CN115873796A

CN115873796A - Nerve cell combination and application thereof

Info

Publication number: CN115873796A
Application number: CN202211191890.XA
Authority: CN
Inventors: 周琪; 李伟; 郝捷; 王昱凯; 胡宝洋; 冯琳; 梁灵敏; 王柳
Original assignee: Beijing Institute Of Stem Cell And Regenerative Medicine; Institute of Zoology of CAS
Current assignee: Beijing Institute Of Stem Cell And Regenerative Medicine; Institute of Zoology of CAS
Priority date: 2021-09-29
Filing date: 2022-09-28
Publication date: 2023-03-31
Anticipated expiration: 2042-09-28
Also published as: CN115873796B

Abstract

The invention relates to a nerve cell combination and application thereof, in particular to a nerve cell sphere, a preparation thereof and application thereof, and more particularly provides a nerve cell sphere which comprises a plurality of nerve-related cells, wherein the nerve-related cells comprise nerve precursor cells, nerve cells, glial cells or any combination thereof, and the diameter of the nerve cell sphere is 30-500 mu m. Compared with single cell suspension, the survival rate of the nerve-related cells can be obviously improved by suspending the nerve-related cells into cell pellets.

Description

Nerve cell combination and application thereof

Technical Field

The invention relates to the field of biology, in particular to the technical field of nerve cell preparation forms, and particularly relates to a preparation method of nerve-related cells and application thereof.

Background

The stem cells have regeneration and reconstruction capabilities, regeneration medicine based on the stem cell technology can supplement the lost nerve cells, and a new way is hopeful to be provided for treating a plurality of histiocyte defect diseases such as degenerative diseases. In recent years, a large number of preclinical research results show that the differentiated dopamine neural cells of human pluripotent stem cells have good treatment effect on a non-human primate Parkinson disease model.

However, at present, neural cell transplantation is mainly performed by digested single cells, the survival of the cells after neural cell transplantation is poor due to the fact that the cells have nerve fiber breakage and micro-environment problems such as oxidative stress, the survival rate is estimated to be only 10%, and how to improve the in vivo survival of the neural cells is the problem which is mainly solved at present.

Disclosure of Invention

Nerve-associated cells (e.g., dopamine nerve cells) are digested, cryopreserved and transplanted as single cell suspensions, and have poor in vivo viability. The inventor of the present application finds that suspending nerve-related cells (such as dopamine nerve cells) into cell pellets in advance can significantly improve the (e.g. in vivo) survival rate of the nerve-related cells (such as dopamine nerve cells).

The nerve-related cell source comprises somatic cell reprogramming, stem cell differentiation, primary separation and the like. Preferably, the stem cells are pluripotent stem cells, such as embryonic stem cells. Cell sources include, but are not limited to, various animals, such as mammals, e.g., bovines, equines, porcines, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans.

To this end, in a first aspect of the present invention, there is provided a neural cell sphere comprising a plurality of neural-related cells, the neural-related cells comprising neural precursor cells, neural cells, glial cells, or any combination thereof, the neural cell sphere having a diameter of 30-500 μm, for example, 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm, 100-110 μm, 110-120 μm, 120-130 μm, 130-140 μm, 140-150 μm, 150-160 μm, 160-170 μm, 170-180 μm, 180-190 μm, 190-200 μm, 200-210 μm, 210-220 μm, 220-230 μm, 230-240 μm, 240-250 μm, 250-260 μm, 260-270 μm, 270-280 μm, 280-290 μm, 290-300 μm, 300-310 μm, 310-320 μm, 320-330 μm, 330-340 μm, 340-350 μm, 350-360 μm, 360-370 μm, 370-380 μm, 380-390 μm, 390-400 μm, 400-410 μm, 410-420 μm, 420-430 μm, 430-440 μm, 440-450 μm, 450-460 μm, 460-470 μm, 470-480 μm, 480-490 μm or 490-500 μm.

In some embodiments, the neural cell sphere has a diameter of 80-300 μm (e.g., 80-120 μm, 120-200 μm, or 180-300 μm, or 80-180 μm).

In some embodiments, the neural cell sphere has a diameter of 80-200 μm (e.g., 80-120 μm or 120-200 μm, or 80-180 μm).

In some embodiments, the neurosphere expresses marker TUJ1.

In some embodiments, at least about 55% (e.g., at least about 57%, at least about 59%, at least about 60%, at least about 61%, at least about 63%, at least about 65%, at least about 67%, at least about 69%, or at least about 70%) of the nerve-associated cells in the nerve cell sphere express marker TUJ1.

In some embodiments, at least about 70% of the nerve-associated cells in the neurosphere express the marker TUJ1.

In some embodiments, the neurosphere does not express or underexpresses the marker caspase3.

In some embodiments, no more than about 40% (e.g., no more than about 35%, no more than about 30%, no more than about 25%, no more than about 20%, no more than about 15%, no more than about 10%, or no more than about 5%) of the neuro-associated cells in the neurosphere express the marker caspase3.

In some embodiments, no more than about 15% (preferably no more than about 10% or no more than about 5%) of the neuro-associated cells in the neurosphere express the marker caspase3.

In some embodiments, the neural-related cells comprise neural precursor cells. In some embodiments, the nerve-associated cell further comprises a neural cell and/or a glial cell.

In some embodiments, the neural-related cell is a neural precursor cell.

In some embodiments, the neural precursor cell is selected from a dopamine neural precursor cell, a gamma-aminobutyric acid (GABA) ergic neuronal precursor cell, a glutamatergic neuronal precursor cell, a cholinergic neuronal precursor cell, a serotonin neuronal precursor cell, a motor neuronal precursor cell, a sensory neuronal precursor cell, or any combination thereof.

In some embodiments, the neural precursor cell is a dopamine neural precursor cell.

In some embodiments, the neural cell is selected from a dopaminergic neuron, a gamma-aminobutyric acid (GABA) energetic neuron, a glutamatergic neuron, a cholinergic neuron, a serotonin neuronal, a motor neuron, a sensory neuron, or any combination thereof.

In some embodiments, wherein the glial cell is selected from the group consisting of an astrocyte, an oligodendrocyte, a microglial cell, or any combination thereof.

In some embodiments, the source of the neural-related cells is somatic cell reprogramming, stem cell differentiation, primary isolation, or the like. Preferably, the stem cells are pluripotent stem cells, such as embryonic stem cells. Sources of cells include, but are not limited to, various animals, such as mammals, e.g., bovines, equines, porcines, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans.

In a second aspect of the present invention, the present invention provides a method for preparing the aforementioned neural cell sphere, comprising:

inoculating a single cell suspension of nerve-associated cells selected from neural precursor cells, neural cells, glial cells, or any combination thereof, into a low-attachment cell culture plate to form the neural cell sphere;

wherein the seeding density of the nerve-associated cells is 5 × 10 ³ /cm ² ～5×10 ⁶ /cm ² E.g. 5X 10 ³ /cm ² ～1×10 ⁴ /cm ² 、1×10 ⁴ /cm ² ～2.5×10 ⁴ /cm ² 、2.5×10 ⁴ /cm ² ～5×10 ⁴ /cm ² 、5×10 ⁴ /cm ² ～1×10 ⁵ /cm ² 、1×10 ⁵ /cm ² ～2.5×10 ⁵ /cm ² 、2.5×10 ⁵ /cm ² ～5×10 ⁵ /cm ² 、5×10 ⁵ /cm ² ～1×10 ⁶ /cm ² 、1×10 ⁶ /cm ² ～2.5×10 ⁶ /cm ² Or 2.5X 10 ⁶ /cm ² ～5×10 ⁶ /cm ² 。

In some embodiments, the seeding density of the neural associated cells is 5 × 10 ⁴ /cm ² ～5×10 ⁵ /cm ² (e.g., 5X 10) ⁴ /cm ² 、2.5×10 ⁵ /cm ² Or 5X 10 ⁵ /cm ² )。

In some embodiments, the seeding density of the neural-associated cells is 5 x 10 ⁴ /cm ² ～2.5×10 ⁵ /cm ² (e.g., 5X 10) ⁴ /cm ² Or 2.5X 10 ⁵ /cm ² )

In some embodiments, the neural-related cell is inoculated in an amount of 1 × 10 ⁴ 1 x 10 of hole ⁷ Per well, e.g. 1X 10 ⁴ pore-5X 10 ⁴ Hole, 5X 10 ⁴ 1 x 10 of hole ⁵ 1X 10 per hole ⁵ pore-5X 10 ⁵ Hole, 5X 10 ⁵ pore-1X 10 ⁶ 1X 10 per hole ⁶ pore-5X 10 ⁶ Per well or 5X 10 ⁶ 1 x 10 of hole ⁷ A/well, and the culture plate is a 24-well plate.

In some embodiments, the neural-related cell is inoculated in an amount of 1 × 10 ⁵ pore-1X 10 ⁶ Per well (e.g. 1X 10) ⁵ Hole, 5X 10 ⁵ Per well or 1X 10 ⁶ Per well) and the culture plate is a 24-well plate.

In some embodiments, the neural-related cell is inoculated in an amount of 1 × 10 ⁵ pore-5X 10 ⁵ Per well (e.g., 1X 10) ⁵ Per well or 5X 10 ⁵ Per well) and the culture plate is a 24-well plate.

