An iPSC-induced neural stem cell injection preparation, preparation method and application thereof

Abstract

The application discloses an iPSC induced neural stem cell injection preparation, a preparation method and application, and belongs to the technical field of biological preparations, and comprises iPSC induced neural stem cells, nutrition factors and micro scaffolds; wherein calcium chloride and nano clay are dissolved in a deionized water solution with a pH of 7-8, and then poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) is added, and a mixed solution is obtained by reacting under the condition of stirring at room temperature for 6-12 hours; the mixed solution is subjected to vacuum freeze drying, and after being ground, is added into deionized water with a pH of 6-7 adjusted by acetic acid to obtain a reaction crosslinking system; laminin is added into the reaction system, and is stirred at low temperature until being uniformly mixed, and vacuum freeze drying is conducted to obtain the micro scaffold. The application can greatly improve the survival rate of neural stem cells by designing the micro scaffold.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to an iPSC-induced neural stem cell injection formulation, its preparation method, and its application. Background Technology

[0002] Damage to the central and peripheral nervous systems is a major cause of long-term disability, commonly seen in trauma (such as spinal cord injury and head injury), stroke, neurodegenerative diseases (such as Parkinson's disease, Alzheimer's disease, and amyotrophic lateral sclerosis), and certain iatrogenic injuries. The inherent regenerative capacity of neurons in the central nervous system is extremely limited. Injury is often accompanied by glial scarring, inflammatory responses, a lack of neurotrophic factors, and an inhibitory microenvironment, severely hindering axonal regeneration and neural circuit reconstruction. While the peripheral nervous system possesses some regenerative capacity, the regeneration rate is slow, the distance is limited, and functional recovery is often incomplete, especially with long-distance defects or proximal injuries. Nerve injury leading to motor and / or sensory loss, chronic pain, and autonomic dysfunction places a heavy burden on the patient's quality of life, their family, and society.

[0003] While neural stem cell transplantation is an effective treatment, it still has several drawbacks: direct injection of neural stem cells results in low survival rates, typically <80% after 48 hours, and potentially <20% over time. The lack of protection in the injected formulation makes neural stem cells susceptible to mechanical damage and microenvironmental stress, further reducing survival. Traditional hydrogel-embedded neural stem cell injections suffer from limited diffusion and inefficient nutrient / metabolic waste exchange, leading to central cell necrosis. Furthermore, existing stem cell formulations generally lack biomimetic structural support, causing neural stem cells to lose 3D microenvironment contact signals, disrupting subsequent differentiation.

[0004] Therefore, how to provide an iPSC-induced neural stem cell injection formulation to improve the survival rate of neural stem cells is a technical problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0005] Therefore, the present invention provides an iPSC-induced neural stem cell injection formulation, its preparation method, and its application to solve the related technical problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] According to a first aspect of the present invention, an iPSC-induced neural stem cell injection formulation is provided, comprising iPSC-induced neural stem cells, trophic factors, and microscaffolds;

[0008] The concentration of iPSC-induced neural stem cells was 10. 6-10 7 cells / mL;

[0009] Among them, the nutritional factors include 10-20 ng / mL EGF, 10-20 ng / mL glial cell-derived neurotrophic factor, 0.4-0.6 mg / mL human serum albumin and 40-60 mg / mL glucose;

[0010] The concentration of the microscaffold added was 10-20 mg / mL.

[0011] Furthermore, the microscaffold comprises the following proportions by weight:

[0012] 7-10 parts calcium chloride

[0013] Poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) 1-1.5 parts

[0014] 1-5 parts nano clay

[0015] 1-3 parts laminin.

[0016] Furthermore, the concentration of iPSC-induced neural stem cells is 10. 6 per mL.

[0017] Furthermore, the concentration of the added microscaffold is 18 mg / mL.

[0018] Furthermore, the concentration of EGF was 16 ng / mL, the concentration of glial cell-derived neurotrophic factor was 13 ng / mL, the concentration of human serum albumin was 0.5 mg / mL, and the concentration of glucose was 55 mg / mL.

