Method for constructing three-dimensional engineered neural tissue, three-dimensional engineered neural tissue and application thereof
阅读说明：本技术 构建三维工程化神经组织的方法、三维工程化神经组织及其用途 (Method for constructing three-dimensional engineered neural tissue, three-dimensional engineered neural tissue and application thereof ) 是由 孟晓婷 王放 董智勇 杜颖珊 于希尧 于 2019-11-10 设计创作，主要内容包括：本发明涉及构建三维工程化神经组织的方法、三维工程化神经组织及其用途。方法。包括以下步骤：一、分离培养动物神经干细胞；二、施加生理电场；将所述步骤一中分离培养出的神经干细胞传代至第5代,取直径100～200μm的神经球离心备用,将基质胶Matrigel冰上预融备用,通过生理电场施加设备连续施加生理电场刺激7天～14天,每天1小时～2小时；三、优化适合工程化神经组织生长的培养液：DMEM/F12,加入1/50 B27,1/100 N2,10～20ng/ml bFGF,1％胎牛血清；四、施加生理电场7～14天,得到三维工程化神经组织。解决了现有神经干细胞在神经组织工程应用过程中存在的神经元分化的数量极少和在三维生长环境中不能诱导神经突起有序生长形成发达的神经网络的技术问题。(The invention relates to a method for constructing a three-dimensional engineered neural tissue, the three-dimensional engineered neural tissue and application thereof. A method. The method comprises the following steps: firstly, separating and culturing animal neural stem cells; secondly, applying a physiological electric field; the neural stem cells separated and cultured in the first step are subcultured to the 5 th generation, neurospheres with the diameter of 100-200 mu m are taken for centrifugation for later use, Matrigel is pre-melted on ice for later use, and physiological electric field stimulation is continuously applied for 7-14 days through physiological electric field applying equipment, wherein the physiological electric field stimulation lasts for 1-2 hours every day; thirdly, optimizing a culture solution suitable for the growth of the engineered nervous tissue: DMEM/F12, adding 1/50B27,1/100N2, 10-20ng/ml bFGF and 1% fetal calf serum; and fourthly, applying the physiological electric field for 7-14 days to obtain the three-dimensional engineering nerve tissue. Solves the technical problems that the quantity of neuron differentiation is very small and neurite can not be induced to grow orderly to form a developed neural network in a three-dimensional growth environment in the application process of the existing neural stem cells in the neural tissue engineering.)
1. A method for constructing three-dimensional engineered neural tissue is characterized by comprising the following steps:
firstly, separating and culturing animal neural stem cells;
secondly, applying a physiological electric field;
the neural stem cells separated and cultured in the first step are passaged to the 5 th generation, neurospheres with the diameter of 100-200 mu m are taken for centrifugation for later use, Matrigel is pre-melted on ice for later use, and physiological electric field stimulation is continuously applied for 7-14 days through physiological electric field applying equipment, wherein the physiological electric field stimulation lasts for 1-2 hours every day;
thirdly, optimizing a culture solution suitable for the growth of the engineered nervous tissue: with "Du's modified Eagle Medium: the nutrient mixture F-12', DMEM/F12, is a basal medium; 1/50 neural cell culture supplement B was added271/100 neural cell culture supplement N210-20ng/ml basic fibroblast growth factor, bFGF and 1% fetal bovine serum, FBS.
And fourthly, applying the physiological electric field for 7-14 days to obtain the three-dimensional engineering nerve tissue.
2. The method of constructing a three-dimensional engineered neural tissue as in claim 1, wherein said animal is a mouse.
3. The method of constructing three-dimensional engineered neural tissue as in claim 2, wherein said first step comprises the steps of:
taking the whole brain of a mouse with 14-16 days of embryos, putting the whole brain into a cold DMEM/F12 basal medium, putting the fetal brain into a flat dish after a dissecting mirror moves down to remove a meningeal membrane, mechanically cutting the brain tissue into tissue fragments by using an ophthalmic fibrous forceps, and transferring the tissue fragments to a centrifuge tube;
and (II) centrifuging to remove meningeal fragments from the supernatant, adding the neural stem cell growth medium into the residual tissue fragments, and gently blowing and beating the cells by using a pipette. Standing, and then passing the cell suspension supernatant through a cell tractor to obtain a single cell suspension;
(III) adjusting the cell concentration to 2-5X 104cells/ml, the cells are seeded into culture flasks, the liquid is changed every 3 days, and the cells are passaged every 6-7 days.