In some embodiments, wherein the neural-related cells comprise neural precursor cells. In some embodiments, the nerve-associated cell further comprises a neural cell and/or a glial cell.

In some embodiments, the neural-related cell is a neural precursor cell.

In some embodiments, the neural precursor cell is selected from a dopamine neural precursor cell, a gamma-aminobutyric acid (GABA) ergic neuron precursor cell, a glutamatergic neuron precursor cell, a cholinergic neuron precursor cell, a serotonin neuron precursor cell, a motor neuron precursor cell, a sensory neuron precursor cell, or any combination thereof.

In some embodiments, the glial cell is selected from the group consisting of an astrocyte, an oligodendrocyte, a microglial cell, or any combination thereof.

In some embodiments, the source of the neural-associated cell is somatic cell reprogramming, stem cell differentiation, primary isolation, or the like. Preferably, the stem cells are pluripotent stem cells, such as embryonic stem cells. Sources of cells include, but are not limited to, various animals, such as mammals, e.g., bovines, equines, porcines, canines, felines, lagomorphs, rodents (e.g., mice or rats), non-human primates (e.g., macaques or cynomolgus monkeys), or humans.

In some embodiments, the culture plate is a round bottom or a sharp bottom microplate.

In some embodiments, the culture plate is AggreWell ^TM And (5) culturing the plate.

In some embodiments, the neural cell sphere is formed after seeding the single cell suspension of neural-related cells into a low-attachment cell culture plate and culturing for 10-30 hours (e.g., 10-12 hours, 12-14 hours, 14-18 hours, 18-20 hours, 20-22 hours, 22-24 hours, 24-26 hours, 26-28 hours, or 28-30 hours, preferably 24 hours).

In some embodiments, the medium used at the time of inoculation is an inoculation medium comprising:

brain-derived neurotrophic factor (BDNF),

glial cell line-derived neurotrophic factor (GDNF),

transforming growth factor beta 3 (TGF-beta 3),

the amount of ascorbic acid is such that,

calcium dibutyryladenosine cyclophosphate (db-cAMP),

DAPT(LY-374973)。

in some embodiments, the inoculation medium further comprises: y27632.

In some embodiments, the concentration of the Brain Derived Neurotrophic Factor (BDNF) is 1-100ng/mL, such as 1-5ng/mL, 5-10ng/mL, 10-15ng/mL, 15-20ng/mL, 20-25ng/mL, 25-30ng/mL, 30-35ng/mL, 35-40ng/mL, 40-45ng/mL, 45-50ng/mL, 50-55ng/mL, 55-60ng/mL, 60-65ng/mL, 65-70ng/mL, 70-75ng/mL, 75-80ng/mL, 80-85ng/mL, 85-90ng/mL, 90-95ng/mL, or 95-100ng/mL.

In some embodiments, the concentration of the Brain Derived Neurotrophic Factor (BDNF) is 10-20ng/mL.

In some embodiments, the concentration of the Brain Derived Neurotrophic Factor (BDNF) is 20ng/mL.

In some embodiments, the concentration of the glial cell line-derived neurotrophic factor (GDNF) is 1-100ng/mL, e.g., 1-5ng/mL, 5-10ng/mL, 10-15ng/mL, 15-20ng/mL, 20-25ng/mL, 25-30ng/mL, 30-35ng/mL, 35-40ng/mL, 40-45ng/mL, 45-50ng/mL, 50-55ng/mL, 55-60ng/mL, 60-65ng/mL, 65-70ng/mL, 70-75ng/mL, 75-80ng/mL, 80-85ng/mL, 85-90ng/mL, 90-95ng/mL, or 95-100ng/mL.

In some embodiments, the concentration of glial cell line-derived neurotrophic factor (GDNF) is 10-20ng/mL.

In some embodiments, the concentration of glial cell line-derived neurotrophic factor (GDNF) is 20ng/mL.

In some embodiments, the transforming growth factor beta 3 (TGF-beta 3) is at a concentration of 0.5-50ng/mL, e.g., 0.5-0.7ng/mL, 0.7-1ng/mL, 1-1.3ng/mL, 1.3-1.5ng/mL, 1.5-1.7ng/mL, 1.7-2ng/mL, 2-2.5ng/mL, 2.5-3ng/mL, 3-3.5ng/mL, 3.5-4ng/mL, 4-4.5ng/mL, 4.5-5ng/mL, 5-10ng/mL, 10-15ng/mL, 15-20ng/mL, 20-25ng/mL, 25-30ng/mL, 30-35ng/mL, 35-40ng/mL, 40-45ng/mL, or 45-50ng/mL.

In some embodiments, the transforming growth factor beta 3 (TGF-beta 3) is at a concentration of 1-10ng/mL.

In some embodiments, the transforming growth factor beta 3 (TGF-beta 3) is at a concentration of 1ng/mL.

In some embodiments, the ascorbic acid is in a concentration of 0.01 to 2mM, such as 0.01 to 0.03mM, 0.03 to 0.05mM, 0.05 to 0.07mM, 0.07 to 0.1mM, 0.1 to 0.13mM, 0.13 to 0.15mM, 0.15 to 0.17mM, 0.17 to 0.2mM, 0.2 to 0.23mM, 0.23 to 0.25mM, 0.25 to 0.27mM, 0.27 to 0.3mM, 0.3 to 0.4mM, 0.4 to 0.5mM, 0.5 to 0.7mM, 0.7 to 0.9mM, 0.9 to 1.1mM, 1.1 to 1.3mM, 1.3 to 1.5mM, 1.5 to 1.7mM, 1.7 to 1.9mM, or 1.9 to 2.0mM.

In some embodiments, the concentration of ascorbic acid is 0.2mM.

In some embodiments, the concentration of calcium dibutyryladenosine cyclophosphate (db-cAMP) is 0.01-5mM, for example, 0.01 to 0.03mM, 0.03 to 0.05mM, 0.05 to 0.07mM, 0.07 to 0.1mM, 0.1 to 0.2mM, 0.2 to 0.3mM, 0.3 to 0.4mM, 0.4 to 0.5mM, 0.5 to 0.7mM, 0.7 to 0.9mM, 0.9 to 1.1mM, 1.1 to 1.3mM, 1.3 to 1.5mM, 1.5 to 1.7mM, 1.7 to 1.9mM, 1.9 to 2.0mM, 2.0 to 2.1mM, 2.0 to 1mM 2.1-2.3mM, 2.3-2.5mM, 2.5-2.7mM, 2.7-2.9mM, 2.9-3.0mM, 3.0-3.1mM, 3.1-3.3mM, 3.3-3.5mM, 3.5-3.7mM, 3.7-3.9mM, 3.9-4.0mM, 4.0-4.1mM, 4.1-4.3mM, 4.3-4.5mM, 4.5-4.7mM, 4.7-4.9mM, or 4.9-5.0mM.

In some embodiments, the concentration of calcium dibutyryladenosine cyclophosphate (db-cAMP) is 0.5mM.

In some embodiments, the DAPT (LY-374973) is at a concentration of 1-100. Mu.M, e.g., 1-5. Mu.M, 5-10. Mu.M, 10-15. Mu.M, 15-20. Mu.M, 20-25. Mu.M, 25-30. Mu.M, 30-35. Mu.M, 35-40. Mu.M, 40-45. Mu.M, 45-50. Mu.M, 50-55. Mu.M, 55-60. Mu.M, 60-65. Mu.M, 65-70. Mu.M, 70-75. Mu.M, 75-80. Mu.M, 80-85. Mu.M, 85-90. Mu.M, 90-95. Mu.M, or 95-100. Mu.M.

In some embodiments, the concentration of DAPT (LY-374973) is 10 μ M.

In some embodiments, the concentration of Y27632 is 1-50 μ M, e.g., 1-2 μ M, 2-3 μ M, 3-4 μ M, 4-5 μ M, 5-6 μ M, 6-7 μ M, 7-8 μ M, 8-9 μ M, 9-10 μ M, 10-11 μ M, 11-12 μ M, 12-13 μ M, 13-14 μ M, 14-15 μ M, 15-16 μ M, 16-17 μ M, 17-18 μ M, 18-19 μ M, 19-20 μ M, 20-25 μ M, 25-30 μ M, 30-35 μ M, 35-40 μ M, 40-45 μ M, or 45-50 μ M.

In some embodiments, the concentration of Y27632 is 10 μ Μ.

In some embodiments, the inoculation medium further comprises: and (3) a basic culture medium.

In some embodiments, the basal medium comprises:

Neurobasal，

B27-supplement，

Glutamax。

in some embodiments, the Neurobasal medium has a volume fraction of 70% -99%, such as 70% -71%, 71% -73%, 73% -75%, 75% -77%, 77% -79%, 79% -80%, 80% -81%, 81% -83%, 83% -85%, 85% -87%, 87% -89%, 89% -90%, 90% -91%, 91% -93%, 93% -95%, 95% -97%, or 97% -99%.

In some embodiments, the volume fraction of Neurobasal in the basal medium is 97%.

