CN113046309A

CN113046309A - Culture medium for suspension culture of brain organoid and application thereof

Info

Publication number: CN113046309A
Application number: CN202110324252.XA
Authority: CN
Inventors: 马峰; 张勇刚; 赖默温
Original assignee: Chengdu Yunce Medical Biotechnology Co ltd
Current assignee: Chengdu Yunce Medical Biotechnology Co ltd
Priority date: 2021-03-26
Filing date: 2021-03-26
Publication date: 2021-06-29
Anticipated expiration: 2041-03-26
Also published as: CN113046309B

Abstract

The invention belongs to the field of biology, and particularly relates to a culture medium for suspension culture of brain organoids and application thereof. The culture medium comprises a first-stage culture medium, a second-stage culture medium, a third-stage culture medium and a fourth-stage culture medium; the culture medium comprises 1-2% of NEAA, 1-2% of Glumax 1-2% and 80-120nM of Mercaptoethanol; the first stage culture medium, the second stage culture medium and the third stage culture medium comprise polyvinyl alcohol. The culture medium is used for successfully separating the mesenchymal stem cells from the brain organoid, the early-stage pluripotent stem cells adopt a suspension culture method, the damage to the cells is small, the yield is high, and the polyvinyl alcohol is creatively added into the later-stage cell culture medium, so that the whole system can stably and efficiently generate the organoid; the obtained mesenchymal stem cells have the application of preparing the medicines in the field of nerve repair, and particularly have wide application prospect in preparing the medicines for treating the Alzheimer's disease.

Description

Culture medium for suspension culture of brain organoid and application thereof

Technical Field

The invention belongs to the field of biology, and particularly relates to a culture medium for suspension culture of brain organoids and application thereof.

Technical Field

Organoids (Organoids) are micro-organs with three-dimensional structures that are cultured in an in vitro environment, possess complex structures similar to real organs, and can partially mimic the physiological functions of the source tissue or organ. By means of organoids, researchers can deeply observe the change of human tissues, better understand the development process, and can be used for regenerative medicine and drug efficacy screening. Therefore, the organoid research has wide development prospect.

Under certain culture conditions, organoids are healthy and capable of developing long enough to produce a wide range of cell types, usually found in the human cerebral cortex. These advances mean that brain organoids can now be used as a viable experimental system to directly study disease in patient tissues and compare the effects of different drugs on human brain tissue.

Brain organoids can form various brain regions. They exhibit functionality and neurons capable of electrical excitation. They are similar to human cortical development at gene expression levels.

Neural (precursor) cell transplantation is an important tool for treating neurodegenerative diseases. The main source of neural (precursor) cells is aborted fetuses, and a number of relevant clinical studies have been carried out in the united states, leading to clinical stage 2, with therapeutic efficacy, and termination of clinical trials for ethical reasons. Human pluripotent stem cells (ES, iPSCs) are induced to differentiate in vitro into new methods for obtaining transplanted cells. Nerve cells have highly complex 3D structures, and two-dimensional planar culture cannot obtain cells with optimal functions. Lacaster in 2012 cultured brain organoids with typical human brain tissue structure and most of the constituent cell types (ischemic vasculature and oligodendrocytes) using human pluripotent stem cells, provided an ethical solution for studying human brain development.

Mesenchymal stem cells are a research branch of stem cells, a type of cells having the ability to self-replicate and differentiate in multiple directions, which can constantly self-renew and under specific conditions transform into one or more cells constituting a tissue or organ of the human body. Stem cells are the cells of origin of the body and are the progenitor cells that form various tissues and organs of the human body.

However, how to select the components of the brain organoid culture medium and how to set the proportion of each component still remain the difficult problems that the skilled person needs to overcome.

Disclosure of Invention

The invention discloses a culture medium for suspension culture of brain organoids, which can be used for separating and purifying a group of mesenchymal stem cells from telencephalon organoids, wherein the mesenchymal stem cells have the potential for the field of nerve repair.