4. The method of constructing three-dimensional engineered neural tissue as in claim 3, wherein said second step comprises the steps of:
(1) the 22mm by 22mm coverslips were cut with a glass knife into the same two 22mm by 11mm coverslips: performing autoclaving on the cover slips 1 and 2;
(2) adhering the cover glass 1 and the cover glass 2 to the middle of the bottom of a plate in parallel by using high-vacuum silicone grease, determining that a thin layer of silicone grease is uniformly paved on the bottom of the cover glass, and keeping the distance between the two cover glass to be 1-2 cm;
(3) adding the neurosphere suspension into Matrigel-Matrigel in a ratio of 1:10, uniformly mixing on ice, then planting into a mold with an inner diameter of 7mm, standing for 30 minutes at 37 ℃, taking out after the Matrigel is solidified into gel, and adding a small chamber to construct;
(4) a 22mm multiplied by 22mm cover glass 3 is put on the cover glass 1 and the cover glass 2 by high vacuum silicone grease to form a top cover, the cover glass 3 is lightly pressed by ophthalmological forceps to ensure that the silicone grease is uniformly stuck on the cover glass 1 and the cover glass 2, and a semi-closed chamber with two open ends is formed between the three cover glasses;
(5) 2 parallel silicone grease walls are built on the edges of the cover glass 3 above the two open ends of the chamber respectively by using silicone grease, and then a small amount of silicone grease is used for sealing gaps and joints, so that no other places except the open ends can pass liquid;
(6) after the chamber architecture is completed, adding a few drops of culture solution to prevent the Matrigel from air drying;
(7) slowly supplementing a proper amount of culture solution at one side of the open end of the small chamber, so that the culture solution slowly flows into the other side of the small chamber from one side of the plate;
(8) covering a flat dish cover with 2 small holes with the diameter of 8mm on a flat dish, and enabling the two small holes to be respectively arranged on two sides of the open end of the small chamber;
(9) preparing a glass bridge: bending a glass tube with the diameter of 6mm and the length of 26cm into an equiarm U-shaped tube at high temperature, and filling 2% agar gel prepared by taking 5-10% calf serum DMEM/F12 as a solution;
(10) preparing 2 100ml glass bottles, and filling 50ml of DMEM/F12 containing 5-10% calf serum, wherein the pH value is 7.4;
(11) one end of each of the 2 glass bridges is respectively connected with the 2 glass bottles, and the other end of each glass bridge is respectively placed in the 2 small holes on the plate cover;
(12) respectively putting the sterile Ag/AgCl electrodes into 2 glass bottles, and respectively connecting the sterile Ag/AgCl electrodes with a direct current power supply through positive and negative electric wires;
(13) setting the voltage of a direct current power supply as '0', turning on the power supply, measuring the voltage at two ends of the small chamber by using a voltmeter, and adjusting the voltage to a required voltage value;
(14) applying a physiological electric field of 100-150 mV/mm for 2 hours every day, and continuously applying for 7-14 days.
5. The method according to claim 3, wherein the culture medium for the growth of the three-dimensional engineered neural tissue is replaced with fresh culture medium every 2 days in the third step.
6. A three-dimensional engineered neural tissue constructed by the method of any one of claims 1-5 for constructing a three-dimensional engineered neural tissue.
7. The use of the three-dimensional engineered neural tissue of claim 6, wherein the three-dimensional engineered neural tissue is used for experimental study of animal three-dimensional engineered neural tissue.
8. Use of the three-dimensionally engineered neural tissue according to claim 6, for in vitro research models of neurological diseases.
9. Use of the three-dimensionally engineered neural tissue according to claim 6, for direct replacement of animal neural tissue.
10. Use of the three-dimensionally engineered neural tissue according to claim 6, for experimental study of human three-dimensionally engineered neural tissue.
Technical Field
The invention relates to a method for constructing a three-dimensional engineered neural tissue, the three-dimensional engineered neural tissue and application thereof.