<xnotran> , , B27-supplement 0.1% -20%, 0.1% -0.15%, 0.15% -0.2%, 0.2% -0.25%, 0.25% -0.3%, 0.3% -0.35%, 0.35% -0.4%, 0.4% -0.45%, 0.45% -0.5%, 0.5% -0.55%, 0.55% -0.6%, 0.6% -0.65%, 0.65% -0.7%, 0.7% -0.75%, 0.75% -0.8%, 0.8% -0.85%, 0.85% -0.9%, 0.9% -0.95%, 0.95% -1.0%, 1.0% -1.1%, 1.1% -1.3%, 1.3% -1.5%, 1.5% -1.7%, 1.7% -1.9%, 1.9% -2.0%, 2.0% -2.1%, 2.1% -2.3%, 2.3% -2.5%, 2.5% -2.7%, 2.7% -2.9%, 2.9% -3.0%, 3.0% -3.1%, 3.1% -3.3%, 3.3% -3.5%, 3.5% -3.7%, 3.7% -3.9%, 3.9% -4.0%, 4.0% -4.1%, 4.1% -4.3%, 4.3% -4.5%, 4.5% -4.7%, 4.7% -4.9%, 4.9% -5.0%, 5.0% -5.5%, 5.5% -6.0%, 6.0% -6.5%, 6.5% -7.0%, 7.0% -7.5%, 7.5% -8.0%, 8.0% -8.5%, 8.5% -9.0%, 9.0% -9.5%, 9.5% -10%, 10% -10.5%, 10.5% -11%, 11% -11.5%, 11.5% -12%, 12% -12.5%, 12.5% -13%, 13% -13.5%, </xnotran> 13.5% -14%, 14% -14.5%, 14.5% -15% or 15% -20%.

In some embodiments, the volume fraction of B27-supplement in the basal medium is 2%.

In some embodiments, the basal medium, the Glutamax has a volume fraction of 0.1% to 10%, for example, 0.1% -0.15%, 0.15% -0.2%, 0.2% -0.25%, 0.25% -0.3%, 0.3% -0.35%, 0.35% -0.4%, 0.4% -0.45%, 0.45% -0.5%, 0.5% -0.55%, 0.55% -0.6%, 0.6% -0.65%, 0.65% -0.7%, 0.7% -0.75%, 0.75% -0.8%, 0.8% -0.85%, 0.85% -0.9%, 0.9% -0.95%, 0.95% -1.0%, 1.0% -1.1%, 1.1% -1.3%, 1.3% -1.5%, 1.5% -1.7%, 1.7% -1.9%, 1.9% -2.0%, 2.0% -2.1% 2.1% -2.3%, 2.3% -2.5%, 2.5% -2.7%, 2.7% -2.9%, 2.9% -3.0%, 3.0% -3.1%, 3.1% -3.3%, 3.3% -3.5%, 3.5% -3.7%, 3.7% -3.9%, 3.9% -4.0%, 4.0% -4.1%, 4.1% -4.3%, 4.3% -4.5%, 4.5% -4.7%, 4.7% -4.9%, 4.9% -5.0%, 5.0% -5.5%, 5.5% -6.0%, 6.0% -6.5%, 6.5% -7.0%, 7.0% -7.5%, 7.5% -8.0%, 8.0% -8.5%, 8.5% -9.0%, 9.0% -9.5% or 9.5% -10%.

In some embodiments, the basal medium has a volume fraction of Glutamax of 1%.

In some embodiments, the sum of the volume fractions of the components in the basal medium is 100%.

In some embodiments, the concentration of each component (i.e., BDNF, GDNF, TGF- β 3, ascorbic acid, db-cAMP, DAPT, and optionally Y27632) in the inoculation medium refers to the concentration of each of the aforementioned components in the basal medium.

In some embodiments, the single cell suspension of the neural-associated cell is prepared by: digesting the nerve-associated cells to obtain a single cell suspension of the nerve-associated cells.

In some embodiments, after said digesting, further comprises: the inoculation medium described above was added.

In some embodiments, the digestion is performed using Tryple or Accutase.

In a third aspect of the invention, the invention provides a nerve cell sphere, which is prepared by the method described above.

In some embodiments, the nerve cell sphere has a diameter of 30-500 μm, for example, 30-40 μm, 40-50 μm, 50-60 μm, 60-70 μm, 70-80 μm, 80-90 μm, 90-100 μm, 100-110 μm, 110-120 μm, 120-130 μm, 130-140 μm, 140-150 μm, 150-160 μm, 160-170 μm, 170-180 μm, 180-190 μm, 190-200 μm, 200-210 μm, 210-220 μm, 220-230 μm, 230-240 μm, 240-250 μm, 250-260 μm, 260-270 μm, 270-280 μm, 280-290 μm, 290-300 μm, 300-310 μm, 310-320 μm, 320-330 μm, 330-340 μm, 340-350 μm, 350-360 μm, 360-370 μm, 370-380 μm, 380-390 μm, 390-400 μm, 400-410 μm, 410-420 μm, 420-430 μm, 430-440 μm, 440-450 μm, 450-460 μm, 460-470 μm, 470-480 μm, 480-490 μm or 490-500 μm.

In some embodiments, the neurosphere expresses marker TUJ1.

In some embodiments, the neurosphere does not express or under-expresses the marker caspase3.

In some embodiments, no more than about 40% (e.g., no more than about 35%, no more than about 30%, no more than about 25%, no more than about 20%, no more than about 15%, no more than about 10%, or no more than about 5%) of the neuro-associated cells express the marker caspase3.

In a fourth aspect of the invention, the invention provides a neural cell sphere cryopreservation formulation comprising: a nerve cell sphere, as described above.

In some embodiments, the neural cell sphere cryopreservation formulation further comprises: freezing and storing the neural cell balls.

In some embodiments, the total cell density of the neural cell sphere in the neural cell sphere cryopreservation solution is 10 ⁵ /mL～10 ⁷ Per mL, e.g. 1X 10 ⁵ /mL～3×10 ⁵ /mL、3×10 ⁵ /mL～5×10 ⁵ /mL、5×10 ⁵ /mL～7×10 ⁵ /mL、7×10 ⁵ /mL～1×10 ⁶ /mL、1×10 ⁶ /mL～3×10 ⁶ /mL、3×10 ⁶ /mL～5×10 ⁶ /mL、5×10 ⁶ /mL～7×10 ⁶ Per mL or 7X 10 ⁶ /mL～1×10 ⁷ /mL。

In some embodiments, the total cell density of the neural cell sphere in the neural cell sphere cryopreservation solution is 3 × 10 ⁶ /mL。

The total cell density of the neurosphere refers to the total number of cells in the cryopreservation solution of the neurosphere per ml.

In some embodiments, the neural cell sphere cryopreservation solution comprises:

the basic components of the mixture are mixed,

B27 Supplement，

N2 Supplement，

Glutamax，

DMSO，

Y27632，

human Serum Albumin (HSA) or fetal bovine serum,

the basic component is selected from Neurobasal, KODMEM/F12 or combination thereof.

In some embodiments, the neural cell sphere cryopreservation solution further comprises: an impermeable protectant.

In some embodiments, the non-osmotic protective agent is selected from dextran, sodium alginate, sodium hyaluronate, gamma-polyglutamic acid, or any combination thereof.

In some embodiments, the base component has a volume fraction of 1% -84%, such as 1% -3%, 3% -5%, 5% -7%, 7% -9%, 9% -11%, 11% -13%, 13% -15%, 15% -17%, 17% -19%, 19% -20%, 20% -21%, 21% -23%, 23% -25%, 25% -27%, 27% -29%, 29% -30%, 30% -31%, 31% -33%, 33% -35%, 35% -37%, 37% -39%, 39% -40%, 40% -41%, 41% -43%, 43% -45%, 45% -47%, 47% -49%, 49% -50%, 50% -51%, 51% -53%, 53% -55% -57%, 57% -59%, 59% -60%, 60% -61%, 61% -63%, 63% -65%, 65% -67%, 67% -69%, 69% -70%, 70% -71%, 71% -73%, 73% -75%, 75% -77%, 77% -79%, 79% -80%, 80% -81%, 81% -83%, or 84% -83%.

In some embodiments, the volume fraction of the base is 79%.

In some embodiments, the Neurobasal has a volume fraction of 1% -84%, such as 1% -3%, 3% -5%, 5% -7%, 7% -9%, 9% -11%, 11% -13%, 13% -15%, 15% -17%, 17% -19%, 19% -20%, 20% -21%, 21% -23%, 23% -25%, 25% -27%, 27% -29%, 29% -30%, 30% -31%, 31% -33%, 33% -35%, 35% -37%, 37% -39%, 39% -40%, 40% -41%, 41% -43%, 43% -45%, 45% -47%, 47% -49%, 49% -50%, 50% -51%, 51% -53%, 53% -55%, 55% -57%, 57% -59%, 59% -60%, 60% -61%, 61% -63%, 63% -65%, 65% -67%, 67% -69%, 69% -70%, 70% -71%, 71% -73%, 73% -75%, 75% -77%, 77% -79%, 79% -80%, 80% -81%, 81% -83%, or 83% -83%.

In some embodiments, the Neurobasal has a volume fraction of 39.5%.

