CN107937344A - A kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier - Google Patents
A kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier Download PDFInfo
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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
- C12N2506/45—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from artificially induced pluripotent stem cells
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- C12N2533/74—Alginate
Abstract
A kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, this method efficiently realize external 3D brain growths using novel biomaterial sodium alginate macaroni yarn as carrier.The 3D brains in HiPSCs sources can simulate the growth course of human brain early stage to a certain extent, and the development in vitro study people provides powerful.Carrier of the macaroni yarn as the 3D brain growths in hiPSCs sources, can not only realize to more convenient and quicker development of a large amount of 3D brains in extracellular matrix, and due to the design feature of silk so that the 3D brains finally developed have consistent size and shape.
Description
Technical field
The present invention relates to by microfluidic chip technology, and in particular to a kind of to realize hiPSCs sources by carrier of macaroni yarn
The method of brain growth.
Background technology
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.According to these features, organoid development technology compensate for
Blank between 2D cell culture differentiation and zoopery, is with a wide range of applications.
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, optimize external organoid development and operation
Still belong to blank.
The content of the invention
The object of the present invention is to provide one kind using sodium alginate macaroni yarn as carrier, the 3D brains in hiPSCs sources are efficiently realized
Development method, this method not only ensure that the mass exchange in growth course, while realize controllable development structure so that final
The 3D brains of development have consistent size and shape, and have the characteristics of monitoring in real time.
The present invention provides a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier high-efficiency, its step
It is broadly divided into three parts:Hair of the preparation, 3D brain inductions early period and 3D brains of sodium alginate macaroni yarn in sodium alginate macaroni yarn
Educate.
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.Replace a subculture 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 Essential Amino 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, into the magnitude range of embryoid body be 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, its basic ingredient are that volume ratio is 1:1 DMEM/F12 and Neuralbasal
Medium, in addition needs addition to account for cumulative volume 1%B27 (50 ×), accounts for cumulative volume 0.5%N2 (100 ×), accounts for cumulative volume 0.5%
NEAA (100 ×), accounts for cumulative volume 1%GlutaMAX (100 ×), accounts 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.
The 3D brain growth methods in the high throughput hiPSCs sources that the present invention establishes, the characterization side of its follow-up 26S Proteasome Structure and Function
Method is as follows:
(1) under the conditions of microscope light field directly monitoring 3D brains the speed of growth and developmental condition;
(2) neural and Different brain region domain gene expression during RT-PCR method detection 3D brain growths;
(3) freezing microtome section technology is used, cell mass is cut into 10-20 μ m thicks, follow-up nerve is carried out and brain structure is related
The Immunofluorescence test of antibody;
(4) cell life or death situation during TUNEL methods detection 3D brain growths;
(5) the apoptosis situation of the scattered individual cells of Live/Dead assay detections;
(6) Ca is carried out using living cells work station2+Imaging detection nerve cells Ca2+Activity, it is ensured that nerve cell
Function.Shooting situation is to open photo per 10s mono-, is continuously shot 5min.
The present invention provides a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier high-efficiency, sodium alginate
Macaroni yarn can be designed to different diameter and pipe thickness according to requirement of experiment.
The present invention provides a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier high-efficiency, is not only applicable in
3D brains in hiPSCs sources, are also applied for the organoid in other different stem cell (including embryonic stem cell) sources, have delivered
Organoid tissue include intestines, stomach, retina, brain etc..
The present invention provides a kind of method that 3D brain growths are realized using sodium alginate silk as carrier high-efficiency, can be applied at the same time
The co-cultivation of other cells and organoid is built, is more nearly simulation physiological status, optimizes organoid development degree.
The present invention provides a kind of method that 3D brain growths are realized using sodium alginate silk as carrier high-efficiency, can be applied to establish
Various pathological models, study the situation of fetus brain growth under pathologic condition.
Brief description of the drawings
PDMS chips of the Fig. 1 with hole shape structure prepares embryoid body
Under the conditions of Fig. 2 light fields of the present invention in sodium alginate silk hiPSCs sources 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.
All reagents of the present invention are purchased in market.
Embodiment 1
Development of the 3D brains in HiPSC sources in sodium alginate silk
Its step is broadly divided into three parts:Preparation, 3D brain inductions early period and the 3D brains of sodium alginate macaroni yarn are in alginic acid
Development in sodium macaroni yarn.