[0019] According to a second aspect of the present invention, a method for preparing an iPSC-induced neural stem cell injection formulation is provided, comprising the following steps:

[0020] Weigh out the microscaffolds according to the proportions, and add EGF, glial cell-derived neurotrophic factor, human serum albumin and glucose from the nutrient factors into the microscaffolds in sequence.

[0021] iPSC-induced neural stem cells were added to the mixture of the above-mentioned nutrient factors and microscaffolds;

[0022] Stir at low temperature until homogeneous to obtain the iPSC-induced neural stem cell injection formulation.

[0023] Furthermore, the microscaffold is prepared as follows:

[0024] Dissolve 7-10 parts calcium chloride and 1-5 parts nano clay in a deionized water solution with a pH of 7-8, and then add 1-1.5 parts poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) and react at room temperature with stirring for 6-12 hours to obtain a mixed solution.

[0025] The above mixed solution was vacuum freeze-dried, ground, and then added to deionized water with pH adjusted to 6-7 by acetic acid to obtain a reaction crosslinking system;

[0026] Add 1-3 parts of laminin to the reaction system, stir at low temperature until uniformly mixed, and freeze-dry under vacuum to obtain microscaffolds.

[0027] Furthermore, the molecular weight of the poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) is 10-20 kDa.

[0028] Furthermore, the method for inducing neural stem cells with iPSCs is as follows:

[0029] Coating: Human pluripotent stem cells were coated in 6-well plates with matrix gel and placed in a 37°C constant temperature carbon dioxide cell culture incubator.

[0030] Cell seeding: Add iPSCs resuspended in iPSC-maintaining medium to the coated well plates so that the cell density can reach 20-40% the next day;

[0031] Washing: Aspirate the supernatant from the culture dish and wash the culture dish with DPBS that does not contain calcium and magnesium ions;

[0032] Neural stem cell induction differentiation: Discard the supernatant and add 2 mL of warmed iPSC neural induction medium to each well. Place the cells in a 5% CO2, 37°C constant temperature carbon dioxide cell culture incubator and record this as Day 0. During the subsequent differentiation process, replace the medium with fresh iPSC neural induction medium that has been restored to room temperature every day and continue differentiation until Day 12-16. After observing a significant rosette, replace the medium with iNPCs Neurobasal™ medium and passage the cells directly or in suspension, or pick rosettes for passage in either form or in suspension.

[0033] According to a third aspect of the present invention, the use of an iPSC-induced neural stem cell injection formulation in the preparation of a neurotrophic drug is provided.

[0034] The present invention has the following advantages:

[0035] Revolutionary improvement in survival rate:

[0036] A biomimetic 3D microenvironment suitable for encapsulating and fixing neural stem cells was constructed using a microscaffold formed from calcium ions, nanoclay, PLA-PEG-PLA, and laminin, which can significantly reduce mechanical damage during injection. Most importantly, due to the structural design of the microscaffold, the survival rate of neural stem cells can be greatly improved, overcoming the bottleneck in transplanted stem cell survival.

[0037] The porous temperature-sensitive gel network constructed from PLA-PEG-PLA and calcium ions can enter the microscaffold formed by calcium ions under low in vitro conditions. After injection, it rapidly gels at body temperature to protect stem cells, ensuring that stem cells can achieve a protective effect both in vitro and in vivo, and improving the problem of central cell necrosis in traditional hydrogels.

[0038] Laminin can not only serve as a microscaffold structure for the adsorption and fixation of stem cells, but also mimic the structure of the neural basement membrane. Combined with glial cell-derived neurotrophic factors GDNF and EGF, it can maintain the stemness of stem cells and guide neural differentiation in a directional manner, thereby reducing the rate of differentiation disorder. Attached Figure Description

[0039] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0040] The structures, proportions, sizes, etc. illustrated in this specification are only for the purpose of assisting those skilled in the art in understanding and reading the content disclosed herein, and are not intended to limit the conditions under which the present invention can be implemented. Therefore, they have no substantial technical significance. Any modifications to the structure, changes in the proportions, or adjustments to the size, without affecting the effects and objectives that the present invention can produce, should still fall within the scope of the technical content disclosed in the present invention.