Specifically, the technical scheme of the invention is as follows:

a culture medium for suspension culture of a brain organoid, the culture medium comprising a first stage medium, a second stage medium, a third stage medium, and a fourth stage medium;

the culture medium comprises 1-2% of NEAA, 1-2% of Glumax 1-2% and 80-120nM of Mercaptoethanol;

the first stage culture medium comprises:

GMEM basal medium, KOSR 12-18%, Dorsomorphin0.8-1.5 mu M, A83-010.8-1.2 mu M and polyvinyl alcohol 0.5-1.5%;

the second stage culture medium comprises: GMEM basic culture medium, N21-2%, SB-4315420.8-1.2 mu M, CHIR-990210.8-1.2 mu M, polyvinyl alcohol 0.8-1.5%;

the third stage medium comprises: MEM basic culture medium, N21-2%, SB-4315420.8-1.2 mu M, CHIR-990210.8-1.2 mu M, Martigel 0.8.8-1.5% and polyvinyl alcohol 0.8-1.5%;

the fourth stage medium comprises: GMEM basic culture medium, N21-2%, B272-3% and insulin 2 mug/mL.

SB 431542 is a potent small molecule inhibitor that selectively inhibits the transforming growth factor β (TGF- β) type I receptor activator receptor-like kinase ALK5(IC50 ═ 94nM), ALK4(IC50 ═ 140nM) and ALK 7. Has no inhibitory activity on other ALK family branch members binding to BMP protein, such as ALK2, ALK3 or ALK 6. SB 431542 inhibits TGF- β induced proliferation of osteosarcoma cells and promotes proliferation, differentiation and sheet formation of Embryonic Stem Cell (ESC) -derived endothelial cells. By specifically blocking the ALK/Smad signaling pathway, SB 431542 is able to maintain not only the self-renewal and embryoid body formation of human embryonic stem cells, but also pluripotent stem cells. SB 431542 induced DC cell maturation in vitro while showing antitumor activity in vivo. Use of SB 431542 in immune tolerant patients associated with TGF β activity may activate an anti-tumor immune response.

CHIR-99021(CT99021) is a potent selective GSK-3 α/β inhibitor with an IC50 of 10nM and 6.7 nM. The selectivity of CHIR-99021 to GSK-3 is more than 500 times higher than that of CDC2, ERK2 and other protein kinases. CHIR-99021 is also a potent activator of the Wnt/beta-catenin signaling pathway. CHIR-99021 enhances the self-renewal of mouse and human embryonic stem cells. CHIR-99021 induces autophagy (autophagy).

Preferably, the culture medium comprises 1% polyvinyl alcohol.

The second aspect of the invention discloses a method for preparing mesenchymal stem cells by using the culture medium, which comprises the following steps:

s1: adopting a first-stage culture medium to culture the human pluripotent stem cells in a suspension manner;

s2: replacing the culture medium with a second stage culture medium, a third stage culture medium and a fourth stage culture medium in sequence D4-D6, D7-D14 and D15-D21;

s3: transferring the cells into a micro organoid bioreactor, culturing for 3-4 days, inoculating into a gelatin-coated culture container, and performing adherent culture by using a first-stage culture medium of N-MSCs to obtain the mesenchymal stem cells.

Preferably, the first-stage culture medium comprises Y-27632, and the concentration of the Y-27632 is 8-12 mu M.

Preferably, the method further comprises step S4: and (3) when the confluence rate of the mesenchymal stem cells in the S3 is 70-80%, carrying out subculture by adopting a serum-free MSCs culture medium.

In some embodiments of the invention, in S1, hPSCs are cultured on 35mm dishes according to the conventional culture method, the culture medium is gradually increased from 2ml to 3ml, and the culture medium is changed every day with the change of culture medium gradually advanced. When the culture was carried out to a density of 90%, the obtained human pluripotent stem cells were digested and designated as day 0.

In some embodiments of the invention, the enzymatic digest of hPSCs is 0.5mM EDTA PBS.

Preferably, Y-27632 is added to the first stage medium.

More preferably, the concentration of Y-27632 is 8-12. mu.M.

In some embodiments of the invention, in S4, the digest is Accutase or collagenase type 1 (1 mg/ml).

The subsequent passaged digestive enzymes of mesenchymal stem cells were recombinant pancreatin (0.05% pancreatin in DHanks).

In some embodiments of the invention, at S4, when the adherent cells are 70% confluent, subculture is performed using commercial serum-free MSCs medium, and the N-MSCs cultured at this time are designated as P1 passages. At P3 passages, cells were harvested for MSCs-associated surface marker identification and pluripotency potential identification.

In some embodiments of the invention, the serum-free MSCs medium is LONZA 12-725FUltracultURE serum-free medium or NcTarget medium from the department of England Biotechnology Ltd, Cat. No. RP 01020.