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 is digested to unicellular, and is transferred in the chip described in (1), centrifuges
1000rpm, 5min, used medium are KSR culture mediums, and add 5 μM of Y27632;2nd day, the embryoid body of formation is shifted
Into the culture dish of low adhesion, suspend culture embryoid body, and used medium is KSR culture mediums.
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.
5th day, induction embryoid body broke up to neural epithelium direction, and KSR culture mediums are replaced with Neuronal induction media;Carefully
Born of the same parents group still keeps suspending and cultivates.Replace a subculture within every 3 days.
The basic ingredient of the 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 Essential Amino Acid, 100
×), 1 μ g/ml heparin, account for cumulative volume 1%penicillin-streptomycin (100 ×).
Development of step (3) the 3D brains in sodium alginate macaroni yarn, is specially:10th day, before being resuspended using Matrigel
The cell mass of phase induction, and injected cell mass in sodium alginate macaroni yarn with syringe, whole process avoids producing bubble, really
Protect operation on ice and maintain low temperature, ensure that Matrigel does not solidify;Sodium alginate silk containing cell mass is placed in 37 DEG C of incubators
25min, solidifies Matrigel;Sodium alginate silk containing cell mass is transferred in 6 orifice plates and carries out follow-up cultivation, training used
It is Neural Differentiation culture medium to support base.
The Neural Differentiation culture medium, its basic ingredient are that volume ratio is 1:1 DMEM/F12 and Neuralbasal
Medium, in addition needs addition to account for cumulative volume 1%B27 (50 ×), accounts for cumulative volume 0.5%N2 (100 ×), accounts for cumulative volume 0.5%
NEAA (100 ×), accounts for cumulative volume 1%GlutaMAX (100 ×), accounts for cumulative volume 1%penicillin-streptomycin (100
×), 0.05mM beta-mercaptoethanol.
Light field tracks the growth course of 3D brains, and the results are shown in Figure 1, Scale bar:500μm.
Embodiment 2
Development method of the 3D brains in HiPSC sources in sodium alginate silk is substantially same as Example 1, and difference 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 is digested to unicellular, and is transferred in the chip described in (1), centrifuges
500rpm, 5min, used medium are KSR culture mediums, and add 5 μM of Y27632;2nd day, the embryoid body of formation is transferred to
In the culture dish of low adhesion, suspend culture embryoid body, and used medium is KSR culture mediums.
Development of step (3) the 3D brains in sodium alginate macaroni yarn, is specially:10th day, before being resuspended using Matrigel
The cell mass of phase induction, and injected cell mass in sodium alginate macaroni yarn with syringe, whole process avoids producing bubble, really
Protect operation on ice and maintain low temperature, ensure that Matrigel does not solidify;Sodium alginate silk containing cell mass is placed in 37 DEG C of incubators
30min, solidifies Matrigel;Sodium alginate silk containing cell mass is transferred in 6 orifice plates and carries out follow-up cultivation, training used
It is Neural Differentiation culture medium to support base.
Embodiment 3
Development method of the 3D brains in HiPSC sources in sodium alginate silk is substantially same as Example 1, and difference 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, depth is 100 μm, the formation for embryoid body.1st
My god, embryoid body is prepared, by 2 × 105-6×105A hiPSCs is digested to unicellular, and is transferred in the chip described in (1), centrifuges
2000rpm, 3min, used medium are KSR culture mediums, and add 5 μM of Y27632;2nd day, the embryoid body of formation is shifted
Into the culture dish of low adhesion, suspend culture embryoid body, and used medium is KSR culture mediums.
Development of step (3) the 3D brains in sodium alginate macaroni yarn, is specially:10th day, before being resuspended using Matrigel
The cell mass of phase induction, and injected cell mass in sodium alginate macaroni yarn with syringe, whole process avoids producing bubble, really
Protect operation on ice and maintain low temperature, ensure that Matrigel does not solidify;Sodium alginate silk containing cell mass is placed in 37 DEG C of incubators
20min, solidifies Matrigel;Sodium alginate silk containing cell mass is transferred in 6 orifice plates and carries out follow-up cultivation, training used
It is Neural Differentiation culture medium to support base.
