CN109880791A - An in vitro construction method of liver fibrosis organoid model - Google Patents
An in vitro construction method of liver fibrosis organoid model Download PDFInfo
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Abstract
The invention discloses a kind of vitro construction methods of liver fibrosis organoid model, and liver cell, hepatic stellate cells, Kupffer Cell and sinusoidal endothelial cell are suspended in culture medium A；Culture was changed to culture medium B and maintains culture to the 4th day；Start within 10th day, is changed to culture medium C, continuous culture six days.Invention is based on liver cellular constituent ratio, and rapid build size structure is uniform, fibrosis characterizes apparent 3D hepatic fibrosis-renal tubular ectasia syndrome organoid external model.The hepatic fibrosis-renal tubular ectasia syndrome organoid model that the present invention constructs, definite principle, stable structure, method is simple, and rapidly, strong operability is suitable for hepatic fibrosis-renal tubular ectasia syndrome occurrence and development Mechanism Study, and anti-fibrosis medicine high flux screening etc. has industrialization meaning for building.
Description
Technical field
The present invention relates to a kind of vitro construction methods of liver fibrosis organoid model.
Background technique
Liver fibrosis is a kind of inflammatory reaction of complexity, is caused by long-term chronic liver injury, and has great risk to send out
Exhibition is cirrhosis or even liver cancer.Aetology shows that many factors are related to liver fibrosis, such as hepatites virus infections, excessive consumption of alcohol,
Autoimmune disease, obesity etc..Liver fibrosis can be defined as the Process of Forming Scar of popularity, and with Inner Constitution and
Function changes.Wherein Extracellular Matrix Secretion caused by hepatic stellate cell activator plays in liver fibrosis of crucial importance extremely
Effect.Therefore, the external model of previous liver fibrosis is often based upon the common 2D culture of hepatic stellate cells.Although for fiber
Change Mechanism Study and drug screening shows certain reference value, but common monolayer cultivation can not good simulated liver physiology
And the microenvironment under pathological state, such as lack cell-ECM, the interaction between cell-extracellular matrix.Accordingly, it is considered to arrive
The complexity of ingredient in liver organization, a kind of 3D model of new simulated liver fibrosis microenvironment are urgently developed.
Organoid technology is a kind of to utilize mammal multipotential stem cell, the stem cell in adult tissue source or adult cell
Characteristic building analogue body internal characteristic, and with many cells type external model.Recently multinomial research report, these have multiple
The organoid of miscellaneous internal structure, in function and has structurally leveled off to corresponding internal structure.This technology is research organizer
The physio-pathological condition of official and drug screening provide ideal platform.
Summary of the invention
The purpose of the present invention is to provide a kind of vitro construction methods of fast simple liver fibrosis organoid model, originally
The method that invention provides can obtain that size structure is uniform, fibrosis characterizes specific liver fibrosis organoid within a short period of time
Model provides platform for the research of further liver fibrosis mechanism and drug screening.
The technical solution of the invention is as follows:
A kind of vitro construction method of liver fibrosis organoid model, it is characterized in that: including the following steps:
By liver cell, hepatic stellate cells, Kupffer Cell and sinusoidal endothelial cell, it is suspended in culture medium A；Culture was changed to the 4th day
Culture medium B maintains culture；Start within 10th day, is changed to culture medium C, continuous culture six days；
The culture medium A includes: bovine insulin, 4- hydroxyethyl piperazineethanesulfonic acid (HEPES), hydrocortisone, glutamine
(GlutaMAX), fetal calf serum, I type rat tail collagen protein and without phenol red WILLIAMS-DARLING Ton E culture medium (William's E
Medium)。
The culture medium B includes: gentamicin, transferrins (Transferrin), vitamin C, bovine insulin, tire ox
Serum, sodium selenite, linoleic acid, hydrocortisone, 4- hydroxyethyl piperazineethanesulfonic acid (HEPES), glutamine (GlutaMAX),
HEGF (HEGF) and HBM liver cell basal medium；
The culture medium C include: gentamicin, transferrins (Transferrin), vitamin C, bovine insulin, fetal calf serum,
Sodium selenite, hydrocortisone, 4- hydroxyethyl piperazineethanesulfonic acid (HEPES), glutamine (GlutaMAX), conversion growth because
Son-β (TGF-β), hEGF (HEGF) and HBM liver cell basal medium.