The third aspect of the invention discloses the mesenchymal stem cells obtained by the method.

In a fourth aspect, the invention discloses a medicament comprising mesenchymal stem cells.

Preferably, the medicament is a medicament for treating alzheimer's disease.

The fifth aspect of the invention discloses the application of the mesenchymal stem cells in the neural restoration field. Preferably, the mesenchymal stem cells are applied to the preparation of the medicine for treating the Alzheimer disease.

The sixth aspect of the invention discloses the application of the culture medium in preparing the mesenchymal stem cells.

Compared with the prior art, the invention has the following beneficial effects:

the invention discloses a culture medium for suspension culture of brain organoids, which can be used for preparing and separating mesenchymal stem cells, wherein the early-stage pluripotent stem cells are subjected to suspension culture by adopting a suspension culture method, so that the damage to the cells is small, the yield is high, polyvinyl alcohol is added in the later-stage cell culture medium in an invasive manner, the shearing force of a culture is changed, the early-stage induced differentiation condition is adapted to the shearing force of the suspension culture, and the use of induction factors can be reduced. The obtained mesenchymal stem cells have the application of preparing the medicines in the field of nerve repair, and particularly have wide application prospect in preparing the medicines for treating the Alzheimer's disease.

Drawings

FIG. 1 is a schematic view of a microscope of the prepared mesenchymal stem cell (A: a morphological diagram of the mesenchymal stem cell under the microscope, B: a schematic view of mesenchymal stem cell adipogenic differentiation, C: a schematic view of mesenchymal stem cell osteogenic differentiation, and D: a schematic view of mesenchymal stem cell chondrogenic differentiation).

FIG. 2 is a diagram of a flow-type positive result of the prepared mesenchymal stem cells;

FIG. 3 is a flow negative result diagram of the prepared mesenchymal stem cells;

FIG. 4 is a graph showing the results of the mouse behavior experiment-open field experiment (centrometer: cm; Total Distance) implanted with mesenchymal stem cells.

FIG. 5 is a graph showing the results of the mouse behavior experiment-open field experiment (experiments in central zone: number of Entries in central zone; Time in central zone: Time in central zone; Distance in central zone: Distance in central zone; rejection amount: feeling of depression) in which mesenchymal stem cells were implanted.

FIG. 6 is a diagram showing the experimental behavior of mice implanted with mesenchymal stem cells-new Object recognition (Novel Object).

FIG. 7 is a graph of conditioned fear (Contextual fear: hippocampal dependency; Cued fear: amygdala dependency) of a mouse implanted with mesenchymal stem cells.

FIG. 8 is a behavioral experiment of mice implanted with mesenchymal stem cells-Barens maze pattern one (escape latency).

FIG. 9 is a mouse behavioral experiment implanted with mesenchymal stem cells-Barens maze two (Latency 1st entry to Target: delay first entry into Target).

FIG. 10 is a mouse behavioral experiment implanted with mesenchymal stem cells-Barnes maze diagram III (Latency 1st entry to Target: delayed first entry into the Target.

FIG. 11 is a graph of the results of sequencing differential gene expression of different mesenchymal stem cells.

FIG. 12 is a flow chart of the calculation of the length of a nascent neuron;

FIG. 13 is a schematic diagram of neurons under a confocal microscope;

FIG. 14 is a graph of neuronal Length of mesenchymal stem cells between different groups (Neuron Length).

FIG. 15 is a graph of mouse brain slice-neuron complexity (interaction Number: Number of segments).

FIG. 16 is a confocal microscope of mouse brain slices.

FIG. 17 is a diagram showing morphological analysis of mouse brain section-Spine (Thin: spindle type, Stubby: dumbbell type, Mushroom: Mushroom type).

FIG. 18 is a graph showing the brain slice-neural stem/progenitor cell count in mice (SOX2 +: SOX2 single positive; SOX2+ GFAP +: SOX2 and GFAP common positive).

FIG. 19 is a graph showing mouse brain slice-microglial morphological analysis (Iba-1positive cells: Iba-1positive cells; interaction Number: Number of segments).

FIG. 20 is a mouse brain slice-microglia morphogram II (Average soma size; Branch length: Branch length; Branch: Branch).

FIG. 21 is a graph comparing the absence and addition of 1% polyvinyl alcohol during brain organoid culture.

