CN117051072A - Liver organoid chip-based screening method for relieving nonalcoholic fatty liver active ingredient and application thereof - Google Patents
Liver organoid chip-based screening method for relieving nonalcoholic fatty liver active ingredient and application thereof Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention provides a screening method for relieving nonalcoholic fatty liver active ingredients based on a liver organoid chip and application thereof. The technical scheme comprises the steps of constructing a high-flux organoid chip, forming 3D non-alcoholic fatty liver and screening ginsenoside bioactivity. The invention utilizes engineering and stem cell developmental principle to remodel the pharmacological action of liver tissue to ginsenoside in human liver pathological state in vitro, and the technology has great application value in aspects of in vitro organ reconstruction, drug efficacy screening and organism response mechanism exploration of drugs, and the method has expansibility and practicability.
Description
Technical Field
The invention relates to the fields of tissue engineering and biomedicine, in particular to a screening method for relieving nonalcoholic fatty liver active ingredients based on a liver organoid chip and application thereof.
Background
The liver is an important and complex organ involved in protein and fat metabolism, bile production, detoxification, immunomodulation, etc. Nonalcoholic fatty liver is a common liver metabolic syndrome and is closely related to dangerous factors such as obesity, insulin resistance, hypertension and the like. The disease is characterized by steatosis, developing hepatitis, cirrhosis and even cancer. The course of the disease is often accompanied by a variety of pathological events including inflammatory cell infiltration, fibrosis, metabolic disorders, and portal hypertension. Liver in vitro models are extremely important for disease research (e.g., profiling of disease occurrence and therapeutic mechanisms) and drug efficacy evaluation. Ginsenoside as main ingredient of Ginseng radix has various biological activities such as reducing blood lipid, lowering blood sugar, and resisting cancer. Due to the diversity of structures (> 150), ginsenosides of different structures showed significant differences in activity. The active ingredients of the ginsenoside are rapidly detected and screened, and the influence of the ginsenoside on the body health is clarified. Currently, in vitro ginsenoside activity evaluation is mainly focused on animal experiments and 2D monolayer cell experiments. Animal models are not sufficiently representative of human biological characteristics due to species differences. Most 2D monolayer cell experiments adopt a static culture mode, lack dynamic reproduction of in-vivo environment, are difficult to simulate key characteristics of organ functions, and have poor pharmaceutical activity prediction capability.
The organoid chip technology is an emerging technology proposed in recent years, namely, stem cells, organoids and organ chips are integrated, so that a brand new platform is provided for simulating human body medication in vitro by virtue of microminiaturization, high fidelity and the like. The method combines the principles of stem cell development and engineering, utilizes the characteristic of self-assembly of adult or multipotent stem cells on a chip, and generates organoids with near physiological functions under the action of biological fluid. The stem cells can fully utilize the microstructure of the organ chip to assemble in situ and induce differentiation to form the 3D liver organoid, and the microfluidic technology can endow a more bionic organ development environment, including fluid shear force, information interaction, medicine gradient and the like. At present, although 3D organoids such as liver, brain, intestine, lung and the like are established in the international range, importance and development potential of the engineering technology in the biomedical and precise medical fields are emphasized. The stem cells, the organoid chip and the microfluid are combined, so that the physiological and pathological characteristics of organs can be remodeled in vitro to the greatest extent, the drug response of organisms is reflected, the defects of animal models and monolayer cell culture in the aspects of ginsenoside activity screening and evaluation are overcome, and the limitations of the existing single technology in-vitro organ remodelling and drug evaluation are solved. The integrated technology is blank in high-throughput screening of ginsenoside bioactivity, and is expected to provide a brand-new technical means for analyzing a non-alcoholic fatty liver disease mechanism and screening ginsenoside components for relieving the disease.
Disclosure of Invention
The invention aims to provide a screening method for relieving nonalcoholic fatty liver active ingredients based on a liver organoid chip and application thereof. The technical scheme effectively realizes 3D liver organoid pathology simulation and ginsenoside bioactivity evaluation. The dynamic perfusion system remodels key microenvironment elements such as in vivo complicated cell components, physiological fluid, 3D tissue morphology and the like, not only realizes in-vitro pathological liver model construction, but also has the potential of high-throughput screening of medicines such as ginsenoside, and reproduces the medicine response of human liver to different medicines such as ginsenoside gradients. The platform is expected to provide a brand new research platform and technical support for in-vitro organ reconstruction, drug efficacy screening and organism response mechanism detection of drugs. The method is not limited to flux screening of ginsenoside components, and can be expanded to evaluation of other molding drugs or lead drugs.
