Method for preparing purified human neural precursor cell culture

Abstract

The invention provides a method for preparing purified human neural precursor cells (hNPCs), which comprises the following steps: two digestive enzymes are sequentially used for respectively treating a rose ring-like structure (Roset), the first digestive enzyme is used for removing parenchyma cells at the edge of the rose ring-like structure, and the second digestive enzyme is used for dispersing the human neural precursor cells.

Description

Technical Field

[0001] This application relates to the field of biomedicine, and specifically to a method for cell culture. The present invention provides a method for preparing a purified human neural progenitor cell (hNPCs) culture, which includes using two enzymes to sequentially treat rosette-like structures (Rosettes) mainly formed by neural fate cells, so as to obtain a highly purified human neural progenitor cell culture without undergoing other sorting steps. Background Art

[0002] Human neural progenitor cells are a type of stem cells with self-renewal ability, and they can differentiate into all types of nerve cells. Research shows that hNPCs and the nerve cells differentiated therefrom can migrate to nerve damaged tissues, re-establish or repair nerve circuits, achieving the purpose of disease remission and cure, and are a novel therapy with great potential.

[0003] Currently, there are two mainstream methods reported in the literature for differentiating human neural progenitor cells from embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs).

[0004] The first method is the embryoid body (EB) method. This method digests ESCs or iPSCs to form EBs, and suspends the EBs in a neural differentiation medium for directed neural differentiation; then transfers to adherent culture, and cells slowly crawl out of the EBs and form rosette-like structures; finally, hNPCs are obtained by manual selection.

[0005] The second method is the monolayer cell culture (monolayer) method. This method is simple to operate and does not require the formation of EBs, and is also called the direct culture method. In this method, the digested ESCs or iPSCs are always cultured adherently in a neural differentiation medium, and the differentiated hNPCs will form rosette-like structures, and hNPCs are amplified by multiple digestions and passages.

[0006] In both methods, neural fate cells differentiated from pluripotent stem cells form rosette-like structures, which contain hNPCs, but are surrounded by non-hNPC heterologous cells on all sides. Therefore, no matter which of these two methods is used, hNPCs need to be selected from the rosette-like structures for subsequent culture or application, such as further culturing to form neurospheres and / or for further differentiation. In existing practices, the process of successfully selecting hNPCs from rosette-like structures largely depends on the experience of the operator performing the selection, resulting in high operation difficulty, low efficiency, and poor repeatability in this step, making it difficult to scale up the entire method.

[0007] Therefore, there is an urgent need in the art to develop a new method that is simple and amenable to process scale-up to obtain high-purity hNPCs. Summary of the invention

[0008] The inventors found through repeated experiments that by using different enzymes in order to digest the rosette-like structure in steps, specifically using a first digestive enzyme to remove the foreign cells at the edge of the rosette-like structure, and then using a second digestive enzyme different from the first digestive enzyme for further digestion, the cells are detached from the culture surface and dispersed from each other, thereby obtaining high-purity hNPCs from the rosette-like structure. The processing steps of this method are simple and standardized, which greatly reduces the dependence on the judgment and proficiency of the operator, making it possible to standardize and scale up a series of culture methods that need to undergo hNPCs, thereby completing the present invention.

[0009] Therefore, in a first aspect, the present invention provides a method for processing rosette-like structures to obtain a purified human neural precursor cell culture, the method comprising:

[0010] (a) treating the rosette-like structure with a first digestive enzyme, preferably the treatment dissociates the foreign cells at the edge of the rosette-like structure and leaves the central portion of the rosette-like structure undissociated; and

[0011] (b) treating the rosette-like structures treated in step (a) with a second digestive enzyme, preferably such that the central portion of the rosette-like structures is detached from the culture surface and optionally dispersed, thereby obtaining a purified human neural precursor cell culture,

[0012] The first digestive enzyme is collagenase.

[0013] Preferably, the method further comprises:

[0014] (c) culturing the purified human neural precursor cell culture obtained in step (b) into neurospheres under appropriate conditions; and

[0015] (d) digesting the neurospheres obtained in step (c) with a third digestive enzyme to obtain a further purified culture containing hNPCs.

[0016] Preferably, the second digestive enzyme is trypsin.

[0017] Preferably, the third digestive enzyme is the same as the second digestive enzyme.

[0018] In a second aspect, the present invention provides a culture comprising hNPCs obtained by the method of the first aspect, wherein PAX6 + FOXG1 + SOX2+ The proportion of hNPCs is at least 80%, preferably at least 85%, of the total number of all cells in the culture.

[0019] In a third aspect, the present invention provides a differentiation method for preparing neural cells or cerebral organoids using the culture containing hNPCs of the second aspect. The neural cells include but are not limited to neurons, neuronal precursor cells, astrocytes, astrocyte precursor cells, oligodendrocytes, and radial glial cells. The cerebral organoids preferably contain cells of all cortices of the brain.

[0020] In a fourth aspect, the present invention provides a combination of enzymes, the combination comprising a first digestive enzyme and a second digestive enzyme, wherein the first digestive enzyme is collagenase and the second digestive enzyme is trypsin.

[0021] Preferably, the first digestive enzyme and the second digestive enzyme are sequentially used to treat the rosette-like structure to obtain a purified culture containing hNPCs.

[0022] In a fifth aspect, the present invention provides a kit, the kit comprising a first digestive enzyme and a second digestive enzyme, wherein the first digestive enzyme is collagenase, the second digestive enzyme is trypsin, and the first digestive enzyme and the second digestive enzyme are contained in separate containers.

