CN107937339B - In-vitro model establishment method for fetal brain injury caused by alcohol exposure in gestation period - Google Patents
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Abstract
An in-vitro model building method for fetal brain injury caused by alcohol exposure in gestation period mainly combines emerging 3D brain tissues derived from hiPSCs and a biological material sodium alginate filament, and simulates the influence of alcoholism in gestation period on fetal early brain development by adding moderate alcohol stimulation. The model is used as an innovative in vitro organ development disease model, is beneficial to visually observing the morphological influence of alcohol on brain development, can also be used for researching the molecular and cytological mechanisms of the influence of the alcohol on the brain development by referring to various detection methods, makes up the defects of the traditional cell experiment and animal experiment, and provides a powerful tool for researching the influence of alcohol in the gestation period on the early brain development of a fetus.
Description
Technical Field
The invention discloses a microfluidic chip technology, and particularly relates to an in-vitro model establishment method for fetal brain injury caused by alcohol exposure in a gestation period.
Background
The study of the molecular and cellular mechanisms of pathological conditions provides the basis for better understanding, control and treatment of diseases. Traditional biological methods mainly include 2D cell culture and animal models. 2D cell culture cannot simulate the microenvironment of cells under physiological conditions, including three-dimensional extracellular matrix, interstitial flow, intercellular interaction and the like, so that the morphological functions of the cells are greatly insufficient compared with those under physiological conditions, and the abnormal changes of the molecular level and the cell level under pathological conditions cannot be completely reflected. Animal experiments mainly use model organisms such as mice, rabbits, fruit flies, nematodes and the like at present, and although the model organisms are convenient in source and simple in culture conditions, the physiological changes of human bodies under various conditions are difficult to completely reflect on the basis of the great difference between humans and animals. On the premise of ensuring minimum damage and convenient observation, the research on human bodies is more commonly used for nuclear magnetic resonance, CT and the like, but the methods have no real-time monitoring characteristic and many methods cannot be applied to fetal development,
Organoid development from various stem cell sources has been actively developed in recent years, including neural stem cells, intestinal stem cells, embryonic stem cells, and iPSCs. The organoid is characterized in that a 3D cell mass structure formed by stem cells or primary cells spontaneously contains a plurality of functional cells of corresponding tissues and organs, the cells are self-assembled into tissues with certain structural function specificity, and the development process of the corresponding tissues and organs is simulated to a certain extent. Wherein the 3D brain derived from hiPSCs in vitro essentially replicates the process of fetal early brain development.
But organoid technology also currently has many deficiencies and needs to be improved. Due to the structural characteristics of the 3D cell mass, internal cells are dead in a large scale due to lack of oxygen and nutrition, the development degree of in vitro organoids, including volume, functional maturity and the like, is greatly limited, and vascular tissues are scattered in tissues and organs to provide required oxygen and nutrition under physiological conditions. Therefore, the combination with other technologies is expected to make up for the deficiency. In addition, the prior traditional cell culture technology has the defects of complicated operation, long time consumption and poor controllability in organ culture. Therefore, the method is hopeful to be combined with the existing bioengineering means to optimize organoid technology.
The filamentous structure as a culture carrier has several advantages: firstly, the micro-fluidic chip technology can be used for flexibly and efficiently producing sodium alginate filaments with different sizes and thicknesses according to practical application. Secondly, the filamentous structure has higher specific surface area ratio, is beneficial to material exchange and provides good living environment for cell culture. Again, the filamentous structures can direct the growth of cell mass, produce brain tissue with a similar structure, and limit its growth during development, mimicking, to some extent, the effects of meninges under physiological conditions. Finally, the sodium alginate material can be dissolved under the condition of high salt or sodium citrate to release cells, thereby meeting different treatment requirements.
But at present, the field of establishing a human disease model by combining bioengineering and hiPSCs-derived organoids is still blank.