In some embodiments, the KODMEM/F12 has a volume fraction of 1% to 84%, such as 1% to 3%, 3% to 5%, 5% to 7%, 7% to 9%, 9% to 11%, 11% to 13%, 13% to 15%, 15% to 17%, 17% to 19%, 19% to 20%, 20% to 21%, 21% to 23%, 23% to 25%, 25% to 27%, 27% to 29%, 29% to 30%, 30% to 31%, 31% to 33%, 33% to 35%, 35% to 37%, 37% to 39%, 39% to 40%, 40% to 41%, 41% to 43%, 43% to 45%, 45% to 47%, 47% to 49%, 49% to 50%, 50% to 51%, 51% to 53%, 53% to 55%, 55% to 57%, 57% to 59% to 60%, 60% to 61%, 61% to 63%, 63% to 65%, 65% to 67%, 67% to 69%, 69% to 70%, 71% to 73%, 73% to 75%, 75% to 77%, 77% to 79%, 79% to 80%, 80% to 81%, 81% to 83%, or 83% to 83%.

In some embodiments, the volume fraction of KODMEM/F12 is 39.5%.

In some embodiments, the volume fraction of the B27Supplement is from 0.1% to 20%, for example, 0.1% -0.5%, 0.5% -1.0%, 1.0% -1.1%, 1.1% -1.3%, 1.3% -1.5%, 1.5% -1.7%, 1.7% -1.9%, 1.9% -2.0%, 2.0% -2.1%, 2.1% -2.3%, 2.3% -2.5%, 2.5% -2.7%, 2.7% -2.9%, 2.9% -3.0%, 3.0% -3.1%, 3.1% -3.3%, 3.3% -3.5%, 3.5% -3.7%, 3.7% -3.9%, 3.9% -4.0%, 4.0% -4.1%, 4.1% -4.3%, 4.3% -4.5% 4.5% -4.7%, 4.7% -4.9%, 4.9% -5.0%, 5.0% -5.5%, 5.5% -6.0%, 6.0% -6.5%, 6.5% -7.0%, 7.0% -7.5%, 7.5% -8.0%, 8.0% -8.5%, 8.5% -9.0%, 9.0% -9.5%, 9.5% -10%, 10% -10.5%, 10.5% -11%, 11% -11.5%, 11.5% -12%, 12% -12.5%, 12.5% -13%, 13% -13.5%, 13.5% -14%, 14% -14.5%, 14.5% -15% or 15% -20%.

In some embodiments, the volume fraction of the B27Supplement is 4%.

In some embodiments, the volume fraction of the N2 Supplement is from 0.1% to 10%, for example, 0.1% -0.15%, 0.15% -0.2%, 0.2% -0.25%, 0.25% -0.3%, 0.3% -0.35%, 0.35% -0.4%, 0.4% -0.45%, 0.45% -0.5%, 0.5% -0.55%, 0.55% -0.6%, 0.6% -0.65%, 0.65% -0.7%, 0.7% -0.75%, 0.75% -0.8%, 0.8% -0.85%, 0.85% -0.9%, 0.9% -0.95%, 0.95% -1.0%, 1.0% -1.1%, 1.1% -1.3%, 1.3% -1.5%, 1.5% -1.7%, 1.7% -1.9%, 1.9% -2.0%, 2.0% -2.1% 2.1% -2.3%, 2.3% -2.5%, 2.5% -2.7%, 2.7% -2.9%, 2.9% -3.0%, 3.0% -3.1%, 3.1% -3.3%, 3.3% -3.5%, 3.5% -3.7%, 3.7% -3.9%, 3.9% -4.0%, 4.0% -4.1%, 4.1% -4.3%, 4.3% -4.5%, 4.5% -4.7%, 4.7% -4.9%, 4.9% -5.0%, 5.0% -5.5%, 5.5% -6.0%, 6.0% -6.5%, 6.5% -7.0%, 7.0% -7.5%, 7.5% -8.0%, 8.0% -8.5%, 8.5% -9.0%, 9.0% -9.5% or 9.5% -10%.

In some embodiments, the volume fraction of the N2 Supplement is 1%.

In some embodiments, the volume fraction of Glutamax is 0.1% to 10%, for example, 0.1% -0.15%, 0.15% -0.2%, 0.2% -0.25%, 0.25% -0.3%, 0.3% -0.35%, 0.35% -0.4%, 0.4% -0.45%, 0.45% -0.5%, 0.5% -0.55%, 0.55% -0.6%, 0.6% -0.65%, 0.65% -0.7%, 0.7% -0.75%, 0.75% -0.8%, 0.8% -0.85%, 0.85% -0.9%, 0.9% -0.95%, 0.95% -1.0%, 1.0% -1.1%, 1.1% -1.3%, 1.3% -1.5%, 1.5% -1.7%, 1.7% -1.9%, 1.9% -2.0%, 2.0% -2.1% 2.1% -2.3%, 2.3% -2.5%, 2.5% -2.7%, 2.7% -2.9%, 2.9% -3.0%, 3.0% -3.1%, 3.1% -3.3%, 3.3% -3.5%, 3.5% -3.7%, 3.7% -3.9%, 3.9% -4.0%, 4.0% -4.1%, 4.1% -4.3%, 4.3% -4.5%, 4.5% -4.7%, 4.7% -4.9%, 4.9% -5.0%, 5.0% -5.5%, 5.5% -6.0%, 6.0% -6.5%, 6.5% -7.0%, 7.0% -7.5%, 7.5% -8.0%, 8.0% -8.5%, 8.5% -9.0%, 9.0% -9.5% or 9.5% -10%.

In some embodiments, the volume fraction of Glutamax is 1%.

In some embodiments, the DMSO has a volume fraction of 5% to 20%, such as 5% to 6%, 6% to 7%, 7% to 8%, 8% to 9%, 9% to 10%, 10% to 11%, 11% to 12%, 12% to 13%, 13% to 14%, 14% to 15%, 15% to 16%, 16% to 17%, 17% to 18%, 18% to 19%, or 19% to 20%.

In some embodiments, the volume fraction of DMSO is 10%.

In some embodiments, the concentration of Y27632 is 5-20 μ M, e.g., 5-6 μ M, 6-7 μ M, 7-8 μ M, 8-9 μ M, 9-10 μ M, 10-11 μ M, 11-12 μ M, 12-13 μ M, 13-14 μ M, 14-15 μ M, 15-16 μ M, 16-17 μ M, 17-18 μ M, 18-19 μ M, or 19-20 μ M.

In some embodiments, the concentration of Y27632 is 10 μ Μ.

In some embodiments, the Human Serum Albumin (HSA) or fetal bovine serum has a volume fraction of 1% -10%, such as 1% -2%, 2% -3%, 3% -4%, 4% -5%, 5% -6%, 6% -7%, 7% -8%, 8% -9%, or 9% -10%.

In some embodiments, the Human Serum Albumin (HSA) or fetal bovine serum is at a volume fraction of 5%.

The concentration of each component in the neural cell sphere cryopreservation solution is the final concentration of each component in the neural cell sphere cryopreservation solution, and the volume fraction of each component in the neural cell sphere cryopreservation solution is the volume of each component/the total volume of the neural cell sphere cryopreservation solution.

In a fifth aspect of the present invention, the present invention provides a method for preparing the aforementioned neural cell sphere cryopreservation preparation, comprising:

centrifuging the nerve cell balls, and removing supernatant to obtain nerve cell ball sediment;

and mixing the neural cell sphere sediment with the neural cell sphere frozen stock solution to enable the neural cell sphere to be resuspended in the neural cell sphere frozen stock solution to obtain a cell sphere heavy suspension, wherein the cell sphere heavy suspension is the neural cell sphere frozen stock preparation.

In some embodiments, the neural cell sphere cryopreservation solution has a total cell density of 10 ⁵ /mL～10 ⁷ Per mL, e.g. 1X 10 ⁵ /mL～3×10 ⁵ /mL、3×10 ⁵ /mL～5×10 ⁵ /mL、5×10 ⁵ /mL～7×10 ⁵ /mL、7×10 ⁵ /mL～1×10 ⁶ /mL、1×10 ⁶ /mL～3×10 ⁶ /mL、3×10 ⁶ /mL～5×10 ⁶ /mL、5×10 ⁶ /mL～7×10 ⁶ Per mL or 7X 10 ⁶ /mL～1×10 ⁷ /mL。

In some embodiments, the neural cell sphere cryopreservation solution is as described above.

In some embodiments, the method further comprises: the cell pellet suspension was subjected to freezing treatment and then stored in liquid nitrogen.

In some embodiments, the freezing is performed at a temperature of-80 ℃ or less for 8-15 hours (overnight).

In some embodiments, the neural cell sphere cryopreservation solution is previously subjected to a cold storage treatment.

In some embodiments, the refrigeration treatment is performed at a temperature of 0-4 ℃.

In a sixth aspect of the invention, the invention provides a neural cell sphere cryopreservation preparation prepared by the method.

In a seventh aspect of the invention, the invention provides the use of a neurosphere as hereinbefore described or a neurosphere preparation as hereinbefore described in the manufacture of an agent or medicament for use in one or more of the following:

(1) For nerve cell transplantation;

(2) Can be used for treating neurodegenerative diseases.

In some embodiments, the neurodegenerative disease is parkinson's disease.

