Preparation method and application of mouse midbrain tissue neural stem cell high-activity single-cell suspension

Abstract

The invention relates to the technical field of neural stem cell tests, in particular to a preparation method of a mouse midbrain tissue neural stem cell high-activity single-cell suspension. The method comprises: obtaining a mouse embryo; treating the mouse midbrain tissue to obtain a cell digestion suspension of the mouse midbrain tissue; filtering and centrifuging to obtain a mouse midbrain single-cell suspension, and performing culture and passage by using a neural stem cell culture medium to obtain P1-generation or P2-generation mouse midbrain neural spheres; resuspending the P1-generation or P2-generation mouse midbrain nerve spheres by using a culture medium, digesting the P1-generation or P2-generation mouse midbrain nerve spheres by using or not using tissue digestive enzymes, and culturing by using the following culture medium to obtain the single-cell suspension of the mouse midbrain tissue neural stem cells. According to the application, the survival rate of the single cells of the neural stem cells is ensured to be more than 95%, the cell library building requirement is met, and efficient living cells are provided for Single-cell sequencing, ATCT-Sequence sequencing and the like of the neural stem cells.

Description

A method for preparing a highly active single-cell suspension of neural stem cells from mouse midbrain tissue and its applications. Technical Field

[0001] This application relates to the field of neural stem cell experimental technology, specifically to a method for preparing a highly active single-cell suspension of neural stem cells from mouse midbrain tissue and its uses. Background Technology

[0002] Stem cell research is a current hot topic in life sciences, and neural stem cells are among the most researched and applied types of stem cells. Initially thought to exist only before and shortly after birth, recent studies have shown that they also exist in the subventricular lateral ventricle (SVZ) and dentate gyrus of the hippocampus in adults. They possess multipotent potential to differentiate into neurons, astrocytes, and oligodendrocytes, exhibit self-renewal, low immunogenicity, and good tissue compatibility. In vitro culture can generate neurospheres. Many characteristics of these neurospheres have been proven usable in cell transplantation for various diseases, leading many neurologists to conduct numerous clinical trials. Because neural stem cells can provide various nerve cells to maintain and repair damaged brain tissue, they are considered a promising tool and a highly valuable natural resource for treating nervous system diseases. Central nervous system damage was once considered an intractable disease; clinical treatments could only improve symptoms and reduce complications, not fundamentally improve function. In recent years, with the continuous development of regenerative medicine, researchers have discovered that transplanting neural stem cells can effectively improve patient symptoms. Currently, basic and clinical research on neural stem cells is a hot topic and a challenge at the forefront of international science and technology.

[0003] Neural stem cell transplantation is an effective method for repairing and replacing damaged brain tissue, capable of reconstructing some circuits and functions. Clinically, there are reports of stem cells being used to treat various human neurological disorders with good results. Furthermore, stem cells can serve as gene vectors for gene therapy of intracranial tumors and other neurological diseases, overcoming some limitations of viral vectors. Because stem cells possess plasticity in generating, repairing, and altering nervous system function, combined with reprogramming technology, human-derived NSCs and their progeny can better mimic neurological diseases. However, several important practical concerns remain to be addressed before neural stem cells can be clinically translated, including the large-scale in vitro expansion of neural stem cells and the uncontrollable heterogeneity of neural stem cells. Therefore, obtaining high-viability single-cell suspensions of neural stem cells in vitro and studying their heterogeneity is crucial.

[0004] Conventional population-based transcriptome analysis methods cannot reveal the heterogeneity of gene expression among individual cells, nor can they analyze extremely rare cells. Single-cell sequencing (scRNA-seq) provides an effective research tool for this purpose. scRNA-seq is a powerful technique for advancing stem cell biology research; it can present a transcriptome map of a single cell, enabling systematic bioinformatics analysis and mining of high-throughput data obtained from sequencing. This reveals the changes in transcription factors among different cell populations during development, and is particularly suitable for revealing the changes in transcriptomes of various cell types during cell fate transitions. Preparing single-cell suspensions of neural stem cells and ensuring they meet cell library standards is crucial for elucidating the development and progression of nervous system diseases.