Background
About 13.3 to 26.2 million spinal cord injury patients are newly added in the world every year, and the economic cost for nursing and treating diseases to disabilities and paralysis is up to billions of dollars, thus causing huge burden to the social development. After the nervous system is damaged, the tissues at the damaged part are disintegrated and the cells are apoptotic, and the functions carried by the corresponding structures are damaged or lost. The engineering nerve tissue is a multi-disciplinary cross field which is emerging in recent years and integrates biology, materials science, informatics and clinical medicine into a whole. The engineered neural tissue is an ordered neural tissue with a three-dimensional tissue structure constructed in vitro by taking neural cells as seed cells and taking bioactive materials as a scaffold. After the engineered neural tissue is transplanted into the damaged part, the transplanted cell components can survive in a large amount to form new tissues and can be integrated into the host neural tissue to fill the defect formed after the damage. Meanwhile, a suitable microenvironment can be created, conditions are provided for the regeneration of tissues or cells of a wounded person and transplanted cells, the pathological change of the damaged nerve tissues of the host is influenced, the reaction of peripheral undamaged nerve tissues to the damage is changed, and the degeneration process is slowed down or reduced. The construction of the engineered nervous tissue and the clinical transplantation treatment bring new eosin for patients and families disabled by central nervous system injury, and have social and economic benefits.
Due to the extreme complexity of neural tissue, the research of engineered neural tissue is still in the initial stage, and many problems to be solved at present are urgently needed, including: the composition of seed cell, the three-dimensional structure construction of bioactive material, the compounding of seed cell and biological material, the formation of three-dimensional ordered nerve tissue structure, etc. Seed cells currently used in engineering neural tissue include embryonic stem cells, neural stem cells, astrocytes, and the like. The scaffold material comprises natural polymers, artificially synthesized high molecular materials, nanometer materials modified by neurotrophic factors and the like.
The neural stem cell has the characteristics of in vitro proliferation and multidirectional differentiation potential, can be differentiated into neurons, astrocytes and oligodendrocytes, and is an ideal seed cell which can be applied to in vitro construction of engineered neural tissues. However, the neural stem cells have two prominent problems in the application process of neural tissue engineering: one is that the natural differentiation direction in vitro is mainly towards astrocytes, while the differentiation quantity towards neurons is very small; the other is how to induce the neurite to grow orderly in a three-dimensional growth environment to form a developed neural network.
Disclosure of Invention
The invention provides a method for constructing a three-dimensional engineered neural tissue, which aims to solve the technical problems that the number of neuron differentiation is very small in the application process of the existing neural stem cells in the neural tissue engineering, and neurite can not be induced to grow orderly in a three-dimensional growth environment to form a developed neural network.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for constructing three-dimensional engineered neural tissue is characterized by comprising the following steps:
firstly, separating and culturing animal neural stem cells;
secondly, applying a physiological electric field;
the neural stem cells separated and cultured in the first step are passaged to the 5 th generation, neurospheres with the diameter of 100-200 mu m are taken for centrifugation for later use, Matrigel is pre-melted on ice for later use, and physiological electric field stimulation is continuously applied for 7-14 days through physiological electric field applying equipment, wherein the physiological electric field stimulation lasts for 1-2 hours every day;
thirdly, optimizing a culture solution suitable for the growth of the engineered nervous tissue: with "Du's modified Eagle Medium: the Nutrient Mixture F-12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12, DMEM/F12) is used as a basic culture Medium, and 1/50 nerve cell culture supplement B is added271/100 neural cell culture supplement N210-20ng/ml of alkaline componentFibroblast growth factor (bFGF), 1% Fetal Bovine Serum (FBS).
And fourthly, applying the physiological electric field for 7-14 days to obtain the three-dimensional engineering nerve tissue.
2. The method of constructing a three-dimensional engineered neural tissue as in
The first step comprises the following steps:
taking the whole brain of a mouse with 14-16 days of embryos, putting the whole brain into a cold DMEM/F12 basal medium, putting the fetal brain into a flat dish after a dissecting mirror moves down to remove a meningeal membrane, mechanically cutting the brain tissue into tissue fragments by using an ophthalmic fibrous forceps, and transferring the tissue fragments to a centrifuge tube;
and (II) centrifuging to remove meningeal fragments from the supernatant, adding the neural stem cell growth medium into the residual tissue fragments, and gently blowing and beating the cells by using a pipette. Standing, and then passing the cell suspension supernatant through a cell tractor to obtain a single cell suspension;
(III) adjusting the cell concentration to 2-5X 104cells/ml, the cells are seeded into culture flasks, the liquid is changed every 3 days, and the cells are passaged every 6-7 days.