In an eighth aspect of the invention, there is provided the aforementioned neurosphere or the aforementioned neurosphere preparation for use in one or more of the following:

(1) For nerve cell transplantation;

(2) Can be used for treating neurodegenerative diseases.

In some embodiments, the neurodegenerative disease is parkinson's disease.

In a ninth aspect of the present invention, the present invention provides a method of transplanting neural cells or a method of treating neurodegenerative diseases, comprising: transplanting an effective amount of the aforementioned neural cell sphere into a mammalian striatum or neural tissue; alternatively, the aforementioned nerve cell sphere preparation is revived to obtain the aforementioned nerve cell sphere, and then an effective amount of the nerve cell sphere is transplanted into striatum or nerve tissue of a mammal.

In some embodiments, the neurodegenerative disease is parkinson's disease.

In a tenth aspect of the invention, the invention provides a method of preparing neurons in vitro, comprising: culturing the aforementioned neurosphere under conditions suitable for differentiation of the aforementioned neurosphere.

In an eleventh aspect of the invention, the invention provides a neuron, prepared by the method described above.

In the present invention, the term "mammal" includes bovine, equine, ovine, porcine, canine, feline, rodent, primate, etc., for example, human, mouse, rat, dog, pig, monkey, etc. In certain embodiments, the mammal refers to a non-human mammal. In other embodiments, the mammal refers to a human.

In the present invention, the term "effective amount" means an amount sufficient to treat the disease in a patient but low enough to avoid serious side effects (at a reasonable benefit/risk ratio), within the scope of sound medical judgment. The therapeutically effective amount will vary depending on the severity of the condition being treated, the age, size, weight and physical condition of the patient being treated, the medical history of the patient being treated, the duration of the treatment, the nature of concurrent therapy, the desired therapeutic effect, and like factors, but can nevertheless be routinely determined by one skilled in the art.

Advantageous effects

The technical scheme of the invention has one or more of the following advantages:

1. one or more of the following objectives can be achieved by means of a formulation that suspends nerve-associated cells (e.g., dopamine nerve cells) into spheres:

(1) Increasing synaptic integrity of a nerve-associated cell (e.g., a dopamine neuron);

(2) Realizing the cryopreservation of new forms of nerve-related cells (such as dopamine nerve cells);

(3) Promoting or increasing survival of nerve-associated cells (e.g., dopamine nerve cells) in vivo;

(4) Possible fields of application: and (4) transplanting nerve cells.

2. When the diameter of the neurospheres of the present invention is controlled within a certain range, neurospheres (e.g., dopamine (DA) neurospheres) can accomplish one or more of the following objectives:

(1) Preserving in frozen stock solution for 12 weeks and maintaining good survival rate (such as more than 80%, or more than 85%);

(2) Preserving in frozen stock solution for 6 weeks and maintaining good survival rate (e.g. more than 85%, or such as more than 90%);

(3) After the cells are stored in the frozen stock solution for 6 weeks or 12 weeks, the cell state is good, the edges of cell spheres are smooth, and almost no dead cells exist;

(4) The activity is still high (for example, more than 90 percent) after the frozen storage for 12 weeks;

(5) After 6 weeks or 12 weeks of storage in the frozen stock solution, the skeleton of the neuron is not damaged, and the proportion of TUJ1 in the frozen neural cell ball is still maintained in a high range (such as about 70%);

(6) After the nerve cell balls are stored in a frozen stock solution for 1 month, the nerve cell balls still have adherence capacity, and the adherence rate of the cells is higher (such as 80%);

(7) The survival ability of the nerve cell ball transplanted in vivo is obviously stronger than that of single cells, and the survival number of the cells of the nerve cell ball transplanted is 4 times or more than that of the single cells.

Drawings

Fig. 1 shows a view under an optical microscope of a nerve cell sphere prepared in an example of the present invention, with scale =200 μm.

Fig. 2 shows the state of the neurosphere (diameter 80-120 μm) after recovery of an example of the present invention, scale =100 μm.

FIG. 3 shows the viability of neural cell spheres (diameter 80-120 μm) cryopreserved for 6 weeks in the example of the present invention.

FIG. 4 shows the viability of neural cell spheres (diameter 80-120 μm) cryopreserved for 12 weeks in the example of the present invention.

Fig. 5 shows the state of the neurosphere (diameter 120-200 μm) after recovery of the example of the invention, with scale =200 μm.

FIG. 6 shows the viability of neural cell spheres (120-200 μm in diameter) frozen for 6 weeks in accordance with an embodiment of the present invention.

FIG. 7 shows the viability of neural cell spheres (120-200 μm in diameter) cryopreserved for 12 weeks in accordance with an embodiment of the present invention.

Fig. 8 shows the state of the neurosphere (diameter 180-300 μm) after recovery of an example of the present invention, scale =200 μm.

FIG. 9 shows the viability of neural cell spheres (diameter 180-300 μm) frozen for 6 weeks in accordance with an embodiment of the present invention.

FIG. 10 shows the viability of neural cell spheres (diameter less than 80 μm) cryopreserved for 6 weeks in accordance with an embodiment of the present invention.

FIG. 11 shows staining of fresh and cryopreserved neural cell sphere (120-200 μm diameter) markers according to an embodiment of the present invention.

FIG. 12 shows adherent culture of neural cell spheres (120-200 μm in diameter) after cryopreservation in an embodiment of the present invention.

FIG. 13 shows the results of transplantation of single cell preparations and neurospheres according to examples of the present invention.

Detailed Description

The following detailed description of embodiments of the invention is intended to be illustrative, and is not to be construed as limiting the invention.

Unless otherwise indicated, the molecular biological experimental methods and immunoassay methods used in the present invention are essentially described by reference to j.sambrook et al, molecular cloning: a laboratory manual, 2 nd edition, cold spring harbor laboratory press, 1989, and f.m. ausubel et al, eds. Molecular biology laboratory guidelines, 3 rd edition, john Wiley & Sons, inc., 1995. Unless otherwise indicated, the reagents used in the present invention are commercially available. It will be appreciated by those skilled in the art that the examples describe the invention by way of example and are not intended to limit the scope of the invention as claimed.

Example 1

1. Experimental procedure

1.1 formation and culture of neural cell spheres:

a) Preparation of a culture medium:

b27 medium preparation: 97% Neurobasal-CTS + 2%;

d17-25 medium preparation: b27 Medium + BDNF (20 ng/mL) + GDNF (20 ng/mL) + TGF-. Beta.3 (1 ng/mL) + AA (0.2 mM) + db-cAMP (0.5 mM) + DAPT (10. Mu.M)

b) Cell digestion: digesting the dopamine nerve precursor cells into single cells by using Tryple or accutase, then adding a fresh culture medium (D17-25 culture medium) into the single cells, blowing the single cells into a cell suspension, adding 10 mu L of the cell suspension into 10 mu L of trypan blue solution, uniformly mixing, and counting by using a countess cell counter;

c) Cell inoculation: the cell suspension was prepared according to the cell size (5X 10) ⁴ 1X 10 per hole ⁵ Hole, 5X 10 ⁵ 1X 10 per hole ⁶ /well) into a low-attachment plate AggreWell with a recessed pattern in the bottom ^TM 400 In the 24-well Plate, the inoculation medium was D17-25 medium containing 10. Mu.M Y27632;

d) Cell sphere formation: seeded dopamine neural precursor cells in AggreWell ^TM 400 Neural cell spheres of different diameter sizes can be formed in a 24-well Plate: 5X 10 ⁴ Pores can form a diameter of less than 80 μmCell pellet, 1X 10 ⁵ The hole can form a nerve cell sphere with the diameter of 80-120 mu m, 5X 10 ⁵ The hole can form a nerve cell sphere with the diameter of 120-200 mu m, 1X 10 ⁶ The hole can form a nerve cell ball with the diameter of 180-300 mu m;

e) Culturing cell balls: the culture medium is changed half a day after another, and the neural precursor cells can continue to differentiate to mature neurons under the treatment of the dopamine maturation culture medium.

1.2 cryopreservation experiment of neural cell balls:

a) Preparing a frozen stock solution: 39.5% KODMEM/F12-CTS + 4%. Blowing and beating for 10 times by using a 1mL liquid transfer gun, uniformly mixing, and placing in a refrigerator for precooling at 4 ℃ for later use; meanwhile, the program cooling box is placed in a refrigerator at 4 ℃ for precooling for standby;

b) Collecting cell balls: collecting the prepared nerve cell balls into a 15mL centrifuge tube, and centrifuging at 800rpm/min at room temperature for 3min;

c) Cell ball resuspension: discarding supernatant, adding pre-cooled above frozen stock solution to resuspend cells, wherein the cell density of resuspended cell ball is about 3 × 10 ⁶ /mL；

d) Freezing the cell balls in a gradient manner: and subpackaging the cell suspension into cryopreservation tubes according to the volume of 1 mL/tube, putting the cryopreservation tubes into a cryopreservation box, keeping the cryopreservation tubes in a refrigerator at the temperature of-80 ℃ overnight, and transferring the cryopreservation tubes into liquid nitrogen for long-term storage the next day.

