CN107937344A - A kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier - Google Patents

Abstract

A method for realizing hiPSCs-derived brain development using hollow filaments as a carrier. This method uses a new biomaterial sodium alginate hollow filament as a carrier to efficiently realize 3D brain development in vitro. The 3D brain derived from HiPSCs can simulate the early development process of human brain to a certain extent, which provides a powerful tool for studying human development in vitro. As the carrier of hiPSCs-derived 3D brain development, hollow filaments can not only realize the development of a large number of 3D brains in the extracellular matrix more conveniently and quickly, but also make the final developed 3D brains have a consistent size and shape due to the structural characteristics of the filaments.

Description

A method to realize hiPSCs-derived brain development using hollow filaments as a carrier

technical field

The invention relates to microfluidic chip technology, in particular to a method for realizing brain development derived from hiPSCs using hollow fibers as carriers.

Background technique

Organoid development from various stem cell sources has been effectively developed in recent years, including neural stem cells, intestinal stem cells, embryonic stem cells, and iPSCs. Organoids are characterized by a 3D cell cluster structure spontaneously formed by stem cells or primary cells, which contain a variety of functional cells of corresponding tissues and organs. The process of organ development. According to these characteristics, organoid development technology bridges the gap between 2D cell culture differentiation and animal experiments, and has broad application prospects.

However, at present, organoid technology still has many shortcomings and needs to be improved. Due to the structural characteristics of its 3D cell cluster, the internal cells die on a large scale due to lack of oxygen and nutrients, which greatly limits the extent of in vitro organoid development, including volume size and functional maturity. Vascular tissue is scattered throughout the organ to provide the oxygen and nutrients it needs. Therefore, the combination of other technologies is expected to make up for this deficiency. In addition, the existing traditional cell culture technology to cultivate organoids is complex, time-consuming and poorly controllable. Therefore, combining with existing bioengineering methods is expected to optimize organoid technology.

As a culture carrier, the filamentous structure has several advantages: First, with the help of microfluidic chip technology, sodium alginate filaments of different sizes and thicknesses can be flexibly and efficiently produced according to actual applications. Secondly, the filamentous structure has a high specific surface area ratio, which is conducive to material exchange and provides a good living environment for cell culture. Thirdly, the filamentous structure can guide the growth of cell clusters, produce structurally similar brain tissue, and limit its growth during development, which to a certain extent mimics the role of meninges in physiological conditions. Finally, the sodium alginate material can be dissolved under high salt or sodium citrate conditions to release cells and meet different processing needs.

However, the combination of bioengineering and hiPSCs-derived organoids to optimize the development and operation of organoids in vitro is still blank.

Contents of the invention

The purpose of the present invention is to provide a 3D brain development method using sodium alginate hollow filaments as a carrier to efficiently realize hiPSCs-derived 3D brain development. The 3D brain has a consistent size and shape and features real-time monitoring.

The invention provides a method for efficiently realizing hiPSCs-derived brain development using hollow fibers as a carrier. The steps are mainly divided into three parts: preparation of sodium alginate hollow fibers, 3D brain pre-induction and 3D brain in sodium alginate hollow fibers internal development.

The preparation of step (1) sodium alginate hollow fiber is specifically:

The hollow fiber of sodium alginate is prepared by using a microfluidic chip in the form of a sleeve. The inner diameter of the prepared hollow fiber is 600-1000 μm, the thickness of the tube wall is 50-200 μm, and the liquid used is treated aseptically.

The sodium alginate silk needs to be soaked in DMEM/F12 medium to complete the swelling process in the medium, and stored at a low temperature of 4°C to facilitate subsequent operations.

Step (2) 3D pre-brain induction, specifically:

(1) Prepare embryoid bodies using a PDMS chip with a pit-like structure: the chip with a pit-like structure is placed in a 24-well plate. The diameter of the small pit structure is 600-800 μm and the depth is 100-300 μm. form.

(2) On the first day, embryoid bodies were prepared, and 2×10 ⁵ -6×10 ⁵ hiPSCs were digested into single cells, and transferred to the chip described in (1), centrifuged at 500-2000rpm for 3-5min, The medium used is KSR medium, and 5 μM Y27632 is added;

The basic component of the KSR medium is DMEM/F12, and in addition, 20% of the total volume of KnockOutReplacement (KSR), 1% of the total volume of NEAA (Non Essential Amino Acid, 100×), and 1% of the total volume of GlutaMax ( 100×), 1% penicillin-streptomycin (100×) in total volume, and 0.1 mM beta-mercaptoethanol, 4 ng/ml bFGF.

