CN107937344B - Method for realizing brain development of hiPSCs source by taking hollow fibers as carrier - Google Patents
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Abstract
A method for realizing brain development of hipsCs sources by taking hollow fibers as a carrier is disclosed, wherein the method takes novel biological material sodium alginate hollow fibers as the carrier, and in-vitro 3D brain development is efficiently realized. The 3D brain derived from the HiPSCs can simulate the early development process of the human brain to a certain extent, and provides a powerful tool for in vitro research on human development. The hollow silk is used as a carrier for 3D brain development of hipSCs sources, so that the development of a large number of 3D brains in an extracellular matrix can be realized more conveniently and rapidly, and the finally developed 3D brains have consistent sizes and shapes due to the structural characteristics of the silk.
Description
Technical Field
The invention relates to a microfluidic chip technology, in particular to a method for realizing brain development of hiPSCs sources by taking hollow fibers as carriers.
Background
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. According to the characteristics, the organoid development technology makes up the blank between 2D cell culture differentiation and animal experiments, and has wide application prospect.
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 combination of bioengineering and hiPSCs-derived organoids optimizes the development and operation of in vitro organoids still leaves a blank.
Disclosure of Invention
The invention aims to provide a method for efficiently realizing 3D brain development from hipsCs by taking sodium alginate hollow fibers as carriers, which not only ensures the material exchange in the development process, but also realizes a controllable development structure, so that the finally developed 3D brain has consistent size and shape and has the characteristic of real-time monitoring.
The invention provides a method for efficiently realizing brain development of hiPSCs sources by taking hollow fibers as carriers, which mainly comprises the following three steps: preparing the sodium alginate hollow fiber, inducing the 3D brain at the early stage and developing the 3D brain in the sodium alginate hollow fiber.
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 remains in suspension culture. The medium was changed 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 penicilin-streptomycin (100 x) accounting for 1% of the total volume are required to be added.
The size of the embryoid body prepared by the PDMS chip with the pit-shaped structure can be adjusted by the change of the volume of the small pit and the number of cells in the cell suspension, and the size of the embryoid body is 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 comprises the basic components of DMEM/F12 and Neuralbasal medium in a volume ratio of 1:1, and additionally needs to be added with 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 0.05 mM.
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 subsequent structural and functional characterization method of the 3D brain development method with the high-throughput hipSCs source established by the invention is as follows:
(1) visually monitoring the growth speed and the development state of the 3D brain under the microscope bright field condition;
(2) detecting the expression of genes in nerves and different brain regions in the 3D brain development process by using an RT-PCR method;
(3) cutting the cell mass into 10-20 μm thickness by using a frozen section technology, and performing subsequent immunofluorescence detection of the neural and brain structure related antibodies;
(4) detecting the cell death and survival condition in the 3D brain development process by a TUNEL method;
(5) Live/Dead assay detects the apoptotic status of scattered single cells;
(6) ca using live cell workstations2+Imaging detection of nerve cell Ca2+And (4) moving to ensure the function of nerve cells. The shooting condition is one picture every 10s, and the pictures are continuously shot for 5 min.
The invention provides a method for efficiently realizing brain development of hiPSCs sources by taking a hollow fiber as a carrier, wherein the sodium alginate hollow fiber can be designed into different diameters and pipe wall thicknesses according to experimental requirements.
The invention provides a method for efficiently realizing brain development of hiPSCs from hollow fibers serving as a carrier, which is not only suitable for 3D brains of hiPSCs, but also suitable for organoids of other different stem cells (including embryonic stem cells), wherein published organoid tissues comprise intestines, stomachs, retinas, brains and the like.
The invention provides a method for efficiently realizing 3D brain development by taking sodium alginate filaments as a carrier, which can be applied to constructing co-culture of other cells and organoids, is closer to the simulated physiological condition and optimizes the organoid development degree.
The invention provides a method for efficiently realizing 3D brain development by taking sodium alginate filaments as a carrier, which can be applied to establishing various pathological models and researching the fetal brain development condition under pathological conditions.
Drawings
FIG. 1 preparation of pseudo-embryo body from PDMS chip with pit-like structure
FIG. 2 shows the 3D brain development of the source of the HIPSCs in the silk of sodium alginate under the bright field condition;
FIG. 3 RT-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 of the present invention are commercially available.
Example 1
Development of HiPSC-derived 3D brain in sodium alginate filaments
The method mainly comprises the following steps: preparing the sodium alginate hollow fiber, inducing the 3D brain at the early stage and developing the 3D brain in the sodium alginate hollow fiber.
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 hiPSCs into the chip in (1), centrifuging the chip at 1000rpm for 5min, wherein the culture medium is KSR culture medium, and adding 5 mu M Y27632; 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.