[0005] Currently, the most common method for obtaining neural stem cell single-cell suspensions in vitro is dilution with PBS. However, after resuspending in PBS, the survival rate of neural stem cells is low and the survival time is short. This obviously has a significant impact on the success of subsequent neural stem cell experiments, leading to serious consequences such as distorted research results.

[0006] Therefore, there is an urgent need to improve existing methods for preparing single-cell suspensions of neural stem cells in order to obtain the survival rate of various neural stem cells quickly and efficiently, so as to obtain real research results and provide technical support for the research of nervous system diseases. Summary of the Invention

[0007] This application addresses at least one of the problems of the related technology in the following aspects.

[0008] Therefore, this application provides a method for preparing a highly active single-cell suspension of mouse midbrain neural stem cells, comprising: obtaining mouse embryos; processing the mouse midbrain tissue to obtain a cell digestion suspension of the mouse midbrain tissue; filtering and centrifuging to obtain a single-cell suspension of the mouse midbrain; culturing and passaged the suspension using a neural stem cell culture medium to obtain P1 or P2 generation mouse midbrain neurospheres; resuspending the P1 or P2 generation mouse midbrain neurospheres in a culture medium; separating the cells of the P1 or P2 generation mouse midbrain neurospheres with or without tissue digestion enzymes; and resuspending the suspension using the following culture medium to obtain a single-cell suspension of neural stem cells, wherein the culture medium includes a basal culture medium, a neurotrophic factor supplement, and additives.

[0009] In some embodiments, the culture medium includes a basal culture medium and a neurotrophic factor supplement selected from one or more of the following: FGF-basic, EGF, GlutaMAX.

[0010] In some embodiments, the neurotrophic factor supplement is 15-30 ng / mL of FGF-basic, 15-30 ng / mL of EGF, and 0.5%-3% of 200 mM GlutaMAX.

[0011] In some embodiments, the neurotrophic factor supplement is 20 ng / mL of FGF-basic, 20 ng / mL of EGF, and 1% of 200 mM GlutaMAX.

[0012] In some embodiments, the culture medium further includes one or more additives selected from the following: N2 supplement, B27 supplement, NEAA, 2-Mercaptoethanol.

[0013] In some embodiments, the basal culture medium includes basal culture media selected from the group consisting of DMEM, MEM, DMEM / F12, Advanced DMEM / F12, and RPMI.

[0014] In some embodiments, the basal culture medium is DMEM / F12.

[0015] In some embodiments, the neurospheres of the brain in P1 or P2 generation mice are digested using 0.125% tissue digestive enzymes.

[0016] In some embodiments, the neurospheres of the midbrain of P1 or P2 generation mice are not digested using 0.125% tissue digestive enzymes.

[0017] In some embodiments, the digestion time is 5 minutes.

[0018] In some embodiments, a 40 μm filter screen is used for filtration.

[0019] In some embodiments, the centrifugation conditions are 600 rpm for 3 minutes.

[0020] This application also proposes a composition suitable for preparing a highly active single-cell suspension of neural stem cells from mouse midbrain tissue, comprising: basal culture medium, neurotrophic factor supplement, and additives.

[0021] In some embodiments, the basal culture medium is DMEM / F12, the neurotrophic factor supplement is 15-30 ng / mL FGF-basic, 15-30 ng / mL EGF and 0.5%-3% 200 mM GlutaMAX; the additives are N2 supplement and B27 supplement.

[0022] In some embodiments, the neurotrophic factor supplement is 20 ng / mL of FGF-basic, 20 ng / mL of EGF and 1% of 200 mM GlutaMAX.