[0023] The purification method of the present application avoids manual selection, is simpler to operate, has higher throughput, and the obtained hNPCs can be differentiated into mature neurons.

[0024] This method replaces the traditional mechanical cutting or manual selection operation, improves the certainty and stability of the operation of selecting human neural precursor cells, and is suitable for large-scale production.

[0025] The advantages of the present invention are at least in the following aspects.

[0026] 1. The cell purification method of the present invention is simple to operate, does not require density gradient centrifugation (for example, compared with the method described in Babu H et al., A protocol for isolation and enriched monolayer cultivation of neural precursor cells from mouse dentate gyrus. Front Neurosci. 2011 Jul 14;5:89), does not require sorting purification based on cell type markers, and only needs to use enzyme treatment to obtain neural precursor cells with a higher purity.

[0027] 2. The high-purity hNPC cultures obtained by using the cell purification method of the present invention can be transferred to subsequent culture steps without manual selection, improving the repeatability and stability of the method and making large-scale and standardized production possible.

[0028] 3. The method of the present invention does not introduce reagents with uncertain chemical composition, such as reagents containing animal-derived components, and is therefore suitable for clinical production.

[0029] 4. In the hNPC cultures obtained by using the method of the present invention, the proportion of PAX6 + FOXG1 + SOX2 + in hNPCs is greater than 85%, and mature neurons with electrophysiological functions can be successfully differentiated from such cultures in vitro, indicating that the cells obtained by the method of the present invention have ideal physiological functions. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] In order to better understand the features and advantages of the present invention, the following description of the drawings and detailed description of the invention are provided. However, those skilled in the art should understand that these are only for illustrative purposes and do not limit the present application. The scope of the present application is defined by the claims.

[0031] Figure 1 Schematic diagram of the process for preparing purified hNPCs from rosettes in Example 1, in which the two-step enzyme digestion method of the present invention was used to treat rosettes with two different enzymes successively.

[0032] Figure 2 Schematic diagram of the process for preparing purified hNPCs from rosettes in Example 2, in which rosettes were treated using a traditional mechanical method.

[0033] Figure 3 Schematic diagram of the process for preparing purified hNPCs from rosettes in Example 3, in which rosettes were treated with a non-enzyme preparation and an enzyme successively.

[0034] Figure 4 Bright-field photographs of cells before and after digestion with the first digestion enzyme in Example 1. (A) Bright-field photograph of cells before digestion; (B) Bright-field photograph of cells after digestion.

[0035] Figure 5 Bright-field photographs of hNPCs prepared by the three methods described in Examples 1, 2, and 3 on P1 Day1 and P4 Day4. (A) Bright-field photograph of the cells in Example 1; (B) Bright-field photograph of the cells in Example 2; (C) Bright-field photograph of the cells in Example 3.

[0036] Figure 6 Raw data of flow cytometry analysis of hNPCs prepared for Examples 1, 2, and 3. (A) Flow cytometry results of Example 1; (B) Flow cytometry results of Example 2; (C) Flow cytometry results of Example 3.

[0037] Figure 7 Bright-field photographs of organoids formed by culturing hNPCs prepared for Examples 1, 2, and 3 for 30 days. (A) Bright-field photograph of Example 1; (B) Bright-field photograph of Example 2; (C) Bright-field photograph of Example 3.

[0038] Figure 8 MEA electrophysiological results of organoids formed by culturing hNPCs prepared for Examples 1, 2, and 3 for 30 days. (A) Results of well B5 in Example 1; (B) Results of well C2 in Example 2; (C) Results of well C4 in Example 3.

[0039] Figure 9 Fluorescence photographs of frozen sections stained with the organoids cultured for 30 days in Example 1.

[0040] Figure 10 Fluorescence photographs of frozen sections stained with the organoids cultured for 30 days in Example 2.

[0041] Figure 11 Fluorescence photographs of frozen sections stained with the organoids cultured for 30 days in Example 3. Detailed implementation manners

[0042] Definition

[0043] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference into this application as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

[0044] Unless otherwise specifically defined elsewhere in this disclosure, all other technical and scientific terms used herein have the meanings commonly understood by those of ordinary skill in the art to which this application belongs.

[0045] In this document, including in the appended claims, unless the context clearly dictates otherwise, the singular forms of words such as "a" and "the" include their corresponding plural references.

[0046] The term "or" means "and / or" and may be used interchangeably therewith unless the context clearly dictates a different meaning.

[0047] In the context of the present invention, unless otherwise specified, "comprising", "including", and "containing" shall be understood to mean including the listed elements, such as a component, a feature, a step, or a group thereof, but excluding any other elements, such as other components, properties, and steps. As used herein, the term "comprising" or any variant thereof may be replaced with "containing", "including", or "having", or a synonymous variant. In certain embodiments, "comprising" also includes the case of "consisting of".

[0048] In the context of the present invention, without otherwise specified, all cells and tissues are of human origin.

[0049] In the context of the present invention, the term "rosette" or "neural rosette" refers to a characteristic neural stem cell aggregate at the neural progenitor stage during the differentiation of ESCs or iPSCs into neural cells, and this aggregate presents a radially organized cellular structure.

