CN107937339A - A kind of external model method for building up of gestational period alcohol exposure to fetus brain damage - Google Patents
A kind of external model method for building up of gestational period alcohol exposure to fetus brain damage Download PDFInfo
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Abstract
A kind of external model method for building up of gestational period alcohol exposure to fetus brain damage, 3D brain tissue and biomaterial sodium alginate silk of this method mainly in combination with emerging hiPSCs sources, stimulated by being subject to appropriate alcohol, the influence that the simulation gestational period indulges in excessive drinking to fetus early stage brain growth.Vitro in organ disorder of development model of the model as innovation, not only it is beneficial to Morphology Effects of the observation alcohol directly perceived to brain growth, various detection methods can also be used for reference, study molecules and cytological mechanism of the alcohol to brain growth, the deficiency of conventional cell experiment and zoopery is compensate for, strong instrument is provided for influence of the research gestational period alcohol to fetus early stage brain growth.
Description
Technical field
A kind of microfluidic chip technology of the present invention, and in particular to external model of gestational period alcohol exposure to fetus brain damage
Method for building up.
Background technology
The molecules and cytological mechanism research for studying pathological conditions are best understood from for us, control and treat disease and carry
Basis is supplied.Traditional biological method mainly includes the cell culture mode and animal model of 2D.The cell culture of 2D can not mould
Intend the microenvironment of cell under physiological condition, including three-dimensional cell epimatrix, a clearance flow, cell-cell interaction etc., therefore with life
Morphological function under the conditions of reason compared to cell has very big deficiency, it is impossible to molecular level and cell aspect completely under reflection pathological conditions
Anomalous variation.And zoopery is presently mainly using mouse, rabbit, drosophila, nematode isotype biology, although model organism source
It is convenient, condition of culture is simple, but the very big difference based on people and animal, therefore be difficult reflect completely it is various under the conditions of human body
Physiological change.It is more common to have nuclear-magnetism to be total to and the research for human body is on the premise of minimum damage and convenient observation is ensured
Shake, CT etc., but these methods do not have real-time monitoring characteristics, and it is many can not all be applied to development of fetus,
The organoid of various source of human stem cell developed to have obtained effective development in recent years, including neural stem cell, intestines are done
Cell, embryonic stem cell and iPSCs.Organoid is characterized in stem cell or the 3D cell unitys that primary cell spontaneously forms
Structure, it contains the multiple functions cell of respective organization organ, these cells are self-assembled into specific with certain structure function
Tissue, simulates the growth course of respective organization organ to a certain extent.The 3D brains in wherein external hiPSCs sources replicate substantially
The process of fetus early stage brain growth.
But organoid technology also has many deficiencies and area for improvement at present.Due to the knot of its 3D cell mass
Structure feature so that internal cell massive mortality occurs, strongly limit external organoid development due to lacking oxygen and nutrition
Degree, including volume size, function maturity etc., and be scattered here and there vascular tissue in physiological conditions undertissue and organ to carry
For required oxygen and nutrition.Therefore it is expected to remedy such and insufficient with reference to other technologies.In addition, existing conventional cell culture skill
Art culture organoid is complicated, expends time length, poor controllability.Therefore combined with existing bioengineering class means be expected to it is excellent
Change organoid technology.
Filament has several big advantages as culture carrier：First, can be according to practical application by microfluidic chip technology
It is flexible and efficient to produce different sizes, the sodium alginate silk of thickness.Secondly, filament has higher specific surface area ratio, is beneficial to
Mass exchange, good living environment is provided for cell culture.Again, filament can be oriented to the growth of cell mass, produce
The similar brain tissue of structure, and its growth is limited in growth course, the work of meninx under physiological conditions is simulated to a certain extent
With.Finally, sodium alginate material can dissolve under the conditions of high salt or sodium citrate, discharge cell, meet different process demands.
But be at present combined bioengineering with the organoid in hiPSCs sources, establish the disease model field of Genus Homo still
Belong to blank.
