CN110438063A - The preparation of humanization class brain organ ischemia's model and application - Google Patents

The preparation of humanization class brain organ ischemia's model and application Download PDF

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CN110438063A
CN110438063A CN201810417891.9A CN201810417891A CN110438063A CN 110438063 A CN110438063 A CN 110438063A CN 201810417891 A CN201810417891 A CN 201810417891A CN 110438063 A CN110438063 A CN 110438063A
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CN110438063B (en
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缪朝玉
王淑娜
徐添颖
王治
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Second Military Medical University SMMU
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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Abstract

The present invention relates to a kind of humanization class brain organ ischemia's models and preparation method thereof, application, the preparation method is that: class brain organ is added in sugar-free DMEM culture medium, DMSO concentration≤1 ‰ in culture medium are then placed into three gas incubator (1%O2, 5%CO2, 94%N2) in culture.Its advantage is shown: the present invention provides a kind of new cerebral ischemic models, and new model construction direction is provided for cerebral ischemia research, provide new optional screening model for brain ischemia medicament screening.The present invention has carried out application study of the class brain in cerebral ischemic model building and drug screening for the first time.

Description

The preparation of humanization class brain organ ischemia's model and application
Technical field
The present invention relates to brain science technical fields, specifically, being humanization class brain organ ischemia model and its preparation side Method, application.
Background technique
In recent years, the research for multipotential stem cell (pluripotent stem cell) gradually increased.It cultivates in vitro Under environment, give multipotential stem cell specific condition of culture, it can be made to develop towards different directions, as ectoderm, mesoderm, Entoderm, and then it is divided into different types of cell, organ sample tissue, including brain tissue.2013, Austrian scientist existed According to the report, first passage 3D culture systems, are class brain organ by multipotential stem cell induction culturing on Nature magazine.This type brain Organ is referred to as " cerebral organoids ", it can successfully simulate cortex development process, in conjunction with RNA perturbation technique and The inductive pluripotent stem cells in patient source successfully construct microcephaly disease model for the first time.Then, in May, 2016, Cell are miscellaneous A current paper in will, the team from Johns Hopkins University, the U.S. pass through the side of rotating biological generator culture Method optimizes the induction culturing method of class brain organ, reduces and cultivates cost, raising can operate repeatability, and realize class brain organ The specific differentiation in brain area domain, respectively obtained class forebrain, midbrain and hypothalamus sample organ.
With the gradually propulsion of research, the application of class brain organ and brain area specificity class brain is gradually promoted, for example, class Microcephaly disease caused by brain analog zika virus；Probe into neurite outgrowth of the antenatal alcohol exposure to class brain, nerve regneration Influence；It is mutated using the inductive pluripotent stem cells combination presenilin-1 in patients with Alzheimer disease source, simulates brain Cortex neural degeneration microenvironment；Utilize class brain chip simulation nicotine exposure etc..But there is no literature reported on class brains in brain at present Application in ischemia model building and drug screening.
Summary of the invention
The first purpose of this invention is aiming at the shortcomings in the prior art, to provide type brain organ ischemia's model Preparation method.
Second object of the present invention is to provide type brain organ ischemia's model.
Third object of the present invention is to provide the application of type brain organ ischemia's model.
To realize above-mentioned first purpose, the technical solution adopted by the present invention is that: the system of type brain organ ischemia's model Preparation Method, the preparation method is that: class brain organ is added in sugar-free DMEM culture medium, DMSO concentration in the culture medium≤ 1 ‰, it is then placed into incubator and cultivates.
The class brain organ is humanization class brain organ.
The humanization class brain organ is the humanization class brain organ of 3D structure.
The humanization class brain organ is successively undergone differentiation of germinal layers, neuroderm differentiation, nerve by human pluripotent stem cells Epithelial tissue is formed, and induction obtains the humanization class brain organ of 3D structure in rotation generating bottle.
The preparation method of the humanization class brain organ the following steps are included:
(1) kind of human pluripotent stem cells are inscribed in ultralow absorption orifice plate, obtain embryoid body like cell cluster；
(2) continue induction differentiation, embryoid body like cell cluster starts differentiation of germinal layers feature occur, formed on initial nerve Skin sample tissue, and be transferred in new ultralow absorption orifice plate and continue to cultivate；
(3) neural epithelium sample tissue continues induction differentiation, and experience Matrigel glue drips coated static culture, forms nerve Epithelial sprout sample tissue starts the differentiation for carrying out class brain organ；
(4) the neural epithelium bud sample tissue for being coated with Matrigel glue drop is transferred in rotation generating bottle, continues to cultivate, obtains The class brain organ that brain function subregion must be formed, express forebrain marker and choroid plexus marker.
The incubator is three gas incubators.
Contain 1%O in the three gas incubator2, 5%CO2, 94%N2。