FIG. 22 is surface marker expression of brain organoids after addition of polyvinyl alcohol.

FIG. 23 shows brain organoids at various culture periods after addition of polyvinyl alcohol.

Detailed Description

The present application is further illustrated by the following detailed examples, which should be construed to be merely illustrative and not limitative of the remainder of the disclosure.

The instruments, equipment, reagents used in the examples are available from various sources, for example, purchased, or may be prepared.

Example 1

The embodiment discloses a culture medium for suspension culture of brain organoids, which comprises a first-stage culture medium, a second-stage culture medium, a third-stage culture medium and a fourth-stage culture medium; the following formulation percentages in the medium are by volume.

The formulation of the first stage medium was as follows:

GMEM basal medium, NEAA 1%, Glumax 1%, Mercaptoethanol100nM, KOSR 15%, Dorsomorphin 1 mu M, A83-011 mu M and polyvinyl alcohol 1%.

The preparation method of the first-stage culture medium comprises the following steps: NEAA 1%, Glumax 1%, Mercaptoethanol100nM, KOSR 15%, Dorsomorphin 1. mu. M, A83-011. mu.M and polyvinyl alcohol 1% were added to GMEM basal medium in one portion.

The second stage culture medium comprises: GMEM basic culture medium, NEAA 1%, Glumax 1%, Mercaptoethanol100nM, N21%, SB-4315421 mu M, CHIR-990211 mu M, polyvinyl alcohol 1%.

The preparation method of the second-stage culture medium comprises the following steps: 1 percent of NEAA, 1 percent of Glumax, 100nM of Mercaptoethanol, 21 percent of N, 4315421 mu of SB, M, CHIR to 990211 mu M and 1 percent of polyvinyl alcohol are added into the GMEM basic culture medium in sequence.

The third stage medium comprises: MEM basal medium, NEAA 1%, Glumax 1%, Mercaptoethanol100nM, N21%, SB-4315421 mu M, CHIR-990211 mu M, Martigel 1%, polyvinyl alcohol 1%.

The preparation method of the third-stage culture medium comprises the following steps: to MEM basal medium were added NEAA 1%, Glumax 1%, Mercaptoethanol100nM, N21%, SB-4315421. mu. M, CHIR-990211. mu. M, Martigel 1%, polyvinyl alcohol 1% in this order.

The fourth stage culture medium comprises: GMEM basal medium, NEAA 1%, Glumax 1%, Mercaptoethanol100nM, N21%, B272%, insulin 2 mug/mL.

The preparation method of the fourth-stage culture medium comprises the following steps: to GMEM basal medium were added NEAA 1%, Glumax 1%, Mercaptoethanol100nM, N21%, B272%, insulin 2. mu.g/mL in this order.

Wherein the product types are respectively:

NEAAGibco 11140050；

GlutaMAX Gibco 35050079；

N2 Gibco A1370701；

B27 Gibco 12587010；

SB-431542MCE HY-10431；

CHIR-99021MCE HY-10182

Martigel Corning 354277

polyvinyl alcohol Sigma-Aldrich, P8136-250G

Insulin Sigma-Aldrich I9278-5 ML.

Example 2

The embodiment discloses a method for preparing mesenchymal stem cells, which specifically comprises the following steps:

the hPSCs are cultured on a 35mm culture dish according to the conventional method, liquid is changed every day, the liquid changing time is gradually advanced, and the culture medium is gradually increased to 3ml from the initial 2 ml. When the density of the culture reached 90%, it was recorded as day 0.

And secondly, washing the cells twice by using DPBS, adding 1 ml/dish of hPSCs enzyme-free digestion solution, and putting the mixture into an incubator at 37 ℃ for 8 minutes. During this time, 3ml of ice-cold brain organoid 1 stage medium was prepared, and Y-27632 was added to a final concentration of 10. mu.M.

Carefully sucking the hPSCs enzyme-free digestive juice, adding 1ml of ice-cold brain organoid 1-stage culture medium containing Y-27632, quickly shaking the culture dish from side to make hPSCs clones shed, transferring the hPSCs clones to one hole of a common 6-hole plate by using a Pasteur pipette (the opening is necessarily large, and a pipette tip cannot be used), and adding the ice-cold brain organoid 1-stage culture medium of Y-27632 to 3 ml.

Four, 6 well plates were placed in an incubator with a horizontal shaker (amplitude 24mm) and incubated overnight at 60 RPM.