The content of the invention
A kind of external model method for building up the object of the present invention is to provide gestational period alcohol exposure to fetus brain damage, should
Method uses sodium alginate macaroni yarn not only to ensure that the matter transportation in growth course, while reality as cell culture vector
Existing controllable development structure, and have the characteristics of monitoring in real time.
A kind of external model method for building up the present invention provides gestational period alcohol exposure to fetus brain damage, its step master
It is divided into four parts：
(1) preparation of sodium alginate macaroni yarn；
(2) 3D brains induction early period；
(3) 3D brains are wrapped in sodium alginate macaroni yarn；
(4) ethanol postincubation 3D brains.
The preparation of step (1) sodium alginate macaroni yarn, is specially：
The preparation of the sodium alginate macaroni yarn is the micro-fluidic chip using barrel forms, and the macaroni yarn internal diameter of preparation is
600-1000 μm, pipe thickness is 50-200 μm, liquid asepsis processing used.
The sodium alginate silk need to be immersed in DMEM/F12 culture mediums, make its swelling process of completion in the medium,
And in 4 DEG C of Cord bloods, easy to subsequent operation.
Step (2) 3D brain inductions early period, are specially：
(1) embryoid body is prepared using the PDMS chips with hole shape structure：The chip of hole shape structure is placed in 24 orifice plates, small
It is 600-800 μm to cheat diameter of movement, and depth is 100-300 μm, the formation for embryoid body.
(2) the 1st days, embryoid body is prepared, by 2 × 105-6×105A hiPSCs is digested to unicellular, and is transferred to (1) institute
In the chip stated, 500-2000rpm is centrifuged, 3-5min, used medium is KSR culture mediums, and adds 5 μM of Y27632；
The basic ingredient of the KSR culture mediums is DMEM/F12, in addition needs addition to account for the KnockOut of cumulative volume 20%
Replacement (KSR), accounts for cumulative volume 1%NEAA (Non Essential Amino Acid, 100 ×), accounts for cumulative volume 1%
GlutaMax (100 ×), accounts for cumulative volume 1%penicillin-streptomycin (100 ×), and 0.1mM beta-
Mercaptoethanol, 4ng/ml bFGF.
(3) the 2nd days, the embryoid body of formation is transferred in the culture dish of low adhesion, suspend culture embryoid body, culture used
Base is KSR culture mediums.
(4) the 5th days, induction embryoid body broke up to neural epithelium direction, and KSR culture mediums are replaced with nerve-inducing culture
Base；Cell mass still keeps suspending and cultivates, and replaces culture medium within every 3 days.
Wherein the basic ingredient of Neuronal induction media is DMEM/F12, in addition needs addition to account for cumulative volume 1%N2 (100
×), account for cumulative volume 1%GlutaMAX (100 ×), account for cumulative volume 1%NEAA (Non EssentialAmino Acid, 100
×), 1 μ g/ml heparin, account for cumulative volume 1%penicillin-streptomycin (100 ×).
Embryoid body is prepared using the PDMS chips of hole shape structure, its big I is hanged by the change and cell of pitting volume
The quantity of cell is adjusted in liquid, and the magnitude range of cell ball is about 50-500 μm.
HiPSCs carries out appropriate digestion using Accutase, and digestion time is about 2-5min, it is ensured that is finally digested to smaller
Cell block.It if digestion excessively becomes the individual cells being dispersed in, can make to contain more dead cell in embryoid body structure, influence later
Differentiation and development；If digestion deficiency, influences the balling-up effect of embryoid body, it is more difficult to form cell mass of the same size.
After HiPSCs is digested to smaller cell block using Accutase, follow-up centrifugally operated is carried out, using up cell can
Energy flocks together, the embryoid body bigger beneficial to volume is formed, and effectively improves the success rate of follow-up brain growth.
Although embryoid body can be formed within several hours, it is the need to ensure that when cell 24 is small and is stood in inherence PDMS chips
Culture, it is ensured that the stabilization of embryoid body structure.