[0050] The term "neural progenitor cell" or "NPC", as used herein, refers to a cell type that can differentiate into cells in the neural lineage, including but not limited to neurons, glial progenitor cells, and glial cells, such as astrocyte progenitor cells, astrocytes, oligodendrocyte progenitor cells, and oligodendrocytes. Progenitor cells are a type of stem cell-like cells, but their replication ability and proliferation ability are weaker than those of stem cells. However, compared with fully differentiated cells, progenitor cells still have the ability to differentiate into different cell types. Therefore, progenitor cells can be used as safer seed cells for cell replacement therapy. Whether a cell (especially NPCs) has successfully differentiated into a specific cell type can be determined by morphological observation, detection of cell-specific biomarkers, and the ability to differentiate into a specific neural cell type with specific biomarkers and electrophysiological activities in vitro.

[0051] The term "embryonic stem cell" or "ESC" as used herein refers to pluripotent stem cells derived from an embryo. The term "induced pluripotent stem cell" or "iPSC" as used herein refers to pluripotent stem cells reprogrammed from differentiated somatic cells. The methods of the present invention can use ESCs or iPSCs as starting materials for directed differentiation. In certain embodiments of the present invention, the ESCs or iPSCs are of mammalian origin, particularly of human origin. The present invention does not limit the sources of stem cells including ESCs and iPSCs, as long as they meet the requirements of clinical applications. The cells can be obtained directly from commercial sources or can be prepared, for example, by reprogramming somatic cells into iPSCs.

[0052] The term "embryoid body" or "EB" as used herein refers to cell aggregates formed by growing ESCs and iPSCs through three-dimensional culture. EBs can be formed by suspension culture. However, it is difficult to obtain EBs of uniform size and shape in the traditional way of culturing EBs.

[0053] In the context of the present application, the term "neurosphere" refers to spherical aggregates formed by suspension culture of neural fate cells separated from rosette-like structures. A neurosphere is a heterogeneous population composed of different types of neural cells, including neural stem cells, neural progenitor cells, and some differentiated neural cells. In the present application, neurospheres are mainly composed of NPCs.

[0054] The term "heterogeneous cell" refers to cells other than the target cells. In the context of the present invention, heterogeneous cells refer to non-neural fate cells. Specific examples of heterogeneous cells include Schwann cells, which will develop towards non-CNS nerves during differentiation.

[0055] The term "culture medium" refers to a mixture containing various nutrients required for the growth of a certain type of cells. The culture medium can be prepared by adding additives to a basal medium. Additives in a broad sense refer to additional components required for cell culture but not included in the basal medium, including proteins, lipids, amino acids, vitamins, hormones, cytokines, growth factors, etc.

[0056] The term "purification" in the context of the present application refers to a method that increases the proportion of target cells in a cell population relative to before purification treatment.

[0057] The term "digestion" in the context of the present application refers to the process by which enzymes degrade macromolecules into small molecules. In the context of cell culture, digestion is usually used to detach cells from the culture surface and / or to separate cells from each other.

[0058] "DAPI" is a dye that shows cell nuclei by binding to DNA.

[0059] "GFAP (glial fibrillary acidic protein)" refers to glial fibrillary acidic protein, which is a marker for the maturation of astrocytes.

[0060] "MAP2 (Microtubule-associated protein-2)" is a marker for nerve cells, especially neurons.

[0061] "TUJ1 (Neuronal Class IIIβ-Tubulin)" refers to class III β-tubulin. Anti-Tuj1 antibodies that recognize only TUJ1 of nerve cells and not TUJ1 of glial cells are widely used for the immunostaining identification of nerve cells, especially neurons.

[0062] "S100β" is a marker for immature astrocytes.

[0063] Rosette structure

[0064] The method of the present invention uses nerve fate cells in the form of rosette-like structures as starting materials, and the obtained cells are human neural progenitor cells (hNPCs).

[0065] As the starting material for the method of the present invention, the rosette-like structure is a characteristic structure formed during the differentiation of pluripotent stem cells into nerve cells, which marks the development of neural progenitor cells. Very similar to the in vivo situation, under suitable conditions for in vitro culture, neural progenitor cells will spontaneously form a radial arrangement in two dimensions, similar to the structure of a flower or a wreath, and continuously increase in height as the culture progresses, forming a highly compact three-dimensional columnar nerve aggregate, that is, the rosette-like structure.

[0066] The rosette-like structure of the present invention can be obtained by differentiating ESC or iPSC. The purification method of the present invention has no limitation on the way of obtaining the rosette-like structure. The rosette-like structure can be obtained by any culture method known in the art, including but not limited to monolayer culture method and culture method via embryoid body. Examples of the EB method can be referred to the content disclosed in CN113604434A. Examples of the monolayer method can be referred to the method described in Stuart M Chambers et al., Nat Biotechnol. 2009 Mar; 27(3):275-80.

[0067] The obtained rosette-like structure should be a cell aggregate with an obvious radial arrangement, which adheres to the culture surface.

[0068] Other cell types with non-neural differentiation fates are also present around the rosette-like structures. To obtain a highly pure hNPC culture from the rosette-like structures, it is necessary to remove these unwanted components as much as possible. At the same time, such treatment should not have an unacceptable negative impact on the function of hNPCs.

[0069] Two-step enzymatic digestion method

[0070] It is desirable to isolate and purify a cell culture containing a high proportion of neural progenitor cells from the rosette-like structures, which ensures the normal progress of subsequent differentiation. Therefore, the present invention provides a method for treating rosette-like structures by two-step enzymatic digestion to obtain a purified human neural progenitor cell culture, the method comprising:

[0071] (a) treating the rosette-like structures with a first digestive enzyme; and

[0072] (b) treating the rosette-like structures treated in step (a) with a second digestive enzyme, thereby obtaining a purified human neural progenitor cell culture,

[0073] wherein the first digestive enzyme is collagenase.