Fifthly, day1, microscopic cell ball with the diameter of about 250-350 μm. The number is about 600. Carefully transferring the culture to a 15ml centrifuge tube by using a Pasteur pipette, naturally settling for 3 minutes, taking about 2.5ml of the clear solution to a new 15ml centrifuge tube, centrifuging for 5 minutes at 3000RPM, removing single cells and cell fragments, adding the single cells and the cell fragments into settled cell spheres, adding a fresh room-temperature brain organoid 1-stage culture medium to 9ml, carefully mixing uniformly, and subpackaging in 3 holes of 6-hole plates, about 200 cell spheres per hole and 3ml of the culture medium. The cells were further placed in a horizontal shaker (amplitude 24mm) of an incubator at 60RPM for cultivation.

Sixthly, day2, half exchange of day3, brain organoid 1 stage medium, incubator horizontal shaker (amplitude 24mm), 60RPM culture.

Seventhly, day4-day6, and half replacing the liquid, and replacing the liquid with the brain organoid 2-stage culture medium. The incubator was incubated with a horizontal shaker (amplitude 24mm), 60 RPM.

Eighthly, day7-day14, half a day of liquid change, change in brain organoid 3-stage culture medium, horizontal shaker (amplitude 24mm) of incubator, 60RPM culture.

Ninthly, changing the culture medium of the brain organoid 4 stage half a day by day15-day21, transferring the culture medium into a micro organoid bioreactor, culturing for 21 days, and adjusting the number of each hole to 20. Wherein a micro organoid bioreactor is disclosed in patent application No. 2017212099229, 2017212027930 or 2017212119025.

And tenthly, taking out the culture from day22-day25, digesting the culture for 15min after sedimentation, blowing the culture into single cells, and performing half-liquid change once every 3 days by using a culture bottle coated with N-MSCs first-stage culture medium gelatin. And removing the suspended cells during the second half-exchange.

Eleven, when the adherent cells are 70% full, subculturing is carried out, a commercial serum-free MSCs culture medium is used, the cultured N-MSCs are recorded as P1 generation, and the schematic diagram under a cell 2 microscope is shown in figure 1. The serum-free MSCs culture medium is a 12-725FULTRACULTURE serum-free culture medium of LONZA company or a ncTarget culture medium of the Shanghai Shenyu Biotech Limited company with the product number RP 01020.

Twelve, at the P3 generation, cells were collected for MSCs-associated surface marker identification and multipotentiality identification, and the schematic representation under the cell microscope is shown in FIG. 1.

The mesenchymal stem cells of P3 generation were then subjected to flow assay, and the obtained cells were further verified to be mesenchymal stem cells, and the results are shown in fig. 2 to fig. 3.

This example also provides a control, which is the only difference from the above method in that no polyvinyl alcohol is added to the first stage medium formulation. Experiments show that the control group has no way to grow into organoids.

Example 3

This example studies the effect of mesenchymal stem cells on mouse neural repair function. This example utilizes AD model (alzheimer's disease) mice (dual transgenes, human presenilin and a β) for treatment efficacy evaluation, with the experiments divided into four groups:

PBS group: a control group;

MSC group: umbilical cord-derived MSCs;

TC group: another subject, genetically modified MSCs, over-expressing TIMP 2N-MSCs in human umbilical cord MSCs using AAV; N-MSC group: telencephalon organoid derived MSCs, i.e. mesenchymal stem cells prepared in example 2.

The experimental design was as follows:

experiment design: and randomly grouping, wherein 9 mice in each group are selected, and three mice are used as GFP retrovirus markers of the neural stem cells in the DG proliferation stage, and are mainly used for evaluating the development of new neurons and the integration capability of a neural network after the MSCs are treated in the future.

Treatment: the treatment period was 8 weeks, tail vein injection was performed once a week, and the cell group dose was 10⁵Cell/mouse. After treatment, performing a mine field experiment and a new object identification experiment at the 9 w; carrying out a Baens maze experiment from 10w to 12 w; conditioned fear experiments were performed at 13 w.

After the mice are sacrificed, the whole body organs are checked, and no obvious tumor is generated; the mice were fixed by perfusion in 6 groups, 3 mice were frozen in liquid nitrogen, and hippocampal tissue was sampled and sequenced.