Step (3) 3D brains are wrapped in sodium alginate macaroni yarn, are specially：
10th day, the cell mass induced early period is resuspended using Matrigel, and cell mass is injected into alginic acid with syringe
In sodium silk, whole process avoids producing bubble, it is ensured that operation maintains low temperature on ice, ensures that Matrigel does not solidify.
Sodium alginate silk containing cell mass is placed in 37 DEG C of incubator 20-30min, solidifies Matrigel.
Sodium alginate silk containing cell mass is transferred in 6 orifice plates and carries out follow-up cultivation, used medium is nerve point
Change culture medium.
The Neural Differentiation culture medium, used medium are Neural Differentiation culture medium, its basic ingredient is that volume ratio is 1:1
DMEM/F12 and Neuralbasal medium, in addition need addition to account for cumulative volume 1%B27 (50 ×), account for cumulative volume 0.5%N2
(100 ×), account for cumulative volume 0.5%NEAA (100 ×), account for cumulative volume 1%GlutaMAX (100 ×), account for cumulative volume 1%
Penicillin-streptomycin (100 ×), 0.05mM beta-mercaptoethanol.
The growth course of 3D brains needs to use Matrigel, Matrigel to provide three as extracellular matrix for 3D brain growths
Medium is tieed up, beneficial to the Rapid development of 3D brains.
Step (4) ethanol postincubation 3D brains concretely comprise the following steps：
11st day, control group used Neural Differentiation medium culture 3D brains, and sample sets are 20-200mM using alcohol concentration
Neural Differentiation medium culture 3D brains, culture medium used needs matching while using.In addition 6 orifice plate gap domestic demands add in sample sets
PBS buffer containing same concentration alcohol, need to be sealed using sealed membrane, reduce alcohol volatilization.Due to alcohol volatilization and ensure
The supply of nutriment, sample sets and control group are all to change a subculture in every two days.
A kind of gestational period alcohol exposure of the present invention is as follows to the external model of fetus brain damage, its follow-up detection method：
(1) under microscope light field in real time monitoring ethanol postincubation in the case of 3D brains the speed of growth and developmental condition；
(2) exception of RT-PCR method detection ethanol postincubation group 3D brain growths.
(3) freezing microtome section technology is used, cell mass is cut into 10-20 μ m thicks, carries out immunofluorescence dyeing, detects alcohol
The exception for the treatment of group 3D brain growths；
(4) Apoptosis during TUNEL methods detection ethanol postincubation group 3D brain growths.
Sodium alginate macaroni yarn can be designed to different diameter and pipe thickness according to requirement of experiment in the present invention.
The material of macaroni yarn of the present invention is not limited only to sodium alginate, further includes the relatively good material of other biological compatibility.
The preparation method of macaroni yarn of the present invention is not limited only to barrel forms, further includes micro-fluidic chip mode.
The present invention provides a kind of external model of gestational period alcohol exposure to fetus brain damage, is also applied for research alcohol pair
The influence of the development of the organoid in other different stem cell (including embryonic stem cell) sources, the organoid tissue delivered include
Intestines, stomach, retina, brain etc..Vitro in organ disorder of development model of the model as innovation, is not only beneficial to observation alcohol pair directly perceived
The Morphology Effects of brain growth, can also use for reference various detection methods, molecules and cell of the research alcohol to brain growth
Mechanism, compensate for the deficiency of conventional cell experiment and zoopery, be to study gestational period alcohol to fetus early stage brain growth
Influence provides strong instrument.
Brief description of the drawings
Control group and the tracking of sample sets 3D brain growths situation under the conditions of Fig. 1 light fields of the present invention；
Fig. 2 immunohistochemistry and freezing microtome section method detection control group and sample sets 3D brain growth situations；
Nerve and Different brain region expression conditions during the detection 3D brain growths of Fig. 3 RT-PCR methods.