[0041] Figure 1 Microscopic image below of human pluripotent stem cells (iPSCs) provided for this invention;

[0042] Figure 2 Microscopic image of neural stem cells (NSCs) provided for this invention. Detailed Implementation

[0043] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0044] In existing technologies, neural stem cell preparations have been widely used to treat nerve injuries. However, the low survival rate of stem cells remains a problem, leading to poor treatment effects and prolonged treatment courses, thus hindering further promotion. This invention provides an iPSC-induced neural stem cell injection preparation, its preparation method, and its application to address the related technical problems existing in the prior art.

[0045] According to a first aspect of the present invention, an iPSC-induced neural stem cell injection formulation is provided, comprising iPSC-induced neural stem cells, trophic factors, and microscaffolds;

[0046] The concentration of iPSC-induced neural stem cells was 10. 6 -10 7 cells / mL;

[0047] Among them, the nutritional factors include 10-20 ng / mL EGF, 10-20 ng / mL glial cell-derived neurotrophic factor, 0.4-0.6 mg / mL human serum albumin and 40-60 mg / mL glucose;

[0048] The concentration of the microscaffold added was 10-20 mg / mL.

[0049] Furthermore, the microscaffold comprises the following proportions by weight:

[0050] 7-10 parts calcium chloride

[0051] Poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) 1-1.5 parts

[0052] 1-5 parts nano clay

[0053] 1-3 parts laminin.

[0054] A biomimetic 3D microenvironment suitable for encapsulating and immobilizing neural stem cells was constructed using a microscaffold formed from calcium ions, nanoclay, PLA-PEG-PLA, and laminin, significantly reducing mechanical damage during injection. Most importantly, the microscaffold's structural design greatly improves the survival rate of neural stem cells, overcoming the bottleneck in transplanted stem cell survival. The porous temperature-sensitive gel network constructed from PLA-PEG-PLA and calcium ions can enter the calcium ion-formed microscaffold under low-temperature conditions in vitro, and rapidly gel at body temperature after injection to protect stem cells, ensuring protective effects both in vitro and in vivo, and improving the central cell necrosis problem of traditional hydrogels.

[0055] Furthermore, the concentration of iPSC-induced neural stem cells is 10. 6 per mL.

[0056] Furthermore, the concentration of the added microscaffold is 18 mg / mL.

[0057] Furthermore, the concentration of EGF was 16 ng / mL, the concentration of glial cell-derived neurotrophic factor was 13 ng / mL, the concentration of human serum albumin was 0.5 mg / mL, and the concentration of glucose was 55 mg / mL.

[0058] Laminin can not only serve as a microscaffold structure for the adsorption and fixation of stem cells, but also mimic the structure of the neural basement membrane. Combined with glial cell-derived neurotrophic factors GDNF and EGF, it can maintain the stemness of stem cells and guide neural differentiation in a directional manner, thereby reducing the rate of differentiation disorder.

[0059] According to a second aspect of the present invention, a method for preparing an iPSC-induced neural stem cell injection formulation is provided, comprising the following steps:

[0060] Weigh out the microscaffolds according to the proportions, and add EGF, glial cell-derived neurotrophic factor, human serum albumin and glucose from the nutrient factors into the microscaffolds in sequence.

[0061] iPSC-induced neural stem cells were added to the mixture of the above-mentioned nutrient factors and microscaffolds;

[0062] Stir at low temperature until homogeneous to obtain the iPSC-induced neural stem cell injection formulation.

[0063] Furthermore, the fabrication method of the microscaffold is as follows:

[0064] Dissolve 7-10 parts calcium chloride and 1-5 parts nano clay in a deionized water solution with a pH of 7-8, and then add 1-1.5 parts poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) and react at room temperature with stirring for 6-12 hours to obtain a mixed solution.

[0065] The above mixed solution was vacuum freeze-dried, ground, and then added to deionized water with pH adjusted to 6-7 by acetic acid to obtain a reaction crosslinking system;

[0066] Add 1-3 parts of laminin to the reaction system, stir at low temperature until uniformly mixed, and freeze-dry under vacuum to obtain microscaffolds.

[0067] The molecular weight of poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) is 10-20 kDa.