The specific test results are as follows:

first, four groups of mice were subjected to the mine field experiment, and the experimental results are shown in fig. 4-5, from which it can be obtained that the four groups of mice have similar activity degrees, and the N-MSC-treated group of mice are more active and aggressive.

And secondly, carrying out a new object identification test on four groups of mice, wherein the score is%. The results are shown in FIG. 6, from which it can be seen that the treatment group scored higher for new object recognition and that the N-MSC-treated mice had better short-term memory.

And thirdly, performing a conditional fear test on four groups of mice to find that the contextual fear of the N-MSC treatment group is obviously improved, the fear test prompted by clues has no obvious difference, and the test result is shown in figure 7.

Fourthly, the Barnes maze test is carried out on the four groups of mice, the first training time is found to have obvious difference on the first day after the adaptation period, the N-MSC treatment group of mice has stronger learning ability, and the result is shown in figure 8.

The results of the training on day four showed that all mice could find the target hole within 30 seconds, indicating that the training was successful and could be used for testing, the results are shown in fig. 9.

The test result on the fifth day shows no obvious difference, which indicates that the short-term memory of four groups of mice is similar; the results at day 12 indicated that the long term memory was improved in the N-MSC treated mice, and the results are shown in fig. 10.

Fifth, the gene expression of the four groups of mice was sequenced by differential gene expression, and the results are shown in FIG. 11.

Sixthly, the length of the neurons in the brain slices of four groups of mice is analyzed, the flow is shown in figure 12, and the result schematic diagram is shown in figures 13-14.

Further study on neuron complexity resulted in longer neurons and more divergent differences with statistical significance in N-MSC group mice as shown in fig. 15.

Seventhly, neuron terminal axons (Spine) of four groups of mice were studied, and 5 μm of the terminal of a newborn neuron was photographed by a confocal microscope, and spines were classified and counted using neurolucosida, as shown in fig. 16 to 17, where Thin means spindle type, Stubby means dumbbell type, and Mushroom means Mushroom type. As can be seen from the figure, the total number of Spine in the mice in the N-MSC group is the largest, and the neuron axons are more mature.

Eighthly, the number of the neural stem/progenitor cells in brain sections of four groups of mice is researched, and the result is shown in figure 18. The treated mouse has more neural stem cells (common positive), which indicates that the MSC has stronger effect of maintaining dryness.

Ninth, the morphology of the microglia in the brain sections of the four groups of mice is analyzed, the result is shown in figure 19, the number of the microglia of the mice is reduced after the treatment that the number of the Iba-1positive cells in the DG and CA1 areas of the mice has no obvious difference, and the difference has statistical significance.

Further studies, as shown in FIG. 20, it was found that the N-MSC-treated mice had smaller glial cell bodies, fewer branches, and shorter branch lengths. FIG. 21 is a graph comparing the absence and addition of 1% polyvinyl alcohol during brain organoid culture. FIG. 22 is surface marker expression of brain organoids after addition of polyvinyl alcohol. FIG. 23 shows brain organoids at various culture periods after addition of polyvinyl alcohol.

In conclusion, the mesenchymal stem cells prepared by the method have the potential of nerve repair, and are possibly used in medicaments for treating Alzheimer's disease in the future.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Claims

1. A culture medium for suspension culture of brain organoids, wherein the culture medium comprises a first-stage medium, a second-stage medium, a third-stage medium, and a fourth-stage medium;

the first stage culture medium comprises:

2. The culture medium of claim 1, wherein the culture medium comprises 1% polyvinyl alcohol.

3. A method for preparing mesenchymal stem cells using the medium of claim 1, comprising:

4. The method of claim 3, wherein the first stage medium comprises Y-27632, and the concentration of Y-27632 is 8-12 μ M.

5. The method according to claim 3, further comprising step S4: and (3) when the confluence rate of the mesenchymal stem cells in the S3 is 70-80%, carrying out subculture by adopting a serum-free MSCs culture medium.

6. Mesenchymal stem cells obtained according to the method of any one of claims 3 to 5.

7. A medicament, comprising mesenchymal stem cells.

8. The medicament of claim 7, wherein the medicament is a medicament for treating alzheimer's disease.

9. Use of mesenchymal stem cells according to claim 6 in the field of nerve repair. Preferably, the mesenchymal stem cells are applied to the preparation of the medicine for treating the Alzheimer disease.

10. Use of the culture medium according to claims 1-2 for the preparation of mesenchymal stem cells.