The second step comprises the following steps:
(1) the 22mm × 22mm coverslips were cut with a glass knife into the same two 22mm × 11mm small coverslips (
(2) adhering the
(3) adding the neurosphere suspension into Matrigel (Matrigel) in a ratio of 1:10, uniformly mixing on ice, then planting into a mold with an inner diameter of 7mm, standing for 30 minutes at 37 ℃, taking out after the Matrigel is solidified into gel, and adding a small chamber to construct;
(4) a cover glass 3(22mm multiplied by 22mm) is arranged above the
(5) 2 parallel silicone grease walls are built on the edges of the cover glass 3 above the two open ends of the chamber respectively by using silicone grease, and then a small amount of silicone grease is used for sealing gaps and joints, so that no other places except the open ends can pass liquid;
(6) after the chamber architecture is completed, adding a few drops of culture solution to prevent the Matrigel from air drying;
(7) slowly supplementing a proper amount of culture solution at one side of the open end of the small chamber, so that the culture solution slowly flows into the other side of the small chamber from one side of the plate;
(8) covering a flat dish cover with 2 small holes with the diameter of 8mm on a flat dish, and enabling the two small holes to be respectively arranged on two sides of the open end of the small chamber;
(9) preparing a glass bridge: bending a glass tube with the diameter of 6mm and the length of 26cm into an equiarm U-shaped tube at high temperature, and filling 2% agar gel prepared by taking 5-10% calf serum DMEM/F12 as a solution;
(10) preparing 2 100ml glass bottles, and filling 50ml of DMEM/F12 containing 5-10% calf serum, wherein the pH value is 7.4;
(11) one end of each of the 2 glass bridges is respectively connected with the 2 glass bottles, and the other end of each glass bridge is respectively placed in the 2 small holes on the plate cover;
(12) respectively putting the sterile Ag/AgCl electrodes into 2 glass bottles, and respectively connecting the sterile Ag/AgCl electrodes with a direct current power supply through positive and negative electric wires;
(13) setting the voltage of a direct current power supply as '0', turning on the power supply, measuring the voltage at two ends of the small chamber by using a voltmeter, and adjusting the voltage to a required voltage value;
(14) applying a physiological electric field of 100-150 mV/mm for 2 hours every day, and continuously applying for 7-14 days.
And in the third step, the fresh culture solution is replaced every 2 days.
After the technical scheme is adopted, the animal neural stem cells with multidirectional differentiation potential are used as seed cells, the Matrigel with good biocompatibility is used as a three-dimensional matrix support, the synergistic effect of a physiological electric field and bFGF is innovatively utilized, the culture condition for obtaining a certain number of functional neurons and the ordered growth of neurites is optimized, the three-dimensional engineering neural tissue with the developed neural network is constructed, and the technical problems that the number of neuron differentiation is very small in the neural tissue engineering application process of the existing neural stem cells, and the neurites cannot be induced to grow in an ordered manner to form the developed neural network in a three-dimensional growth environment are solved. In order to further promote the growth of the neurite, the technical solution adopted by the invention is to construct an in-vitro culture system suitable for the growth of the three-dimensional nerve tissue according to the growth characteristics of nerve cells, so that the culture system can cooperate with the stimulation of a physiological electric field: the two nerve cell culture supplements B27 and N2 are respectively prepared according to the proportion of 1:50 and 1:100, and a cell growth factor capable of promoting the growth of the processes and a basic fibroblast growth factor of 10-20ng/ml are added into a culture system. And then the new neuron protrusions grow orderly in the three-dimensional biological material to a certain degree by utilizing the guidance function of the physiological electric field, so as to construct the engineered neural tissue with a physiological structure. Matrigel matrix was chosen to fulfill this concept in terms of the problem of building three-dimensional structures from biomaterials. Because Matrigel can be polymerized to form a three-dimensional matrix with bioactivity at room temperature, the structure, the composition, the physical characteristics and the function of an in vivo cell basement membrane can be simulated, and the culture and the differentiation of in vitro cells are facilitated. Matrigel has porous structures with various diameters and has lower surface tension; the structure stability is stronger, and the porous structure ensures that the porous structure has larger surface/volume ratio and higher void ratio; viscoelasticity is similar to central nervous tissue; can be arbitrarily shaped according to the condition of the repaired tissue, and can form a living tissue structure and the like. At present, Matrigel is widely applied to cell growth, differentiation and tissue damage repair research, and the technology is mature and is beneficial to the rapid formation of products.