By liver cell, hepatic stellate cells, Kupffer Cell and sinusoidal endothelial cell, counted using calculating instrument, by 7:1.5:
The special ratios of 0.5:1, are suspended in culture medium A.
Liver cell, hepatic stellate cells, Kupffer Cell and sinusoidal endothelial cell are mixed after A in specific proportions, after counting
Ultralow absorption round bottom cell plates, the body of every hole inoculating cell suspension are equably inoculated in using the volley of rifle fire by 1200, every hole cell
Product is 100 μ L.
Liver cell, hepatic stellate cells, Kupffer Cell and sinusoidal endothelial cell are suspended in culture medium A in proportion, are inoculated in super
Low adsorption round bottom cell plates are then placed in 37 DEG C of carbon dioxide incubators, stand 3 days, avoid moving as far as possible.
Culture, using micropipette rifle, was close to culture medium liquid level, old culture medium is sucked out to the 4th day；Slowly add along hole wall again
Enter 100 μ L culture medium B, the next day inhale and abandon 50 μ L culture mediums in hole, and equivalent culture medium B is added, persistently cultivates 6 days.
It cultivates to after the 10th day, using micropipette rifle, is close to culture medium liquid level, old culture medium is sucked out；It is slow along hole wall again
It is slow to be added 100 μ L culture medium Cs, the next day inhale and abandon 50 μ L culture mediums, equivalent culture medium C is added, persistently cultivates 6 days.
Culture medium A includes: 2 μ g/mL I type rat tail collagen proteins, 10% fetal calf serum, 8 μ g/mL bovine insulins, 10ng/mL
Hydrocortisone, 2 μm of ol/mL glutamine (GlutaMAX), 2 μm of ol/mL 4- hydroxyethyl piperazineethanesulfonic acids (HEPES), nothing
Phenol red WILLIAMS-DARLING Ton E culture medium (William's E Medium)；Culture medium B includes: 100 μ g/mL gentamicins, 10% tire ox
Serum, 10 μ g/mL transferrins (Transferrin), 10 μ g/mL sodium selenites, 10 μ g/mL linoleic acid, 1 μ g/mL vitamin
C, 8 μ g/mL bovine insulins, 10ng/mL hydrocortisone, 2 μm of ol/mL glutamine (GlutaMAX), 2 μm of ol/mL 4- hydroxyls
Ethyl piperazidine ethanesulfonic acid (HEPES), 2ng/mL hEGF (HEGF), HBM liver cell basal medium；Culture medium C
It include: 5ng/mL transforming growth factor-β (TGF-β), 2% fetal calf serum, 100 μ g/mL gentamicins, 10 μ g/mL transferrins
(Transferrin), 10 μ g/mL sodium selenite, 1 μ g/mL vitamin C, 8 μ g/mL bovine insulins, 10ng/mL hydrogenation can
Pine, 2 μm of ol/mL glutamine (GlutaMAX), 2ng/mL hEGF (HEGF), 2 μm of ol/mL 4- ethoxy piperazines
Piperazine ethanesulfonic acid (HEPES), HBM liver cell basal medium.
The experimental results showed that the present invention can construct rapidly liver fibrosis organoid model in 16 days, and mould shapes are uniform,
Size is 200 μm or so, avoids the situation of the excessive centrocyte anoxic necrosis of volume.The organoid can secrete white egg simultaneously
It is white, and CYP4A3 is expressed, the livers specific markers such as CYP450.It is used culture medium C successive induction six days after the tenth day, Ke Yi
Stable fibrosis phenotype is obtained in liver organoid, such as expresses Fibrosis Markers α-actin in albumen and gene level
And I-type collagen.