[0074] In step (a), it is necessary to select an enzyme with moderate digestive ability to treat the cells, so as to achieve different degrees of digestion and dissociation of different types of cells as required. Ideally, the enzyme treatment in step (a) will keep target cells such as neural progenitor cells attached to the culture surface, while the heterogeneous cells will detach from the culture surface and dissociate from the target cells. To achieve this purpose, the inventors found that digestive enzymes commonly used in neural cell differentiation culture, such as trypsin, are not suitable because they will detach and dissociate all target cells and non-target cells indiscriminately. In the present invention, collagenase is innovatively used to achieve this purpose.

[0075] Collagenase is an enzyme that breaks peptide bonds in collagen. Collagenase can disrupt the extracellular matrix and is therefore commonly used for dissociation and detachment in cell culture. According to the differences in collagenase activity, it is divided into multiple types and is suitable for different uses.

[0076] The collagenase that can be used as the first digestive enzyme in the present invention can be selected from one or more of type I, type II, type III, and type IV collagenases. In some embodiments, a mixture of multiple collagenases can be used, such as a mixture of two, three, or four of type I, type II, type III, and type IV collagenases. In a preferred embodiment, a non-animal-derived collagenase is preferred, for example, the collagenase is derived from a microorganism such as a bacterium. In a specific embodiment, the first digestive enzyme of the present invention is type IV collagenase. In a specific embodiment, the first digestive enzyme is a mixture containing type IV collagenase. An example of a specific type IV collagenase is Collagenase (Gibco#17104019). In another specific embodiment, the collagenase is a mixture of two collagenases, such as a mixture of type I collagenase and type II collagenase, such as Collagenase NB 6GMP Grade (Amsbio#N0002779). When using a commercially available collagenase product, the collagenase may have other digestive enzyme activities. Therefore, it is preferred that the collagenase product has high activity of the corresponding type of collagenase and has low or substantially no other digestive enzyme activities such as trypsin activity.

[0077] In a preferred embodiment, the collagenase used as the first digestive enzyme is used at a concentration of 0.1 - 10 mg / mL, preferably 1 - 8 mg / mL, more preferably 3 - 5 mg / mL.

[0078] The time and conditions for treatment with collagenase should be sufficient to loosen the heterogeneous cells at the outer edge of the rosette-like structure and detach them from the adherent plane, while the part mainly composed of neural progenitor cells in the center of the rosette-like structure still remains in the adherent state. For example, the temperature for treatment with collagenase is about 37°C. For example, the cell culture can be placed in a shaker to make the digestion more complete. In a preferred embodiment, when treating with the first digestive enzyme, the cell culture is placed in a shaker, and the shaker speed is 50 - 120 rpm, preferably 70 - 100 rpm. In a preferred embodiment, especially in the case of using type IV collagenase, the digestion time of the collagenase is 5 - 20 minutes, more preferably 5 - 15 minutes, still more preferably 10 - 15 minutes.

[0079] In a preferred embodiment, before treatment with the first digestive enzyme, the rosette-like structure is washed, for example, washed one or more times with a buffer or a culture medium. For example, PBS can be used for washing. Preferably, as much liquid as possible is discarded after washing, and only the cells are retained.

[0080] After treatment with the first digestive enzyme, the detached cells and the enzyme-containing solution are removed. Preferably, after removing the contaminating cells and the enzyme-containing solution, the remaining cells are washed one or more times. For example, washing can be performed using PBS. Preferably, after washing, as much of the liquid as possible is discarded, leaving only the cells.

[0081] As the second digestive enzyme, an enzyme that is routinely used for detaching, dissociating, and / or dispersing cells in the culture of neural stem cells or neural progenitor cells can be selected. In a preferred embodiment, the second digestive enzyme has tryptic activity (EC 3.4.*.*) or has a function similar to trypsin. In a preferred embodiment, a non-animal-derived trypsin is preferred, for example, the trypsin is derived from a microorganism such as a bacterium. Such an enzyme can be a commercially available enzyme, for example, a commercially available preparation containing one or more enzymes. The second digestive enzyme of the present invention includes, but is not limited to: trypsin, Accutase, TrypLE, Versene, CTS TM TrypLE, CTS TM Versene. In a specific embodiment, the second digestive enzyme is TrypLE or CTS TM TrypLE. When using a commercially available trypsin product, the trypsin may have other digestive enzyme activities. Therefore, it is preferred that the trypsin product has high activity corresponding to the type of trypsin and has low or substantially no other digestive enzyme activities such as collagenase activity.

[0082] After treatment with the first digestive enzyme, the part mainly composed of neural progenitor cells located in the center of the rosette structure remains in an attached state. The time and conditions for treatment with the second digestive enzyme should be sufficient to detach the central part of the still-attached rosette structure from the attached plane and preferably make it loose and dispersed. For example, the temperature for treatment with trypsin is about 37 °C. For example, the cell culture can be placed in a shaker to make the digestion more complete. In a preferred embodiment, when treating with the second digestive enzyme, the cell culture is placed in a shaker, and the shaker speed is 50 - 120 rpm, preferably 70 - 100 rpm. In a preferred embodiment, the digestion time of trypsin is 8 - 10 min.

[0083] In a preferred embodiment, CTS is used TM TrypLE (Gibco#A1285901) is used as the second digestive enzyme. Specifically, the second digestive enzyme is used at the concentration recommended in the instructions of the commercially available product, for example, at a 1X concentration.