Disclosure of Invention
The invention aims to provide an in vitro model building method for fetal brain injury caused by alcohol exposure in gestation period, which uses sodium alginate hollow fibers as cell culture carriers, ensures material transportation in a development process, realizes a controllable development structure and has the characteristic of real-time monitoring.
The invention provides an in vitro model building method for fetal brain injury caused by alcohol exposure in gestation period, which mainly comprises the following four steps:
(1) preparing sodium alginate hollow fibers;
(2)3D brain prophase induction;
(3) the 3D brain is wrapped in the sodium alginate hollow fiber;
(4) alcohol treatment of 3D brain.
The preparation method of the sodium alginate hollow fiber comprises the following steps:
the sodium alginate hollow fiber is prepared by utilizing a micro-fluidic chip in a sleeve form, the inner diameter of the prepared hollow fiber is 600-1000 mu m, the wall thickness of the tube is 50-200 mu m, and the used liquid is subjected to aseptic treatment.
The sodium alginate filaments are required to be soaked in a DMEM/F12 culture medium, so that the swelling process of the sodium alginate filaments in the culture medium is completed, and the sodium alginate filaments are stored at a low temperature of 4 ℃ and are convenient for subsequent operation.
Step (2)3D brain prophase induction, which specifically comprises the following steps:
(1) preparing a pseudo-blank by using a PDMS chip with a pit-shaped structure: the chip with the pit-shaped structure is arranged in a 24-hole plate, the diameter of the small pit structure is 600-.
(2) On day 1, embryoid bodies were prepared and 2X 10 cells were used5-6×105Digesting the hiPSCs into single cells, transferring the single cells to the chip in (1), centrifuging at 500-2000rpm for 3-5min, wherein the used culture medium is KSR culture medium, and adding 5 mu M Y27632;
the KSR medium comprises DMEM/F12 as a basic component, and is additionally added with KnockOut Replacement (KSR) accounting for 20% of the total volume, NEAA (Non Essential Amino Acid, 100 x) accounting for 1% of the total volume, GlutaMax (100 x) accounting for 1% of the total volume, peniciln-streptomycin (100 x) accounting for 1% of the total volume, and bFGF of 0.1mM beta-mercaptoethanol and 4 ng/ml.
(3) On day 2, the embryoid bodies formed were transferred to a low-adhesion petri dish and embryoid bodies were cultured in suspension using KSR medium.
(4) On the 5 th day, inducing the embryoid bodies to differentiate towards the neuroepithelial direction, and replacing the KSR culture medium with a neural induction culture medium; the cell pellet was still maintained in suspension culture with medium changes every 3 days.
The basic component of the nerve induction culture medium is DMEM/F12, and N2(100 x) accounting for 1% of the total volume, GlutaMAX (100 x) accounting for 1% of the total volume, NEAA (Non essential amino Acid, 100 x) accounting for 1% of the total volume, heparin (1 mu g/ml) accounting for 1% of the total volume and penicillin-streptomycin (100 x) accounting for 1% of the total volume are required to be added.
The embryoid bodies are prepared by using PDMS chips with pit-like structures, the size of which can be adjusted by the change of the volume of the small pits and the number of cells in the cell suspension, and the size of the cell balls is in the range of about 50-500 μm.
The HiPSCs were mildly digested with Accutase for about 2-5min, ensuring final digestion into smaller cell clumps. If the digestion is excessive and scattered single cells are formed, the embryoid body structure contains more dead cells, and the subsequent differentiation and development are influenced; if the digestion is insufficient, the balling effect of the embryoid bodies is affected, and cell clusters with consistent sizes are difficult to form.
After the HiPSCs are digested into small cell masses by using Accutase, subsequent centrifugation is carried out, so that the cells are gathered together as much as possible, a pseudoembryoid body with a large volume is formed, and the success rate of subsequent brain development is effectively improved.
Although the embryoid body can be formed in several hours, the cell needs to be kept still and cultured in the PDMS chip within 24 hours, so as to ensure the stability of the structure of the embryoid body.
