Construction method of cytotoxicity assessment model based on chip and application and method of cytotoxicity assessment model in in-vitro cytotoxicity assessment
By designing a multi-channel chip for cytotoxicity evaluation, combining AI to analyze cell image chromaticity, and calculating cell relative activity, it solves the problems of cumbersome operation, long cycles and unstable data of the existing method, and achieves a fast and repeatable cytotoxicity detection effect.
Patent Information
- Application Number
- CN202411615715.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-13
- Publication Date
- 2025-03-21
AI Technical Summary
The existing cytotoxicity assessment methods have problems such as cumbersome operation, long cycles, and unstable data. Especially when using MTT method, dimethyl sulfoxide (DMSO) is harmful to cells and experimenters, with low sensitivity, narrow linear range, unstable data and poor repetition.
A multi-channel chip is designed for the culture of multiple cells in an in vitro cytotoxicity assessment. By taking pictures of the cells and performing picture chromaticity analysis in combination with AI analysis, the relative activity of the cells was calculated, and the toxicity of the substance to be tested to cells was evaluated.
The cytotoxicity detection of compounds is achieved quickly, standardized and repeatable, and the dispersed and grouped culture of cells is achieved through the principle of fluid mechanics, and the cells in the chip are directly stained with formazan crystals, achieving rapid and accurate detection results.
Smart Images
Figure CN119662766A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microfluidic chips, and in particular relates to a method for constructing a chip-based cytotoxicity assessment model and an application and method thereof in in vitro cytotoxicity assessment. Background Art
[0002] Cytotoxicity assessment is an important means to evaluate the toxic effects of compounds or drugs on cells in vivo. Cytotoxicity evaluation criteria are used to statistically analyze the results of cytotoxicity assessment to determine the degree of cytotoxicity of compounds or drugs. Common evaluation criteria include cell survival rate, cell survival curve, IC 50 In addition, in the evaluation of cytotoxicity, comprehensive evaluation can be performed by measuring indicators such as cell apoptosis, cell cycle and cell damage. Cytotoxicity assessment methods and evaluation criteria have important application significance in the fields of drug development, environmental toxicology, etc.
[0003] Cytotoxicity assessment methods are mainly divided into two categories: in vitro cytotoxicity assessment and in vivo cytotoxicity assessment. In vitro cytotoxicity assessment is the evaluation of compounds or drugs using a cell culture system. Common methods include the MTT method and the CCK8 method. In addition, cell imaging can be performed using the Live / dead double staining method to more intuitively record cell survival. In vivo cytotoxicity assessment is the evaluation of compounds or drugs through in vivo animal experiments, such as the mouse lethal dose 50% (LD50) test and the mouse bone marrow micronucleus rate test.
[0004] The detection principle of MTT method is that succinate dehydrogenase in the mitochondria of living cells can reduce exogenous MTT (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide) to water-insoluble blue-purple crystalline formazan and deposit it in the cells, while dead cells do not have this function. Dimethyl sulfoxide (DMSO) can dissolve the formazan in the cells, and its light absorption value is measured at a specific wavelength using an enzyme-linked immunosorbent assay, which can indirectly reflect the number of living cells. Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells.
[0005] In the prior art, it has been proposed that the MTT staining method is the preferred method for toxicity assessment of cosmetic chemical raw materials in in vitro corneal epithelial cell short-term exposure tests. The MTT method for detecting cytotoxicity is applicable to various types of cells, including suspended cells and adherent cells, but dimethyl sulfoxide (DMSO) is required to dissolve the formazan during the operation. DMSO is harmful to both cells and experimenters, has low sensitivity, narrow linear range, unstable data, and poor repeatability. Cytotoxicity experiments are usually performed in 96-well plates, which have problems such as cumbersome operations and long experimental cycles.