[0084] Preferably, the first and / or second digestive enzyme of the present invention does not contain animal-derived components. Preferably, the first and / or second digestive enzyme of the present invention is clinical grade, GMP grade, cGMP grade or CTS TM grade. The first digestive enzyme and the second digestive enzyme can be used in the amounts recommended by their manufacturers.

[0085] Further purification after two-step enzymatic digestion method

[0086] The two-step enzyme digestion method of the present invention can directly obtain a relatively pure culture of neural progenitor cells from the rosette-like structure. The culture containing neural progenitor cells obtained in this way can be used without further additional purification steps, or in some cases, it can also be further purified as needed to obtain a culture with higher purity.

[0087] In some embodiments, further purification is carried out by sorting the neural progenitor cells. The sorting can be carried out by magnetic bead sorting or flow cytometry by means of specific markers on the neural progenitor cells.

[0088] In a specific embodiment, further enrichment of neural progenitor cells can be carried out by forming neurospheres from the purified neural progenitor cell culture, because neural progenitor cells have a stronger tendency to spontaneously form aggregates compared to other cell types. For example, after step (b) of the method of the present invention, the following steps are further included:

[0089] (c) Culturing the purified human neural progenitor cell culture obtained in step (b) into neurospheres under suitable conditions; and

[0090] (d) Digesting the neurospheres obtained in step (c) with a third digestive enzyme to obtain a further purified culture containing hNPCs.

[0091] Preferably, the culture in step (c) is carried out in a low-attachment culture plate. This is because the low-attachment culture plate can promote the formation of neurospheres.

[0092] Preferably, the third digestive enzyme can be selected from enzymes that are routinely used for detaching, dissociating and / or dispersing cells in the culture of neural stem cells or neural progenitor cells. The third digestive enzyme can be the same as or different from the second digestive enzyme. In a preferred embodiment, the second digestive enzyme has trypsinolytic activity (EC 3.4.*.*) or has a function similar to that of trypsin. Such enzymes can be commercially available enzymes, such as commercially available preparations containing one or more enzymes. The third digestive enzyme of the present invention includes but is not limited to: trypsin, Accutase, TrypLE, Versene, CTS TM TrypLE, CTS TMVersene. In a specific embodiment, the third digestive enzyme is TrypLE or CTS TM TrypLE.

[0093] In some embodiments, during neurosphere culture, the cell seeding density is 1×10 5 -5×10 5 / cm 2 , preferably the seeding density is 2×10 5 / cm 2 .

[0094] In some embodiments, when the neurospheres are digested, the digestion time used is 10 - 15 min, preferably 12 min. After digestion, they are seeded at 1×10 5 -5×10 5 / cm 2 .

[0095] In some embodiments, the neurospheres are seeded in a well plate coated with Matrigel, preferably the Matrigel is BIOLAMININ521MX (Biolamina #MX521 / 05).

[0096] Culture medium

[0097] In the purification process of the present invention, a medium suitable for culturing human neural progenitor cells can be used. The medium can be composed of a basal medium, additives, and essential nutrients and factors for maintaining the growth and function of human neural progenitor cells.

[0098] Suitable basal media include but are not limited to: Neurobasal TM Medium (Gibco), BrainPhys TM Neuronal Medium (STEMCELL). More media suitable for human neural progenitor cells are known to those skilled in the art.

[0099] Examples of additives include B27 nutritional additive.

[0100] Nutrients and factors for maintaining the growth and function of human neural progenitor cells include but are not limited to one or more of the following: brain-derived neurotrophic factor (BDNF), glial cell-derived neurotrophic factor (GDNF), L-ascorbic acid, dibutyryl-cAMP (db-cAMP) or its salts such as sodium salt, glutamine.

[0101] In a specific embodiment, the basal medium of the human neural progenitor cell medium (hereinafter referred to as hNPC medium) is Neurobasal TMMedium (Gibco), with the additive being B-27 TM Additives, other nutritional components and factors are BDNF, GDNF, L-ascorbic acid, db-cAMP and GlutaMAX TM -I additive.

[0102] In a more specific embodiment, the hNPC medium consists of the following components: CTS TM Neurobasal TM Medium (Gibco) as the basal medium, and animal-component-free recombinant human / mouse / rat BDNF (PeproTech), animal-component-free recombinant human GDNF (PeproTech), L-ascorbic acid (VC) (Sigma), N 6 ,O 2 ’-dibutyryladenosine-3’,5’-cyclic monophosphate sodium salt (DB-cAMP) (Sigma), CTS TM GlutaMAX TM -I additive (Gibco) and CTS TM Vitamin A-free and xenogeneic-free B-27 TM additive (Gibco).

[0103] In a further more specific embodiment, the hNPC medium consists of the following components: CTS TM Neurobasal TM Medium (Gibco) as the basal medium, and 20 ng / mL animal-component-free recombinant human / mouse / rat BDNF (PeproTech), 20 ng / mL animal-component-free recombinant human GDNF (PeproTech), 0.2 mM L-ascorbic acid (VC) (Sigma), 0.5 mM N 6 ,O 2 ’-dibutyryladenosine-3’,5’-cyclic monophosphate sodium salt (DB-cAMP) (Sigma), 1 vol% CTS TM GlutaMAX TM -I additive (100×) (Gibco) and 2 vol% CTS TM Vitamin A-free and xenogeneic-free B-27 TM additive (50×) (Gibco).

[0104] Preferably, the medium used in the method of the present invention is an animal-source-free medium.