(3) On the second day, the formed embryoid bodies were transferred to a low-adhesion culture dish, and the embryoid bodies were cultured in suspension, and the medium used was KSR medium.

(4) On the 5th day, the embryoid bodies were induced to differentiate toward the neuroepithelium, and the KSR medium was replaced with the neural induction medium; the cell mass remained in suspension culture. Medium was changed every 3 days.

The basic component of the neural induction medium is DMEM/F12, in addition, 1% N2 (100×) of the total volume, 1% GlutaMAX (100×) of the total volume, and 1% NEAA (Non Essential Amino Acid, 100×), 1 μg/ml heparin, 1% penicillin-streptomycin (100×) in the total volume.

The PDMS chip with a pit-like structure was used to prepare embryoid bodies. The size of the embryoid bodies can be adjusted by changing the volume of the pits and the number of cells in the cell suspension. The size range of the resulting embryoid bodies is about 50-500 μm.

HiPSCs are moderately digested with Accutase, and the digestion time is about 2-5 minutes to ensure that they are finally digested into smaller cell masses. If the digestion is over-digested into scattered single cells, there will be more dead cells in the embryoid body structure, which will affect the subsequent differentiation and development; if the digestion is insufficient, the effect of forming a ball into the embryoid body will be affected, and it will be difficult to form cell clusters of uniform size .

After HiPSCs are digested into relatively small cell blocks with Accutase, subsequent centrifugation is performed to make the cells gather together as much as possible, which is conducive to the formation of relatively large embryoid bodies and effectively improves the success rate of subsequent brain development.

Although embryoid bodies can be formed within a few hours, it is necessary to ensure that the cells are statically cultured in the PDMS chip within 24 hours to ensure the stability of the embryoid body structure.

Step (3) The 3D brain is wrapped in the sodium alginate hollow fiber, specifically:

On the 10th day, use Matrigel to resuspend the pre-induced cell mass, and use a syringe to inject the cell mass into the sodium alginate wire. During the whole process, avoid air bubbles and ensure that the operation on ice is maintained at a low temperature to ensure that the Matrigel does not solidify.

Place the sodium alginate wire containing the cell mass in a 37°C incubator for 20-30min to solidify the Matrigel.

The sodium alginate filaments containing the cell clusters were transferred to a 6-well plate for subsequent culture, and the medium used was neural differentiation medium.

The neural differentiation medium, whose basic components are DMEM/F12 and Neuralbasalmedium with a volume ratio of 1:1, needs to add 1% B27 (50×) of the total volume and 0.5% N2 (100×) of the total volume, accounting for 0.5% NEAA (100×) in total volume, 1% GlutaMAX (100×) in total volume, 1% penicillin-streptomycin (100×) in total volume, 0.05 mM beta-mercaptoethanol.

The development process of 3D brain requires the use of Matrigel, which provides a three-dimensional medium for 3D brain development as an extracellular matrix, which is conducive to the rapid development of 3D brain.

The 3D brain development method derived from high-throughput hiPSCs established by the present invention, its subsequent structural and functional characterization methods are as follows:

(1) Visually monitor the growth rate and developmental state of the 3D brain under microscope bright field conditions;

(2) RT-PCR method to detect the expression of genes in nerves and different brain regions during 3D brain development;

(3) Using frozen section technology, cut the cell mass into 10-20 μm thickness, and perform subsequent immunofluorescence detection of antibodies related to nerve and brain structure;

(4) TUNEL method to detect the life and death of cells in the process of 3D brain development;

(5) Live/Dead assay detects the apoptosis status of scattered single cells;

(6) Ca ²⁺ Imaging was used to detect the Ca ²⁺ activity of nerve cells by live cell workstation to ensure the function of nerve cells. The shooting condition is one photo every 10s, and the continuous shooting is 5 minutes.

The invention provides a method for efficiently realizing brain development derived from hiPSCs using hollow fibers as a carrier. The sodium alginate hollow fibers can be designed with different diameters and tube wall thicknesses according to experimental requirements.

The present invention provides a method for efficiently realizing hiPSCs-derived brain development using hollow filaments as a carrier, which is not only applicable to hiPSCs-derived 3D brains, but also to other organoids derived from different stem cells (including embryonic stem cells). Published organoids Tissues include intestine, stomach, retina, brain, etc.

The invention provides a method for efficiently realizing 3D brain development using sodium alginate silk as a carrier, and can be applied to co-cultivation of other cells and organoids, which is closer to simulating physiological conditions and optimizing the degree of organoid development.