[0006] With the rapid development of cell biology and toxicology, modern cytotoxicity assessment methods are also constantly updated and improved. Summary of the invention
[0007] In view of the problems existing in the prior art, the present invention aims to provide a method for constructing a cytotoxicity assessment model and its application, so as to solve the problems of cumbersome operation, long cycle, unstable data, etc. existing methods of using cytotoxicity to assess cytotoxicity. The present invention designs a multi-channel chip in which a variety of cells can be cultured. By taking pictures of the cells and combining AI analysis with image colorimetric analysis, the relative activity of the cells is calculated, and then the toxicity of the test substance to the cells is evaluated.
[0008] The technical solution adopted by the present invention is as follows:
[0009] A method for constructing a chip-based cytotoxicity assessment model, the method comprising the following steps:
[0010] Step 1. Structural design and fabrication of cytotoxicity assessment chip.
[0011] Step 2. In-chip cytotoxicity assay.
[0012] Specifically, the cytotoxicity assessment chip described in step 1 comprises a plurality of independent units, each of which is provided with a plurality of parallel pipes or cell culture areas of arbitrary shapes, and these pipes or areas can be connected to a total inlet or a total outlet, or the inlets and outlets can be independently provided in parallel. The area and volume of all cell culture areas are equal to ensure that the fluid can be evenly distributed during the test.
[0013] Preferably, in the independent unit, the parallel straight pipes have an input diameter of 0.1 mm, an output diameter of 0.1 mm, an inner diameter of 0.5 mm, and a length of 2.5 mm.
[0014] Specifically, step 2 includes:
[0015] Step 2-1. Prepare cell suspension and chip pretreatment;
[0016] Step 2-2. Inject the cell suspension into the cytotoxicity assessment chip and culture until the cells adhere to the wall;
[0017] Step 2-3. Inject the test substance into the cytotoxicity assessment chip and incubate;
[0018] Step 2-4. After the incubation, detect the cell viability.
[0019] Preferably, in step 2-1, the cells in the cell suspension are selected from cells derived from human skin.
[0020] Further preferably, in step 2-1, the cells in the cell suspension include but are not limited to any one or more of fibroblasts, melanocytes, and keratinocytes.
[0021] Specifically, in step 2-2, the cell density of the cell suspension is 1×10 6 -5×10 6 / mL, and the injection volume of the cell suspension is 10-30μL.
[0022] Preferably, in step 2-2, the cell density of the cell suspension to be tested is 2×10 6 / mL, and the injection volume of the cell suspension to be tested is 20μL.
[0023] In some specific embodiments, step 2 comprises:
[0024] S21. Separating, extracting, and culturing the fibroblasts on a large scale to obtain a cell suspension of a certain density.
[0025] S22. Inject a cell suspension of a certain density into the channel of the cytotoxicity evaluation chip, transfer the chip to a 37° C., 5% CO 2 incubator for static culture, and wait for the cells to adhere to the wall.
[0026] S23. Inject a solution of the test compound of a certain concentration into the channel of the cytotoxicity evaluation chip, and transfer the chip to a 37° C., 5% CO 2 incubator for static culture.
[0027] S24. After the compound incubation is completed, the MTT solution is injected into the channel of the cytotoxicity evaluation chip, and the chip is transferred to a 37° C., 5% CO 2 incubator for 4 hours and then photographed and counted.
[0028] Optionally, the cells tested in this cytotoxicity assessment chip are not limited to fibroblasts, melanocytes, keratinocytes or other cells.
[0029] Optionally, the cells tested in this cytotoxicity assessment chip are cells within 5 generations of recovery.
[0030] Optionally, the time for the cells to adhere to the wall during S22 may be determined according to the cell type and cell characteristics.
[0031] Preferably, in steps 2-4, the method for detecting cell viability is the MTT method.
[0032] In step 2-4, the cell viability detection is combined with AI analysis to perform image colorimetric analysis and then calculate the relative activity of the cells, thereby evaluating the toxicity of the test substance to the cells.
[0033] In another aspect, the present invention provides a cytotoxicity assessment model constructed by the aforementioned construction method and related applications.
[0034] Said applications include, but are not limited to, in vitro cytotoxicity assessment.
