S3.1 Adjust the laser intensity to 532nm and irradiate the single cells in chamber 2 for 1s within a range of 50mW; introduce air into chip channel 1 to drain all the culture medium in chip channel 1, and divide each chamber 2 into an independent culture unit; S3.2 Seal the chip and place it in a water-filled culture dish to prevent chamber 2 from drying out. Incubate at 37°C for 18 hours, then calculate the single-cell growth rate using the following formula. .

[0026] The laser energy and single-cell growth rate of Examples 1-4 and Comparative Examples 1-2 are shown in Table 1 and Appendix. Figures 1-4 As shown.

[0027] Table 1. Laser energy and single-cell growth rate in Examples 1-2 and Comparative Examples 1-2

[0028] Figure 1 The graph shows a comparison between the cell viability of Example 1 (with vitamin C (VC) working solution) and Comparative Example 1 (CG1). When the laser energy is 25 mJ, compared with Comparative Example 1, the single cell viability of Example 1 increased from 12.9% to 48.15% after adding 5 μg / mL of vitamin C to the culture medium. That is, the cell viability of Example 1 (with vitamin C working solution) is 3.73 times that of Comparative Example 1.

[0029] Figure 2 Ferrous ions (Fe) were added to Example 2 2+The graph shows a comparison between the cell survival rate of the working solution and the cell survival rate of Comparative Example 1 (CG1). When the laser energy is 25 mJ, compared with Comparative Example 1, the single cell survival rate increased from 12.9% to 31.2% after adding 27.8 μg / mL of ferrous ions to the culture medium of Example 2. That is, the cell survival rate of the cell with added ferrous ions increased by 2.42 times compared with Comparative Example 1.

[0030] Figure 3 For Example 3, a high concentration of ferrous ions (Fe) was added. 2+ The graph compares the cell viability of cell cultured in the (high) working solution with that of Comparative Example 2 (CG2). When the laser energy is 50 mJ, compared with Comparative Example 2, the single cell viability increased from 8.3% to 25.6% after adding 278 μg / mL of ferrous ions to the culture medium in Example 3. This shows that adding a high concentration of ferrous ions can improve cell viability under high laser energy irradiation.

[0031] Figure 4 This is a comparison of cell viability in Example 4 (with tetrahydropyrimidine (Ectoin) working solution and cell viability in Comparative Example 2 (CG2). When the laser energy is 50 mJ, compared with Comparative Example 2, the single cell viability in Example 4 increased from 8.3% to 28.4% after adding 1 mg / mL of tetrahydropyrimidine to the culture medium. That is, the cell viability in Example 4 (with tetrahydropyrimidine working solution) is 3.42 times that of Comparative Example 2.

[0032] As can be seen from the above, this invention can improve the success rate of single-cell culture by adding a specific concentration of exogenous substances to the culture medium and using laser irradiation. Vitamin C, ferrous ions, and tetrahydropyrimidine can all improve the survival rate of single cells to varying degrees. Among them, vitamin C can scavenge ROS in the environment, protect cells from oxidative stress damage, maintain redox balance, and can be used as a carbon source to promote growth and metabolism. When added to the culture medium as an exogenous substance working solution for single-cell culture, the survival rate of single cells can reach 48.15%. Ferrous ions can promote cellular energy metabolism, increase the energy supply of cells, and induce the expression of antioxidant defense systems to reduce ROS damage to cells.

[0033] The above-described embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, evolutions, or improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A method for improving the success rate of single-cell culture under external stimuli, characterized in that, Includes the following steps: Prepare a working solution containing exogenous substances; The experimental strains were pretreated with a culture medium containing the exogenous substance to obtain a cell suspension; The cell suspension is introduced into the chambers of the microfluidic chip and adjusted so that each chamber contains at most one cell; The single cell is cultured in the presence of the exogenous substance; The exogenous substance is selected from one or more of antioxidants, anti-radiation substances, anti-apoptotic substances, and repair-promoting substances.

2. The method according to claim 1, characterized in that, The exogenous substance is selected from one or more of the following categories: Antioxidants include vitamin C, glutathione, ferrous salts, coenzyme Q10, catalase, superoxide dismutase, reduced coenzyme II, astaxanthin, and N-acetylcysteine. Anti-radiation substances include tetrahydropyrimidine, melanin, microbial polysaccharides, and chitosan oligosaccharides; Anti-apoptotic substances, including ferroptosis inhibitors; Repair-promoting substances include growth factors, cytokines, heat shock protein inducers, and DNA repair enzyme cofactors; And derivatives, precursors or combinations thereof of the aforementioned substances.

3. The method according to claim 1, characterized in that, The pretreatment step includes: inoculating a single colony of the experimental strain into a culture medium for activation to obtain a cell suspension; and then diluting the cell suspension with a culture medium containing the exogenous substance to bring the sample concentration to a predetermined range.

4. The method according to claim 1, characterized in that, The step of adjusting each chamber to contain at most one cell includes: using fluid drive to infuse a pretreated cell suspension into multiple chambers of the microfluidic chip; and then removing excess cells so that each chamber contains at most one cell.

5. The method according to claim 4, characterized in that, After adjusting each chamber to contain at most one cell, the occupancy rate of single cells in each chamber is detected. When the occupancy rate reaches or exceeds a preset target value, subsequent culture steps are performed. If the target value is not reached, the cell suspension addition and adjustment steps are repeated until the requirements are met.

6. The method according to claim 1, characterized in that, The step of culturing the single cells includes: isolating each chamber containing the single cells from the chip channels to form an independent micro-culture unit; and then culturing them under suitable cell growth conditions.

7. The method according to any one of claims 1-6, characterized in that, The method improves cell survival rate to 48.15% after exposure to external stimuli.

8. The method according to any one of claims 1-6, characterized in that, The external stimulus is selected from one or more of physical, chemical, or biological stimuli.

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