The invention also provides the three-dimensional engineered neural tissue obtained by the method.
The invention also provides the application of the three-dimensional engineered neural tissue, which is used for experimental research of animal three-dimensional engineered neural tissue.
The invention also provides the application of the three-dimensional engineered neural tissue, which is used for an in-vitro research model of nervous system diseases.
The invention also provides the application of the three-dimensional engineered neural tissue, which is used for directly replacing animal neural tissue.
The invention also provides the application of the three-dimensional engineered neural tissue, and the three-dimensional engineered neural tissue is used for experimental research of human three-dimensional engineered neural tissue.
Drawings
FIG. 1 shows the state of cell growth and cell network formation in a three-dimensional Matrigel scaffold under different conditions.
FIG. 2 shows the state of cell growth in three-dimensional Matrigel scaffolds under different conditions as measured by Nissl Stains staining.
FIG. 3 is a cell viability assay in a physiological electric field stimulated three-dimensional Matrigel scaffold.
FIG. 4 is an apoptosis assay in a physiological electric field stimulated three-dimensional Matrigel scaffold.
FIG. 5 is the immunofluorescence detection and layer-by-layer scan reconstruction of three-dimensional neural tissue structure 7 days after the physiological electric field is applied.
Fig. 6 is a three-dimensional nerve tissue structure reconstructed by layer-by-layer scanning under a high power mirror.
FIG. 7 is a transmission electron microscopy technique to detect ultrastructures of engineered neural tissue.
Detailed Description
The invention provides a three-dimensional engineering nerve tissue which can be constructed in vitro and has a certain number of functional neurons and neurites growing in order: inducing the neural stem cells to differentiate towards the neurons under the action of a physiological electric field of 100 mV/mm-150 mV/mm to obtain a certain number of neurons; an in vitro culture system suitable for three-dimensional nerve tissue growth is constructed, and the brief method comprises the following steps:
1. isolated culture of mouse neural stem cells
2. Physiological electric field application
And (3) passaging the neural stem cells to the 5 th generation, centrifuging neurospheres with the diameters of 100-200 mu m for later use, and pre-melting the Matrigel on ice for later use. A physiological electric field application device is provided.
3. The physiological electric field stimulation was applied continuously for 14 days, 2 hours a day.
4. Engineering nerve tissue culture solution: DMEM/F12, added with 1/50B27,1/100N2, 10-20ng/ml bFGF and 1% fetal calf serum.
5. And applying a physiological electric field for 7-14 days, and detecting the cell structure and tissue structure characteristics of the engineered neural tissue by applying immunofluorescence chemical staining and an electron microscope technology.
6. The results of the invention are: the method takes mouse neural stem cells as seed cells, takes Matrigel matrix hydrogel as a bracket, applies stimulation of a physiological electric field to induce the neural stem cells in Matrigel to differentiate towards neurons, and utilizes the guidance effect of the physiological electric field to enable the neonatal neuron protrusion to grow orderly in the three-dimensional hydrogel to a certain extent so as to construct the engineered neural tissue with a physiological structure. The mouse neural stem cells with the multidirectional differentiation potential are used as seed cells, the Matrigel with good biocompatibility is used as a three-dimensional matrix support, the construction of the engineered neural tissue is innovatively guided by the physiological electric field guidance effect and the bFGF in a synergetic mode, the active three-dimensional cultured mouse neural tissue is obtained, and the mouse neural tissue can be applied to in-vitro research models of nervous system diseases and lays a foundation for experimental research of human three-dimensional engineered neural tissue.