[0035] The substances to be tested in the in vitro cytotoxicity assessment application include, but are not limited to, any one or more of food, medicine, health care products and cosmetics.
[0036] Preferably, the substance to be tested includes but is not limited to small molecule compounds. Further preferably, the application is used for screening or quality control of small molecule compounds.
[0037] Based on the above, the present invention provides an in vitro cytotoxicity assessment method, which includes the construction method of the aforementioned cytotoxicity assessment model
[0038] The beneficial effects of the present invention are as follows:
[0039] The present invention integrates a microfluidic control system with a cell culture mode, and realizes a dynamic culture and a sufficient exchange of nutrients based on a chip, which more simulates the real physiological conditions of cells in the human body. The multi-channel cytotoxicity assessment chip of the present invention can be applied to a variety of cells for rapid, standardized, and repeatable cytotoxicity detection of compounds. By using a cytotoxicity assessment chip to construct an MTT cytotoxicity assessment model, the dispersed grouping culture of cells is realized by relying on the principle of fluid mechanics; by directly staining the cells in the chip with formazan crystals, rapid and accurate detection results are achieved. BRIEF DESCRIPTION OF THE DRAWINGS
[0040] Figure 1 This is a schematic diagram of an independent unit in the cytotoxicity assessment chip of Example 1.
[0041] Figure 2 Bright field schematic of fibroblasts seeded within the chip for multichannel cytotoxicity assessment.
[0042] Figure 3 This is a bright field schematic diagram of cell morphology after incubation with different concentrations of test substances in the multi-channel cytotoxicity assessment chip and then adding MTT solution for incubation. DETAILED DESCRIPTION
[0043] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.
[0044] In the present invention:
[0045] “PBS” refers to phosphate buffered saline, “FBS” refers to fetal bovine serum, “Low-Glucose-DMEM” refers to low-glucose DMEM medium, “P / S” refers to penicillin / streptomycin, and “MTT” refers to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide.
[0046] Some of the raw materials and sources involved in the specific implementation of the present invention are as follows:
[0047] Fibroblast (human primary fibroblasts) were purchased from Shaanxi Boxi General Detection Technology Co., Ltd.; DMEM medium, P / S, and trypsin-EDTA digestion solution were all purchased from Gibco; FBS was purchased from Lanzhou Rongye Biotechnology Co., Ltd.; PBS was purchased from Beijing Solebow Technology Co., Ltd.; 95% ethanol was purchased from Hebei Ruikang Pharmaceutical Technology Co., Ltd.; MTT was purchased from Beyotime Biotechnology Co., Ltd.
[0048] The optical imaging instrument involved in the specific implementation of the present invention is: Olympus high-resolution inverted microscope, which is used for cell photography.
[0049] Example 1 Construction of a cytotoxicity assessment model based on a multi-channel chip
[0050] The following steps are involved:
[0051] S1, Structural design and production of cytotoxicity assessment chip, including:
[0052] S11, each chip contains multiple independent units, each unit has a number of parallel channels or cell culture areas of arbitrary shapes;
[0053] S12, the pipes or areas in each unit can be connected to a main inlet or a main outlet, or the inlets and outlets can be set up in parallel and independently. The area and volume of all cell culture areas are equal to ensure that the fluid can be evenly distributed during the test. In the independent unit, the input diameter of the parallel straight pipe is 0.1mm, the output diameter is 0.1mm, the inner diameter is 0.5mm, and the length is 2.5mm. As an example, the schematic diagram of the independent unit of this embodiment is as follows Figure 1 shown.