The present invention is based on liver cellular constituent ratio, rapid build size structure is uniform, fibrosis characterizes apparent 3D
Hepatic fibrosis-renal tubular ectasia syndrome organoid external model.The hepatic fibrosis-renal tubular ectasia syndrome organoid model that the present invention constructs, definite principle, stable structure,
Method is simple, and rapidly, strong operability is suitable for hepatic fibrosis-renal tubular ectasia syndrome occurrence and development Mechanism Study, and anti-fibrosis medicine is high for building
Flux screening etc. has industrialization meaning.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1 is the preparation technology flow chart of liver fibrosis organoid.
Fig. 2 is the light microscopic shooting figure of different time points.
Fig. 3 is the expression schematic diagram of confocal detection liver fibrosis organoid liver specific markers.
Fig. 4 is Live/dead fluorescence detection liver fibrosis organoid growth conditions schematic diagram.
Fig. 5 is that ELISA detection liver fibrosis organoid model albumin (A) and inflammatory factor (B and C) secretion level shows
It is intended to.
Fig. 6 is RT-PCR detection liver fibrosis organoid model albumin (A) and Fibrosis Markers (B and C) mRNA water
Flat schematic diagram.
Fig. 7 is the expression schematic diagram of confocal detection liver fibrosis organoid Fibrosis Markers.
Fig. 8 is the expression schematic diagram of immunohistochemistry detection liver fibrosis organoid Fibrosis Markers.
Specific embodiment
Referring to Fig. 1, Fig. 1 is the preparation technology flow chart of liver fibrosis organoid provided by the invention.
Step 1, the primary a variety of liver components cells of sorting, passes through liquid nitrogen cryopreservation conservation；Or directly purchase a variety of livers frozen
Component cells.By liver cell, hepatic stellate cells, Kupffer Cell, sinusoidal endothelial cell is carefully multiple using automated cell resuscitation system
Soviet Union, 800rps centrifugation, is resuspended using culture medium A, is counted respectively using cell counter.According to 7:1.5:0.5:1 ratio, with
1200 cell per wells/100 μ L are carefully equably planted the round bottom tissue culture plate in ultralow absorption using the volley of rifle fire, persistently cultivate 3
It.Culture medium A ingredient includes: 2 μ g/mL I type rat tail collagen proteins, 10% fetal calf serum, 8 μ g/mL bovine insulins, 10ng/mL
Hydrocortisone, 2 μm of ol/mL glutamine (GlutaMAX), 2 μm of ol/mL 4- hydroxyethyl piperazineethanesulfonic acids (HEPES), nothing
Phenol red WILLIAMS-DARLING Ton E culture medium (William's E Medium).
Step 2 is placed in 37 DEG C, 5% CO in culture medium A after inoculation2It is cultivated in incubator, stationary culture 3 days
It observes each hole and forms uniform cell mass.Culture medium B is replaced on day 4, and equal conditions continue to cultivate.Culture medium B at
Divide includes 100 μ g/mL gentamicins, 10 μ g/mL transferrins (Transferrin), 10% fetal calf serum, 10 μ g/mL selenous acid
Sodium, 10 μ g/mL linoleic acid, 1 μ g/mL vitamin C, 8 μ g/mL bovine insulins, 10ng/mL hydrocortisone, 2 μm of ol/mL paddy
Glutamine (GlutaMAX), 2 μm of ol/mL 4- hydroxyethyl piperazineethanesulfonic acids (HEPES), 2ng/mL hEGF
(HEGF), HBM liver cell basal medium.
Step 3 is close to culture medium liquid level using micropipette rifle, the next day inhale and abandon the 50 old culture mediums of μ L in hole, slowly plus
Enter equivalent culture medium B, persistently cultivates 6 days.The organoid that visible size is 200 μm or so in each hole of tissue culture plate.?