Embodiment 4
Nerve and Different brain region expression conditions during RT-PCR method detection 3D brain growths
The 3D brains that embodiment 1 develops different times are collected, PBS buffer is cleaned 1 time, and is centrifuged.Carried using Trizol methods
Full RNA is taken, method is 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 chloroforms, mix 5min up and down, are stored at room temperature 3min, 15000rpm centrifugations 15min;Solution is layered, and takes supernatant liquid extremely
In new pipe, whole process careful operation avoids contact with intermediate layer white precipitate as far as possible;Equivalent isopropanol is added in liquid draw, on
Lower reverse mixing, -20 DEG C stand overnight, and are separated out beneficial to RNA;15000rpm centrifuges 10min;The white RNA for retaining bottom of the tube sinks
Form sediment, carefully remove supernatant;75% ethanol cleans white precipitate, 15000rpm centrifugations 5min;75% ethanol is eliminated as much as, room temperature allows
Its 5min that volatilizees, in case ethanol pollution sample, influences subsequent experimental;DEPC water dissolves RNA precipitate;RNA concentration and purity are surveyed, and
Diluted accordingly, 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, PCR, ligand solution is required according to kit, and whole system is 20ul, and annealing temperature Tm is unified for 58 DEG C, and 4
DEG C preserve.
2ul reaction solutions are taken, add loading buffer, water and ethidium bromide, common 6ul, marker 4ul, carry out agar
Sugared gel electrophoresis, and related data is gathered, as shown in Figure 2.RT-PCR detects hiPSCs coherency gene deregulations, and neural
Related and Different brain region gene expression rises.
Embodiment 5
The expression of nerve and brain area GAP-associated protein GAP in growth course
The 3D brains that Example 1 develops different times carry out freezing microtome section, and 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 (PAX6,
PAX2, NESTIN, TUJ1, SOX2, TBR1, CTIP2) 1:100 or 1:400 dilutions, 4 DEG C are incubated overnight, and PBS buffer rinses 1
It is secondary, 5min;Secondary antibody (goat antirabbit or mouse IgG of Fluorescence488/594 marks) 1:100 dilutions, room temperature are incubated 1h,
PBS buffer is rinsed 1 time, 5min;1 is added after flushing:2000 diluted DAPI working solutions, take pictures under fluorescence microscope,
The expression of corresponding albumen is recorded, the results are shown in Figure 3.Neural related and Different brain region albumen all shows high expression, table
Bright hiPSCs in this system can Successful development into 3D brains.
Claims (8)
- A kind of 1. method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, it is characterised in that:This method mainly walks Suddenly it is:(1) preparation of sodium alginate macaroni yarn;(2) 3D brains induction early period;(3) development of the 3D brains in sodium alginate macaroni yarn.
- 2. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 1 It is: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. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 2 Being that sodium alginate silk prepares the instrument that is related to and solution all must be sterile, and corresponding disinfecting action can be carried out before experiment, is had Body is autoclave sterilization, UV irradiates or 0.2 μm of aperture membrane filtration.
- 4. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 1 It is: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, 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 is centrifuged, 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. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 4 It is to prepare embryoid body using the PDMS chips of hole shape structure, change and cell of its big I by pitting diameter and depth The quantity of cell is adjusted in suspension, into the magnitude range of embryoid body be about 50-500 μm.
- 6. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 4 The cell derived for being hiPSCs is transformed by human skin fibroblasts virus infection.
- 7. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 1 It is:Development of step (3) the 3D brains in sodium alginate macaroni yarn, is 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.
- 8. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 7 It is that the Matrigel concentration is 2-10mg/ml.
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刘祎: "海藻酸钠水凝胶用于神经干细胞定向诱导分化的研究", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
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CN111254107A (en) * | 2018-11-30 | 2020-06-09 | 中国科学院大连化学物理研究所 | Method for establishing autism-like model with hiPSCs (hiPSCs) source |
CN114214267A (en) * | 2021-12-06 | 2022-03-22 | 大连大学 | Organoid matrigel microspheres and preparation method and application thereof |
CN114214267B (en) * | 2021-12-06 | 2024-04-09 | 大连大学 | Organoid matrigel microsphere and preparation method and application thereof |
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