Embodiment
The following examples will be further described the present invention, but not thereby limiting the invention.The present invention
Used all reagents are purchased in market.
Embodiment 1
Gestational period alcohol exposure establishes the external model of fetus brain damage
Its step is broadly divided into four parts：The preparation of sodium alginate macaroni yarn, induction early period of 3D brains, 3D brains are wrapped in seaweed
In sour sodium macaroni yarn and ethanol postincubation 3D brains.
The preparation of step (1) sodium alginate macaroni yarn, is specially：The preparation of the sodium alginate macaroni yarn is to utilize casing
The micro-fluidic chip of form, the macaroni yarn internal diameter of preparation is 600-1000 μm, and pipe thickness is 50-200 μm, liquid asepsis used
Processing.The sodium alginate silk need to be immersed in DMEM/F12 culture mediums, its is completed swelling process in the medium, and
4 DEG C of Cord bloods, easy to subsequent operation.
Step (2) 3D brain inductions early period, are specially：Embryoid body is prepared using the PDMS chips with hole shape structure：Cheat shape
The chip of structure is placed in 24 orifice plates, and pitting diameter of movement is 800 μm, and depth is 300 μm, the formation for embryoid body.1st
My god, embryoid body is prepared, by 2 × 105-6×105A hiPSCs be digested to it is unicellular, and be transferred to hole shape structure PDMS cores
In piece, 1000rpm is centrifuged, 5min, used medium is KSR culture mediums, and adds 5 μM of Y27632；
The basic ingredient of KSR culture mediums is DMEM/F12, in addition needs addition to account for the KnockOut of cumulative volume 20%
Replacement (KSR), accounts for cumulative volume 1%NEAA (Non Essential Amino Acid, 100 ×), accounts for cumulative volume 1%
GlutaMax (100 ×), accounts for cumulative volume 1%penicillin-streptomycin (100 ×), and 0.1mM beta-
Mercaptoethanol, 4ng/ml bFGF.
2nd day, the embryoid body of formation is transferred in the culture dish of low adhesion, suspend culture embryoid body, used medium
For KSR culture mediums.5th day, induction embryoid body broke up to neural epithelium direction, and KSR culture mediums are replaced with nerve-inducing culture
Base.Cell mass still keeps suspending and cultivates.Replace culture medium within every 3 days.
Wherein the basic ingredient of Neuronal induction media is DMEM/F12, in addition needs addition to account for the N2 (100 of cumulative volume 1%
×), account for the GlutaMAX (100 ×) of cumulative volume 1%, account for cumulative volume 1% NEAA (Non Essential Amino Acid,
100 ×), 1 μ g/ml heparin, account for the penicillin-streptomycin (100 ×) of cumulative volume 1%.
Step (3) 3D brains are wrapped in sodium alginate macaroni yarn, are specially：10th day, early period is resuspended using Matrigel
The cell mass of induction, and injected cell mass in sodium alginate silk with syringe, whole process avoids producing bubble, operates on ice
Low temperature is maintained, ensures that Matrigel does not solidify.Sodium alginate silk containing cell mass is placed in 37 DEG C of incubator 30min, is made
Matrigel solidifies.Sodium alginate silk containing cell mass is transferred in 6 orifice plates and carries out follow-up cultivation, used medium is god
Cultivated through differentiation, its basic ingredient is that volume ratio is 1:1 DMEM/F12 and Neuralbasal medium, in addition need to add
The B27 (50 ×) of cumulative volume volume 1%, the N2 (100 ×) of cumulative volume 0.5%, the 0.5%NEAA (100 ×) of cumulative volume are accounted for, always
The GlutaMAX (100 ×) of volume 1%, the 1%penicillin-streptomycin (100 ×) of cumulative volume volume, 0.05mM
beta-mercaptoethanol。
Step (4) ethanol postincubation 3D brains concretely comprise the following steps：
11st day, control group used Neural Differentiation medium culture 3D brains, and sample sets use the god that alcohol concentration is 100mM
Through differential medium culture 3D brains, culture medium used needs matching while using.In addition 6 orifice plate gap domestic demands are added containing same in sample sets
The PBS buffer of concentration alcohol, need to be sealed using sealed membrane, reduce alcohol volatilization.Due to alcohol volatilization and ensure nutrition
The supply of material, sample sets and control group are all to change a subculture in every two days.