[0068] Furthermore, the method for inducing neural stem cells with iPSCs is as follows:

[0069] Coating: Human pluripotent stem cells were coated in 6-well plates with matrix gel and placed in a 37°C constant temperature carbon dioxide cell culture incubator.

[0070] Cell seeding: Add iPSCs resuspended in iPSC-maintaining medium to the coated well plates so that the cell density can reach 20-40% the next day;

[0071] Washing: Aspirate the supernatant from the culture dish and wash the culture dish with DPBS that does not contain calcium and magnesium ions;

[0072] Neural stem cell induction differentiation: Discard the supernatant and add 2 mL of warmed iPSC neural induction medium to each well. Place the cells in a 5% CO2, 37°C constant temperature carbon dioxide cell culture incubator and record this as Day 0. During the subsequent differentiation process, replace the medium with fresh iPSC neural induction medium that has been restored to room temperature every day and continue differentiation until Day 12-16. After observing a significant rosette, replace the medium with iNPCs Neurobasal™ medium and passage the cells directly or in suspension, or pick rosettes for passage in either form or in suspension.

[0073] According to a third aspect of the present invention, the use of an iPSC-induced neural stem cell injection formulation in the preparation of a neurotrophic drug is provided.

[0074] To better illustrate the technical effects of this application, the following embodiments are provided.

[0075] Preparation Example 1

[0076] The experimental steps for inducing pluripotent stem cells into neural stem cells are as follows:

[0077] I. Differentiation from human pluripotent stem cells (iPSCs) to neural stem cells (NSCs) (using a six-well plate as an example)

[0078] 1. Coating: Human pluripotent stem cells are sourced from Guangzhou Ruizhen Regenerative Medicine Technology Co., Ltd., such as... Figure 1As shown, Matrigel (using Bispetech in our experiments) was added to the 6-well plates and placed in a 37°C constant temperature carbon dioxide cell culture incubator for coating (100×Matrigel coating for 30 min, 200×Matrigel coating for 2 h).

[0079] 2. Cell seeding: Add iPSCs to the coated well plate, maintaining the medium resuspended with iPSCs, so that the density can reach 20-40% on the second day.

[0080] 3. Cleaning: Aspirate the supernatant from the culture dish and clean the culture dish with DPBS that does not contain calcium and magnesium ions.

[0081] 4. Neural stem cell induction differentiation: Discard the supernatant and add 2 mL of warmed iPSC Neural Induction Medium to each well (our experiment uses STEMdiff™ Neural Induction Medium / STEMdiff™ Neural Induction Medium). Incubate the cells in a 5% CO2, 37°C constant temperature carbon dioxide cell culture incubator. This is recorded as Day 0. During the subsequent differentiation process, replace the medium daily with fresh iPSC Neural Induction Medium that has been brought to room temperature (adjust the volume of medium added according to cell proliferation). Continue differentiation until Day 12-16. Figure 2 As shown, after observing significant rosettes during the period, the culture medium was replaced with iNPCs Maintenance Medium (Neurobasal™ medium was used in our experiments), and the culture was directly cultured or suspended (digested with Acutase), or the rosettes were picked for cultured or suspended.

[0082] II. Suspension passage amplification of NPCs (taking T25 bottles as an example)

[0083] 1. Choose a rose wreath (optional).

[0084] 2. Digestion: After the neural rosettes differentiate, discard the culture medium, wash the plate twice with DPBS without calcium and magnesium ions, add 1.5 mL of preheated cell digestion solution (Accutase) to the plate, and digest at 37°C for 5 min until most cells separate from the surface of the culture container. Gently pipette to remove any remaining cells.

[0085] 3. Washing: Transfer the cell suspension to a centrifuge tube and add 10 times the volume of DMEM / F12 medium. Mix well and pipette three times. Centrifuge at 300×g for 5 min. Discard the supernatant, add DMEM / F12 medium for washing, centrifuge at 300×g for 5 min, and discard the supernatant. Repeat the washing process twice.

[0086] 4. Seeding: The cell pellet is resuspended in preheated iNPCs Maintenance Medium and seeded into T25 vials with ultra-low adhesion or no coating.

[0087] 5. Culture: Incubate the cells in a 5% CO2, 37°C incubator for 2 days. During this period, the cells will float in the culture dish and form aggregates.