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.
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 remains in suspension culture. The medium was changed every 3 days.
The basic component of the nerve induction culture medium is DMEM/F12, and additionally, 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 peniciln-streptomycin (100 x) accounting for 1% of the total volume need to be added.
And (3) the development of the 3D brain in the sodium alginate hollow fiber is specifically as follows: 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 25min 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 neural differentiation medium comprises the basic components of DMEM/F12 and Neuralbasal medium in a volume ratio of 1:1, and additionally needs to be added with 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 0.05 mM.
The development process of 3D brain was followed brightfield, and the results are shown in fig. 1, Scale bar: 500 μm.
Example 2
The method for developing the 3D brain derived from HiPSC in the sodium alginate filaments is basically the same as that in example 1, but 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 hiPSCs into the chip in (1), centrifuging at 500rpm for 5min, wherein the culture medium is KSR culture medium, and adding 5 mu M Y27632; 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.
And (3) the development of the 3D brain in the sodium alginate hollow fiber is specifically as follows: 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 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.
Example 3
The development method of the 3D brain derived from the HiPSC in the sodium alginate filaments is basically the same as that of the example 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 pit structure is placed in a 24-hole plate, the diameter of the small pit structure is 700 mu m, the depth is 100 mu m, and the small pit structure is used for forming the pseudo-embryo body. On day 1, embryoid bodies were prepared and 2X 10 cells were used5-6×105Digesting the hiPSCs into single cells, transferring the hiPSCs into the chip in (1), centrifuging at 2000rpm for 3min, wherein the culture medium is KSR culture medium, and adding 5 mu M Y27632; 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.
And (3) the development of the 3D brain in the sodium alginate hollow fiber is specifically as follows: 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 20min 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.
Example 4
RT-PCR method for detecting gene expression conditions of nerves and different brain regions in 3D brain development process
The 3D brains of example 1, which were developed at different stages, 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, PCR, namely, according to the requirement of the kit on ligand solution, wherein the final system is 20ul, the annealing temperatures Tm are unified to 58 ℃, and the ligand solution is stored at 4 ℃.
2ul of the reaction solution was added with loading buffer, water and ethidium bromide, 6ul in total, and 4ul of marker, and subjected to agarose gel electrophoresis, and relevant data were collected, as shown in FIG. 2. RT-PCR detected that the hipSCs-associated sternness gene was down-regulated, while the gene expression of neuro-related and different brain regions was elevated.
Example 5
Expression of neural and brain region-related proteins during development
The 3D brains of example 1, developed at different stages, were cryosectioned 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, primary antibodies (PAX6, PAX2, NESTIN, TUJ1, SOX2, TBR1 and CTIP2) are diluted at 1:100 or 1:400, incubated at 4 ℃ overnight, 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 the washing, 1:2000 diluted DAPI working solution was added, and the expression of the corresponding protein was recorded by taking a photograph under a fluorescent microscope, the result is shown in FIG. 3. Proteins in both neuro-related and different brain regions showed high expression, indicating that hiPSCs could successfully develop into 3D brain in this system.
Claims (5)
1. A method for realizing brain development of hipsCs by taking hollow fibers as carriers is characterized by comprising the following steps: the method mainly comprises the following steps:
preparing sodium alginate hollow fibers;
preparing the sodium alginate hollow fiber: 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;
development of 3D brain within sodium alginate hollow filaments;
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 the hiPSCs into single cells, transferring the single cells to a PDMS chip, centrifuging at 500-2000rpm for 3-5min, wherein the used culture medium is KSR culture medium, and adding 5 mu M Y27632; 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 development of the 3D brain in the sodium alginate hollow fiber specifically comprises the following steps: 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.
2. The method for realizing brain development of hiPSCs origin by using hollow filaments as carriers according to claim 1, characterized in that instruments and solutions involved in the preparation of the sodium alginate filaments are necessarily sterile, and corresponding sterilization operations are performed before experiments, specifically high-temperature high-pressure sterilization, UV irradiation or filtration through a 0.2 μm-pore size filter membrane.
3. The method for realizing brain development from hiPSCs by using a hollow wire as a carrier according to claim 1, characterized in that a pit-shaped structured PDMS chip is used to prepare an embryoid body, the size of which is adjusted by the change of the diameter and the depth of the pit and the number of cells in the cell suspension, and the size of the embryoid body is 50-500 μm.
4. The method of claim 1, wherein the source of hiPSCs is transformed from human skin fibroblast virus infection, using hollow fibers as vectors for brain development.
5. The method of claim 1, wherein the Matrigel is present in a concentration of 2 to 10 mg/ml.
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