And (3) wrapping the 3D brain in the sodium alginate hollow fiber, specifically:
on the 10 th day, the Matrigel is used for re-suspending the cell clusters induced in the early stage, and the cell clusters are injected into the sodium alginate filaments by using an injector, so that bubbles are prevented from being generated in the whole process, the operation on ice is ensured to be kept at low temperature, and the Matrigel is ensured not to be solidified.
And (3) placing the sodium alginate filaments containing the cell clusters in an incubator at 37 ℃ for 20-30min to solidify the Matrigel.
Transferring the sodium alginate filaments containing the cell clusters to a 6-hole plate for subsequent culture, wherein the used culture medium is a neural differentiation culture medium.
The neural differentiation medium is a neural differentiation medium, the basic components of the neural differentiation medium are DMEM/F12 and Neuralbasal medium in a volume ratio of 1:1, and additionally, B27(50 x) accounting for 1% of the total volume, N2(100 x) accounting for 0.5% of the total volume, NEAA (100 x) accounting for 0.5% of the total volume, GlutaMAX (100 x) accounting for 1% of the total volume, peniciln-streptomycin (100 x) accounting for 1% of the total volume and alpha-beta-meroethanol accounting for 0.05mM are added.
The development process of the 3D brain needs to use Matrigel, and the Matrigel is used as an extracellular matrix to provide a three-dimensional medium for the development of the 3D brain, so that the rapid development of the 3D brain is facilitated.
The step (4) of alcohol treatment of the 3D brain comprises the following specific steps:
on day 11, 3D brain was cultured in the control group using neural differentiation medium, and 3D brain was cultured in the sample group using neural differentiation medium with alcohol concentration of 20-200mM, using the medium as prepared. In addition, PBS buffer solution containing alcohol with the same concentration needs to be added into the gap of the 6-pore plate in the sample group, and a sealing film needs to be used for sealing, so that the volatilization of the alcohol is reduced. Due to the evaporation of alcohol and the supply of nutrients, the culture medium was changed every two days for both the sample group and the control group.
The invention relates to an in vitro model of fetal brain injury caused by alcohol exposure in the gestation period, and a subsequent detection method of the model is as follows:
(1) monitoring the growth speed and the development state of the 3D brain in real time under the condition of alcohol treatment under a microscope bright field;
(2) RT-PCR method detects the abnormal development of 3D brain in alcohol treatment group.
(3) Cutting the cell mass into 10-20 μm thickness by using cryosectioning technology, performing immunofluorescence staining, and detecting abnormal 3D brain development of the alcohol-treated group;
(4) TUNEL method detects apoptosis during 3D brain development in alcohol-treated groups.
The sodium alginate hollow fiber can be designed into different diameters and tube wall thicknesses according to experimental requirements.
The hollow silk material is not limited to sodium alginate, but also comprises other materials with good biocompatibility.
The preparation method of the hollow wire is not limited to a sleeve form, but also comprises a micro-fluidic chip form.
The invention provides an in vitro model of alcohol exposure to fetal brain injury in gestation period, which is also suitable for researching the influence of alcohol on the development of organoids from other different stem cells (including embryonic stem cells), wherein published organoid tissues comprise intestines, stomach, retina, brain and the like. The model is used as an innovative in vitro organ development disease model, is beneficial to visually observing the morphological influence of alcohol on brain development, can also be used for researching the molecular and cytological mechanisms of the influence of the alcohol on the brain development by referring to various detection methods, makes up the defects of the traditional cell experiment and animal experiment, and provides a powerful tool for researching the influence of alcohol in the gestation period on the early brain development of a fetus.
Drawings
FIG. 1 tracking 3D brain development of control and sample groups under light field conditions according to the invention;
FIG. 2 immunohistochemistry and cryosectioning methods to detect 3D brain development in control and sample groups;
FIG. 3RT-PCR method for detecting gene expression of nerves and different brain regions in 3D brain development process.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention thereto. All reagents used in the present invention are commercially available.