The invention provides a screening method for relieving nonalcoholic fatty liver disease drug active ingredients based on a liver organoid chip, which comprises the following steps: first, forming a 3D non-alcoholic fatty liver organoid in a high throughput organoid chip; second, a pharmacological bioactivity screen is performed, which is to intervene in the 3D non-alcoholic fatty liver organoid with a drug.
The organoid chip mainly comprises a plurality of microarray units, wherein each array unit comprises a cell and culture medium inlet, a perfusion channel, a microarray structure and a culture medium outlet, cells are retained in the microarray structure after entering from the cell and culture medium inlet, organoids are formed by in-situ assembly and induction, and the culture medium enters from the cell and culture medium inlet according to a certain flow rate and flows out from the culture medium outlet through the perfusion channel containing the microarray structure. The array units can be communicated through perfusion tubes and can also be independently controlled, and the chip construction has the characteristics of flexibility, controllability and high flux.
The organoid chip is composed of an upper layer and a lower layer, and the material is Polydimethylsiloxane (PDMS). The number of array units is 1-8, the micro array structure can be pit-shaped or columnar, the diameter of the micro pit/column is 200 μm-1 mm, the depth is 200 μm-800 μm, and the interval is 50-500 μm.
The flow rate of the culture medium in the organoid chip is 30-100 mu L/h.
The construction of the high-flux organoid chip comprises the following specific steps: preparing a chip by adopting a conventional soft lithography technology, pouring PDMS prepolymer into a formed template to obtain an upper layer and a lower layer, sealing after oxygen plasma treatment of 10-90 s, and then modifying the chip by PF127 with a certain concentration in a hydrophobic manner for later use.
The concentration range of the PF127 modified chip is 0.1% -1%, and the action time is 1-24 h.
The invention provides the formation of 3D non-alcoholic fatty liver organoids, which comprises the following specific steps:
(1) Differentiation of human pluripotent stem cells into liver-specific endoderm: the culture medium is RPMI-1640 culture medium, glutaMAX, KSR, B and ActivinA are added in a certain concentration, and after 5 days of treatment, the culture medium is changed into the culture medium containing GlutaMAX, KSR, B, BMP4 and bFGF in the RPMI-1640 for 3 days.
The GlutaMAX, KSR and B27 are all in the range of 0.1-5%, the ActivinA concentration is in the range of 50-100 ng/mL, the BMP4 concentration is in the range of 10-40 ng/mL, and the bFGF concentration is in the range of 5-20 ng/mL.
(2) Differentiation into hepatic progenitors: the cells were treated with RPMI-1640 medium supplemented with GlutaMAX, KSR, B and HGF for 5 days.
The GlutaMAX, KSR and B27 are all in the range of 0.1-5%, and the HGF concentration is in the range of 10-40 ng/mL.
(3) Hepatocyte differentiation and organoid formation: treatment with HCM medium supplemented with OSM and Dex medium was carried out for 5 days.
The OSM concentration range is 5-20 ng/mL, and the Dex concentration range is 10 -8 -10 -7 M。
(4) Long-term culture of liver organoids: treatment with Dex-added medium in HCM medium was performed for several days.
The Dex concentration range is 10 -8 -10 -7 M, the treatment time is 2-10 days.
(5) Free fatty acid exposure: treatment with medium supplemented with Dex and free fatty acids in HCM medium is carried out for 1-7 days.
The free fatty acid concentration is 100-1000 μm, and Dex concentration is 10 -8 -10 -7 M。
The medicine is anti-inflammatory, lipid-lowering, anti-fibrosis, anticancer or antioxidant. Specifically, the medicine is ginsenoside with concentration of 0.01 μm-1 mM, ginsenoside action time of 1-7 days, and Dex concentration of 10 -8 -10 -7 M。
The invention has the advantages that aiming at the bottleneck problem of lack of proper in vitro model in early activity evaluation of drug development, a multidisciplinary crossing research method is adopted, an emerging organoid chip technology is taken as a core, a high-flux platform integrating a non-alcoholic fatty liver model and a drug evaluation system is innovatively built and developed, the building of a high-bionic pathological model is realized, a key technology is provided for early efficacy evaluation and activity screening of the drug, a screening method for relieving the non-alcoholic fatty liver active ingredient based on the liver organoid chip and application thereof are finally provided, and the verification shows that the platform is suitable for high-flux ginsenoside biological activity evaluation and is used for effectively screening candidate drugs for relieving the non-alcoholic fatty liver disease.