The invention provides a method for efficiently realizing 3D brain development using sodium alginate silk as a carrier, which can be applied to establish various pathological models and study the status of fetal brain development under pathological conditions.

Description of drawings

Figure 1 Preparation of embryoid bodies with PDMS chips with pit-like structures

Fig. 2 The 3D brain development of hiPSCs derived from sodium alginate filaments under the bright field conditions of the present invention;

Figure 3 RT-PCR method to detect gene expression in nerves and different brain regions during 3D brain development.

Detailed ways

The following examples will further illustrate the present invention, but do not limit the present invention thereby.

All reagents of the present invention are commercially available.

Example 1

Development of HiPSC-derived 3D brains within alginate filaments

The steps are mainly divided into three parts: the preparation of the sodium alginate hollow fiber, the pre-induction of the 3D brain and the development of the 3D brain in the sodium alginate hollow fiber.

Step (1) Preparation of sodium alginate hollow fiber, specifically: the preparation of the sodium alginate hollow fiber is to use a microfluidic chip in the form of a sleeve, the inner diameter of the prepared hollow fiber is 600-1000 μm, and the thickness of the tube wall is 50 μm. -200μm, the liquid used is sterile. The sodium alginate silk needs to be soaked in DMEM/F12 medium to complete the swelling process in the medium, and stored at a low temperature of 4°C to facilitate subsequent operations.

Step (2) Pre-induction of 3D brain, specifically: using a PDMS chip with a pit-like structure to prepare an embryoid body: the chip with a pit-like structure is placed in a 24-well plate, the diameter of the pit structure is 800 μm, and the depth is 300 μm. in the formation of embryoid bodies. On day 1, prepare embryoid bodies, digest 2×10 ⁵ -6×10 ⁵ hiPSCs into single cells, and transfer them to the chip described in (1), centrifuge at 1000 rpm for 5 min, and the medium used is KSR medium , and 5 μM Y27632 was added; on the second day, the formed embryoid bodies were transferred to a low-adhesion petri dish, and the embryoid bodies were cultured in suspension, and the medium used was KSR medium.

The basic component of the KSR medium is DMEM/F12, and in addition, 20% of the total volume of KnockOutReplacement (KSR), 1% of the total volume of NEAA (Non Essential Amino Acid, 100×), and 1% of the total volume of GlutaMax ( 100×), 1% penicillin-streptomycin (100×) in total volume, and 0.1 mM beta-mercaptoethanol, 4 ng/ml bFGF.

On day 5, embryoid bodies were induced to differentiate toward the neuroepithelium, and KSR medium was replaced with neural induction medium; cell clusters remained in suspension culture. Medium was changed every 3 days.

The basic component of the neural induction medium is DMEM/F12, in addition, 1% N2 (100×) of the total volume, 1% GlutaMAX (100×) of the total volume, and 1% NEAA (Non Essential Amino Acid , 100×), 1 μg/ml heparin, 1% penicillin-streptomycin (100×) in the total volume.

Step (3) 3D brain development in sodium alginate hollow filaments, specifically: on the 10th day, use Matrigel to resuspend the pre-induced cell clusters, and inject the cell clusters into the sodium alginate hollow filaments with a syringe, avoiding the generation of Air bubbles to ensure that the operation on ice is maintained at a low temperature to ensure that Matrigel does not solidify; place the sodium alginate wire containing cell clusters in a 37°C incubator for 25 minutes to solidify Matrigel; transfer the sodium alginate wire containing cell clusters to a 6-well plate Subsequent culture was carried out, and the medium used was neural differentiation medium.

The neural differentiation medium, whose basic components are DMEM/F12 and Neuralbasalmedium with a volume ratio of 1:1, needs to add 1% B27 (50×) of the total volume and 0.5% N2 (100×) of the total volume, accounting for 0.5% NEAA (100×) in total volume, 1% GlutaMAX (100×) in total volume, 1% penicillin-streptomycin (100×) in total volume, 0.05 mM beta-mercaptoethanol.

The development process of the 3D brain was tracked in bright field, and the results are shown in Figure 1, Scale bar: 500 μm.

Example 2

The method for the development of HiPSC-derived 3D brains in sodium alginate filaments is basically the same as in Example 1, with the difference that

Step (2) Pre-induction of 3D brain, specifically: using a PDMS chip with a pit-like structure to prepare an embryoid body: the chip with a pit-like structure is placed in a 24-well plate, the diameter of the pit structure is 600 μm, and the depth is 200 μm. in the formation of embryoid bodies. On day 1, prepare embryoid bodies, digest 2×10 ⁵ -6×10 ⁵ hiPSCs into single cells, and transfer them to the chip described in (1), centrifuge at 500 rpm for 5 min, and the medium used is KSR medium , and 5 μM Y27632 was added; on the second day, the formed embryoid bodies were transferred to a low-adhesion petri dish, and the embryoid bodies were cultured in suspension, and the medium used was KSR medium.