The detailed method comprises the following steps:
first, separate culture of animal neural stem cells
Taking the whole brain of a mouse with 14-16 days of embryos, putting the whole brain into a cold DMEM/F12 basal medium, putting the fetal brain into a 35mm dish after removing meninges under a dissecting mirror, mechanically cutting the brain tissue fragments by using an ophthalmic fibrous forceps, transferring the tissue fragments into a 15ml centrifuge tube, and centrifuging at 800rpm for 3min to remove the meninges fragments by removing supernatant. The remaining tissue fragment was added with 2ml growth medium: DMEM/F12 containing 20ng/ml bFGF, 20ng/ml EGF, 1/100N2 nerve culture supplement was gently pipetted into separate cells. Standing for 1 minute, obtaining single cell suspension by passing cell suspension supernatant through a cell purifier, and adjusting the cell concentration to 2-5 × 104cells/ml, cells were seeded into 25ml flasks and the medium changed every 3 days and passaged every 6 days.
Secondly, applying physiological electric field
(1) Cutting the cover glass with the size of 22x 22mm into the size of 22x 11mm by using a glass cutter, and autoclaving;
(2) adhering two 22x 11mm cover slips in parallel to the middle of the bottom of a 100mm plate by using high-vacuum silicone grease, and determining that the thin silicone grease is uniformly paved at the bottom of each cover slip, wherein the distance between every two 2 cover slips is 1 cm-2 cm;
(3) adding the neurosphere suspension into Matrigel in a ratio of 1:10, uniformly mixing on ice, then planting the mixture into a mold with the diameter of 5mm, carrying out 30 minutes at 37 ℃, taking out after the Matrigel is solidified into gel, and adding a small chamber to construct;
(4) using high vacuum silicone grease to support a 22x 22mm cover glass on 2 22x 11mm cover glass to form a top cover, using an ophthalmic forceps to lightly press the 22x 22mm cover glass, so that the silicone grease is uniformly adhered on the 2 22x 11mm cover glass, and forming a closed chamber with two open ends among the three cover glass;
(5) 2 parallel silicone grease walls are built on the edges of 22x 22mm cover glass above the two open ends of the chamber respectively by using silicone grease, and then a small amount of silicone grease is used for sealing gaps and joints, so that no other places except the open ends can pass through liquid;
(6) after the chamber architecture is completed, adding a few drops of culture solution to prevent the Matrigel from air drying;
(7) slowly supplementing a proper amount of culture solution at one side of the open end of the small chamber, so that the culture solution slowly flows into the other side of the small chamber from one side of the plate;
(8) covering a flat dish cover with 2 small holes with the diameter of 8mm on a flat dish, and enabling the two small holes to be respectively arranged on two sides of the open end of the small chamber;
(9) preparing a glass bridge: bending a glass tube with the diameter of 6mm and the length of 26cm into an equiarm U-shaped tube at high temperature, and filling 2% agar gel prepared by taking 5-10% calf serum DMEM/F12 as a solution;
(10) preparing 2 100ml glass bottles, and filling 50ml of DMEM/F12 containing 5-10% calf serum, wherein the pH value is 7.4;
(11) one end of each of the 2 glass bridges is respectively connected with the 2 glass bottles, and the other end of each glass bridge is respectively placed in the 2 small holes on the plate cover;
(12) respectively putting the sterile Ag/AgCl electrodes into 2 glass bottles, and respectively connecting the sterile Ag/AgCl electrodes with a direct current power supply through positive and negative electric wires;
(13) setting the voltage of a direct current power supply as '0', turning on the power supply, measuring the voltage at two ends of the small chamber by using a voltmeter, and adjusting the voltage to a required voltage value;
(14) applying the mixture for 2 hours at a concentration of 100-150 mV/mm every day for 7-14 days;
thirdly, optimizing a culture solution suitable for the growth of the engineered nervous tissue: with "Du's modified Eagle Medium: the Nutrient Mixture F-12 (Dulbecco's Modified Eagle Medium: Nutrient Mixture F-12, DMEM/F12) is used as a basic culture Medium, and 1/50 nerve cell culture supplement B is added271/100 neural cell culture supplement N210-20ng/ml basic fibroblast growth factor (bFGF), 1% Fetal Bovine Serum (FBS). Fresh medium was replaced every 2 days.
And fourthly, applying the physiological electric field for 7-14 days to obtain the three-dimensional engineering nerve tissue.
Constructing results
1. Optimizing the neurosphere planting density: screening out the cell planting density and the optimal thickness of Matrigel for constructing the three-dimensional neural tissue by applying the neural stem cells in vitro.