Drawings
FIG. 1 is a schematic diagram of the structure of an organoid chip. Wherein A is a high throughput array chip; b is a single array element of the organoid chip. Reference numerals meaning: 1 is a cell and culture medium inlet, 2 is a perfusion channel, 3 is a micro array structure, and 4 is a culture medium outlet.
FIG. 2 non-alcoholic fatty liver organogenesis and Rb 1 And (5) an intervention result graph. Wherein A is the accumulation of lipid droplets in physiological conditions; b is the accumulation of lipid droplets in pathological conditions.
FIG. 3 is a graph of Rb at various concentrations 1 Triglyceride (a) and cholesterol (B) levels under intervention.
FIG. 4 is ginsenoside Rb 1 And interfering with the identification result of inflammatory and fibrotic markers of the non-alcoholic fatty liver. Wherein A is an anti-inflammatory efficacy evaluation characterization graph; b is ginsenoside Rb 1 Characterization graph of anti-fibrosis efficacy evaluation.
Fig. 5 is a representation of obeticholic acid (Ob) intervention in non-alcoholic fatty liver disease. Wherein a is triglyceride levels under Ob intervention; b is cholesterol level; c is an anti-inflammatory efficacy evaluation characterization graph; d is ginsenoside Rb 1 Characterization graph of anti-fibrosis efficacy evaluation.
Detailed Description
The following examples further illustrate the invention, but are not intended to limit it.
Example 1:3D non-alcoholic fatty liver organoid construction and ginsenoside bioactivity screening.
The invention provides a screening method for relieving nonalcoholic fatty liver active ingredients based on a liver organoid chip and application thereof, which is characterized in that: the method for constructing the model mainly comprises the following steps: high-flux organoid chip construction, 3D nonalcoholic fatty liver organoid formation and ginsenoside bioactivity screening.
1. Construction of high throughput organoid chip
The organoid chip of this embodiment is mainly composed of a plurality of microarray units. As shown in FIG. 1, each array unit comprises a cell and culture medium inlet 1, a perfusion channel 2, a micro-array structure 3 and a culture medium outlet 4, wherein cells are retained in the micro-array structure after entering from the cell and culture medium inlet 1, are assembled in situ and induced to form an organoid, and the culture medium enters from the cell and culture medium inlet 1 at a certain flow rate, passes through the perfusion channel containing the micro-array structure 3 and then flows out from the culture medium outlet 4. The array units can be communicated through perfusion tubes and can also be independently controlled, and the chip construction has the characteristics of flexibility, controllability and high flux.
The organoid chip is composed of an upper layer and a lower layer, and the material is Polydimethylsiloxane (PDMS). The number of array units is 4, the micro array structure is columnar, the diameter of the micro column is 500 μm, the depth is 600 μm, and the interval is 50 μm.
The construction of the high-flux organoid chip comprises the following specific steps: the chip is prepared by adopting a conventional soft lithography technology, a PDMS prepolymer is poured into a formed template to obtain an upper layer and a lower layer, the upper layer and the lower layer are sealed after being treated by oxygen plasma 15 s, and then the chip 4 h is modified by PF127 with the water repellency of 0.2 percent for standby.
2. 3D non-alcoholic fatty liver organogenesis
Human pluripotent stem cells are induced to 10 days, digested into single cells or small cell clusters, resuspended by a culture medium, introduced from the cells of the chip and the culture medium inlet 1, self-assembled to form cell spheres, and continuously replaced and cultured according to steps.
The culture medium was introduced in order at a medium flow rate of 40. Mu.L/h.
Specifically, the 3D non-alcoholic fatty liver organogenesis procedure is:
(1) Differentiation of human pluripotent stem cells into liver-specific endoderm: the culture medium is prepared by adding GlutaMAX, KSR, B and ActivinA with a certain concentration into RPMI-1640 culture medium for 5 days, and then changing into the culture medium containing GlutaMAX, KSR, B, BMP4 and bFGF in RPMI-1640 for 3 days. Wherein RPMI 1640 medium was purchased from Instratagem (Invitrogen) under the designation C11875500BT.