[0088] 6. Filtration (optional): When the aggregates reach a size of approximately 70-200 μm, use a pipette to aspirate all the culture medium from the culture dish and pass it through a cell filter (40 μM) to collect the NPC aggregates.

[0089] 7. Digestion: Carefully aspirate the old culture medium from the culture flask or dish using a pipette. Add an appropriate amount of calcium- and magnesium-free DPBS. Gently agitate the culture flask to thoroughly rinse the surface of the cell spheroids with the DPBS buffer to remove residual culture medium and metabolic products. After rinsing, aspirate the DPBS buffer again. Then, add an appropriate amount of pre-warmed cell digestion buffer (Accutase) to the culture flask, ensuring that the digestive enzyme completely covers the cell spheroids. Place the culture flask in a cell culture incubator and incubate for 3-5 minutes. During this time, you can remove the flask and observe the digestion of the cell spheroids under a microscope. When the cell spheroids begin to loosen and cells at the edges begin to detach from the spheroids, the digestion is appropriate.

[0090] 8. Termination of Digestion: After digestion, quickly add 10 times the volume of DMEM / F12 medium to the culture flask and mix well. Gently pipette the cell suspension repeatedly to completely disperse the cell spheres into single cells or small cell clusters. Be careful to use moderate force when pipetting to avoid generating too many air bubbles and causing mechanical damage to the cells. Generally, pipette about 10-20 times until the cell spheres in the cell suspension are completely dispersed, then centrifuge at 300×g for 5 min. After discarding the supernatant, add DMEM / F12 medium for washing, centrifuge at 300×g for 5 min, and discard the supernatant again. Repeat this washing process twice.

[0091] 9. Passage or cryopreservation: Passage or cryopreservation shall be carried out as needed for the experiment. Passage shall be carried out by suspension expansion of neural stem and progenitor cells in accordance with the above steps. The cryopreservation solution shall be prepared in the ratio of 8:2 = FBS:DMSO for cryopreservation.

[0092] III. Flow cytometry detection (at 1×10⁻⁶) 6 (Taking cells as an example)

[0093] 1. Digestion and washing: Same as "Step 2" above.

[0094] Additional procedure: Cell counting, take 1×10⁻⁶ cells. 6 The cells were then processed in 1.5 mL centrifuge tubes.

[0095] 2. Fixation: Add 250 μL of cell fixative to the cell pellet, mix immediately, and fix at 4°C for 30 min.

[0096] 3. Washing: Add 1 ml of PBS to the centrifuge tube and mix well. Centrifuge at 400×g for 5 min.

[0097] 4. Permeabilization: Add 200 μL of pre-cooled cell permeabilization solution to the cell pellet, mix immediately, and place in a 4°C refrigerator for permeabilization for 25-30 min.

[0098] Add the ingredients one tube at a time, mixing them thoroughly immediately after each addition, and be gentle in your movements.

[0099] 5. Washing: Add 1 ml of PBS to the centrifuge tube, mix well, centrifuge at 400×g for 5 min, and wash twice.

[0100] 6. Antibody incubation: Add 100 μL of "PBS + 1% FBS" buffer to the precipitate, and add the corresponding antibody and ISO according to the instructions. Incubate at 4°C or on ice in the dark for 30 min.

[0101] 7. Sample loading: Wash once with 1 mL PBS, resuspend the precipitate with 100-200 μL PBS, and then perform analysis.

[0102] iPSC-induced neural stem cells were prepared through the above process to prepare for subsequent experiments.

[0103] Preparation Example 2

[0104] The fabrication method of the microscaffold is as follows:

[0105] Prepare 500 mL of deionized water and adjust the pH of the deionized water to 7-8 with sodium hydroxide aqueous solution. Weigh 40 g of calcium chloride and 20 g of nano clay (montmorillonite is used in this preparation example) and dissolve them in the above deionized water. Stir at room temperature for 10-15 min until well mixed.

[0106] Subsequently, 7.5g of poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) was weighed out and stirred at 100 rpm at room temperature until well mixed. The reaction was carried out for 8 hours to obtain a mixed solution, wherein the molecular weight of poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) was 10-20kDa.