Step (3) 3D brain development in sodium alginate hollow filaments, specifically: on the 10th day, use Matrigel to resuspend the pre-induced cell clusters, and inject the cell clusters into the sodium alginate hollow filaments with a syringe, avoiding the generation of Air bubbles to ensure that the operation on ice is maintained at a low temperature to ensure that Matrigel does not solidify; place the sodium alginate wire containing cell clusters in a 37°C incubator for 30 minutes to solidify Matrigel; transfer the sodium alginate wire containing cell clusters to a 6-well plate Subsequent culture was carried out, and the medium used was neural differentiation medium.

Example 3

The method for the development of HiPSC-derived 3D brains in sodium alginate filaments is basically the same as in Example 1, with the following differences:

Step (2) 3D brain pre-induction, specifically: using a PDMS chip with a pit-like structure to prepare an embryoid body: the chip with a pit-like structure is placed in a 24-well plate, the diameter of the pit structure is: 700 μm, and the depth is 100 μm. For the formation of embryoid bodies. On day 1, prepare embryoid bodies, digest 2×10 ⁵ -6×10 ⁵ hiPSCs into single cells, and transfer them to the chip described in (1), centrifuge at 2000 rpm for 3 min, and the medium used is KSR medium , and 5 μM Y27632 was added; on the second day, the formed embryoid bodies were transferred to a low-adhesion petri dish, and the embryoid bodies were cultured in suspension, and the medium used was KSR medium.

Step (3) 3D brain development in sodium alginate hollow filaments, specifically: on the 10th day, use Matrigel to resuspend the pre-induced cell clusters, and inject the cell clusters into the sodium alginate hollow filaments with a syringe, avoiding the generation of Air bubbles to ensure that the operation on ice is maintained at a low temperature to ensure that Matrigel does not solidify; place the sodium alginate wire containing cell clusters in a 37°C incubator for 20 minutes to solidify Matrigel; transfer the sodium alginate wire containing cell clusters to a 6-well plate Subsequent culture was carried out, and the medium used was neural differentiation medium.

Example 4

RT-PCR method to detect gene expression in nerves and different brain regions during 3D brain development

The 3D brains of Example 1 at different stages of development were collected, washed once with PBS buffer, and centrifuged. Use the Trizol method to extract total RNA. The method is as follows: resuspend the cells in 1ml Trizol, pipette up and down until there is no cell mass, and the whole solution is clear; add 200μl chloroform, mix up and down for 5min, let stand at room temperature for 3min, and centrifuge at 15000rpm for 15min; Take the upper layer liquid into a new tube, and carefully operate the whole process to avoid contact with the white precipitate in the middle layer; add an equal amount of isopropanol to the absorption liquid, mix it upside down, and let stand at -20°C overnight to facilitate RNA precipitation; centrifuge at 15,000rpm for 10min ; Keep the white RNA precipitate at the bottom of the tube, carefully remove the supernatant; wash the white precipitate with 75% ethanol, centrifuge at 15,000 rpm for 5 minutes; remove 75% ethanol as much as possible, and let it evaporate at room temperature for 5 minutes, so as not to contaminate the sample with ethanol and affect subsequent experiments; dissolve DEPC in water RNA precipitation; measure RNA concentration and purity, and make corresponding dilutions, the final concentration is 500ng/ml.

The second step is to reverse transcribe mRNA into cDNA, the system is 50μl, the specific ratio is as follows: 500ng/ml RNA 10μl, Buffer 10μl, Oligo dT 2.5μl, Random 6mers 2.5μl, Enzyme 2.5μl, DEPC water 27.5μl, 37 ℃ 20min, 4 ℃ preservation.

The third step, PCR, according to the requirements of the kit, the ligand solution, the final system is 20ul, the annealing temperature Tm is unified at 58°C, and stored at 4°C.

Take 2ul of the reaction solution, add loading buffer, water and ethidium bromide, a total of 6ul, marker 4ul, conduct agarose gel electrophoresis, and collect relevant data, as shown in Figure 2. RT-PCR detected the downregulation of stemness-related genes in hiPSCs, while the expression of genes related to neural and different brain regions was upregulated.