[0023] This application also proposes the use of a single-cell suspension of brain tissue neural stem cells prepared according to any embodiment of this application for single-cell sequencing.

[0024] The embodiments of this application achieve the following beneficial effects: The method of this application is simple to operate and does not require digestion of mouse midbrain neurospheres by tissue digestive enzymes, resulting in mouse midbrain neural stem cells with high survival rates. This application identifies an effective culture medium for preparing neural stem cell single-cell suspensions, including basal culture medium, neurotrophic factor supplements and additives, as well as their corresponding types and concentrations, ensuring high activity and low cell death rates of neural stem cells. This application ensures that the survival rate of neural stem cell single cells is greater than 95%, meeting the requirements for cell library construction, and providing highly efficient live cells for neural stem cell single-cell sequencing and ATCT-Sequence sequencing; furthermore, systematic bioinformatics analysis and mining of the high-throughput data obtained from sequencing can reveal the changes in transcription factors in different cell populations during neural stem cell development and the changes in transcriptomes of various cells during cell fate transitions. The neural stem cell single cells obtained by the method of this application have high survival rates, providing effectiveness and accuracy for various single-cell sequencing methods, providing technical support for basic and clinical research on neural stem cells in the brain, and providing a crucial material basis for elucidating neural stem cell development and the occurrence and development of brain diseases. Attached Figure Description

[0025] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0026] Figure 1 shows the results of single-cell suspension of neural stem cells resuspended in PBS for analysis; Figure 2 shows the results of single-cell suspension of neural stem cells resuspended in PBS with neurotrophic factor and additives for analysis; Figure 3 shows the results of single-cell suspension of neural stem cells resuspended in DMEM / F12 for analysis; Figure 4 shows the results of single-cell suspension of neural stem cells resuspended in DMEM / F12 with neurotrophic factor and additives for analysis; Figure 5 shows the results of single-cell suspension of neural stem cells resuspended in PBS after trypsin pre-digestion for analysis; Figure 6 shows the results of single-cell suspension of neural stem cells resuspended in PBS after trypsin pre-digestion for analysis. Figure 7 shows the results of single-cell suspension of neural stem cells resuspended in PBS with neurotrophic factor and additives for analysis; Figure 8 shows the results of single-cell suspension of neural stem cells resuspended in DMEM / F12 after trypsin pre-digestion for analysis; Figure 9 compares the cell viability of the experimental group treated with DMEM / F12 + neurotrophic factor and additives before and after trypsin pre-digestion (*p≤ 0.05, p= 0.0125); Figure 10 compares the cell clumping rate of the experimental group treated with DMEM / F12 + neurotrophic factor and additives before and after trypsin pre-digestion (**p≤ 0.01, p= 0.0036). Detailed Implementation

[0027] The present invention will now be described in further detail with reference to specific embodiments. The embodiments given are merely illustrative of the invention and are not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0028] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0030] Unless otherwise specified, the quantitative analysis experiments in the following examples are all repeated three times, and the results are averaged.

[0031] Example 1 Preparation of single-cell suspension of neural stem cells from mouse brain tissue 1.1 Acquisition of embryonic mice 12.5-day-old pregnant mouse (C57BL / 6N, from Spiford (Beijing) Biotechnology Co., Ltd.) embryos were rinsed three times with pre-chilled 1X D-Hank's. The embryos were cut open with clean scissors and the mice were placed in pre-chilled 1X D-Hank's and rinsed three times.

[0032] 1.2 Digestion of Mouse Midbrain Tissue Under a dissecting microscope, the embryonic mouse brain was exposed. The meninges and vascular tissue on the surface of the brain were dissected using ophthalmic forceps, and the midbrain tissue was isolated and quickly placed into pre-chilled 1X D-Hank's solutions. In a 1.5 mL D-Hank's solution containing the midbrain tissue, 1.5 mL of 0.25% trypsin was added, and the solution was incubated at 37°C for 30 min, agitated every 8 min. Once a single round cell was observed under the microscope, the cell suspension containing the midbrain tissue was immediately transferred to a 15 mL centrifuge tube. An equal volume of cell culture medium containing 10% FBS was added to the centrifuge tube to terminate the digestion. The supernatant was discarded after centrifugation.