Unlike two-dimensional culture, the culture of engineered neural tissue with certain three-dimensional structures in vitro must address the supply of nutrients to cells within the scaffold and the excretion of cellular metabolites. Cell seeding density plays a key role. Either too low or too high cell density can affect the survival and viability of cells within the scaffold.
We culture the neurospheres with the diameter of 100-200 μm at the rate of 10 neurospheres/
2. Optimizing the culture condition of the engineered neural tissue:
to obtain neural tissue with three-dimensional ordered growth, we first screened a medium suitable for three-dimensional neural tissue. Experimental observations have shown that a medium containing only 10% fetal bovine serum does not provide sufficient nutrition for three-dimensional tissue with a certain thickness. Therefore, we added the supplement for neural cell culture and fibroblast growth factor bFGF-BNb to the culture medium for the growth characteristics of neural tissue cells: the culture system containing the bFGF can be used for promoting differentiation and protrusion growth of nerve cells under the synergistic effect of a physiological electric field, and can obtain a three-dimensional nerve tissue with a developed nerve network.
(1) Cell viability and apoptosis assay: and detecting the cell activity and apoptosis condition in the engineered neural tissue by using Live/Dead and TUNEL detection kits.
A physiological electric field of 150mV/mm is applied to stimulate three-dimensional nerve tissues to detect whether the stimulation of the physiological electric field influences the survival and growth of cells, and a fluorescent live/dead detection kit and an apoptosis detection kit are applied to detect the cell viability and the apoptosis condition after 7 days. The results show that the cell viability is shown in fig. 3 and the apoptosis is shown in fig. 4, and the cell viability and the apoptosis are not obviously different from those of the group without the physiological electric field stimulation, which indicates that the stimulation of the physiological electric field does not influence the survival and the growth of the nerve cells.
In FIG. 1, red fluorescence indicates dead cells, and green fluorescence indicates live cells
FIG. 2 shows DAPI-labeled nuclei in blue fluorescence and FITC-labeled apoptotic cells in green fluorescence
(2) And (3) histological detection:
tuj1, Tubulin β 3 is a neuron specific marker, GFAP, glial fibroblastic protein is a astrocyte specific marker, after
Scanning and reconstructing a three-dimensional nerve tissue structure layer by using a laser confocal microscope, detecting the distribution, the form and the protrusion extension degree of cells in the bracket and the formation of a nerve network: it can be seen that after 7 days of physiological electric field stimulation, the number of positive neurons differentiated into Tuj1 by the neural stem cells is obviously higher than that of the groups without physiological electric field stimulation, and 10% of FBS cells in the control group are more differentiated into GFAP positive astrocytes; however, although a large number of neurons can be obtained by a single physiological electric field stimulation, developed neurites are not formed. When the cells stimulated by the physiological electric field grow in the BNb culture solution, the physiological electric field and the bFGF act synergistically to obtain developed cell processes, and developed neural networks are formed among the cells, as shown in FIG. 5. After three-dimensional reconstruction, cultured neural tissue was measured between 100 μm and 150 μm, as shown in FIG. 6.
(3) Transmission electron microscope technology for detecting ultra-micro structure of engineered nervous tissue
The three-dimensional engineered neural tissue constructed under the synergistic action of the physiological electric field and the bFGF is observed under a transmission electron microscope, and the connection between the cell ultrastructure and the cell in the tissue can be seen. We observed cells with ultrastructures characteristic of neurons, with thickened cell membranes as shown in fig. 7A; synaptic connections are formed between cells, and a large number of synaptic vesicles are shown in FIG. 7B.
The invention also provides a three-dimensional engineered neural tissue constructed by the method for constructing the three-dimensional engineered neural tissue.
The invention also provides the application of the three-dimensional engineered neural tissue, which is applied to an in-vitro research model of nervous system diseases.
The invention also provides the application of the three-dimensional engineered neural tissue, which is used for experimental research of animal three-dimensional engineered neural tissue.
The invention also provides the application of the three-dimensional engineered neural tissue, which is used for directly replacing animal neural tissue.
The invention also provides the application of the three-dimensional engineered neural tissue, and the three-dimensional engineered neural tissue is used for experimental research of human three-dimensional engineered neural tissue.