Example 1
In-vitro model establishment of fetal brain injury caused by alcohol exposure in gestation period
The method mainly comprises the following four steps: preparing sodium alginate hollow fibers, inducing the 3D brain at the early stage, wrapping the 3D brain in the sodium alginate hollow fibers and treating the 3D brain with alcohol.
The preparation method of the sodium alginate hollow fiber comprises the following steps: the sodium alginate hollow fiber is prepared by utilizing a micro-fluidic chip in a sleeve form, the inner diameter of the prepared hollow fiber is 600-1000 mu m, the wall thickness of the tube is 50-200 mu m, and the used liquid is subjected to aseptic treatment. The sodium alginate filaments are required to be soaked in a DMEM/F12 culture medium, so that the swelling process of the sodium alginate filaments in the culture medium is completed, and the sodium alginate filaments are stored at a low temperature of 4 ℃ and are convenient for subsequent operation.
Step (2)3D brain prophase induction, which specifically comprises the following steps: preparing a pseudo-blank by using a PDMS chip with a pit-shaped structure: the chip with the pit-shaped structure is placed in a 24-hole plate, the diameter of the pit-shaped structure is 800 mu m, the depth of the pit-shaped structure is 300 mu m, and the pit-shaped structure is used for forming the embryoid body. On day 1, embryoid bodies were prepared and 2X 10 cells were used5-6×105Digesting the hiPSCs into single cells, transferring the single cells into a PDMS chip with a pit-shaped structure, centrifuging the single cells at 1000rpm for 5min, wherein the used culture medium is KSR culture medium, and adding 5 mu M Y27632;
the KSR medium comprises DMEM/F12 as the basic component, and further comprises KnockOut Replacement (KSR) accounting for 20% of the total volume, NEAA (Non Essential Amino Acid, 100 x) accounting for 1% of the total volume, GlutaMax (100 x) accounting for 1% of the total volume, peniciln-streptomycin (100 x) accounting for 1% of the total volume, and beta-mercaptoethanol, 4ng/ml bFGF.
On day 2, the embryoid bodies formed were transferred to a low-adhesion petri dish and embryoid bodies were cultured in suspension using KSR medium. On day 5, the embryoid bodies were induced to differentiate in the neuroepithelial direction, and the KSR medium was replaced with the neural induction medium. The cell pellet remains in suspension culture. The medium was changed every 3 days.
The basic component of the nerve induction medium is DMEM/F12, and N2(100 x) accounting for 1% of the total volume, GlutaMAX (100 x) accounting for 1% of the total volume, NEAA (Non Essential Amino Acid, 100 x) accounting for 1% of the total volume, 1 mu g/ml heparin and peniciln-streptomycin (100 x) accounting for 1% of the total volume are added.
And (3) wrapping the 3D brain in the sodium alginate hollow fiber, specifically: and on the 10 th day, the Matrigel is used for re-suspending the cell mass induced in the early stage, and the cell mass is injected into the sodium alginate filaments by using an injector, so that bubbles are prevented from being generated in the whole process, and the operation on ice is kept at low temperature, so that the Matrigel is not solidified. The sodium alginate filaments containing the cell clusters were placed in an incubator at 37 ℃ for 30min to coagulate Matrigel. Transferring the sodium alginate filaments containing the cell clusters into a 6-well plate for subsequent culture, wherein the used culture medium is used for neural differentiation culture, the basic components of the medium are DMEM/F12 and Neuralbasal medium in a volume ratio of 1:1, and B27 (50X) accounting for 1% of the total volume, N2 (100X) accounting for 0.5% of the total volume, NEAA (100X) accounting for 0.5% of the total volume, GlutaMAX (100X) accounting for 1% of the total volume, penicilin-streptomycin (100X) accounting for 1% of the total volume and beta-mercaptoethanol accounting for 0.05mM are additionally added.
