Construction and application of human pluripotent stem cell-derived heart organoid traditional Chinese medicine extract toxicity test chip

Abstract

The invention provides construction and application of a human pluripotent stem cell-derived heart organoid traditional Chinese medicine extract toxicity test chip. The chip comprises a plurality of micro-cell culture chambers and a micro-fluidic pore channel, heart organoid culture is carried out in the micro-cell culture room, culture media are added in the micro-fluidic channel to maintain the activity of the heart organoid, and traditional Chinese medicine extracts are added to evaluate the toxicity of traditional Chinese medicine. By simulating the reaction of the heart organ, the toxicity of the traditional Chinese medicine can be evaluated, the safety and effectiveness information about the traditional Chinese medicine can be provided, finally, the use of experimental animals can be reduced, and the animal welfare can be improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug toxicity testing, and relates to toxicity testing of Chinese medicine extracts of cardiac organoids, and specifically to the construction and application of a toxicity testing chip for Chinese medicine extracts of cardiac organoids derived from human pluripotent stem cells. Background Art

[0002] Cardiac organoids are a type of three-dimensional culture composed of multiple types of heart cells, including cardiomyocytes, which are highly similar to the heart in vivo and can simulate the three-dimensional structure and physiological function of the heart. Human pluripotent stem cells have unlimited proliferation capacity and can differentiate into almost all human cells in vitro, including cardiac constituent cells. Therefore, human pluripotent stem cells are an ideal source of seed cells for constructing cardiac organoids.

[0003] Chinese herbal medicine extracts are widely used in the field of traditional Chinese medicine, and safety assessment is an indispensable part. In the existing technology, most of them are carried out through animal experiments. By observing the poisoning symptoms and deaths of experimental animals, the acute toxicity level of the extract can be determined, and its possible lethal dose can be understood, thereby evaluating its potential harm to the human body. Cardiac organoids can simulate the three-dimensional structure and physiological functions of the heart. Based on this, the toxicity of different Chinese herbal drugs to cardiac organoids can be evaluated, which will help reduce the use of experimental animals and improve detection efficiency and success rate. Summary of the invention

[0004] The present invention is based on the above ideas and aims to provide a toxicity test chip for Chinese medicine extracts of heart organoids derived from human pluripotent stem cells and its application.

[0005] In order to achieve this purpose, the technical solution provided by the present invention is as follows:

[0006] In the first aspect of the present invention, a component for toxicity testing of Chinese medicine extracts of cardiac organoids derived from human pluripotent stem cells is provided, comprising: a plurality of microfluidic channels, each of which is provided with medium injection or outflow holes at both ends, and a plurality of drug delivery holes in the middle; a plurality of cardiac organoid culture chambers, which are respectively provided on the drug delivery holes, and the bottom ends are sealed with a sterile mixed cellulose ester semipermeable membrane. The micro cell culture chamber is used for cardiac organoid culture, and the microfluidic channel is used for adding culture medium to maintain the activity of cardiac organoids and adding Chinese medicine extracts to evaluate the toxicity of Chinese medicine.

[0007] Preferably, a plurality of microfluidic channels are connected in series and externally connected to a peristaltic pump to circulate and provide culture medium.

[0008] Preferably, the cardiac organoid culture chamber is a PDMS tissue chip, PDMS is patterned using a photolithography pattern mold, and then PDMS is poured and cured, PDMS is cured at 60° C. for 3 hours, the cured PDMS mold is peeled and cut from the silicon wafer, and the PDMS chip is plasma treated;

[0009] The sterile mixed cellulose ester semipermeable membrane is a 0.45 μm sterile mixed cellulose ester membrane.

[0010] In a second aspect, the present invention provides a method for constructing a toxicity test chip of Chinese medicine extracts of human pluripotent stem cell-derived heart organoids, which is achieved by combining the construction of heart organoids with the above-mentioned components, and comprises the following steps:

[0011] (1) Construction of cardiac organoids

[0012] The human pluripotent stem cell spheres are sequentially cultured with cardiac organoid differentiation medium A for the first differentiation culture, cardiac organoid differentiation medium B for the second differentiation culture, and cardiac organoid differentiation medium C for the third differentiation culture to obtain cardiac organoids, wherein the culture process is all carried out by a matrix gel embedding culture method;

[0013] (2) Cardiac organoid culture

[0014] The cardiac organoids constructed in step (1) are collected, resuspended in matrix gel, and placed on a sterile mixed cellulose ester semipermeable membrane; the chip is placed in a 37° C., 5% CO2 incubator for culture, cardiac organoid culture medium is injected from the culture medium, and a peristaltic pump is started to circulate the culture medium, and the cardiac organoids self-assemble in the organoid culture chamber and gradually grow into mature organoids.