1.3 recovery of the neural cell sphere

a) Opening a cell resuscitator in advance for preheating;

b) Preparing a 15mL centrifuge tube in a biological safety cabinet, and adding 9mL physiological saline for later use;

c) Taking out one frozen neural cell ball from liquid nitrogen, and quickly putting the neural cell ball into a cell recovery instrument for quick thawing;

d) Wiping the freezing tube with 75% alcohol, sucking the freezing solution, adding into prepared physiological saline, and centrifuging at room temperature of 800r/min for 3min; the supernatant was discarded and the solution was washed twice with physiological saline.

e) Discarding the supernatant, adding the above D17-25 medium + Y27632 (10. Mu.M) medium to resuspend the nervesThe cell pellet was inoculated into six well plates with low adherence, 3mL of D17-25 medium + Y27632 (10. Mu.M) medium was added to each well, the mixture was incubated at 37 ℃ and 5% CO ₂ Culturing in an incubator.

1.4 Activity Rate detection after Resuscitation of neural cell spheres

a) Opening a cell resuscitator in advance for preheating;

c) Taking out a frozen neural cell ball from the liquid nitrogen, and quickly putting the neural cell ball into a cell recovery instrument for quick thawing;

d) Wiping the freezing tube with 75% alcohol, sucking the freezing solution, adding into prepared physiological saline, and centrifuging at room temperature of 800r/min for 3min; the supernatant was discarded, and the solution was washed twice with physiological saline.

e) After washing, the supernatant was aspirated and Tryple was added, the reaction was carried out at 37 ℃ with 5% CO ₂ Incubating for 15min in incubator (to prolong or shorten digestion time); gently blowing and beating DA cell balls into single cells by using a pipette gun, stopping digestion by using normal saline, and centrifuging for 3min at room temperature of 1200 r/min;

f) Discarding the supernatant, and resuspending the cells with physiological saline; and adding 10 mu L of cell suspension into 10 mu L of trypan blue solution, mixing uniformly, and detecting the cell density and the cell viability by using a countess counter.

1.5 anchorage rate detection experiment after recovery of nerve cell sphere

a) Preparing a Vitronectin substrate: at room temperature, 6mL of DPBS is sucked and added into a 15mL centrifuge tube, then 60 muL of Vitronectin is sucked and added into the DPBS, and a pipetting gun is blown and beaten for 10 times to be mixed uniformly to obtain Vitronectin matrix which is prepared on site. Adding 1ml of prepared substrate into each hole of the six-hole plate, and incubating for 1h at room temperature for later use.

b) Preparing a 15mL centrifuge tube in a biological safety cabinet, and adding 9mL of the B27 culture medium for later use;

d) Wiping the cryopreservation tube with 75% alcohol, sucking the cryopreservation liquid, adding into the prepared B27 culture medium, and centrifuging at room temperature of 800r/min for 3min;

e) Discarding supernatant, adding the above D17-25 medium + Y27632 (10 μ M) medium to suspend neurospheres, inoculating in six-well plates with low adherence, adding 3mL D17-25 medium + Y27632 (10 μ M) medium per well, placing at 37 deg.C, 5% CO ₂ Culturing in an incubator, and changing the culture solution half every day.

f) The cells to be recovered are completely spread on a culture plate, the culture medium is discarded, after DPBS is washed once, tryple is used for digestion for 5min (the digestion time can be properly prolonged or shortened), the number of adherent cells is detected by a countess cell counter after collection, and the cell adherence rate is further calculated (adherence rate = number of adherent cells/number of inoculated cells).

1.6 immunofluorescence staining

a, sample treatment:

a) Fixing the nerve cell ball to be identified by using a solution containing 4% paraformaldehyde at room temperature for 30 minutes;

washing with PBS for 3 times, and dehydrating in 30% sucrose solution at 4 deg.C overnight;

b) OCT embedding: the surface sucrose was removed by washing with PBS once. Then the sample is placed into an embedding box filled with OCT for embedding, and the position of the sample is noticed, so that bubbles are avoided as much as possible.

c) Liquid nitrogen quick freezing: the embedding box is clamped by tweezers, the bottom surface of the embedding box is carefully contacted with liquid nitrogen, and the embedding box is not directly immersed so as to avoid the fragmentation of a sample caused by the over-fast temperature reduction and uneven temperature. And after OCT solidification, putting the embedded block into a refrigerator at the temperature of minus 80 ℃ to cool for 30 minutes or storing for later use.

d) Slicing: the sample was equilibrated by taking it to a cryomicrotome, which was adjusted to a temperature of-20 ℃. Then, the slide was sliced with a Leica SM2010R slicer, and the polylysine-coated slide was mounted on the slide and finally stored in a refrigerator at-80 ℃ or stained.

b, dyeing:

a) After sectioning, immunofluorescent staining was performed. The sections were first washed 3 times with PBS for 10 minutes each;

b) And (3) sealing: adding 320. Mu.L of a mixed solution of 2% BSA and 0.3% Triton per well, and blocking at room temperature for 2 hours;

c) Adding a primary antibody: diluting the primary antibody with a mixed solution containing 2% BSA and 0.3% Triton (antibody information: mouse anti-TUBB3 (BioLegend, 801202,1, 500), rabbitanti-caspase-3 (cell signalling, 9661S,1, 400)), blowing, mixing, discarding the blocking solution, adding 320. Mu.L of the primary antibody to each well, sealing, and standing overnight at 4 ℃;

d) Adding a secondary antibody: after the primary antibody was discarded by pipetting, washing with PBS for 2 times; 320. Mu.L of 2-percent BSA and 0.3-percent Triton mixed solution diluted secondary antibody (mouse cy3 (1;

e) Dyeing the core: discarding the secondary antibody, washing with PBS 2 times; add 320. Mu.L of Hoechst33342 diluent (1 ml PBS plus 1ul Hoechst 33342) to each well and incubate cells for 15min;

f) And (3) sealing: after PBS is washed once, the piece is sealed by using a sealing agent;

g) Image shooting: all images were taken with a confocal microscope (Zeiss LSM 780).

1.7 cell transplantation and viability assay

a. In vivo transplantation of neural cell spheres

a) Collecting nerve cell balls: collecting all the nerve cell balls subjected to suspension culture into a 50ml centrifuge tube, washing the holes with PBS if residual nerve cell balls still remain in the holes, collecting the washing liquid into the centrifuge tube together, and centrifuging the centrifuge tube at the room temperature of 800rpm/min for 3min;

b) Resuspending the nerve cell balls: discarding the supernatant, and adding cell injection to gently resuspend the cells;

c) Transplanting the nerve cell ball: 2.5X 10 injections per animal ⁵ The nerve cell sphere is arranged in a striatum, and the coordinate of the striatum is antigen (A) and is 0.6mm; lateral (L), -2mm; ventral (V), -4mm; the injection speed is 1 mul/min, the needle is left in situ for 5min after injection, and the wound is sutured.

b. Testing of the ability of neural cell spheres to survive in vivo

Sample treatment:

a) Material taking: three months later, animals were sacrificed and brains were removed;

b) Fixing: placing fresh rat brain tissue into 4% paraformaldehyde, and fixing at 4 deg.C overnight;

c) Gradient dehydration: washing with PBS for 3 times, 5min each time; then, using 15 percent and 30 percent sucrose solution prepared by PBS to dehydrate in a gradient way at the temperature of 4 ℃ until the sample sinks;

d) OCT embedding: the surface sucrose was removed by washing with PBS once. The sample was then embedded in an embedding cassette containing OCT.

e) And (3) quick freezing by liquid nitrogen: clamping the embedding box by using a forceps, contacting with liquid nitrogen, and after OCT is solidified, putting the embedding block into a refrigerator at minus 80 ℃ for cooling for 30 minutes or storing for later use.

Slicing: the sample was equilibrated by taking it to a cryomicrotome, which was adjusted to a temperature of-20 ℃. Then slicing with a Leica SM2010R slicer to slice thickness of 12-15 μm, pasting the glass slide coated with polylysine, and storing or dyeing in a refrigerator at-80 deg.C.

Tissue staining:

a) Taking out the slices from the refrigerator, and drying at room temperature for 15-30min;

b) Antigen retrieval: first, wash with PBS 3 times for 5min each time. Then, the slide is immersed in the repairing solution and heated for 10min by a microwave oven. Taking out, and cooling at room temperature for 30min;

c) Then washing with PBS for 5min for 3 times; wiping PBS around the tissue, and circling around the tissue by using a grouping pen;

d) And (3) sealing: adding 320. Mu.L of a mixed solution of 2% BSA and 0.3% Triton per well, and blocking at room temperature for 2 hours;

e) Adding a primary antibody: diluting primary antibody with mixed solution containing 2% BSA and 0.3% Triton, blowing, mixing, removing blocking solution, adding 320 μ L primary antibody dilution solution into each well, sealing, and standing at 4 deg.C overnight;

f) Adding a secondary antibody: after the primary antibody was discarded by pipetting, washing with PBS for 2 times; adding 320. Mu.L of 2% BSA and 0.3% Triton mixed solution diluted secondary antibody per well, and leaving the mixture at room temperature in the dark for 2 hours;

g) Dyeing the core: discarding the secondary antibody, washing with PBS 2 times; add 320. Mu.L of Hoechst33342 diluent (1ul Hoechst33342 in 1ml PBS) to each well, incubate cells for 10-15min;

h) And (3) sealing: after PBS is washed once, the piece is sealed by using a sealing agent; mu.l of an anti-fluorescence quencher was added dropwise to the tissue, and the slide was covered and fixed.

i) Image shooting: all images were taken with a confocal microscope (Zeiss LSM 780).