Light field tracks 3D brain growth situations in sample sets and control group, and the results are shown in Figure 1.
Embodiment 2
Gestational period alcohol exposure is substantially same as Example 1 to the external model method for building up of fetus brain damage, difference
It is：
Step (2) 3D brain inductions early period, are specially：Embryoid body is prepared using the PDMS chips with hole shape structure：Cheat shape
The chip of structure is placed in 24 orifice plates, and pitting diameter of movement is 600 μm, and depth is 200 μm, the formation for embryoid body.1st
My god, embryoid body is prepared, by 2 × 105-6×105A hiPSCs be digested to it is unicellular, and be transferred to hole shape structure PDMS cores
In piece, 2000rpm is centrifuged, 3min, used medium is KSR culture mediums, and adds 5 μM of Y27632；
Step (3) 3D brains are wrapped in sodium alginate macaroni yarn, are specially：10th day, early period is resuspended using Matrigel
The cell mass of induction, and injected cell mass in sodium alginate silk with syringe, whole process avoids producing bubble, operates on ice
Low temperature is maintained, ensures that Matrigel does not solidify.Sodium alginate silk containing cell mass is placed in 37 DEG C of incubator 25min, is made
Matrigel solidifies.Sodium alginate silk containing cell mass is transferred in 6 orifice plates and carries out follow-up cultivation,
Step (4) ethanol postincubation 3D brains concretely comprise the following steps：
11st day, control group used Neural Differentiation medium culture 3D brains, and sample sets use the god that alcohol concentration is 200mM
Through differential medium culture 3D brains, culture medium used needs matching while using.In addition 6 orifice plate gap domestic demands are added containing same in sample sets
The PBS buffer of concentration alcohol, need to be sealed using sealed membrane, reduce alcohol volatilization.Due to alcohol volatilization and ensure nutrition
The supply of material, sample sets and control group are all to change a subculture in every two days.
Embodiment 3
Gestational period alcohol exposure is substantially same as Example 1 to the external model method for building up of fetus brain damage, difference
It is：
Step (2) 3D brain inductions early period, are specially：Embryoid body is prepared using the PDMS chips with hole shape structure：Cheat shape
The chip of structure is placed in 24 orifice plates, and pitting diameter of movement is 700 μm, and depth is 200 μm, the formation for embryoid body.1st
My god, embryoid body is prepared, by 2 × 105-6×105A hiPSCs be digested to it is unicellular, and be transferred to hole shape structure PDMS cores
In piece, 500rpm is centrifuged, 5min, used medium is KSR culture mediums, and adds 5 μM of Y27632；
Step (3) 3D brains are wrapped in sodium alginate macaroni yarn, are specially：10th day, early period is resuspended using Matrigel
The cell mass of induction, and injected cell mass in sodium alginate silk with syringe, whole process avoids producing bubble, operates on ice
Low temperature is maintained, ensures that Matrigel does not solidify.Sodium alginate silk containing cell mass is placed in 37 DEG C of incubator 20min, is made
Matrigel solidifies.
Step (4) ethanol postincubation 3D brains concretely comprise the following steps：
11st day, control group used Neural Differentiation medium culture 3D brains, and sample sets use the god that alcohol concentration is 20mM
Through differential medium culture 3D brains, culture medium used needs matching while using.In addition 6 orifice plate gap domestic demands are added containing same in sample sets
The PBS buffer of concentration alcohol, need to be sealed using sealed membrane, reduce alcohol volatilization.Due to alcohol volatilization and ensure nutrition
The supply of material, sample sets and control group are all to change a subculture in every two days.