Example 5

Expression of related proteins in nerve and brain regions during development

The 3D brains of Example 1 at different stages of development were taken for cryosectioning, and the method was as follows: cells were fixed with 4% paraformaldehyde for 20 minutes, washed three times with PBS buffer for 10 minutes each time; 30% sucrose was dehydrated overnight at 4°C; OCT was embedded at room temperature Store for 30 minutes, solidify at 80°C; cryosections with a thickness of 10-20 μm, and attach them to electrostatically adsorbed glass slides. Immunofluorescence staining was then carried out. The method was as follows: soak the slides with sections in PBS buffer for 5 minutes; act with 0.1% triton X-100 porogen for 10 minutes, rinse once with PBS buffer for 5 minutes; goat blocking serum Effect at room temperature for 1 hour, primary antibody (PAX6, PAX2, NESTIN, TUJ1, SOX2, TBR1, CTIP2) diluted 1:100 or 1:400, incubated overnight at 4°C, washed once with PBS buffer, 5min; secondary antibody (Fluorescence488/594 Labeled goat anti-rabbit or mouse IgG) diluted 1:100, incubated at room temperature for 1 hour, washed once with PBS buffer for 5 minutes; after washing, added 1:2000 diluted DAPI working solution, photographed under a fluorescent microscope, and recorded the expression of the corresponding protein situation, the results are shown in Figure 3. Proteins related to nerves and different brain regions showed high expression, indicating that hiPSCs could successfully develop into 3D brains in this system.

Claims (8)

1. A kind of 1. method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, it is characterised in that：This method mainly walks Suddenly it is：

   (1) preparation of sodium alginate macaroni yarn；

   (2) 3D brains induction early period；

   (3) development of the 3D brains in sodium alginate macaroni yarn.
2. 2. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 1 It is：The preparation of step (1) sodium alginate macaroni yarn, is specially：

   (1) the sodium alginate silk with hollow structure being prepared using the micro-fluidic chip of barrel forms, internal diameter is 600-1000 μm, Pipe thickness is 50-200 μm, liquid asepsis processing used；

   (2) the sodium alginate silk prepared need to be immersed in DMEM/F12 culture mediums, make its swelling process of completion in the medium, And in 4 DEG C of Cord bloods, easy to subsequent operation.
3. 3. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 2 Being that sodium alginate silk prepares the instrument that is related to and solution all must be sterile, and corresponding disinfecting action can be carried out before experiment, is had Body is autoclave sterilization, UV irradiates or 0.2 μm of aperture membrane filtration.
4. 4. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 1 It is：Step (2) 3D brain inductions early period, are specially：

   (1) embryoid body is prepared using the PDMS chips with hole shape structure：The chip of hole shape structure is placed in 24 orifice plates, pitting knot A diameter of 600-800 μm of structure, depth are 100-300 μm, the formation for embryoid body；

   (2) the 1st days, embryoid body is prepared, by 2 × 10⁵-6×10⁵A hiPSCs is digested to unicellular, and is transferred to described in (1) In chip, 500-2000rpm is centrifuged, 3-5min, used medium is KSR culture mediums, and adds 5 μM of Y27632；

   (3) the 2nd days, the embryoid body of formation is transferred in the culture dish of low adhesion, suspend culture embryoid body, and used medium is KSR culture mediums；

   (4) the 5th days, induction embryoid body broke up to neural epithelium direction, and KSR culture mediums are replaced with Neuronal induction media.
5. 5. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 4 It is to prepare embryoid body using the PDMS chips of hole shape structure, change and cell of its big I by pitting diameter and depth The quantity of cell is adjusted in suspension, into the magnitude range of embryoid body be about 50-500 μm.
6. 6. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 4 The cell derived for being hiPSCs is transformed by human skin fibroblasts virus infection.
7. 7. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 1 It is：Development of step (3) the 3D brains in sodium alginate macaroni yarn, is specially：

   (1) the 10th day, the cell mass induced early period is resuspended using Matrigel, and cell mass is injected into sodium alginate with syringe In macaroni yarn, whole process avoids producing bubble, it is ensured that operation maintains low temperature on ice, ensures that Matrigel does not solidify；

   (2) the sodium alginate silk containing cell mass is placed in 37 DEG C of incubator 20-30min, solidifies Matrigel；

   (3) the sodium alginate silk containing cell mass is transferred in 6 orifice plates and carries out follow-up cultivation, used medium is Neural Differentiation Culture medium.
8. 8. according to a kind of method for the brain growth that hiPSCs sources are realized using macaroni yarn as carrier, its feature described in claim 7 It is that the Matrigel concentration is 2-10mg/ml.