1.8 detection of cell status

And placing the inoculated 24h or recovered 24h nerve cell spheres under an inverted optical microscope to visually observe the state of the nerve cell spheres, and taking pictures to record.

2. Results of the experiment

2.1 preparation of neural cell spheres of different diameters and sizes

In AggreWell by the above method ^TM 400 Different cell volumes were seeded in 24-well plates to prepare nerve cell spheres of different diameter sizes: 5X 10 ⁴ The hole can form a nerve cell sphere with the diameter of less than 80 mu m, 1X 10 ⁵ The hole can form a nerve cell sphere with the diameter of 80-120 mu m, 5X 10 ⁵ The hole can form a nerve cell sphere with the diameter of 120-200 mu m, 1X 10 ⁶ The hole can form a nerve cell ball with the diameter of 180-300 mu m. The prepared neural cell sphere is shown in fig. 1.

2.2 cell status after recovery of the neurocyte sphere (diameter 80-120 μm)

The cell state of the above-mentioned nerve cell sphere (diameter 80-120 μm) after 6 weeks of cryopreservation is shown in FIG. 2, and the results show that the cell state is good, the cell sphere edge is smooth, and almost no dead cells are present.

2.3 detection result of viability of neural cell balls (diameter 80-120 μm) after 6 weeks of cryopreservation

After digesting the cryopreserved neural cell balls (diameter 80-120 μm) of 6 weeks, the cell number and cell viability rate were measured by a countess cell counter, and the results are shown in fig. 3, which indicates that the viability rate of the neural cells of 6 weeks was 90% or more.

2.4 detection result of viability of neural cell balls (diameter 80-120 μm) after 12 weeks of cryopreservation

After digesting the neural cell balls (diameter 80-120 μm) cryopreserved for 12 weeks, the number of cells and the cell viability were measured using a countess cell counter. The results of the examination are shown in FIG. 4, which shows that the survival rate of the cryopreserved neural cell balls for 12 weeks is 85% or more.

2.5 cell status after recovery of the neurocyte sphere (diameter 120-200 μm)

The cell state of the nerve cell sphere (diameter 120-200 μm) after 6 weeks of cryopreservation is shown in FIG. 5, and the results show that the cell state is good, the cell sphere has smooth edges, and almost no dead cells.

2.6 detection results of viability of neural cell spheres (diameter 120-200 μm) after 6 weeks of cryopreservation

After digesting the nerve cell balls (diameter 120-200 μm) cryopreserved for 6 weeks, the number of cells and the cell viability rate were measured by a countess cell counter, and the results are shown in fig. 6, which indicates that the viability rate of the nerve cells cryopreserved for 6 weeks was 85% or more.

2.7 detection result of viability of neural cell spheres (diameter 120-200 μm) after 12 weeks of cryopreservation

After digesting the cryopreserved neural cell balls (diameter 120-200 μm) of 12 weeks, the cell number and cell viability rate were measured by a countess cell counter, and the results are shown in fig. 7, which indicates that the viability rate of the neural cells of 12 weeks was 80% or more.

2.8 cell status after recovery of the neurocyte sphere (diameter 180-300 μm)

The state of cells after 6 weeks of cryopreservation of the neurocyte beads (diameter 180-300 μm) is shown in FIG. 8, which indicates that the color of the middle of the cell beads is darker and that some cells may die.

2.9 detection result of viability of neural cell balls (diameter 180-300 μm) after 6 weeks of cryopreservation

After digesting the cryopreserved neural cell balls (diameter 180-300 μm) for 6 weeks, the cell number and cell viability rate were measured by countess cell counter, and the results are shown in fig. 9, which indicates that the viability rate of the neural cells for 6 weeks was less than 60%.

2.10 detection result of viability of nerve cell spheres (diameter less than 80 μm) after 6 weeks of cryopreservation

After tryple digestion, frozen neural cell balls (diameter less than 80 μm) were used to test the cell number and cell viability using a countess cell counter, the results are shown in fig. 10, and the results show that the viability of the neural cells in 6 weeks of frozen storage is less than 65%.

2.11 comparison of fresh and cryopreserved neural cell sphere (120-200 μm in diameter) markers

With fresh neurospheres as control, the number of dead cells (caspase 3 ratio) in the neurospheres frozen for 12 weeks was less than 5% (fig. 11), indicating that the neurospheres could maintain over 90% of viability after being frozen for 12 weeks.

In addition, TUJ1 is a tubulin believed to be involved in neuronal cell type-specific differentiation. Tubulin is the major structure of microtubules, is a component of the cytoskeleton, and plays a role in maintenance of cell structure, mitosis, meiosis, intracellular transport, and the like. Therefore, immunofluorescence was identified for the marker TUJ1 of neuronal tubulin before and after cryopreservation, and the results demonstrated that the proportion of nerve cells in the nerve cell sphere, TUJ1+, was about 70%, and the proportion of TUJ1 in the nerve cell sphere after cryopreservation was still maintained at about 70% (fig. 11), indicating that the neuronal cytoskeleton was not damaged by cryopreservation of the nerve cell sphere.

2.12 detection of adherence Rate after Resuscitation of neural cell spheres (diameter 120-200 μm)

Adherent culture of the neural cell balls after being frozen for 1 month shows that the neural cell balls after being frozen still have the adherent capacity, and the cell adherence rate is 80% (fig. 12).

2.13 determination of the viability of the neurocyte spheres (diameter 120-200 μm) in the animal

Single cell preparation: i.e.the cell suspension obtained in step b) of 1.1 above.

Neurosphere preparation: i.e. the nerve cell sphere of diameter 120-200 μm obtained in step d) of 1.1 above.

And the total number of cells in the single cell preparation and the neurosphere preparation is substantially the same.

The survival ability of the single cell preparation and the preparation of the nerve cell ball in vivo is compared, the slicing result shows that the preparation form of the nerve cell ball is favorable for the survival of the nerve cells in vivo, and the counting result shows that the survival number of the cells transplanted by the nerve cell ball is 4 times that of the single cell transplantation (figure 13).

The above-mentioned embodiments are provided to further explain the objects, technical solutions and advantages of the present invention in detail, and it should be understood that the above-mentioned embodiments are only examples of the present invention and should not be used to limit the scope of the present invention, and any modifications, equivalents, improvements and the like made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. A neural cell sphere comprising a plurality of nerve-associated cells comprising neural precursor cells, neural cells, glial cells, or any combination thereof, the neural cell sphere having a diameter of 30-500 μm, preferably 80-300 μm (such as 80-120 μm, 120-200 μm, or 180-300 μm), more preferably 80-200 μm (such as 80-120 μm or 120-200 μm).

2. The neurosphere of claim 1, wherein the neurosphere expresses the marker TUJ1, and/or the neurosphere does not express or underexpresses the marker caspase3;

preferably, at least about 55% (e.g., at least about 60%, at least about 65%, or at least about 70%) of the nerve-associated cells in the neurosphere express marker TUJ1;

preferably, at least about 70% of the nerve-associated cells in the neurosphere express the marker TUJ1;

preferably, no more than about 40% (e.g., no more than about 30%, no more than about 20%, no more than about 15%, no more than about 10%, or no more than about 5%) of the nerve-associated cells in the neurosphere express the marker caspase3;

preferably, no more than about 15% (preferably no more than about 10% or no more than about 5%) of the neuro-relevant cells in the neurosphere express the marker caspase3.

3. The neural cell sphere of any one of claims 1-2, wherein the neural-related cells comprise neural precursor cells;

optionally, the nerve-associated cells further comprise nerve cells and/or glial cells.

4. The nerve cell sphere of any one of claims 1-2, wherein the nerve-associated cells are neural precursor cells.

5. The neural cell sphere of any one of claims 1-4, wherein the neural precursor cell is selected from the group consisting of a dopamine neural precursor cell, a gamma-aminobutyric acid (GABA) ergic neuronal precursor cell, a glutamatergic neuronal precursor cell, a cholinergic neuronal precursor cell, a serotonin neuronal precursor cell, a motor neuronal precursor cell, a sensory neuronal precursor cell, or any combination thereof;

preferably, the neural precursor cells are dopamine neural precursor cells.

6. The neural cell sphere of any one of claims 1-3, wherein the neural cell is selected from a dopaminergic neuron, a gamma-aminobutyric acid (GABA) ergic neuron, a glutamatergic neuron, a cholinergic neuron, a serotonin neuron, a motor neuron, a sensory neuron, or any combination thereof.

7. The neurosphere of any one of claims 1-3, wherein the glial cells are selected from the group consisting of astrocytes, oligodendrocytes, microglia, or any combination thereof.