The class brain organ is added before sugar-free DMEM culture medium, first inhales and abandons cell culture medium, then is cleaned with PBS.
To realize above-mentioned second purpose, the technical solution adopted by the present invention is that: the class brain device of above-mentioned preparation method preparation Official's ischemia model.
To realize above-mentioned third purpose, the technical solution adopted by the present invention is that: class brain organ ischemia's model exists Application in drug screening.
The invention has the advantages that:
1, the present invention provides a kind of new cerebral ischemic model, with the extension of scarce sugared anoxic (OGD) time, Caspase 3activity increases, LDH Release increases；Give the related autophagy inhibitor 3-MA of cerebral ischemia access, 3 inhibitors of apoptosis Z-VAD-FMK, nNOS inhibitor L-NAME, AMPA inhibitor C NQX of Caspase can significantly reduce OGD and draw The Caspase 3activity risen increases, that is, effectively reduces cell death.
2, the present invention provides new model construction direction for cerebral ischemia research, provides for brain ischemia medicament screening new Screening model may be selected.
3, the present invention has carried out application study of the class brain in cerebral ischemic model building and drug screening for the first time.
Detailed description of the invention
Attached drawing 1 is the schematic diagram (shooting under inverted microscope) of class brain organ difference cultivation stage.
Attached drawing 2 is the class brain organ immunofluorescence (shooting under laser confocal microscope) of different development times.In class The different phase that brain organ is cultivated, take culture 15 days, 30 days, 60 days class brains carry out frozen section and immunofluorescence dyeing.It is logical Labeled neural stem cells marker SOX2 and neuron marker Tuj-1 are crossed, display is with the extension for cultivating the time, stem cell ratio Example is reduced, and neuron ratio increases, i.e., class brain organ is gradually reached maturity.
Attached drawing 3 is that (laser co-focusing is micro- for class brain orga- nogenesis " forebrain and choroid plexus " brain function subregion immunofluorescence It is shot under mirror).Class brain organ culture carried out frozen section and immunofluorescence dyeing to 75 days, and forebrain and train of thought have been expressed in display Clump marker prompts to have formed brain function subregion, i.e., successfully constructs class brain organ.
Specific embodiment
It elaborates below with reference to embodiment to specific embodiment provided by the invention.
Embodiment 1: the culture of class brain organ
According to bibliography (Nature 2013；501:373-379 and Nat Protoc 2014；9:2329-2340), it passes through It is a series of to grope and improve, ultralow absorption orifice plate and rotating biological generator culture systems are successively used, human pluripotent stem cells are first By go through differentiation of germinal layers, neuroderm differentiation, neuroepithelial tissue formed, rotation generating bottle in induction obtain 3D knot The humanization class brain organ of structure, cultivation stage are as follows.
First stage: in 96 orifice plates of ultralow absorption, according to viable cell density 6 × 104A/mL, every hole are inoculated with 150 μ L, obtain Obtain embryoid body like cell cluster (shown in Fig. 1, Day 2).
Second stage: continuing induction differentiation, and embryoid body starts differentiation of germinal layers feature occur, carries out inducing initial nerve Epithelial tissue (shown in Fig. 1, Day 5) is transferred in 24 orifice plates of ultralow absorption and continues to cultivate.
Phase III: neural epithelium sample tissue continues induction differentiation, and experience Matrigel glue drips coated static culture, shape At neural epithelium bud sample tissue, that is, start the differentiation for carrying out class brain organ (shown in Fig. 1, Day 15).
Fourth stage: the tissue that above-mentioned coating Matrigel glue drips is transferred in rotation generating bottle, with the speed of 85rpm Degree, continues to cultivate.Class brain organ-tissue is further differentiated into ripe, and volume becomes larger (shown in Fig. 1, Day 30).With the cultivation time Extension, neural stem cell ratio gradually decreases in class brain organ-tissue, and neuron ratio increases (Fig. 2), finally, class brain organ Interior formation brain function subregion expresses forebrain marker and choroid plexus marker (Fig. 3), successfully obtains class brain organ.
Embodiment 2: class brain OGD modeling cerebral ischemia
Experimental method:
1. being grown to the 75th day to class brain, inhale and abandon cell culture medium, after being cleaned twice with 1 × PBS, under naked eyes and microscope Class brain volume size is observed, and is made marks.
2. being grouped according to class brain volume to it, class brain of the same size is evenly distributed in each group.
3. sugar-free DMEM culture medium is added, and control group is set, DMSO concentration≤1 ‰ in culture medium.
4. being placed in three gas incubator (1%O2, 5%CO2, 94%N2) in culture.
5. surveying Caspase 3activity or LDH Release after culture to 4h, 6h, 8h.
Test method:
1.Caspase 3activity measurement
The measuring method uses green 3 activity detection kit of skies Caspase (article No.: C1115), includes: C1115-1 Lysate 8ml；C1115-2 detects buffer 8ml；C1115-3Ac-DEVD-pNA(2mM)200μl；C1115-4pNA(10mM) 200μl.- 20 DEG C of preservations, Ac-DEVD-pNA and pNA need to be kept in dark place.
(1) preparation:
A. mixing is placed in spare on ice bath after lysate dissolves.
B. detect buffer solution after mix be placed in it is spare on ice bath.
(2) measures pNA standard curve:
A. it the preparation of standard dilutions: is prepared according to the ratio that 0.1ml lysate is added in every 0.9ml detection buffer suitable The standard dilutions of amount.
B. the pNA (10mM) that kit provides is diluted to 0,10,20,50,100 and 200 μM with standard dilutions, made For standard items.