[0033] 1.3 Obtaining neural stem cells from the brain: Add 5 mL of neural stem cell culture medium to a centrifuge tube containing midbrain cell precipitate and suspend the cells. Filter the midbrain single-cell suspension through a 40 μm filter. Centrifuge the obtained single-cell suspension (600 rpm, 3 min), remove the supernatant, add 5 mL of neural stem cell culture medium, mix well, and obtain a neural stem cell single-cell suspension. Transfer the suspension to a 5 mL culture flask and culture and passage to P1 or P2 generation.

[0034] 1.4 Preparation of highly active neural stem cell single-cell suspension: Transfer the culture medium containing P1 or P2 generation neurospheres to a 15 mL centrifuge tube and centrifuge at 600 rpm for 3 min. After centrifugation, discard the supernatant, wash once with PBS, centrifuge at 600 rpm for 3 min, and discard the supernatant after centrifugation.

[0035] The cell pellet obtained above was divided into four equal parts and resuspended in the following four groups of liquids: Group 1 (control group A): pure PBS.

[0036] Group 2 (Experimental Group B): PBS + neurotrophic factor supplements and additives. The supplements included: FGF-basic (final concentration 20 ng / mL), EGF (final concentration 20 ng / mL), and GlutaMAX (0.2 mL stock solution, 1% of 200 mM GlutaMAX). The additives included N2 supplement and B27 supplement.

[0037] Group 3 (Control Group C): Pure DMEM / F12 medium.

[0038] Group 4 (Experimental Group D): DMEM / F12 culture medium + neurotrophic factor supplements and additives with the same composition and concentration as Group 2.

[0039] After resuspending each group, the cells were pipetted 20 times to ensure dispersion. The four resuspended cell suspensions were then filtered through a 40 μm cell filter to remove any cell clumps, thus obtaining four corresponding "neural stem cell single-cell suspensions".

[0040] 1.5 Effect Verification: Cell viability was immediately measured in four suspensions using a MONWEI cell counter, repeated four times. The results are shown in Figures 1-4. Figure 1 shows that the control group A had a low concentration of viable cells and a low survival rate, failing to meet the conditions for scRNA-seq library construction, and also exhibited a high cell clumping rate. Figure 2 shows that the experimental group B had a low concentration of viable cells, a lower survival rate compared to the experimental group D, and a higher cell clumping rate. Figure 3 shows that the control group C had a low concentration of viable cells, a lower survival rate compared to the experimental group D, and did not reach 90%. Figure 4 shows that the experimental group D had a high concentration of viable cells, a high survival rate, and a low cell clumping rate.

[0041] Furthermore, the test results were statistically analyzed, as shown in Table 1 below. It was determined that adding neurotrophic factor supplements and additives (i.e., experimental group D) to DMEM / F12 culture medium could stably maintain a cell viability of over 95%, with the lowest clumping rate and the best effect.

[0042] The comparative example used a similar method and steps as Example 1 to prepare a suspension of highly active single-cell neural stem cells from mouse midbrain tissue, except for a pre-digestion step involving digestion with a mixture of 0.125% tissue digestive enzyme trypsin and an equal volume of PBS for 5 minutes. The statistical results are shown in Table 1 below.