[0105] Preferably, in order to obtain neural progenitor cells suitable for clinical applications, any basal medium and additives used are of clinical grade, preferably GMP grade, cGMP grade or CTSTM Level

[0106] Preferably, the various culture media used in the method of the present invention are all chemically defined culture media. "Chemically defined culture media" means that all the components contained in the culture media are known, and thus are distinguished from culture media containing components of animal origin, especially serum. Therefore, chemically defined culture media do not contain any serum products as a source of nutrients. The advantage of using chemically defined culture media is to reduce the potential risks brought by uncertain components and unclear safety. Another advantage is that it is beneficial to adapt the whole method to the production standards of cells for clinical use.

[0107] Cell adhesion medium

[0108] The term "cell adhesion medium" refers to a medium that can help cells that do not adhere easily to grow adherently. Such a medium should be non-toxic to cells and can be a medium commonly used in cell culture, such as a Matrigel-like medium.

[0109] The term "Matrigel" is used in the same sense as "extracellular matrix (ECM)". For neural progenitor cells, an extracellular matrix is used to support the attachment and growth of cells. ECM is the extracellular surface matrix, mainly composed of proteins such as collagen, elastin, and laminin. ECM is widely used in the culture of mammalian cells and is known to those skilled in the art.

[0110] Non-limiting examples of ECM that can be used to coat a solid support or a culture surface (such as a culture plate) include: Matrigel TM , laminin, polylysine, a combination of polyornithine (PO) / fibronectin (FN) / laminin, fibronectin (FN), vitronectin, etc.

[0111] In one embodiment, the Matrigel can be a type commonly used in cell culture. For example, Matrigel, vitronectin (such as VTN-N), laminin (such as LN521, MX521) can be used. In a preferred embodiment, the culture surface is coated with laminin, such as coated with MX521. The covering concentration of laminin such as MX521 can be 5 - 20 μg / mL, preferably 6 - 8 μg / mL.

[0112] The thickness and dosage of the cell adhesion medium in a cell culture plate or dish depend on specific culture conditions, such as the type of cells being cultured. Generally speaking, the cell adhesion medium is used in an amount that enables (the vast majority of) cells to grow in an adherent state and not detach or float in the culture medium under microscopic observation. When using Matrigel as the cell adhesion medium, its dosage is much lower than that used for 3D culture.

[0113] For example, the culture surface can be coated with the cell adhesion medium. Specifically, after adding the cell adhesion medium to the culture container, it is left for a period of time and then discarded, thereby forming a thin layer. It should be understood that the thickness of the cell adhesion medium can be uneven, which helps cell attachment. The time left after adding Matrigel can be 1 to 3 hours, preferably 1.5 to 2.5 hours, for example, about 2 hours.

[0114] For example, the cell adhesion medium can be mixed with the cells to be inoculated, and the mixture is used for inoculation.

[0115] Examples

[0116] The present application is described in detail by the following examples, but the present application is not limited to the embodiments shown in these examples.

[0117] Example 1. Purification of hNPCs using a two-step enzyme digestion method

[0118] In this example, the purification method of the present invention using two enzymes for digestion in sequence is described exemplarily.

[0119] Figure 1 A general flow of the method of this example is shown. As Figure 1 shown, before the cells are inoculated in a low-attachment porous culture plate to form neurospheres, two different enzymes, namely the first digestion enzyme and the second digestion enzyme, are used to treat the cultured cells successively. The first digestion enzyme is used to remove contaminating cells, while the second digestion enzyme digests cell clusters into single cells. Then, the cells treated with the two enzymes are cultured into neurospheres, and then undergo digestion, culture, and finally form highly pure hNPCs. The specific method steps of this example are described as follows.

[0120] Step 1: Preparation of rosette-like structures

[0121] The rosette-like structures used in this example are obtained from ESCs according to the method disclosed in Example 1 of the applicant's prior application publication text CN113604434A.

[0122] Specifically, the commercially obtained human embryonic stem cell line H1 was digested with trypsin and cultured in a pluripotent stem cell medium containing 10 μM Rock inhibitor (Y-27632, Wako) in an ultra-low attachment 96-well culture plate to promote the formation of embryoid bodies.

[0123] After embryoid bodies were formed the next day, the embryoid bodies were continuously cultured for 2 days and then transferred to a low attachment culture plate and cultured in embryoid body medium for 5 days. The embryoid body medium consisted of the following components: CTS TM KnockOut TM DMEM / F-12 medium (Gibco) and CTS TM Neurobasal TM medium (Gibco) were mixed at a ratio of 1:1 (vol / vol) as the basal medium, and 50 ng / mL Noggin GMP (R&D), 1 μM Dorsomorphin (Tocris), 10 μM SB431542 (Tocris), 1 vol% of CTS TM N-2 supplement (100×) (Gibco) and 0.5 vol% of CTS TM GlutaMAX TM -I supplement (200×) (Gibco).

[0124] After that, the embryoid bodies were transferred to a 6-well culture plate coated with Laminin-521 (BioLamina, MX521 / CT521) to allow them to fully attach, and then cultured using a serum-free neural induction medium. The serum-free neural induction medium consisted of the following components: CTS TM KnockOut TM DMEM / F-12 medium (Gibco) and CTS TM Neurobasal TM medium (Gibco) were mixed at a ratio of 1:1 (vol / vol) as the basal medium, and 1 vol% of CTS TM N-2 supplement (100×) (Gibco) and 0.5 vol% of CTS TM GlutaMAX TM -I supplement (200×) (Gibco). The embryoid bodies attached to the culture plate and gradually formed adherent cell aggregates. The formation of neural-specific rosette-like structures could usually be observed on the first day after adherent culture and became more obvious on the third day, indicating the formation of neural stem cell aggregates.