The step (4) of alcohol treatment of the 3D brain comprises the following specific steps:
on day 11, 3D brains were cultured in the control group using neural differentiation medium, and 3D brains were cultured in the sample group using neural differentiation medium with alcohol concentration of 100mM, using the medium as prepared. In addition, PBS buffer solution containing alcohol with the same concentration needs to be added into the gap of the 6-pore plate in the sample group, and a sealing film needs to be used for sealing, so that the volatilization of the alcohol is reduced. Due to the evaporation of alcohol and the supply of nutrients, the culture medium was changed every two days for both the sample group and the control group.
The 3D brain development status in the sample group and the control group was followed by the bright field, and the results are shown in fig. 1.
Example 2
The method for establishing the in vitro model of the fetal brain injury caused by alcohol exposure in the gestation period is basically the same as that in the embodiment 1, and the difference is that:
step (2)3D brain prophase induction, which specifically comprises the following steps: preparing a pseudo-blank by using a PDMS chip with a pit-shaped structure: the chip with the pit-shaped structure is placed in a 24-hole plate, and the diameter of the small pit structure is 600 mu m, the depth of the small pit structure is 200 mu m, and the small pit structure is used for forming the embryoid body. On day 1, embryoid bodies were prepared and 2X 10 cells were used5-6×105Digesting the hiPSCs into single cells, transferring the single cells into a PDMS chip with a pit-shaped structure, centrifuging at 2000rpm for 3min, wherein the used culture medium is KSR culture medium, and adding 5 mu M Y27632;
and (3) wrapping the 3D brain in the sodium alginate hollow fiber, specifically: and on the 10 th day, the Matrigel is used for re-suspending the cell mass induced in the early stage, and the cell mass is injected into the sodium alginate filaments by using an injector, so that bubbles are prevented from being generated in the whole process, and the operation on ice is kept at low temperature, so that the Matrigel is not solidified. The sodium alginate filaments containing the cell clusters were placed in an incubator at 37 ℃ for 25min to coagulate Matrigel. Transferring the sodium alginate filaments containing the cell clusters to a 6-hole plate for subsequent culture,
the step (4) of alcohol treatment of the 3D brain comprises the following specific steps:
on day 11, 3D brains were cultured in the control group using neural differentiation medium, and 3D brains were cultured in the sample group using neural differentiation medium with alcohol concentration of 200mM, using medium prepared as needed. In addition, PBS buffer solution containing alcohol with the same concentration needs to be added into the gap of the 6-pore plate in the sample group, and a sealing film needs to be used for sealing, so that the volatilization of the alcohol is reduced. Due to the evaporation of alcohol and the supply of nutrients, the culture medium was changed every two days for both the sample group and the control group.
Example 3
The method for establishing the in vitro model of the fetal brain injury caused by alcohol exposure in the gestation period is basically the same as that in the embodiment 1, and the difference is that:
step (2)3D brain prophase induction, which specifically comprises the following steps: preparing a pseudo-blank by using a PDMS chip with a pit-shaped structure: the chip with the pit-shaped structure is placed in a 24-hole plate, and the diameter of the pit-shaped structure is 700 mu m, the depth of the pit-shaped structure is 200 mu m, and the pit-shaped structure is used for forming the embryoid body. On day 1, embryoid bodies were prepared and 2X 10 cells were used5-6×105Digesting the hiPSCs into single cells, transferring the single cells into a PDMS chip with a pit-shaped structure, centrifuging the single cells at 500rpm for 5min, wherein the used culture medium is KSR culture medium, and adding 5 mu M Y27632;
and (3) wrapping the 3D brain in the sodium alginate hollow fiber, specifically: and on the 10 th day, the Matrigel is used for re-suspending the cell mass induced in the early stage, and the cell mass is injected into the sodium alginate filaments by using an injector, so that bubbles are prevented from being generated in the whole process, and the operation on ice is kept at low temperature, so that the Matrigel is not solidified. The sodium alginate filaments containing the cell clusters were placed in an incubator at 37 ℃ for 20min to coagulate Matrigel.