The concentrations of Glutamax, KSR and B27 are all 1%, the concentration of ActivinA is 100 ng/mL, the concentration of BMP4 is 20 ng/mL, and the concentration of bFGF is 10 ng/mL.
(2) Differentiation into hepatic progenitors: the cells were treated with RPMI-1640 medium containing GlutaMAX, KSR, B and HGF for 5 days.
The GlutaMAX, KSR and B27 were all 1% and HGF concentration was 20 ng/mL.
(3) Hepatocyte differentiation and organoid formation: the medium composition was HCM medium supplemented with OSM and Dex for 5 days. Wherein, HCM medium is purchased from scientific company under the product number 5201.
The OSM concentration is 10 ng/mL, and the Dex concentration is 10 -7 M。
(4) Long-term culture of liver organoids: treatment with Dex-containing medium in HCM medium was carried out for 2 days.
The Dex concentration is 10 -7 M, treatment time was 2 days.
(5) Free fatty acid exposure: treatment with Dex and free fatty acids in HCM medium was carried out for 5 days. The non-alcoholic fatty liver organoids had a large accumulation of lipid droplets relative to normal tissue as shown in fig. 2 a and fig. 2B.
The free fatty acid concentration was 600. Mu.M, and the Dex concentration was 10 -7 M。
3. Ginsenoside biological activity screening
The method comprises the following specific steps: preparing a ginsenoside working solution with a certain concentration to intervene in 3D non-alcoholic fatty liver organoids, namely dissolving ginsenoside in a HCM culture medium containing Dex.
The ginsenoside is Rb 1 Since the nonalcoholic fatty liver is characterized by steatosis and accompanied with a certain degree of inflammation and fibrosis, the therapeutic effect of ginsenoside on the disease is evaluated, namely, the lipid-lowering activity, anti-inflammatory activity and anti-fibrosis activity of ginsenoside are evaluated, the concentration of ginsenoside is 0.05 mu M, 1 mu M, 10 mu M, 50 mu M and 100 mu M, the action time of ginsenoside is 5 days, and the Dex concentration is 10 -7 M. As shown in fig. 2 a, the accumulation of lipid droplets in normal liver (FFAs without free fatty acid and drug group, denoted as Ctrl group) is shown, and in fig. 2B, the accumulation of lipid droplets in non-alcoholic fatty liver group (denoted as FFAs group) is shown, and significantly more lipid droplet accumulation indicates successful construction of the non-alcoholic fatty liver model. Similarly, it can be seen from fig. 3 that the FFAs group has higher Triglyceride (TG) and cholesterol (TC) levels relative to the Ctrl group. As can be seen from FIG. 3, in the absence ofGinsenoside Rb with same concentration gradient 1 Low concentration (0.05. Mu.M) of ginsenoside Rb under the intervention of (C) 1 No obvious difference is caused to the accumulation of non-alcoholic fatty liver fat drops, and the TG and TC levels are obviously reduced at higher concentration (1-100 mu M), which indicates that the concentration range has good lipid-lowering effect. As can be seen from FIG. 4A, inflammatory factor IL8 was elevated relative to mRNA expression (Relative mRNA expression) in the FFAs group, but showed reduced IL8 expression at both 1. Mu.M and 100. Mu.M systems, suggesting Rb 1 Has good therapeutic effect on inflammation. Similarly, FIG. 4B shows that the fibrosis factor a-SMA in the FFAs group is elevated relative to mRNA expression, but shows reduced a-SMA expression in both 1. Mu.M and 100. Mu.M systems, suggesting Rb 1 Has good therapeutic effect on fibrosis. The platform is suitable for evaluating the bioactivity of the high-throughput ginsenoside, and effectively screening candidate medicines for relieving the non-alcoholic fatty liver disease.
Example 2:3D non-alcoholic fatty liver organoid construction and obeticholic acid activity evaluation and verification.
1. The specific procedure for the construction of the high throughput organoid chip was the same as in example 1.
2. The specific procedure for the formation of the 3D non-alcoholic fatty liver organoid was the same as in example 1.
3. Unlike example 1, the clinically usual drug obeticholic acid (Ob) was selected for evaluation at an action concentration of 5 μm. As shown in fig. 5, the levels of TG and TC, as well as the expression of inflammatory IL8 and fibrotic a-SMA in non-alcoholic fatty liver after Ob intervention can be significantly down-regulated, suggesting that the model is suitable for activity assessment and validation of shaped drugs.