10 days, replace culture medium C, equal conditions continue culture six days, the next day half amount change liquid.Culture medium C includes: 5ng/mL conversion life
The long factor-β (TGF-β), 100 μ g/mL gentamicins, 10 μ g/mL transferrins (Transferrin), 2% fetal calf serum, 10 μ
G/mL sodium selenite, 1 μ g/mL vitamin C, 8 μ g/mL bovine insulins, 10ng/mL hydrocortisone, 2 μm of ol/mL glutamine
(GlutaMAX), 2ng/mL hEGF (HEGF), 2 μm of ol/mL 4- hydroxyethyl piperazineethanesulfonic acids (HEPES), HBM
Liver cell basal medium.
Referring to fig. 2, Fig. 2 is according to step 1 to three, from 4h to the 16th day in the organoid mould of different time nodes shooting
Type light microscopic figure.It is cultivated 3 days in culture medium A, being initially placed in culture medium B on day 4 makes organoid model form simultaneously maturation,
Start within 10th day to carry out fibrosis induction, the 16th day formation liver fibrosis organoid model.Liver reality is broadly divided into human liver
Cell plastid and nonparenchymal cell, wherein nonparenchymal cell mainly has hepatic stellate cells, sinusoidal endothelial cell and Kupffer Cell.By this
Cell proportion combined inoculation is in round 96 orifice plates of ultralow absorption in four kinds of cell simulation livers, by being self-assembly of organoid sample
After model, morphological feature can be preferably kept in next cultivation cycle.The liver organoid that the present invention constructs is stablized
Within 200 μm of diameter, this can preferably avoid as the excessive center anoxic of volume and caused by meronecrosis.Meanwhile
After carrying out fibrosis induction using culture medium C, from form and no abnormal variation.
Referring to Fig. 3, on day 10 of culture, liver specific markers albumin in identified by immunofluorescence organoid, and spy are used
Different enzyme CYP4A3 and CYP450.Specifically, organoid ball is carefully sucked out using suction nozzle, is placed in the poly that volume fraction is 4%
30 min are fixed in formaldehyde, then use 0.1% Triton-X permeable membrane, 15 min.Be added 5% BSA solution closing 2h after be incubated in
It is stayed overnight for 4 DEG C in specific primary antibody.The next day wash 3 times using PBS after corresponding fluorescence secondary antibody is added, be protected from light incubation at room temperature one hour.Make
After washing 3 times with PBS, room temperature is protected from light DAPI dyeing 30min.The confocal detection three luciferase expression intensity under particular excitation light.
Liver marker albumin and metabolic enzyme CYP4A3 and CYP450 have specifically expressing in the 10th day, liver organoid model, mention
Show that liver organoid model has liver organization similar characteristics.
Referring to fig. 4, it according to process flow, took out part liver organoid respectively at the 10th day of culture and the 16th day, uses
Live/dead method analyzes survival condition.Specifically, two groups of organoid balls are carefully sucked out using suction nozzle, embathe 3 using PBS
Time.It is pre-configured with dyeing liquor (calcein AM/ethidium homodimer-1,1:4), organoid ball is added and is protected from light dyeing
30min, the confocal detection under particular excitation light.As shown in figure 4, after culture medium C inducing fibrosis, compared to the 10th day,
Dead cell in liver organoid is increased slightly, but there is no apparent differences for overall survival state, have no obvious a large amount of liver cells
It is dead.This is also that a kind of concept of chronic liver injury disease is consistent with liver fibrosis.
Referring to Fig. 5, the medium supernatant of the 10th day and the 16th day liver organoid is taken, with enzyme-linked absorption method is immunized
(ELISA), the secretion situation of liver fibrosis organoid albumin and inflammatory factor is detected.Specifically, standard items and not are taken
It is added in particular bore with 100 μ L of time point liver organoid supernatant, is incubated at room temperature 2.5h.4 are rinsed using 300 μ L cleaning buffer solutions
Secondary, each 5min is inverted detection plate and pats removal extraction raffinate.100 μ L antibody diluents are added, are incubated at room temperature 1h.It is clear using 300 μ L
After wash buffer rinses 4 times, 100 μ L Streptavidins are added and are incubated at room temperature 45min.Continue to use the punching of 300 μ L cleaning buffer solutions
It washes 4 times, tmb substrate solution room temperature is added and is protected from light incubation 30min.50 μ L terminate liquids are added in each hole, with 450nm wave in plate reader
Long detection.As shown in figure 5, liver specific markers albumin contains in supernatant after using culture medium C continuous induction culture 6 days
Amount is decreased obviously, and the function of albumin secretion of liver organoid is prompted to be suppressed, thus it is speculated that liver cell in liver organoid by
Certain damage.Meanwhile organoid inflammatory factor TNF-α and IL-1 β secretion increased significantly.The liver fiber that the prompt present invention constructs
Change the feature that organoid external model there can be liver damage and inflammation within a short period of time.