Embodiment 4
RT-PCR method detects control group and sample sets brain growth related gene expression situation
The 3D brains developed in embodiment 1 30 days are collected, PBS buffer is cleaned 1 time, and is centrifuged.Extracted using Trizol methods
Full RNA, method are as follows：Cell is resuspended in 1ml Trizol, and piping and druming is up to acellular piece up and down, whole solution clarification；Add 200 μ l
Chloroform, mixes 5min up and down, is stored at room temperature 3min, 15000rpm centrifugations 15min；Solution is layered, and takes supernatant liquid to new
Guan Zhong, whole process careful operation avoid contact with intermediate layer white precipitate as far as possible；Equivalent isopropanol is added in liquid draw, up and down
Reverse to mix, -20 DEG C stand overnight, and are separated out beneficial to RNA；15000rpm centrifuges 10min；Retain the white RNA precipitate of bottom of the tube,
Carefully remove supernatant；75% ethanol cleans white precipitate, 15000rpm centrifugations 5min；75% ethanol is eliminated as much as, room temperature allows it
Volatilize 5min, in case ethanol pollution sample, influences subsequent experimental；DEPC water dissolves RNA precipitate；RNA concentration and purity are surveyed, is gone forward side by side
The corresponding dilution of row, final concentration of 500ng/ml.
Second step, is cDNA by mRNA reverse transcriptions, and system is 50 μ l, and specific proportioning is as follows：10 μ l of 500ng/ml RNA,
10 μ l, Oligo dT of Buffer, 2.5 μ l, Random 6mers, 2.5 2.5 μ l, DEPC water of μ l, Enzyme 27.5 μ l, 37 DEG C
20min, 4 DEG C of preservations.
3rd step, Real-time PCR, require ligand solution, whole system is 5ul, and annealing temperature Tm unites according to kit
One is 58 DEG C.
The expression of control group and sample sets brain growth related gene is finally counted, the results are shown in Figure 2.
Embodiment 5
ImmunohistochemistryMethods Methods detect control group and sample sets 3D brain growth situations
Collect the 3D brains of the 15th and 24 day of embodiment 1 and carry out freezing microtome section, method is as follows：4% paraformaldehyde carries out cell
Fixed 20min, PBS buffer flush three times, each 10min；30% 4 DEG C of sucrose is dehydrated overnight；OCT is embedded, room temperature storage
30min, 80 DEG C of solidifications；Freezing microtome section, thickness is 10-20 μm, and is attached on the glass slide of Electrostatic Absorption.Then carry out
Immunofluorescence dyeing, method are：Glass slide with section is placed in PBS buffer and soaks 5min；0.1%triton X-
100 pore-foaming agents act on 10min, and PBS buffer is rinsed 1 time, 5min；Goat closing serum room temperature effect 1h, primary antibody (TUJ1,
SOX2)1:400 dilutions, 4 DEG C are incubated overnight, and PBS buffer is rinsed 1 time, 5min；(Fluorescence488/594 is marked secondary antibody
Goat antirabbit or mouse IgG) 1:100 dilutions, room temperature are incubated 1h, and PBS buffer is rinsed 1 time, 5min；1 is added after flushing:
2000 diluted DAPI working solutions, take pictures under fluorescence microscope, record the expression of SOX2 and TUJ1, the results are shown in Figure 3.
The results are shown in Figure 3.The expression of neuron (TUJ1) is apparently higher than control group in sample sets, it was demonstrated that ethanol postincubation promotes 3D brains
Neurodevelopment.
Claims (8)
1. a kind of gestational period alcohol exposure is to the external model method for building up of fetus brain damage, it is characterised in that its key step point
For：
(1) preparation of sodium alginate macaroni yarn；
(2) 3D brains induction early period；
(3) 3D brains are wrapped in sodium alginate macaroni yarn；
(4) ethanol postincubation 3D brains.