[0043] The results are shown in Figures 5-8. Compared with the four groups in Example 1 that were not pre-digested with trypsin, the four groups in the comparative examples with trypsin pre-digestion showed lower cell viability than the corresponding treatment groups in Example 1. However, the four groups in the comparative examples showed higher clumping rates than the corresponding treatment groups in Example 1. In other words, the cell state obtained in Example 1 without trypsin pre-digestion was better than the cell state obtained in the comparative examples with trypsin pre-digestion. Since the cell state obtained by the treatment regimen of DMEM / F12 medium supplemented with neurotrophic factor and additives (i.e., experimental group D) was the best among the four treatments in the comparative example (pre-digested with trypsin) and the four treatments in Example 1 (unpre-digested with trypsin), the relevant indicators of cells in experimental group D in the comparative example (pre-digested with trypsin) and experimental group D in Example 1 (unpre-digested with trypsin) were further compared, as shown in Figures 9 and 10 and Table 1 below. Among them, compared with experimental group D pre-digested with trypsin, the cell survival rate of experimental group D (unpre-digested with trypsin) was significantly increased (p = 0.0125) (Figure 9); compared with experimental group D pre-digested with trypsin, the cell clumping rate of experimental group D (unpre-digested with trypsin) was significantly reduced (p = 0.0036) (Figure 10).

[0044] The results show that the method of this application can, on the one hand, eliminate the step of enzyme digestion in conventional processing, saving steps and thus improving efficiency; on the other hand, it can achieve excellent results. Compared with cells obtained by pretreatment with the tissue digestive enzyme trypsin, the cell state obtained by the method of this application is better, with a cell survival rate of over 95% and the lowest clumping rate, thus efficiently preparing highly active single-cell suspensions of neural stem cells.

[0045] Table 1

[0046] Note: Experimental groups D-4', D-3', D-2' and D-1' correspond to the experimental groups D-4, D-3, D-2 and D-1 in Example 1, respectively, with the addition of the tissue digestive enzyme trypsin pretreatment step.

[0047] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0048] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for preparing a highly active single-cell suspension of neural stem cells from mouse midbrain tissue, characterized in that, include: Mouse embryos were obtained; the mouse midbrain tissue was processed to obtain a cell digestion suspension of the mouse midbrain tissue; the suspension was filtered and centrifuged to obtain a single-cell suspension of the mouse midbrain, which was cultured and passaged using neural stem cell culture medium to obtain P1 or P2 generation mouse midbrain neurospheres; the P1 or P2 generation mouse midbrain neurospheres were resuspended in culture medium, and cell separation was performed on the P1 or P2 generation mouse midbrain neurospheres with or without the use of tissue digestive enzymes, and resuspended in the following culture medium to obtain a single-cell suspension of neural stem cells, wherein the culture medium includes a basal culture medium, a neurotrophic factor supplement, and additives; the basal culture medium is DMEM / F12, the neurotrophic factor supplement is 20 ng / mL FGF-basic, 20 ng / mL EGF, and 1% 200 mM GlutaMAX, and the additives are N2 supplement and B27 supplement.

2. The method according to claim 1, characterized in that, The neurospheres of the midbrain of the P1 or P2 generation mice were digested using 0.125% tissue digestive enzymes.

3. The method according to claim 1, characterized in that, The neurospheres of the midbrain of the P1 or P2 generation mice were not digested using 0.125% tissue digestive enzymes.

4. The method according to claim 2, characterized in that, The digestion time is 5 minutes.

5. The method according to any one of claims 1-4, characterized in that, The filtration was performed using a 40 μm filter.

6. The method according to any one of claims 1-5, characterized in that, The centrifugation conditions were 600 rpm for 3 minutes.

7. A composition suitable for preparing a single-cell suspension of neural stem cells from mouse midbrain tissue, characterized in that, include: The basal culture medium, neurotrophic factor supplement, and additives are provided; wherein the basal culture medium is DMEM / F12, the neurotrophic factor supplement is 20 ng / mL FGF-basic, 20 ng / mL EGF, and 1% 200 mM GlutaMAX; and the additives are N2 supplement and B27 supplement.

8. Use of the brain tissue neural stem cell single-cell suspension prepared by the method according to any one of claims 1-6 for single-cell sequencing.

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