Referring to Fig. 6, albumin and Fibrosis Markers mRNA expression in RT-PCR detection liver organoid are used.It uses
RNeasy Mini kit (Qiagen) extracts the 10th day and the 16th day liver organoid total serum IgE, uses High-Capacity cDNA
Reverse Transcription kit (Applied Biosystems) reverse transcription is cDNA.Use PowerUp SYBR
Green Master Mix(Thermo Fisher Scientific), according to primer (ACTA2, F:
AAAAGACAGCTACGTGGGTGA; R: GCCATGTTCTATCGGGTACTTC. ALB, F:
TGCAACTCTTCGTGAAACCTATG; R:ACATCAACCTCTGGTCTCACC. COL1A1, F:
GAGGGCCAAGACGAAGACATC;R:CAGATCACGTCATCGCACAAC.GAPDH, F: GGAGCGAGATCCCTCCAAAAT,
R:GGCTGTTGTCATACTTCTCATGG RT-PCR) is carried out on real-time PCR.As shown in fig. 6, with ELISA result
Unanimously, the ALB gene expression of encoding albumin is obviously lowered after continuously being cultivated using culture medium C 6 days.Meanwhile encoding fiber
The COL1A1 expression of the ACAT2 gene and encoding Type I collagen of marker alpha Actinin significantly rises, and prompts structure of the present invention
The liver fibrosis organoid external model built is provided with fibrosis characterization within a short period of time.
Referring to Fig. 7, Fibrosis Markers alpha Actinin and I type in immunofluorescence/laser co-focusing identification organoid are used
Collagen expression.The same Fig. 3 of specific method.As shown in fig. 7, Fibrosis Markers α-flesh after continuously being cultivated using culture medium C 6 days
Filamentous actin and the obvious rising of I- collagen type expression, illustrate that the liver fibrosis organoid that the present invention constructs has typical fiber
Change phenotype.
Referring to Fig. 8, Fibrosis Markers alpha Actinin and I-type collagen table in immunohistochemistry detection organoid are used
It reaches.The 10th day and the 16th day liver organoid are taken, using the fixed 1h of 4% formalin, paraffin embedding cuts 5 μm of slices.Dewax water
Change, is put into 3% hydrogen peroxide to reduce endogenous catalase.Citrate buffer antigen retrieval 30min is used at 100 DEG C,
Room temperature closes 1h in BSA solution.After TBST is cleaned three times, secondary antibody dilution is added in 4 DEG C of the primary antibody dilution overnight incubations in wet box
Liquid is incubated at room temperature 1h.After the processing of DAB solution, haematoxylin redyeing is used.Mounting is simultaneously taken pictures in microscopically observation.With the above copolymerization
Burnt fluorescence analysis is consistent, and such as Fig. 8 immunohistochemical staining, Fibrosis Markers α-flesh moves egg after culture medium C continuous induction culture 6 days
The expression of white and I- collagen type is obvious to be risen, and further illustrates that the present invention successfully constructs liver fibrosis organoid model.
Described above is only the preferred embodiment of the present invention, under the premise of following the principle of the invention, can also be made
Several improvement, these improvement should also be considered as protection scope of the present invention.