[0125] Step 2: Remove heterogeneous cells using the first digestive enzyme

[0126] Before digestion, heterologous cells were observed by microscopy adhering to the edge of the rosette-like structure ( Figure 4 A). Discard the old medium for culturing the rosette-like structure, and add PBS for washing. After washing, discard the excess liquid and retain the cells. Then, add 1 mL of 2 mg / mL Collagenase (Gibco#17104019) to each well, and transfer the culture plate to a carbon dioxide-resistant horizontal shaker in an incubator at 37 °C for digestion. The shaker speed is 100 rpm, and the digestion time is 15 min. After digestion, most of the heterologous cells at the edge of the rosette-like structure were observed to have detached by microscopy ( Figure 4 B), while the human neural progenitor cells in the middle remained adherent.

[0127] Transfer the culture plate to a biosafety cabinet, and use a negative pressure pump to discard the culture solution containing Collagenase and the detached heterologous cells. Then, add 2 mL of PBS to each well for washing once to remove the remaining heterologous cells.

[0128] Step 3: Digest into single cells using a second digestive enzyme

[0129] Add 1 mL of CTS TM TrypLE (Gibco#A1285901) to each well of the above culture plate, and transfer the culture plate to a carbon dioxide-resistant horizontal shaker in an incubator at 37 °C for digestion. The shaker speed is 100 rpm, and the digestion time is 10 min. After digestion, it was observed by microscopy that the neural progenitor cells in the middle had detached in large areas and dispersed from the state of being tightly connected to each other into round single-cell forms separated from each other.

[0130] Transfer the culture plate to a biosafety cabinet, and further add hNPC medium to terminate the digestion to obtain a purified neural progenitor cell culture.

[0131] The hNPC medium consists of the following components: CTS TM Neurobasal TM medium (Gibco) as the basal medium, and 20 ng / mL animal component-free recombinant human / mouse / rat BDNF (PeproTech), 20 ng / mL animal component-free recombinant human GDNF (PeproTech), 0.2 mM L-ascorbic acid (VC) (Sigma), 0.5 mM N 6 ,O 2 ’-dibutyryladenosine-3’,5’-cyclic monophosphate sodium salt (DB-cAMP) (Sigma), 1 vol% CTS TM GlutaMAX TM -I additive (100×) (Gibco) and 2 vol% CTSTM Vitamin A-free xeno-free B-27 TM Additive (50×) (Gibco).

[0132] Step 4: Neural sphere culture

[0133] Centrifuge the cell culture obtained in Step 3 and remove the supernatant. Resuspend the cells with 2 mL of hNPC medium and pipette 3 - 5 times. Count the cells, and then seed them in a low-attachment well plate at a density of 2×10 5 cells / cm 2 of the bottom area of the culture plate. Transfer the cell culture medium into a 37 °C incubator and let it stand overnight to form neural spheres.

[0134] Step 5: Digest neural spheres to obtain hNPCs

[0135] Collect the neural spheres in Step 4 and place them together with the medium in a centrifuge tube. Let it stand for 5 min. After the neural spheres settle to the bottom of the tube, discard the supernatant. Add 2 mL of PBS, let it stand for 5 min. After the neural spheres settle to the bottom of the tube, discard the supernatant. Add 0.5 mL of CTS TM TrypLE and transfer it back to a 6-well plate. Let it stand in a 37 °C incubator for 12 min for digestion. Then add 1 mL of fresh hNPC medium to terminate the digestion, thereby obtaining a culture of further purified neural progenitor cells. Count the cells and seed them in a well plate coated with Matrigel Biolaminin 521MX (MX521) (BioLamina #MX521 / 05) at a density of 2×10 5 cells / cm 2 of the bottom area of the culture plate. The cells at this time are designated as the first passage (P1), so this time is designated as hNPCs P1 Day0.

[0136] Example 2. Purification of hNPCs by manual selection method

[0137] This example is a comparative example, Figure 2 which is a schematic flow chart of the purification method of this comparative example. As Figure 2 shown, after obtaining the rosette-like structure according to Step 1 of Example 1, only mechanical pipetting is used to detach the cells for neural sphere culture.

[0138] Specifically, rosette-like structures begin to form around the 7th day after the start of EB differentiation in the same manner as described in Example 1. On the 21st day of EB differentiation, select and isolate neural aggregates rich in neural progenitor cells under a stereomicroscope, and transfer the selected neural aggregates to a low-attachment 6-well culture plate, where each well contains 2.5 mL of hNPC medium. After culturing for 1 day, the formation of neural spheres can be observed.

[0139] The formed neurospheres were processed in the same manner as in Step 5 of Example 1 to obtain high-purity P1Day0hNPCs.

[0140] Example 3. Purification of hNPCs by conventional dissociation method

[0141] This example is a comparative example that describes a conventional dissociation method that uses a reagent specifically for selectively detaching neural rosette clusters from adherent cultured neural aggregates differentiated from pluripotent stem cells to obtain a culture rich in neural progenitor cells.

[0142] Figure 3 This is the method flow of this comparative example. As Figure 3 shown, the rosette-like structure was first treated with the enzyme-free dissociation agent STEMdiff TM Neural Rosette Select Reagent (STEMCELL Technologies; Catalog#05832), and then treated with CTS TM TrypLE for enzymatic treatment. Then, the treated cells were further purified through neurosphere formation, digestion, and culture. The specific steps are described as follows.

[0143] Step 1: Preparation of rosette-like structure

[0144] The rosette-like structure was prepared in the same manner as in Step 1 of Example 1.