[0107] The above mixed solution was freeze-dried at -25°C, ground, and then added to deionized water with pH adjusted to 6-7 by acetic acid to obtain a reaction crosslinking system.

[0108] Add 10g of laminin to the reaction system, stir at 5-10℃ until homogeneous, and freeze-dry under vacuum at -25℃ to obtain the microscaffold. If the microscaffold exhibits clumping, it needs to be ground.

[0109] Example 1

[0110] This embodiment provides a method for preparing an iPSC-induced neural stem cell injection formulation, using the neural stem cells prepared in Example 1 and the microscaffold prepared in Example 2 as described above, including the following steps:

[0111] Prepare deionized water and adjust the pH to 6-7. The preparation temperature is 10-15℃. Weigh the microscaffold according to the ratio and add it to the above deionized water to make the final concentration 11mg / mL. Then, add the following nutrients to the above deionized water in sequence: EGF (final concentration 10ng / mL), glial cell-derived neurotrophic factor (final concentration 20ng / mL), human serum albumin (final concentration 0.4mg / mL), and glucose (final concentration 45mg / mL) to work with the microscaffold.

[0112] The final concentration of iPSC-induced neural stem cells was 10. 6 The iPSC-induced neural stem cell injection formulation was obtained by adding 1 / mL of the above-mentioned nutrient factor and microscaffold mixture and stirring at low temperature until homogeneous.

[0113] Example 2

[0114] This embodiment provides a method for preparing an iPSC-induced neural stem cell injection formulation, using the neural stem cells prepared in Example 1 and the microscaffold prepared in Example 2 as described above, including the following steps:

[0115] Prepare deionized water and adjust the pH to 6-7. The preparation temperature is 10-15℃. Weigh the microscaffold according to the ratio and add it to the above deionized water to make a final concentration of 15mg / mL. Then, add the following nutrients to the above deionized water in sequence: EGF (final concentration 20ng / mL), glial cell-derived neurotrophic factor (final concentration 15ng / mL), human serum albumin (final concentration 0.5mg / mL), and glucose (final concentration 40mg / mL) to work with the microscaffold.

[0116] The final concentration of iPSC-induced neural stem cells was 10. 6 The iPSC-induced neural stem cell injection formulation was obtained by adding 1 / mL of the above-mentioned nutrient factor and microscaffold mixture and stirring at low temperature until homogeneous.

[0117] Example 3

[0118] This embodiment provides a method for preparing an iPSC-induced neural stem cell injection formulation, using the neural stem cells prepared in Example 1 and the microscaffold prepared in Example 2 as described above, including the following steps:

[0119] Prepare deionized water and adjust the pH to 6-7. The preparation temperature is 10-15℃. Weigh the microscaffold according to the ratio and add it to the above deionized water to make the final concentration 18mg / mL. Then, add the following nutrients to the above deionized water in sequence: EGF (final concentration 16ng / mL), glial cell-derived neurotrophic factor (final concentration 13ng / mL), human serum albumin (final concentration 0.5mg / mL), and glucose (final concentration 55mg / mL) to work with the microscaffold.

[0120] The final concentration of iPSC-induced neural stem cells was 10. 6 The iPSC-induced neural stem cell injection formulation was obtained by adding 1 / mL of the above-mentioned nutrient factor and microscaffold mixture and stirring at low temperature until homogeneous.

[0121] Example 4

[0122] This embodiment provides a method for preparing an iPSC-induced neural stem cell injection formulation, using the neural stem cells prepared in Example 1 and the microscaffold prepared in Example 2 as described above, including the following steps:

[0123] Prepare deionized water and adjust the pH to 6-7. The preparation temperature is 10-15℃. Weigh the microscaffold according to the ratio and add it to the above deionized water to make the final concentration 16mg / mL. Then, add the following nutrients to the above deionized water in sequence: EGF (final concentration 15ng / mL), glial cell-derived neurotrophic factor (final concentration 17ng / mL), human serum albumin (final concentration 0.5mg / mL), and glucose (final concentration 60mg / mL) to work with the microscaffold.