4. The remaining unexplained steps are the same as those of example 1.
Claims (11)
1. A screening method for relieving nonalcoholic fatty liver disease drug active ingredients based on liver organoid chip is characterized by comprising the following steps: the method comprises the following steps:
first, forming a 3D non-alcoholic fatty liver organoid in a high throughput organoid chip;
secondly, screening the biological activity of the medicine, which is to intervene the 3D non-alcoholic fatty liver organoid with the medicine;
the high-flux organoid chip consists of one or more microarray units, wherein each array unit consists of a cell and culture medium inlet (1), a perfusion channel (2), a microarray structure (3) and a culture medium outlet (4), cells stay in the microarray structure after entering from the cell and culture medium inlet (1), are assembled in situ and are induced to form organoids, and the culture medium enters from the cell and culture medium inlet (1) at a certain flow rate and flows out from the culture medium outlet (4) through the perfusion channel containing the microarray structure (3); the array units are communicated through perfusion tubes or the units are independently controlled;
the specific steps of the 3D non-alcoholic fatty liver organoid formation are as follows:
s1 differentiation of human pluripotent stem cells into liver-specific endoderm: the culture medium is RPMI-1640 culture medium, glutaMAX, KSR, B and ActivinA are added, and after 5 days of treatment, the culture medium is replaced by the culture medium containing GlutaMAX, KSR, B, BMP4 and bFGF in the RPMI-1640 for 3 days;
s2 differentiation to hepatic progenitors: treating with RPMI-1640 medium supplemented with GlutaMAX, KSR, B and HGF for 4-6 days;
s3 hepatocyte differentiation and organoid formation: treating with HCM medium supplemented with OSM and Dex medium for 4-6 days;
s4, long-term culture of liver organoids: culturing for a long period by adding Dex culture medium into HCM culture medium;
s5 free fatty acid exposure: treatment with medium supplemented with Dex and free fatty acids in HCM medium is carried out for 1-7 days.
2. A screening method according to claim 1, characterized in that: the organoid chip is composed of an upper layer and a lower layer, and is made of polydimethylsiloxane; the number of array units is 1-8, and the micro array structure (3) is pit-shaped or columnar.
3. A screening method according to claim 2, characterized in that: the diameter of the micro array structure ranges from 200 mu m to 1 mu m mm, the depth ranges from 200 mu m to 800 mu m, and the interval ranges from 50 mu m to 500 mu m.
4. A screening method according to claim 2, characterized in that: the manufacturing steps of the high-flux organoid chip are as follows: the chip is prepared by adopting a soft lithography technology, a formed template is poured with PDMS prepolymer to obtain an upper layer and a lower layer, the upper layer and the lower layer are sealed after being treated by oxygen plasma to 10-90 s, and the chip is modified by PF127 with a certain concentration in a hydrophobic manner for standby.
5. The screening method according to claim 4, wherein: the concentration range of the PF127 hydrophobic modification chip is 0.1% -1%, and the action time is 1-24 h.
6. A screening method according to claim 1, characterized in that: the second step of drug activity screening comprises the following specific steps: preparing a medicine working solution with a preset concentration to intervene in the 3D non-alcoholic fatty liver organoid, wherein Dex is added into a HCM basic culture medium.
7. The screening method according to claim 6, wherein the concentration of Glutamax, KSR and B27 in S1 and S2 is 0.1-5%.
8. The screening method according to claim 6, wherein the concentration of ActivinA in S1 is in the range of 50-100 ng/mL, the concentration of BMP4 is in the range of 10-40 ng/mL, and the concentration of bFGF is in the range of 5-20 ng/mL.
9. The method of claim 6, wherein the concentration of HGF in S2 is in the range of 10 to 40 ng/mL; the concentration of OSM in S3 is 5-20 ng/mL, and the concentration of Dex is 10 -8 -10 -7 M; dex concentration in S4 was in the range of 10 -8 -10 -7 M, the treatment time is 2-10 days; the concentration of free fatty acid in S5 is 100-1000 μm, and the concentration of Dex is 10 -8 -10 -7 M。
10. The screening method according to claim 7, wherein the drug is an anti-inflammatory, lipid-lowering, anti-fibrosis, anti-cancer or anti-oxidant drug.
11. The screening method according to claim 10, wherein the drug is ginsenoside with concentration of 0.01 μm-1 mM, ginsenoside action time of 1-7 days, and Dex concentration of 10 -8 -10 -7 M。
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