[0015] Preferably, in step (1), the components of the three differentiation culture media are as follows:

[0016] Cardiac organoid differentiation medium A consisted of the following components: 6 μg / mL CHIR99021, 1% 50× B27 supplement, 500 μg / mL L-ascorbic acid-2-phosphate trisodium salt, RPMI-1640 medium;

[0017] Cardiac organoid differentiation medium B consisted of the following components: 5.5 μg / mL KY02111, 1% 50×B27 supplement, 500 ug / mL L-ascorbic acid-2-phosphate trisodium salt, RPMI-1640 medium;

[0018] Cardiac organoid differentiation medium C consists of the following components: 1% 50×B27 supplement, 500 ug / mL L-ascorbic acid-2-phosphate trisodium salt, and RPMI-1640 medium.

[0019] More preferably, the construction of cardiac organoids in step (1) comprises the following steps:

[0020] 1) Culture in cardiac organoid differentiation medium A: The culture product obtained from the stem cells was aspirated from the culture medium, and 5-10 μl of matrix gel was added to each well to encapsulate the pluripotent stem cells in the matrix gel. After the matrix gel solidified, 200 μl of cardiac organoid differentiation medium A was added to each well, and the wells were returned to the CO2 incubator and cultured for 48 h to obtain the first differentiation culture product;

[0021] 2) Culture in cardiac organoid differentiation medium B: discard the first differentiation culture product obtained in 1) above, and then add 5 μl of matrix gel to each well to ensure that the cell spheres are wrapped in the matrix gel; then put the cell plate back into the CO2 incubator for 30 minutes to allow the matrix gel to solidify; add 200 μl of organoid differentiation medium B to each well, return to the CO2 incubator, and culture for 48 hours to obtain the second differentiation culture product;

[0022] 3) Culturing in cardiac organoid differentiation medium C: discard the second differentiation culture product obtained in 2) above, and then add 200 μl of organoid differentiation medium C to each well, return to the CO2 incubator, and culture for 48 hours; thereafter, replace the organoid differentiation medium C every 2 days to obtain the third differentiation culture product, and cardiac organoids will be obtained when beating occurs on the 7th to 10th day of cardiac organoid differentiation.

[0023] The unique advantage of the present invention is that the success rate of constructing cardiac organoids according to the method is greater than 85%, and can be directly used for drug toxicity detection.

[0024] In a third aspect, the present invention provides a human pluripotent stem cell-derived cardiac organoid Chinese medicine extract toxicity test chip, which is prepared using the method described above and can be used for drug toxicity detection including Chinese medicine extracts.

[0025] In a fourth aspect, the present invention provides a method for toxicity testing of Chinese medicine extracts in cardiac organoids using a chip, comprising the following steps: extracting the Chinese medicine extract, adding it to the cardiac organoid culture medium at different drug concentrations, re-culturing the chip in a 37°C, 5% CO2 incubator, observing the cell conditions under a microscope at specific time points, and measuring the toxicity of the Chinese medicine extract to the cardiac organoids after the last observation time point.

[0026] Preferably, during the test, the cell conditions are observed under a microscope at 24h, 48h, 72h, and 96h. After 96h, the toxicity of the Chinese herbal extract to the cardiac organoids is measured, including the detection of cell survival rate, cell membrane integrity, changes in intracellular enzyme activity, and cell apoptosis indicators; the chip can be photographed with a fluorescence microscope, confocal microscope, or high-resolution camera equipment, and can observe and record the morphological structure, activity state, and changes in specific fluorescent markers in the cells of cardiac cells.

[0027] The unique advantage of the present invention is that the toxicity evaluation indexes of the Chinese herbal medicine extract evaluated by the human pluripotent stem cell-derived heart organoid toxicity test chip are diverse and have high clinical consistency.