[0124] The final concentration of iPSC-induced neural stem cells was 10. 6 The iPSC-induced neural stem cell injection formulation was obtained by adding 1 / mL of the above-mentioned nutrient factor and microscaffold mixture and stirring at low temperature until homogeneous.

[0125] Example 5

[0126] This embodiment provides a method for preparing an iPSC-induced neural stem cell injection formulation, using the neural stem cells prepared in Example 1 and the microscaffold prepared in Example 2 as described above, including the following steps:

[0127] Prepare deionized water and adjust the pH to 6-7. The preparation temperature is 10-15℃. Weigh the microscaffold according to the ratio and add it to the above deionized water to make the final concentration 13mg / mL. Then, add the following nutrients to the above deionized water in sequence: EGF (final concentration 18ng / mL), glial cell-derived neurotrophic factor (final concentration 11ng / mL), human serum albumin (final concentration 0.6mg / mL), and glucose (final concentration 50mg / mL) to work with the microscaffold.

[0128] The final concentration of iPSC-induced neural stem cells was 10. 6 The iPSC-induced neural stem cell injection formulation was obtained by adding 1 / mL of the above-mentioned nutrient factor and microscaffold mixture and stirring at low temperature until homogeneous.

[0129] Comparative Example 1

[0130] The preparation method of the iPSC-induced neural stem cell injection formulation in Comparative Example 1 is the same as that in Example 3, but the microscaffold from Preparation Example 2 is not added.

[0131] Comparative Example 2

[0132] The preparation method of the iPSC-induced neural stem cell injection formulation in Comparative Example 1 is the same as that in Example 3, except that the microscaffold from Preparation Example 2 is added, wherein poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) is not added in the preparation of the microscaffold.

[0133] Comparative Example 3

[0134] The preparation method of the iPSC-induced neural stem cell injection formulation in Comparative Example 1 is the same as that in Example 3, except that the microscaffold from Preparation Example 2 is added, wherein no nano-clay is added during the preparation of the microscaffold.

[0135] Comparative Example 4

[0136] The preparation method of the iPSC-induced neural stem cell injection formulation in Comparative Example 1 is the same as that in Example 3, except that the microscaffold from Preparation Example 2 is added, wherein no calcium ions are added during the preparation of the microscaffold.

[0137] Comparative Example 5

[0138] The preparation method of the iPSC-induced neural stem cell injection formulation in Comparative Example 1 is the same as that in Example 3, except that the microscaffold from Preparation Example 2 is added, wherein laminin is not added during the preparation of the microscaffold.

[0139] Detection example

[0140] Dissociation treatment: Take 500 μL of injection formulation, add 500 μL of dissociation solution (0.1% BSA-PBS buffer containing 0.05% collagenase IV, 0.1% hyaluronidase and 2 mM EDTA, pH 7.4), and incubate at 37°C with shaking for 25 min;

[0141] To terminate the reaction: Add 5 mL of DMEM / F12 ice-pre-cooled termination solution containing 10% FBS, centrifuge at 400×g for 5 min (4℃), and discard the supernatant;

[0142] Calcium ion treatment: Add 2 mL of calcium chelation buffer (5 mM EDTA-PBS) to the precipitate, incubate on ice for 10 minutes, centrifuge at 400×g for 5 minutes, discard the supernatant, add 2 mL of membrane repair buffer (2 mM CaCl2 and 1 mM MgCl2-PBS), and incubate at 37°C for 5 minutes.

[0143] Cell recovery: Centrifuge at 400×g for 5 min, discard the supernatant, resuspend the cells in 1 mL of 0.1% BSA-PBS, filter through a 40 μm nylon filter, and collect the cell suspension;

[0144] Flow cytometry was performed using the detection steps described in Preparation Example 1 to detect the survival rate of neural stem cells in the injected formulation at 12h, 24h, and 48h, respectively. The results are shown in the table below:

[0145]

[0146] Note: Data are the mean of three independent experiments ± SD (n=9)

[0147] In summary, compared with Comparative Example 1, Example 3 of this application shows that the calcium ion cross-linking layer can resist injection shear force, which increases the survival rate by 32%. At the same time, PLA-PEG-PLA thermogelation forms a physical barrier to protect neural stem cells, and the nano-clay porous network ensures nutrient / waste exchange.