Claims (7)
1. a kind of vitro construction method of liver fibrosis organoid model, it is characterized in that: including the following steps:
By liver cell, hepatic stellate cells, Kupffer Cell and sinusoidal endothelial cell, it is suspended in culture medium A；Culture was changed to the 4th day
Culture medium B maintains culture；Start within 10th day, is changed to culture medium C, continuous culture six days；
The culture medium A include: bovine insulin, 4- hydroxyethyl piperazineethanesulfonic acid, hydrocortisone, glutamine, fetal calf serum,
I type rat tail collagen protein and without phenol red WILLIAMS-DARLING Ton E culture medium；
The culture medium B includes: gentamicin, transferrins, vitamin C, bovine insulin, fetal calf serum, sodium selenite, sub- oil
Acid, hydrocortisone, 4- hydroxyethyl piperazineethanesulfonic acid, glutamine, hEGF and the culture of HBM liver cell basis
Base；
The culture medium C includes: gentamicin, transferrins, vitamin C, bovine insulin, fetal calf serum, sodium selenite, hydrogenation
Cortisone, 4- hydroxyethyl piperazineethanesulfonic acid, glutamine, transforming growth factor-β, hEGF and HBM liver cell base
Basal culture medium.
2. the vitro construction method of a kind of liver fibrosis organoid model according to claim 1, it is characterized in that: liver is thin
Born of the same parents, hepatic stellate cells, Kupffer Cell and sinusoidal endothelial cell, are counted using calculating instrument, by the special ratios of 7:1.5:0.5:1,
It is suspended in culture medium A.
3. the vitro construction method of a kind of liver fibrosis organoid model according to claim 2, it is characterized in that: by liver
Cell, hepatic stellate cells, Kupffer Cell and sinusoidal endothelial cell mix after A in specific proportions, and 1200, every hole is pressed after counting
Cell is equably inoculated in ultralow absorption round bottom cell plates using the volley of rifle fire, and the volume of every hole inoculating cell suspension is 100 μ L.
4. according to a kind of vitro construction method of liver fibrosis organoid model of claims 3, it is characterized in that: liver cell,
Hepatic stellate cells, Kupffer Cell and sinusoidal endothelial cell are suspended in culture medium A in proportion, are inoculated in ultralow absorption round bottom cell
Plate is then placed in 37 DEG C of carbon dioxide incubators, stands 3 days, avoids moving as far as possible.
5. a kind of vitro construction method of liver fibrosis organoid model, feature according to claims 1,2,3 or 4
Be: culture, using micropipette rifle, was close to culture medium liquid level, old culture medium is sucked out to the 4th day；100 are slowly added to along hole wall again
μ L culture medium B, the next day inhale and abandon 50 μ L culture mediums in hole, and equivalent culture medium B is added, persistently cultivates 6 days.
6. a kind of vitro construction method of liver fibrosis organoid model, feature according to claims 1,2,3 or 4
It is: cultivates to after the 10th day, using micropipette rifle, is close to culture medium liquid level, old culture medium is sucked out；It is slowly added to again along hole wall
100 μ L culture medium Cs, the next day inhale abandon 50 μ L culture mediums, be added equivalent culture medium C, persistently cultivate 6 days.