[0028] Functions and Effects of the Invention

[0029] The present invention discloses a toxicity test chip for Chinese medicine extracts of heart organoids derived from multifunctional stem cells, which includes multiple micro cell culture chambers and microfluidic channels, can simulate the response of heart organs, help evaluate the toxicity of Chinese medicines and provide information on the safety and effectiveness of Chinese medicines, and ultimately reduce the use of experimental animals and improve animal welfare. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The structural diagram of the human pluripotent stem cell-derived cardiac organoid Chinese medicine extract toxicity test chip is shown;

[0031] Figure 2 Bright field images, HE images, and histochemical identification images of cardiac organoids are shown. DETAILED DESCRIPTION

[0032] The present invention is described in detail below in conjunction with the embodiments and the accompanying drawings. However, the following embodiments should not be considered as limiting the scope of the present invention.

[0033] Example 1: Components for toxicity testing of Chinese medicine extracts of human pluripotent stem cell-derived cardiac organoids

[0034] See also Figure 1 The component includes a plurality of microfluidic channels 1 and a plurality of cardiac organoid culture chambers 2 arranged thereon. The microfluidic channels are in the shape of long strips, and the plurality of microfluidic channels are connected in series; a medium injection hole 11 and an outflow hole 12 are respectively arranged at both ends of each microfluidic channel, and an external peristaltic pump is connected to circulate and provide the medium; a plurality of drug delivery holes are arranged in the middle part.

[0035] A plurality of cardiac organoid culture chambers 12 are respectively arranged on the drug administration holes, and the bottom ends are sealed with a 0.45 μm sterile mixed cellulose ester semipermeable membrane 3 to separate the cardiac organoids from the test drugs.

[0036] Specifically, the cardiac organoid culture chamber is a PDMS tissue chip. The PDMS is patterned using a photolithography pattern mold and then poured and cured. The PDMS is cured at 60°C for 3 hours. The cured PDMS mold is peeled off and cut from the silicon wafer, the inlet and outlet are punched, and the PDMS chip is plasma treated for self-assembly, growth and maturation of cardiac organoids.

[0037] Example 2 Construction of cardiac organoids

[0038] The specific operations are as follows:

[0039] 1) Cultivate in cardiac organoid differentiation medium A. Aspirate the medium in the culture product obtained from the above stem cells, add 5-10ul of matrix gel to each well to encapsulate the pluripotent stem cells in the matrix gel, add 200ul of cardiac organoid differentiation medium A to each well after the matrix gel solidifies, return to the CO2 incubator, and culture for 48h to obtain the first differentiation culture product.

[0040] 2) Culture in cardiac organoid differentiation medium B. The first differentiation culture product obtained in 1) above was discarded by aspirating the organoid differentiation medium A; 5ul of matrix gel was added to each well to ensure that the cell balls were wrapped in the matrix gel; then the cell plate was returned to the CO2 incubator for 30 minutes to allow the matrix gel to solidify. 200ul of organoid differentiation medium B was added to each well, and the well was returned to the CO2 incubator for 48 hours to obtain the second differentiation culture product.

[0041] 3) Culture in cardiac organoid differentiation medium C. The second differentiation culture product is obtained in 2) above, and the organoid differentiation medium B is discarded; 200ul of organoid differentiation medium C is added to each well, and the well is returned to the CO2 incubator for 48h; thereafter, the organoid differentiation medium C is replaced every 2 days to obtain the third differentiation culture product. The cardiac organoids are obtained when beating occurs on the 7th to 10th day of cardiac organoid differentiation.

[0042] Among them, the components of the three differentiation medium are as follows:

[0043] Cardiac organoid differentiation medium A consisted of the following components: 6 μg / mL CHIR99021, 1% 50× B27 supplement, 500 μg / mL L-ascorbic acid-2-phosphate trisodium salt, RPMI-1640 medium;

[0044] Cardiac organoid differentiation medium B consisted of the following components: 5.5 μg / mL KY02111, 1% 50×B27 supplement, 500 ug / mL L-ascorbic acid-2-phosphate trisodium salt, RPMI-1640 medium;

[0045] Cardiac organoid differentiation medium C consists of the following components: 1% 50×B27 supplement, 500 ug / mL L-ascorbic acid-2-phosphate trisodium salt, and RPMI-1640 medium.