[0054] S2, on-chip cytotoxicity testing applications, including:
[0055] S21, Isolation and culture of human primary fibroblasts: Place human skin tissue in a culture dish and wash it 4-5 times with 4°C precooled PBS solution containing 1% P / S to remove blood stains on the skin surface. Cut subcutaneous tissue with ophthalmic scissors and wash it 4-5 times with 4°C precooled PBS solution (containing 1% P / S). Use scissors to cut the tissue into 1×1 mm 2 The size of the cells was added to 2U / mL of dispase digestion solution and digested overnight (24h) at 4℃. The dermis was separated from the epidermis using surgical forceps, and the separated dermal tissue was placed in a culture dish. After digestion at 37℃ for 30min with 0.25% trypsin-EDTA digestion solution, the digestion was terminated with DMEM culture solution containing 10% fetal bovine serum, filtered with a 200-mesh sieve, and the supernatant was taken after centrifugation to collect the precipitated cells. The cell pellet was washed and resuspended with PBS, and the cells were resuspended with fibroblast culture medium, inoculated into a T75 bottle, and cultured in a 37℃, 5% CO2 incubator. The medium was changed every 24h, and the cells were passaged when the plating rate was about 80%-85%. The cells were expanded to the third to fifth generations and can be used for model construction.
[0056] The above-mentioned fibroblast culture medium: 5 ng / mL insulin, 1 μg / mL thyroxine, 18 μg / mL basic fibroblast growth factor and 1.5 μg / mL recombinant human transforming growth factor (TGF) were added to the DMEM / F12 mixed culture medium.
[0057] S22, the chip is sterilized by high pressure, and the outer package of the pre-cooled chip is disinfected with 75% alcohol, and then quickly placed in a sterile operating table, the outer package is torn open and taken out, and modified and rinsed with 95% ethanol and PBS in turn, and all pipelines are checked under a microscope to be intact and set aside.
[0058] S23, digest the qualified fibroblasts under microscopic examination with trypsin, centrifuge, count, etc., and adjust the cell suspension density to 2×10 6 100 μL / mL, injected into the channel of the cytotoxicity evaluation chip, 20 μL of cell suspension was injected into each chip, and the chip was transferred to a 37°C, 5% CO2 incubator for static culture to wait for the cells to adhere to the wall.
[0059] S24, prepare the test substance with DMSO and culture medium. In this embodiment, the test substance is resveratrol (Synonyms: trans-Resveratrol; SRT501; CAS No.: 501-36-0), and the test concentration is 0.00625, 0.0125, 0.025, 0.05, 0.1, 0.2, 0.4 (mg / mL), and the DMSO content in the compound solution must be less than 0.1%. At the same time, prepare 10% DMSO solution as a positive control.
[0060] S25, after the cells have attached to the wall (4 hours in this embodiment), the prepared test substance solution is injected into the channels of the cell toxicity evaluation chip, and the chip is transferred to a 37°C, 5% CO2 incubator for static culture. Figure 2 As shown, the fibroblasts injected into the chip can completely adhere to the wall within 4 hours, and the number of cells in each chip is basically the same.
[0061] S26, after the compound incubation is completed, the MTT solution is injected into the channels of the cell toxicity evaluation chip, and the chip is transferred to a 37°C, 5% CO2 incubator for static incubation. After 4 hours, the chip is placed under a microscope for photography. Figure 2 As shown, different concentrations of compounds produce different cytotoxicity to fibroblasts. After incubation with MTT solution, the content of formazan crystals produced in the cells is different, which is reflected in the difference in the number and degree of coloring of the colored cells.
[0062] S27, the obtained images are analyzed by algorithms, the cells are accurately segmented and outlined, and then four classifications are performed based on the cell morphology and color intensity: true positive (normal morphology and dark purple), true negative (abnormal morphology and no obvious purple), false positive (abnormal morphology but dark purple) and false negative (normal morphology but no obvious purple). Through the comprehensive analysis of the morphological characteristics and color intensity of the cells, the cell viability under the action conditions of each concentration of the compound can be obtained.
[0063] The results were compared with the traditional method in 96-well plates (industry standard "Nanomaterials for Biological Evaluation of Medical Devices: In Vitro Cytotoxicity Test (MTT Test and LDH Test)" YY / T 0993-2015).
[0064] The results are shown in Table 1 below:
[0065] Table 1
[0066]
[0067]
[0068] Note: The above results are presented in the form of mean ± standard deviation (SD).