8. A method of making the neural cell sphere of any one of claims 1-7, comprising:

inoculating a single cell suspension of nerve-associated cells into a low-attachment cell culture plate to form the nerve cell sphere, wherein the nerve-associated cells are selected from neural precursor cells, nerve cells, glial cells or any combination thereof;

wherein the seeding density of the nerve-associated cells is 5 × 10 ³ /cm ² ～5×10 ⁶ /cm ² Preferably 5X 10 ⁴ /cm ² ～5×10 ⁵ /cm ² (e.g., 5X 10) ⁴ /cm ² 、2.5×10 ⁵ /cm ² Or 5X 10 ⁵ /cm ² ) More preferably 5X 10 ⁴ /cm ² ～2.5×10 ⁵ /cm ² (e.g., 5X 10) ⁴ /cm ² Or 2.5X 10 ⁵ /cm ² )。

9. The method of claim 8, wherein the amount of the neural-related cells inoculated is 1 x 10 ⁴ 1 x 10 of hole ⁷ Per well, preferably 1X 10 ⁵ 1 x 10 of hole ⁶ Per well (e.g. 1X 10) ⁵ 5X 10 per hole ⁵ Per well or 1X 10 ⁶ Per hole), more preferably 1X 10 ⁵ pore-5X 10 ⁵ Per well (e.g., 1X 10) ⁵ Per well or 5X 10 ⁵ Per well) and the culture plate is a 24-well plate.

10. The method of any one of claims 8-9, wherein the neural-related cells comprise neural precursor cells; optionally, the nerve-associated cells further comprise nerve cells and/or glial cells.

11. The method of any one of claims 8-9, wherein the neural-related cells are neural precursor cells.

12. The method of any one of claims 8-11, wherein the neural precursor cell is selected from a dopamine neural precursor cell, a gamma-aminobutyric acid (GABA) ergic neuronal precursor cell, a glutamatergic neuronal precursor cell, a cholinergic neuronal precursor cell, a serotonin neuronal precursor cell, a motor neuronal precursor cell, a sensory neuronal precursor cell, or any combination thereof;

preferably, the neural precursor cells are dopamine neural precursor cells.

13. The method of any one of claims 8-10, wherein the neural cell is selected from a dopaminergic neuron, a gamma-aminobutyric acid (GABA) energetic neuron, a glutamatergic neuron, a cholinergic neuron, a serotonin neuron, a motor neuron, a sensory neuron, or any combination thereof.

14. The method of any one of claims 8-10, wherein the glial cells are selected from the group consisting of astrocytes, oligodendrocytes, microglia, or any combination thereof.

15. The method according to any of claims 8-14, wherein the culture plate is a round or sharp-bottomed microplate, such as AggreWell ^TM And (5) culturing the plate.

16. The method of any one of claims 8-15, wherein the medium used at the time of inoculation is an inoculation medium comprising:

brain-derived neurotrophic factor (BDNF),

glial cell line-derived neurotrophic factor (GDNF),

transforming growth factor beta 3 (TGF-beta 3),

the amount of ascorbic acid is such that,

calcium dibutyryladenosine cyclophosphate (db-cAMP),

DAPT(LY-374973)；

optionally, the inoculation medium further comprises:

Y27632。

17. the method of claim 16, wherein the inoculation medium has one or more technical characteristics selected from the group consisting of (1) - (7) below:

(1) The concentration of the brain-derived neurotrophic factor (BDNF) is 1-100ng/mL, preferably 10-20ng/mL;

(2) The concentration of the glial cell line-derived neurotrophic factor (GDNF) is 1-100ng/mL, preferably 10-20ng/mL;

(3) The concentration of the transforming growth factor beta 3 (TGF-beta 3) is 0.5-50ng/mL, preferably 1-10ng/mL;

(4) The concentration of the ascorbic acid is 0.01-2mM, preferably 0.2mM;

(5) The concentration of calcium dibutyryladenosine cyclophosphate (db-cAMP) is 0.01-5mM, preferably 0.5mM;

(6) The concentration of the DAPT (LY-374973) is 1-100 μ M, preferably 10 μ M;

(7) The concentration of Y27632 is 1-50. Mu.M, preferably 10. Mu.M.

18. The method of any one of claims 16-17, wherein the inoculation medium further comprises: a basal medium;

preferably, the basal medium comprises:

Neurobasal，

B27-supplement，

Glutamax；

preferably, the basal medium has one or more technical features selected from the following (1) to (4):

(1) In the basic culture medium, the volume fraction of Neurobasal is 70-99%, preferably 97%;

(2) In the basic culture medium, the volume fraction of the B27-supplement is 0.1-20%, and preferably 2%;

(3) In the basic culture medium, the volume fraction of Glutamax is 0.1-10%, preferably 1%;

(4) The sum of the volume fractions of Neurobasal, B27-supplement and Glutamax is 100%.

19. The method of any one of claims 8-18, wherein the single cell suspension of neural-associated cells is prepared by:

digesting the nerve-associated cells to obtain a single cell suspension of the nerve-associated cells;

preferably, after the digestion, the method further comprises: adding the inoculation medium of any one of claims 16-18;

preferably, the digestion is performed using Tryple or Accutase.

20. A neural cell sphere produced by the method of any one of claims 8-19;

preferably, the diameter of the nerve cell sphere is 30-500. Mu.m, preferably 80-300. Mu.m (e.g., 80-120. Mu.m, 120-200. Mu.m, or 180-300. Mu.m), more preferably 80-200. Mu.m (e.g., 80-120. Mu.m, or 120-200. Mu.m);

preferably, said neurosphere expresses marker TUJ1, and/or said neurosphere does not express or under express marker caspase3;

21. A neural cell sphere cryopreservation formulation comprising:

a nerve cell sphere as defined in any one of claims 1 to 7 or claim 20.

22. The preparation of claim 21, wherein the neural cell sphere cryopreservation preparation further comprises:

freezing and storing the neural cell balls;

preferably, in the frozen stock solution of the nerve cell ball, the total cell density of the nerve cell ball is 10 ⁵ /mL～10 ⁷ mL, preferably 3X 10 ⁶ /mL。

23. The formulation of claim 22, wherein the neural cell sphere cryopreservation solution comprises:

the basic components of the mixture are mixed,

B27 Supplement，

N2 Supplement，

Glutamax，

DMSO，

Y27632，

human Serum Albumin (HSA) or fetal bovine serum,

the basic component is selected from Neurobasal, KODMEM/F12 or the combination thereof;

preferably, the neural cell sphere cryopreservation solution further comprises: an impermeable protectant;

preferably, the non-osmotic protective agent is selected from dextran, sodium alginate, sodium hyaluronate, gamma-polyglutamic acid, or any combination thereof.

24. The preparation of claim 23, wherein the neural cell sphere cryopreservation solution has one or more technical characteristics selected from the following (1) to (9):

(1) The volume fraction of the base component is 1-84%, preferably 79%;

(2) The volume fraction of the Neurobasal is 1-84%, preferably 39.5%;

(3) The volume fraction of the KODMEM/F12 is 1% -84%, and the preferred volume fraction is 39.5%;

(4) The volume fraction of the B27Supplement is 0.1-20%, and the volume fraction is preferably 4%;

(5) The volume fraction of the N2 Supplement is 0.1-10%, preferably 1%;

(6) The volume fraction of the Glutamax is 0.1-10%, preferably 1%;

(7) The volume fraction of DMSO is 5% -20%, preferably 10%;

(8) The concentration of the Y27632 is 5-20 mu M, preferably 10 mu M;

(9) The volume fraction of the Human Serum Albumin (HSA) or the fetal bovine serum is 1-10%, preferably 5%.

25. A method of preparing the neural cell sphere cryopreservation formulation of any one of claims 22-24, comprising:

centrifuging the neural cell sphere of any one of claims 1-7 or claim 20, discarding the supernatant to obtain a neural cell sphere pellet;

mixing the neural cell sphere sediment with a neural cell sphere cryopreservation solution to enable the neural cell sphere to be resuspended in the neural cell sphere cryopreservation solution to obtain a cell sphere resuspension solution, wherein the cell sphere resuspension solution is the neural cell sphere cryopreservation preparation;

26. The method of claim 25, wherein the neural cell sphere cryopreservation solution is as defined in any one of claims 23 to 24.

27. The method of any one of claims 25-26, wherein the method further comprises:

freezing the cell ball heavy suspension, and then placing the cell ball heavy suspension in liquid nitrogen for preservation;

preferably, the freezing treatment is carried out at a temperature of-80 ℃ or less for 8 to 15 hours.

28. The method according to any one of claims 25 to 27, wherein the neural cell sphere cryopreservation solution is previously subjected to cold storage treatment;

preferably, the refrigeration treatment is carried out at a temperature of 0-4 ℃.

29. A neuronal cell pellet cryopreservation formulation prepared by the method of any one of claims 25 to 28.

30. Use of a neurosphere according to any one of claims 1 to 7 or 20 or a neurosphere preparation according to any one of claims 21 to 24 or 29 in the manufacture of an agent or medicament for use in one or more of:

(1) For nerve cell transplantation;

(2) For the treatment of neurodegenerative diseases, preferably Parkinson's disease.

31. A method of making neurons in vitro comprising: culturing the neurosphere of any one of claims 1-7 or 20 under conditions suitable for differentiation of the neurosphere of any one of claims 1-7 or 20.

32. A neuron prepared by the method of claim 31.