The step (4) of alcohol treatment of the 3D brain comprises the following specific steps:
on day 11, 3D brains were cultured in the control group using neural differentiation medium, and 3D brains were cultured in the sample group using neural differentiation medium with alcohol concentration of 20mM, using the medium as prepared. In addition, PBS buffer solution containing alcohol with the same concentration needs to be added into the gap of the 6-pore plate in the sample group, and a sealing film needs to be used for sealing, so that the volatilization of the alcohol is reduced. Due to the evaporation of alcohol and the supply of nutrients, the culture medium was changed every two days for both the sample group and the control group.
Example 4
RT-PCR method for detecting brain development related gene expression conditions of control group and sample group
The 30-day-old 3D brains from example 1 were collected, washed 1 time with PBS buffer and centrifuged. The Trizol method is used for extracting the whole RNA and comprises the following steps: 1ml Trizol is used for resuspending the cells, the cells are blown up and down until no cell block exists, and the whole solution is clarified; adding 200 μ l chloroform, mixing for 5min, standing at room temperature for 3min, and centrifuging at 15000rpm for 15 min; layering the solution, taking the upper layer liquid into a new pipe, and carefully operating the whole process to avoid contacting white precipitate in the middle layer as much as possible; adding equivalent isopropanol into the absorbed solution, turning upside down and mixing uniformly, standing overnight at-20 ℃ to facilitate RNA precipitation; centrifuging at 15000rpm for 10 min; the white RNA precipitate at the bottom of the tube was retained and the supernatant carefully removed; cleaning the white precipitate with 75% ethanol, and centrifuging at 15000rpm for 5 min; removing 75% of ethanol as far as possible, and volatilizing the ethanol at room temperature for 5min to prevent the ethanol from polluting a sample and influencing subsequent experiments; dissolving RNA precipitate by DEPC water; RNA concentration and purity were determined and corresponding dilutions were made to a final concentration of 500 ng/ml.
Secondly, mRNA is reversely transcribed into cDNA, the system is 50 mu l, and the specific mixture ratio is as follows: 10. mu.l of 500ng/ml RNA, 10. mu.l of Buffer, 2.5. mu.l of Oligo dT, 2.5. mu.l of Random 6mers, 2.5. mu.l of Enzyme, 27.5. mu.l of DEPC water, 20min at 37 ℃ and 4 ℃.
And thirdly, Real-time PCR, according to the requirement of the kit on ligand solution, wherein the final system is 5ul, and the annealing temperatures Tm are unified to 58 ℃.
Finally, the expression of the genes related to brain development in the control group and the sample group is counted, and the result is shown in FIG. 2.
Example 5
Immunohistochemical method for detecting 3D brain development of control group and sample group
The 3D brains from days 15 and 24 of example 1 were collected for cryosectioning as follows: fixing cells with 4% paraformaldehyde for 20min, washing with PBS buffer solution for three times, each time for 10 min; dehydrating 30% sucrose overnight at 4 deg.C; OCT embedding, storing for 30min at room temperature, and solidifying at 80 deg.C; the sections were frozen to a thickness of 10-20 μm and attached to an electrostatically adsorbed slide. Then carrying out immunofluorescence staining, wherein the method comprises the following steps: placing the slide with the slices in a PBS buffer solution to soak for 5 min; allowing 0.1% triton X-100 pore-forming agent to act for 10min, washing with PBS buffer solution for 1 time and 5 min; goat blocking serum is acted for 1h at room temperature, a primary antibody (TUJ1, SOX2) is diluted at 1:400, incubated overnight at 4 ℃, and washed for 1 time and 5min by PBS buffer; diluting a secondary antibody (goat anti-rabbit or mouse IgG labeled by Fluorescence 488/594) at a ratio of 1:100, incubating at normal temperature for 1h, washing for 1 time in PBS buffer solution for 5 min; after washing, 1:2000 diluted DAPI working solution was added, pictures were taken under a fluorescent microscope, and the expression of SOX2 and TUJ1 were recorded, and the results are shown in FIG. 3. The results are shown in FIG. 3. The expression of neurons (TUJ1) in the sample group was significantly higher than the control group, demonstrating that alcohol treatment promoted neurological development of the 3D brain.