2. exist according to gestational period alcohol exposure described in claim 1 to the external model method for building up of fetus brain damage, its feature
In：The preparation of step (1) sodium alginate macaroni yarn, is specially：
(1) the sodium alginate silk with hollow structure being prepared using the micro-fluidic chip of barrel forms, internal diameter is 600-1000 μm,
Pipe thickness is 50-200 μm, liquid asepsis processing used；
(2) the sodium alginate silk prepared need to be immersed in DMEM/F12 culture mediums, make its swelling process of completion in the medium,
And in 4 DEG C of Cord bloods, easy to subsequent operation.
3. exist according to gestational period alcohol exposure described in claim 2 to the external model method for building up of fetus brain damage, its feature
Preparing the instrument being related to and solution in sodium alginate silk all must be sterile, corresponding disinfecting action can be carried out before experiment, specifically
For autoclave sterilization, UV irradiations or 0.2 μm of aperture membrane filtration.
4. the external model of fetus brain damage is established according to gestational period alcohol exposure described in claim 1, it is characterised in that：Step
Suddenly (2) 3D brains induction early period, is specially：
(1) embryoid body is prepared using the PDMS chips with hole shape structure：The chip of hole shape structure is placed in 24 orifice plates, pitting knot
A diameter of 600-800 μm of structure, depth are 100-300 μm, the formation for embryoid body；
(2) the 1st days, embryoid body is prepared, by 2 × 105-6×105A hiPSCs is digested to unicellular, and is transferred to described in (1)
In chip, 500-2000rpm centrifugation 3-5min, used medium is KSR culture mediums, and adds 5 μM of Y27632；
(3) the 2nd days, the embryoid body of formation is transferred in the culture dish of low adhesion, suspend culture embryoid body, and used medium is
KSR culture mediums；
(4) the 5th days, induction embryoid body broke up to neural epithelium direction, and KSR culture mediums are replaced with Neuronal induction media.
5. exist according to gestational period alcohol exposure described in claim 1 to the external model method for building up of fetus brain damage, its feature
In：Step (3) the 3D brains are wrapped in sodium alginate macaroni yarn, are specially：
(1) the 10th day, the cell mass induced early period is resuspended using Matrigel, and cell mass is injected into sodium alginate with syringe
In macaroni yarn, whole process avoids producing bubble, it is ensured that operation maintains low temperature on ice, ensures that Matrigel does not solidify；
(2) the sodium alginate silk containing cell mass is placed in 37 DEG C of incubator 20-30min, solidifies Matrigel；
(3) the sodium alginate silk containing cell mass is transferred in 6 orifice plates and carries out follow-up cultivation, used medium is Neural Differentiation
Culture medium.
6. the external model of fetus brain damage is established according to gestational period alcohol exposure described in claim 5, it is characterised in that institute
The Matrigel concentration stated is 2-10mg/ml.
7. the external model of fetus brain damage is established according to gestational period alcohol exposure described in claim 1, it is characterised in that：Step
Suddenly (4) ethanol postincubation 3D brains, are specially：
(1) the 11st day, control group used Neural Differentiation medium culture 3D brains, and sample sets are 20-200mM's using alcohol concentration
Neural Differentiation medium culture 3D brains, culture medium used need matching while using；
(2) gap of 6 orifice plates adds the PBS buffer of equal alcohol concentration in sample sets；
(3) 6 orifice plates need to be sealed using sealed membrane in sample sets, reduce alcohol volatilization；
(4) ethanol postincubation was to 3D brain growths 30 days, and carried out subsequent detection.
8. the external model of fetus brain damage is established according to gestational period alcohol exposure described in claim 7, it is characterised in that：Wine
The concentration range of essence is 20-200mM, to simulate the alcohol concentration under physiological conditions.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610891029.2A CN107937339B (en) | 2016-10-13 | 2016-10-13 | In-vitro model establishment method for fetal brain injury caused by alcohol exposure in gestation period |
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