C. each concentration takes 100 μ l to be detected with microplate reader, or the light splitting light for taking appropriate amount capacity to be no more than 100 μ l Degree detection cup is detected, and A405 is measured.
D. the A405 that the A405 of each standard items subtracts the blank control without pNA is calculated actual to be led because of pNA The absorbance of cause, and produce standard curve of the pNA concentration relative to A405.
(3) collection of sample
A. for class brain tissue sample: lysate is added according to the ratio that 100 microlitres of lysates are added in every 3-10mg tissue, It is homogenized on ice bath with glass homogenizer.Then homogenate is transferred in 1.5ml centrifuge tube, ice bath cracks 5 minutes again.
B.4 DEG C 16,000-20,000g is centrifuged 10-15 minutes.
C. supernatant is transferred in the centrifuge tube of ice bath pre-cooling.
D. the enzymatic activity or -70 DEG C of preservation samples of caspase 3 are measured immediately.A small amount of sample can be taken to use simultaneously Bradford method measures protein concentration, so that protein concentration is reached 1-3mg/ml as far as possible, is equivalent in every 10 microlitres of samples to be tested extremely Contain 10-30 μ g albumen less.If cell is smaller, it can suitably increase the dosage of cell.
2.Bradford measures protein concentration
The measuring method uses green skies Bradford determination of protein concentration kit (article No.: P0006), includes: P0006-1G250 dyeing liquor 125ml × 2；P0006-2 protein standard (5mg/ml BSA) 1.5ml.4 DEG C of preservations, a Nian Youxiao. Protein standard can be with -20 DEG C of long-term preservations.
(1) preparation of protein standard
A. for protein sample in what solution, standard items also preferably use any solution dilution.But for simplicity, if Solution where protein sample can also use 0.9%NaCl, PBS or water dilution mark without containing the substance for interfering the detection of this kit Quasi- product.Protein standard (5mg/ml BSA) uses after please melting and mix completely if frozen.
B. 0,0.125,0.25,0.5,0.75,1,1.5mg/ml protein standard are prepared according to following table.Pay attention to when dilution every time It mixes well.The protein standard of setting 0.0625mg/ml can be increased if necessary.
Number Dilution volume Standard items volume Ultimate density
A 70μl 5mg/ml BSA 30μl 1.5mg/ml
B 30μl 60 μ l are taken from A pipe 1mg/ml
C 20μl 60 μ l are taken from B pipe 0.75mg/ml
D 30μl 60 μ l are taken from C pipe 0.5mg/ml
E 60μl 60 μ l are taken from D pipe 0.25mg/ml
F 60μl 60 μ l are taken from E pipe 0.125mg/ml
G 60μl 0μl 0mg/ml
(2) determination of protein concentration
A. 5 μ l various concentration protein standards is taken to be added in the protein standard hole of 96 orifice plates.
B. in the sample well for taking 5 μ l samples to 96 orifice plates.If sample need to add standard dilutions to supply to 5 less than 5 μ l μl.Please note that record sample volume.
C. 250 μ l G250 dyeing liquors are added in each hole.
D. with the absorbance of other wavelength between microplate reader measurement A595 or 560-610nm.Extinction can be measured immediately Degree, can also measure, detection data is without significant changes in 2 hours in 2 hours.
E. the protein concentration in sample is calculated according to standard curve and the sample volume used.
3.LDH Release measurement
The measuring method uses Japan DOJINDO Cytotoxicity LDH Assay Kit-WST assay kit (goods Number: it CK12), includes: Mixture × 1 Dye；Assay Buffer 11ml×1；Lysis Buffer 1.1ml×1；Stop Solution 5.5ml×1.4 DEG C of preservations, validity period 6 months.
(1) prepares Working Solution
1ml Assay Buffer is added into Dye Mixture bottle, is sufficiently mixed.It is transferred to after being completely dissolved In the bottle of Assay Buffer, it is sufficiently mixed.Working Solution is please kept in dark place at 0-5 DEG C, and stability is up to 6 Month.
(2) .LDH Release is measured
A. class brain takes 100 μ l of its supernatant in 96 orifice plates after OGD, and blank control wells are arranged.
B. after 100 μ l Working Solution are added in each hole, be protected from light, room temperature under conditions of cultivate.
C. it is incubated at room temperature after twenty minutes, 50 μ l Stop Solution are added in every hole, at once with microplate reader measurement 490nm's Absorbance.
Experimental result:
With the extension of OGD time, the Caspase 3activity of class brain organ increases.
With the extension of OGD time, the LDH Release of class brain organ increases.
Embodiment 3: effect of the cerebral ischemia access related inhibitors in class brain OGD model
Experimental method:
1. being grown to the 75th day to class brain, inhale and abandon cell culture medium, after being cleaned twice with 1 × PBS, under naked eyes and microscope Class brain volume size is observed, and is made marks.
2. being grouped according to class brain volume to it, class brain of the same size is evenly distributed in each group.
3. the sugar-free DMEM culture medium of drug containing or drug solvent is added, DMSO concentration≤1 ‰ in culture medium.
4. being placed in three gas incubator (1%O2, 5%CO2, 94%N2) in culture.
5. surveying Caspase 3activity after culture to 8h (test method is shown in embodiment 2).
Experimental result:
Give cerebral ischemia access correlation autophagy inhibitor 3-MA, Caspase 3 inhibitors of apoptosis Z-VAD-FMK, nNOS suppression Effectively preparation L-NAME, AMPA inhibitor C NQX can significantly reduce the Caspase 3activity increase of class brain after OGD8h, i.e., Reduce cell death in class brain.
Caspase 3acticity(Fold)
1 ‰ DMSO of control group 1.00
50 μM of 3-MA of autophagy inhibitor 0.69
3 inhibitor of Caspase, 20 μM of Z-VAD-FMK 0.63
20 μM of CNQX of AMPA inhibitor 0.71
Control group H2O 1.00
200 μM of L-NAME of nNOS inhibitor 0.71
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art Member, under the premise of not departing from the method for the present invention, can also make several improvement and supplement, these are improved and supplement also should be regarded as Protection scope of the present invention.