[0046] The constructed heart organoids were tested and the results were as follows Figure 2 As shown, the morphology, size and arrangement of the cells can be clearly seen, and the nuclei, actin and troponin of the differentiated cardiac cells are also clearly visible.

[0047] Example 3 Construction of cardiac organoid chips

[0048] Cardiac organoids were harvested, resuspended in Matrigel, and placed on a custom-made polydimethylsiloxane (PDMS) tissue chip separated from the microfluidic channels by a 0.45 μm sterile mixed cellulose ester membrane.

[0049] The unique advantage of the present invention is that it uses customized PDMS chip materials and Whatman sterile mixed cellulose ester membrane to separate cardiac organoids from drugs. The specific operation is as follows:

[0050] 1) making a PDMS tissue chip, patterning the PDMS using a photolithography pattern mold, pouring and curing the PDMS, curing the PDMS at 60° C. for 3 hours, peeling and cutting the cured PDMS mold from the silicon wafer; punching holes at the inlet and outlet of the cardiac organoid chip, and plasma treating the PDMS chip model;

[0051] 2) Seal the bottom of the cardiac organoid chip with a 0.45 um sterile mixed cellulose ester membrane;

[0052] 3) Collect cardiac organoids, resuspend in matrix gel, and place on sterile mixed cellulose ester membrane

[0053] 4) Cardiac organoid culture medium is perfused from the perfusion channel, and an external peristaltic pump is used to circulate the culture medium. The cardiac organoids self-assemble in the organoid culture chamber and gradually grow into mature organoids.

[0054] Example 4: Adding Chinese herbal medicine extracts to heart organoid chips for drug toxicity testing and evaluation

[0055] 1) Taking the preparation of the Chinese medicine extract of Taohong Siwu Decoction as an example, Taohong Siwu Decoction is made from the following raw materials: 15 grams each of Angelica sinensis, Rehmannia glutinosa, Chuanxiong, White Peony Root, Peach Kernel, and Safflower. Wash and soak the raw materials separately, put the soaked medicinal materials together with the soaking water into the decoction pot, heat to boiling over high heat, then switch to low heat and continue to decoct for 30-40 minutes, and absorb the supernatant;

[0056] 2) According to the method of adding 0.2ml, 1ml and 10ml of culture medium C to every 100ml of cardiac organoid, three concentration gradients of Chinese herbal extracts were set. The culture medium of cardiac organoid chip was replaced with different concentrations of Chinese herbal culture medium, and the chip was placed in a 37°C, 5% CO2 incubator for culture. The cells were observed under a microscope for 24h, 48h, 72h and 96h. After 96h, the toxicity of Chinese herbal extracts on cardiac organoids was measured, including the detection of cell survival rate, cell membrane integrity, changes in intracellular enzyme activity, and cell apoptosis indicators; at the same time, microscopic observation and photography were taken to observe and record the morphological structure of cardiac cells and the killing of Chinese herbal medicines. The results showed that it had no toxic side effects on cardiac cells.

[0057] The basic principles, main features and significant advantages of the present invention have been described in detail above. The purpose of the description in the embodiments and the specification is only to further explain the operating principle of the present invention. Without violating the core idea of ​​the present invention and the statutory protection scope, the present invention allows and covers all kinds of reasonable changes and improvements. These changes and improvements will be deemed to be within the scope of protection requested by the present invention. Specifically, the scope of protection of the present invention will be clearly and strictly defined by the attached claims and their equivalents with equal legal effect.

Claims

1. A component for toxicity testing of Chinese medicine extracts of cardiac organoids derived from human pluripotent stem cells, characterized in that: include: A plurality of microfluidic channels, each of which is provided with medium injection or outflow holes at both ends and a plurality of drug delivery holes in the middle; A plurality of cardiac organoid culture chambers are respectively arranged on the drug administration holes, and the bottom ends are sealed with a sterile mixed cellulose ester semipermeable membrane.

2. The component for toxicity testing of Chinese medicine extracts of human pluripotent stem cell-derived heart organoids according to claim 1, characterized in that: The multiple microfluidic channels are connected in series and externally connected to a peristaltic pump to circulate and provide culture medium.