7. a kind of vitro construction method of liver fibrosis organoid model according to claims 1,2,3 or 4, special
Sign is: culture medium A includes: 2 μ g/mL I type rat tail collagen proteins, 10% fetal calf serum, 8 μ g/mL bovine insulins, 10ng/mL hydrogen
Change cortisone, 2 μm of ol/mL glutamine, 2 μm of ol/mL 4- hydroxyethyl piperazineethanesulfonic acids, without phenol red WILLIAMS-DARLING Ton E culture medium；
Culture medium B includes: 100 μ g/mL gentamicins, 10% fetal calf serum, 10 μ g/mL transferrins, 10 μ g/mL sodium selenites, 10 μ
G/mL linoleic acid, 1 μ g/mL vitamin C, 8 μ g/mL bovine insulins, 10ng/mL hydrocortisone, 2 μm of ol/mL glutamine,
2 μm of ol/mL 4- hydroxyethyl piperazineethanesulfonic acids, 2ng/mL hEGF, HBM liver cell basal medium；Culture medium C
It include: 5ng/mL transforming growth factor-β, 2% fetal calf serum, 100 μ g/mL gentamicins, 10 μ g/mL transferrins, 10 μ g/mL
Sodium selenite, 1 μ g/mL vitamin C, 8 μ g/mL bovine insulins, 10ng/mL hydrocortisone, 2 μm of ol/mL glutamine,
2ng/mL hEGF, 2 μm of ol/mL 4- hydroxyethyl piperazineethanesulfonic acids, HBM liver cell basal medium.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111411084A (en) * | 2020-04-28 | 2020-07-14 | 江苏信安佳医疗科技有限公司 | Culture medium and culture method for constructing liver tumor stent-free organoid |
| CN113337454A (en) * | 2021-07-21 | 2021-09-03 | 中国人民解放军总医院第五医学中心 | Construction method and application of 3D hepatic fibrosis model |
| CN113667633A (en) * | 2021-07-29 | 2021-11-19 | 四川大学华西医院 | A method for constructing a cortical organoid model of dysplasia in schizophrenia |
| CN113717928A (en) * | 2021-09-03 | 2021-11-30 | 复旦大学附属中山医院 | Method for constructing 3D liver bud organoid based on frame nucleic acid material and application |
| WO2023019760A1 (en) * | 2021-08-19 | 2023-02-23 | 清华大学 | Artificial liver organoid, and preparation method therefor and application thereof |
| WO2023206763A1 (en) * | 2022-04-28 | 2023-11-02 | 苏州大学 | Hepatic fibrosis chip and use thereof in development and treatment of hepatic fibrosis drug |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018115533A1 (en) * | 2016-12-23 | 2018-06-28 | Insphero Ag | Screenable liver disease models and methods |
| CN109337860A (en) * | 2018-10-29 | 2019-02-15 | 妙顺（上海）生物科技有限公司 | A kind of hepatic fibrosis in vitro 3D model building method |
-
2019
- 2019-02-20 CN CN201910124700.4A patent/CN109880791B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2018115533A1 (en) * | 2016-12-23 | 2018-06-28 | Insphero Ag | Screenable liver disease models and methods |
| CN109337860A (en) * | 2018-10-29 | 2019-02-15 | 妙顺（上海）生物科技有限公司 | A kind of hepatic fibrosis in vitro 3D model building method |
Non-Patent Citations (1)
| Title |
|---|
| VINCENZO PRESTIGIACOMO ET AL: "pro-fibrotic compounds induce stellate cell activation, ECM-remodelling and Nrf2 activation in a human 3D-multicellular model of liver fibrosis", 《PLOS ONE》 * |
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| CN111411084A (en) * | 2020-04-28 | 2020-07-14 | 江苏信安佳医疗科技有限公司 | Culture medium and culture method for constructing liver tumor stent-free organoid |
| CN113337454A (en) * | 2021-07-21 | 2021-09-03 | 中国人民解放军总医院第五医学中心 | Construction method and application of 3D hepatic fibrosis model |
| CN113337454B (en) * | 2021-07-21 | 2023-02-21 | 中国人民解放军总医院第五医学中心 | Construction method and application of 3D hepatic fibrosis model |
| CN113667633A (en) * | 2021-07-29 | 2021-11-19 | 四川大学华西医院 | A method for constructing a cortical organoid model of dysplasia in schizophrenia |
| WO2023019760A1 (en) * | 2021-08-19 | 2023-02-23 | 清华大学 | Artificial liver organoid, and preparation method therefor and application thereof |
| CN113717928A (en) * | 2021-09-03 | 2021-11-30 | 复旦大学附属中山医院 | Method for constructing 3D liver bud organoid based on frame nucleic acid material and application |
| CN113717928B (en) * | 2021-09-03 | 2024-01-26 | 苏州朴衡科技有限公司 | Method for constructing 3D liver bud organoid based on framework nucleic acid material and application |
| WO2023206763A1 (en) * | 2022-04-28 | 2023-11-02 | 苏州大学 | Hepatic fibrosis chip and use thereof in development and treatment of hepatic fibrosis drug |
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