[0069] The above results show that the cell viability obtained by the test under various concentrations of the compound is close to the cell viability measured by the chip and the traditional method, indicating that the test results in the chip can achieve similar or even consistent results with the traditional 96-well plate test, verifying the reliability of the cytotoxicity evaluation chip results. At the same time, the SD values presented in the results, except for the 0.2 mg / mL condition, the two methods are relatively close, and the chip method of the present invention is generally lower than the SD value of the 96-well plate results under other conditions, further verifying that the chip cytotoxicity test method has better repeatability and stability.
Claims
1. A method for constructing a chip-based cytotoxicity assessment model, characterized in that: The construction method comprises the following steps: Step 1. Structural design and fabrication of cytotoxicity assessment chip; Step 2. Cytotoxicity assessment within the chip; The cytotoxicity assessment chip described in step 1 includes independent units, each of which includes parallel straight pipes, and each independent unit includes an inlet (1) and an outlet (2); the entire length of each parallel straight pipe connecting the inlet and the outlet has the same volume, so that the fluid is evenly distributed in the test.
2. The construction method according to claim 1, characterized in that: The cell culture platform or tool comprises a plurality of independent units, each of which is provided with a plurality of parallel pipes or cell culture areas of arbitrary shapes. These pipes or areas can be connected to a main inlet or a main outlet, or the inlets and outlets can be independently arranged in parallel; the area and volume of all cell culture areas are equal to ensure that the fluid can be evenly distributed during the test.
3. The construction method according to claim 3, characterized in that: In the independent unit, the parallel straight pipes have an input diameter of 0.1-1 mm, an output diameter of 0.1-1 mm, an inner diameter of 0.5-1 mm, and a length of 0.5-4 mm.
4. The construction method according to claim 1, characterized in that: Step 2 includes: Step 2-1. Prepare cell suspension and chip pretreatment; Step 2-2. Inject the cell suspension into the cytotoxicity assessment chip and culture until the cells adhere to the wall; Step 2-3. Inject the test substance into the cytotoxicity assessment chip and incubate; Step 2-4. After the incubation, detect the cell viability.
5. The construction method according to claim 4, characterized in that: In step 2-1, the cells in the cell suspension are selected from cells derived from human skin.
6. The construction method according to claim 5, characterized in that: In step 2-1, the cells in the cell suspension are selected from any one or more of fibroblasts, melanocytes, and keratinocytes.
7. The construction method according to claim 4, characterized in that: In step 2-1, the chip pretreatment includes: after sterilization, modifying and rinsing with 95% ethanol and PBS in sequence, and checking under a microscope that each channel is intact.
8. The construction method according to claim 4, characterized in that: In step 2-2, the cell density of the cell suspension is 1×10 6 -5×10 6 Pieces / mL.
9. The construction method according to claim 8, characterized in that: In step 2-2, the injection volume of the cell suspension is 10-30 μL.
10. The construction method according to claim 9, characterized in that: In step 2-2, the cell density of the cell suspension to be tested is 2×10 6 / mL, and the injection volume of the cell suspension to be tested is 20μL.
11. The construction method according to claim 4, characterized in that: In step 2-4, the method for detecting cell viability is the MTT method.
12. The construction method according to claim 11, characterized in that: In step 2-4, the cell viability detection is combined with AI analysis to perform image colorimetric analysis and then calculate the relative activity of the cells, thereby evaluating the toxicity of the test substance to the cells.
13. A cytotoxicity assessment model constructed according to the construction method according to any one of claims 1 to 12.
14. Use of the cytotoxicity assessment model according to claim 13 in in vitro cytotoxicity assessment.
15. The use according to claim 14, characterized in that: The substances to be tested for the application include: any one or more of food, medicine, health care products and cosmetics.
16. The use according to claim 15, characterized in that The substances to be tested include small compounds.
17. The use according to claim 16, characterized in that Used for screening or quality control of small molecule compounds.
18. An in vitro cytotoxicity assessment method, characterized in that: The invention comprises the construction method according to any one of claims 1 to 12.