[0148] As shown in Comparative Examples 2-5, the absence of any component in the microscaffold resulted in a >23% decrease in 48-hour survival rate. This indicates that the four components together form the excellent structure of the microscaffold, improving the encapsulation and fixation efficiency of stem cells, thereby increasing survival rate. In particular, Example 3 provides the optimal ratio of stem cells, microscaffold, and nutrient factors.

[0149] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. An iPSC-induced neural stem cell injection formulation, characterized in that, Including iPSC-induced neural stem cells, nutritional factors, and microscaffolds; The concentration of iPSC-induced neural stem cells was 10. 6 -10 7 cells / mL; Among them, the nutritional factors include 10-20 ng / mL epidermal growth factor (EGF), 10-20 ng / mL glial cell-derived neurotrophic factor (GGF), 0.4-0.6 mg / mL human serum albumin, and 40-60 mg / mL glucose. The concentration of the microscaffold added was 10-20 mg / mL; The microscaffold is prepared as follows: 7-10 parts by weight of calcium chloride and 1-5 parts by weight of nano-clay were dissolved in a deionized water solution with a pH of 7-8. Then, 1-1.5 parts by weight of poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) were added and reacted at room temperature with stirring for 6-12 hours to obtain a mixed solution. The above mixed solution was vacuum freeze-dried, ground, and then added to deionized water with pH adjusted to 6-7 by acetic acid to obtain a reaction crosslinking system; Add 1-3 parts by weight of laminin to the reaction crosslinking system, stir at low temperature until uniformly mixed, and freeze-dry under vacuum to obtain microscaffolds.

2. The iPSC-induced neural stem cell injection formulation as described in claim 1, characterized in that, in, The concentration of iPSC-induced neural stem cells was 10. 6 per mL.

3. The iPSC-induced neural stem cell injection formulation as described in claim 1, characterized in that, in, The concentration of the microscaffold added was 18 mg / mL.

4. The iPSC-induced neural stem cell injection formulation as described in claim 1, characterized in that, in, The concentrations of EGF, glial cell-derived neurotrophic factor, human serum albumin, and glucose were 16 ng / mL, 13 ng / mL, 0.5 mg / mL, and 55 mg / mL, respectively.

5. A method for preparing the iPSC-induced neural stem cell injection formulation as described in claim 1, characterized in that, Includes the following steps: Weigh out the microscaffold according to the proportion and add it to deionized water. Then add EGF, glial cell-derived neurotrophic factor, human serum albumin and glucose from the nutrient factors to the deionized water in sequence to work with the microscaffold. iPSC-induced neural stem cells were added to the mixture of the above-mentioned nutrient factors and microscaffolds; Stir at low temperature until homogeneous to obtain the iPSC-induced neural stem cell injection formulation.

6. The preparation method according to claim 5, characterized in that, The molecular weight of the poly(D,L-lactide)-poly(ethylene glycol)-poly(D,L-lactide) is 10-20 kDa.

7. The preparation method according to claim 5, characterized in that, The method for preparing iPSC-induced neural stem cells is as follows: Coating: Add matrix gel to 6-well plates and place them in a 37°C constant temperature carbon dioxide cell culture incubator for coating; Cell seeding: Add iPSCs resuspended in iPSC-maintaining medium to the coated well plates so that the cell density can reach 20-40% the next day; Washing: Aspirate the supernatant from the culture plate and wash the culture plate with DPBS that does not contain calcium and magnesium ions; Neural stem cell induction differentiation: Discard the supernatant, add 2 mL of warmed iPSC neural induction medium to each well, and place the cells in a 5% CO2, 37°C constant temperature carbon dioxide cell culture incubator. This point is recorded as Day 0. During the subsequent differentiation process, replace the medium with fresh iPSC neural induction medium that has been restored to room temperature every day. Continue differentiation until Day 12-16. After observing a significant rosette, replace the medium with induced neural stem cell Neurobasal™ medium and passage the cells directly or in suspension, or pick rosettes for passage in either form or in suspension.

8. The use of the iPSC-induced neural stem cell injection formulation according to any one of claims 1-4 in the preparation of a drug for repairing nerve damage.

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