Claims (4)
1. An in vitro model building method of fetal brain injury caused by alcohol exposure in gestation period is characterized by mainly comprising the following steps:
preparing sodium alginate hollow fibers;
preparing a sodium alginate wire with a hollow structure by using a micro-fluidic chip in a sleeve form, wherein the inner diameter is 600-1000 mu m, the thickness of a tube wall is 50-200 mu m, and the used liquid is subjected to aseptic treatment; the prepared sodium alginate filaments need to be soaked in a DMEM/F12 culture medium, so that the swelling process of the sodium alginate filaments in the culture medium is completed, and the sodium alginate filaments are stored at a low temperature of 4 ℃ to facilitate subsequent operation;
3D brain prophase induction;
the 3D brain is wrapped in the sodium alginate hollow fiber;
treating the 3D brain with alcohol;
the 3D brain prophase induction specifically comprises the following steps: preparing a pseudo-blank by using a PDMS chip with a pit-shaped structure: the chip with the pit-shaped structure is placed in a 24-hole plate, the diameter of the small pit structure is 800 mu m through 600 plus one year, the depth is 300 mu m through 100 plus one year, and the small pit structure is used for forming an embryoid body; on day 1, embryoid bodies were prepared and 2X 10 cells were used5-6×105Digesting individual hipSCs into single cells, transferring the single cells to a PDMS chip, centrifuging at 500-5min, the culture medium is KSR culture medium, and 5 mu M Y27632 is added; on the 2 nd day, transferring the formed embryoid body to a culture dish with low adhesion, and performing suspension culture on the embryoid body, wherein the culture medium is a KSR culture medium; on the 5 th day, inducing the embryoid bodies to differentiate towards the neuroepithelial direction, and replacing the KSR culture medium with a neural induction culture medium;
the 3D brain is wrapped in the sodium alginate hollow filament and specifically comprises the following components: on the 10 th day, the Matrigel is used for re-suspending the cell clusters induced in the early stage, and the cell clusters are injected into the sodium alginate hollow filament by using an injector, so that bubbles are prevented from being generated in the whole process, the operation on ice is ensured to be maintained at low temperature, and the Matrigel is ensured not to be solidified; placing the sodium alginate filaments containing the cell clusters in an incubator at 37 ℃ for 20-30min to solidify Matrigel; transferring the sodium alginate filaments containing the cell clusters to a 6-hole plate for subsequent culture, wherein the used culture medium is a neural differentiation culture medium;
the alcohol treatment of the 3D brain specifically comprises the following steps: on day 11, the control group uses the neural differentiation culture medium to culture the 3D brain, the sample group uses the neural differentiation culture medium with the alcohol concentration of 20-200mM to culture the 3D brain, and the used culture medium needs to be prepared in situ; adding PBS buffer solution with the same alcohol concentration into the gap of the 6-hole plate in the sample group; sealing 6 pore plates in the sample group by using a sealing film to reduce alcohol volatilization; alcohol treatment was carried out until 3D brain development was 30 days and follow-up testing was performed.
2. The method for establishing the in-vitro model of the fetal brain injury caused by alcohol exposure in the gestation period according to claim 1, wherein instruments and solutions involved in sodium alginate thread preparation are necessarily sterile, and corresponding sterilization operations are performed before experiments, specifically high-temperature high-pressure sterilization, UV irradiation or 0.2 μm-aperture filter membrane filtration.
3. The method for in vitro modeling of fetal brain injury from alcohol exposure during pregnancy according to claim 1, wherein said Matrigel concentration is 2-10 mg/ml.
4. The method of establishing an in vitro model of fetal brain injury from alcohol exposure during pregnancy according to claim 1, wherein: the concentration of alcohol ranged from 20-200mM to mimic the alcohol concentration in physiological conditions.
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