3. The component for toxicity testing of Chinese medicine extracts of human pluripotent stem cell-derived heart organoids according to claim 1, characterized in that: The cardiac organoid culture chamber is a PDMS tissue chip. The PDMS is patterned using a photolithography pattern mold and then poured and cured. The PDMS is cured at 60°C for 3 hours. The cured PDMS mold is peeled and cut from the silicon wafer, and the PDMS chip is plasma treated. The sterile mixed cellulose ester semipermeable membrane is a 0.45 μm sterile mixed cellulose ester membrane.

4. A method for constructing a toxicity test chip for Chinese medicine extracts of heart organoids derived from human pluripotent stem cells, characterized in that: The method is achieved by combining cardiac organoid construction with the assembly according to any one of claims 1 to 3, comprising the following steps: (1) Construction of cardiac organoids The human pluripotent stem cell spheres are sequentially cultured with cardiac organoid differentiation medium A for the first differentiation culture, cardiac organoid differentiation medium B for the second differentiation culture, and cardiac organoid differentiation medium C for the third differentiation culture to obtain cardiac organoids, wherein the culture process is all carried out by a matrix gel embedding culture method; (2) Cardiac organoid culture The cardiac organoids constructed in step (1) are collected, resuspended in matrix gel, and placed on a sterile mixed cellulose ester semipermeable membrane; the chip is placed in a 37° C., 5% CO2 incubator for culture, cardiac organoid culture medium is injected from the culture medium, and a peristaltic pump is started to circulate the culture medium, and the cardiac organoids self-assemble in the organoid culture chamber and gradually grow into mature organoids.

5. The method according to claim 4, characterized in that In step (1), the components of the three differentiation culture media are as follows: Cardiac organoid differentiation medium A consisted of the following components: 6 μg / mL CHIR99021, 1% 50× B27 supplement, 500 μg / mL L-ascorbic acid-2-phosphate trisodium salt, RPMI-1640 medium; Cardiac organoid differentiation medium B consisted of the following components: 5.5 μg / mL KY02111, 1% 50×B27 supplement, 500 ug / mL L-ascorbic acid-2-phosphate trisodium salt, RPMI-1640 medium; Cardiac organoid differentiation medium C consists of the following components: 1% 50×B27 supplement, 500 ug / mL L-ascorbic acid-2-phosphate trisodium salt, and RPMI-1640 medium.

6. The method according to claim 5, characterized in that The construction of cardiac organoids in step (1) includes the following steps: 1) Culture in cardiac organoid differentiation medium A: The culture product obtained from the stem cells was aspirated from the culture medium, and 5-10 μl of matrix gel was added to each well to encapsulate the pluripotent stem cells in the matrix gel. After the matrix gel solidified, 200 μl of cardiac organoid differentiation medium A was added to each well, and the wells were returned to the CO2 incubator and cultured for 48 h to obtain the first differentiation culture product; 2) Culture in cardiac organoid differentiation medium B: discard the first differentiation culture product obtained in 1) above, and then add 5 μl of matrix gel to each well to ensure that the cell spheres are wrapped in the matrix gel; then put the cell plate back into the CO2 incubator for 30 minutes to allow the matrix gel to solidify; add 200 μl of organoid differentiation medium B to each well, return to the CO2 incubator, and culture for 48 hours to obtain the second differentiation culture product; 3) Culturing in cardiac organoid differentiation medium C: discard the second differentiation culture product obtained in 2) above, and then add 200 μl of organoid differentiation medium C to each well, return to the CO2 incubator, and culture for 48 hours; thereafter, replace the organoid differentiation medium C every 2 days to obtain the third differentiation culture product, and cardiac organoids will be obtained when beating occurs on the 7th to 10th day of cardiac organoid differentiation.

7. A toxicity test chip for Chinese medicine extracts of heart organoids derived from human pluripotent stem cells, characterized in that The method is used to prepare the product according to any one of claims 4 to 6.

8. A method for testing the toxicity of Chinese medicine extracts in cardiac organoids using the chip of claim 7, characterized in that: The steps include: The Chinese herbal extracts were extracted and added to the cardiac organoid culture medium at different drug concentrations. The chip was placed back in a 37°C, 5% CO2 incubator for culture. The cell conditions were observed under a microscope at specific time points, and the toxicity of the Chinese herbal extracts on the cardiac organoids was measured after the last observation time point.

9. The method according to claim 8, characterized in that The cells were observed under a microscope at 24 h, 48 h, 72 h, and 96 h, and the toxicity of the Chinese medicine extract on cardiac organoids was measured after 96 h.

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