Claims (10)

1. the preparation method of type brain organ ischemia's model, which is characterized in that the preparation method is that: class brain organ is added In sugar-free DMEM culture medium, DMSO concentration≤1 ‰, are then placed into incubator and cultivate in the culture medium.
2. preparation method according to claim 1, which is characterized in that the class brain organ is humanization class brain organ.
3. preparation method according to claim 2, which is characterized in that the humanization class brain organ is the source of people of 3D structure Change class brain organ.
4. preparation method according to claim 3, which is characterized in that the humanization class brain organ is by human pluripotent stem cells Successively experience differentiation of germinal layers, neuroderm differentiation, neuroepithelial tissue are formed, and induction obtains 3D knot in rotation generating bottle The humanization class brain organ of structure.
5. the preparation method according to claim 4, which is characterized in that the preparation method of the humanization class brain organ includes Following steps:
(1) kind of human pluripotent stem cells are inscribed in ultralow absorption orifice plate, obtain embryoid body like cell cluster；
(2) continue induction differentiation, embryoid body like cell cluster starts differentiation of germinal layers feature occur, forms initial neural epithelium sample Tissue, and be transferred in new ultralow absorption orifice plate and continue to cultivate；
(3) neural epithelium sample tissue continues induction differentiation, and experience Matrigel glue drips coated static culture, forms neural epithelium Bud sample tissue starts the differentiation for carrying out class brain organ；
(4) the neural epithelium bud sample tissue for being coated with Matrigel glue drop is transferred in rotation generating bottle, continues to cultivate, obtain shape At brain function subregion, the class brain organ of expression forebrain marker and choroid plexus marker.
6. preparation method according to claim 1, which is characterized in that the incubator is three gas incubators.
7. preparation method according to claim 6, which is characterized in that contain 1%O in the three gas incubator2, 5%CO2、 94%N2。
8. preparation method according to claim 1, which is characterized in that the class brain organ be added sugar-free DMEM culture medium it Before, it first inhales and abandons cell culture medium, then cleaned with PBS.
9. class brain organ ischemia's model of any preparation method preparation according to claim 1~8.
10. application of the class brain organ ischemia's model according to claim 9 in drug screening.
CN201810417891.9A 2018-05-04 2018-05-04 Preparation and application of humanized brain organ ischemia model Active CN110438063B (en)

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CN114958746B (en) * 2022-06-08 2024-05-31 中国科学院动物研究所 Method and kit for inducing pluripotent stem cells to generate 3D brain-like bodies

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