[0145] Step 2: Treatment of rosette-like structure with enzyme-free dissociation agent

[0146] Discard the old medium for culturing the rosette-like structure, add 2 mL / well of PBS to the wells for one wash to remove the residue of the old medium. Then add 1 mL / well of STEMdiff TM Neural Rosette Select Reagent to the wells and operate according to the product instructions. Specifically, transfer the multi-well plate to a carbon dioxide-resistant horizontal shaker in a 37 °C incubator, with the shaker speed at 100 rpm and the incubation time at 100 min. It can be observed through a microscope that the heterogeneous cells at the edge of the rosette-like structure do not fall off, while the cell fragments in the middle become loose but cannot fall off. At this time, transfer the above culture plate to a biosafety cabinet, pipette the loose cell fragments in the middle to obtain a cell suspension and collect it into a centrifuge tube, and add medium to terminate the digestion.

[0147] Step 3: Treatment of cells with enzyme

[0148] Centrifuge to discard the supernatant, then add PBS for one wash. After centrifuging to remove the PBS, add CTS TM Add TrypLE at 1-2 mL / tube to cover the cells, transfer to a carbon dioxide-resistant horizontal shaker in a 37 °C incubator for digestion. The shaker speed is 100 rpm and the digestion time is 10 min. Observe under the microscope that the cell fragments become loose. At this time, transfer the above centrifuge tube to a biosafety cabinet, gently pipette the detached cell clusters 3-5 times with a pipette tip to disperse the cells as much as possible, and then add the old medium from step one to terminate the digestion to obtain a cell suspension.

[0149] Step four: Neurosphere culture

[0150] Perform according to the same method as step four of Example 1 to obtain neurospheres.

[0151] Step five: Digest neurospheres to obtain hNPCs

[0152] Perform according to the same method as step five of Example 1 to obtain high-purity P1 Day0 hNPCs.

[0153] Example 4. Characterization test of hNPCs obtained by different purification methods

[0154] In this example, the hNPCs obtained in Examples 1-3 were detected by various methods.

[0155] 4.1 Flow cytometry identification

[0156] Perform flow cytometry on the P1 Day0 hNPCs prepared in Examples 1-3 to observe the expression levels of FOXG1 (Forkhead box protein G1, a marker of human forebrain neural progenitor cells), SOX2 (a member of the SOX region Y-related HMG (High Mobility Group) protein family, widely expressed in neural stem cells), and PAX6 (Paired box protein Pax-6, an important transcription factor in neural development). The specific operation is as follows.

[0157] Take 3×10 6 cells of P1 Day0 hNPCs obtained by three methods. After staining with a flow cytometry live / dead dye (LIVE / DEAD TM Fixable Far Red Dead Cell Stain Kit, Thermo #L34973), fix and permeabilize, then incubate with anti-FOXG1 antibody, anti-SOX2 antibody, and anti-PAX6 antibody after blocking, and finally perform detection on the machine. The flow cytometry results are shown in Table 1 and Figure 6 .

[0158] Table 1. Positive ratios of cell markers (P1 Day0) in flow cytometry results

[0159]

[0160] The results showed that in hNPCs of P1 Day0 obtained by three different methods, the proportions of cells expressing FOXG1, SOX2, and PAX6 were all above 85%, indicating that the cell cultures obtained from rosette-like structures by the three methods all contained hNPCs at a high proportion.

[0161] 4.2 Formation of organoids

[0162] Take the hNPCs of P1 Day0 prepared in Examples 1-3 and inoculate them in a multi-well plate at a density of 2×10 5 cells / cm 2 of the bottom area of the culture plate. After inoculation, the medium was replaced semi-every 2 days, and subcultured once every 4 days using CTS TM TrypLE digestion. The media used for culture and medium replacement were both hNPC medium. The inoculation density after subculture was 2×10 5 cells / cm 2 . Repeat the operation until P4 Day4. Take bright-field photos at P4 Day4, as shown in Figure 5 .

[0163] Then use CTS TM TrypLE to digest and inoculate into a low-attachment 96-well plate to form organoids (this day is regarded as the first day of organoid culture). After that, the medium was changed 2 times a week, and the media used for culture and medium replacement were hNPC medium. Take bright-field photos 30 days after organoid culture, as shown in Figure 7 .

[0164] Figure 7 The bright-field photo results of

[0165] showed that the organoids formed by hNPCs obtained by the three isolation methods were all in good condition, with round edges and good refractive index, and there was no significant difference among the three groups, indicating that the neural progenitor cells obtained by the three methods could all form organoids with normal morphology.

[0166] The method for forming organoids was the same as that in Example 4.2. After 30 days of organoid culture, it was fixed with 4% paraformaldehyde, dehydrated successively with 30% sucrose solution, embedded in OCT (optimal cutting temperature compound), then quickly frozen in a cryostat, and the sample was sectioned with a microtome with a thickness of 20 μm.

[0167] After slicing, the expressions of DAPI, GFAP, MAP2, TUJ1, and S100β in the organoids were examined by immunofluorescence staining. Photographs of the organoids obtained from the cells prepared in the three examples (day 30) under a fluorescence microscope are shown in sequence in Figure 9 - Figure 11 .

[0168] The results showed that Figure 9 - Figure 11 the expressions of DAPI, MAP2, TUJ1, S100β, and GFAP were all observed in the cells in

[0169] Since MAP2 and TUJ1 are markers of neurons, and S100β and GFAP are markers of astrocytes, it means that neurons and astrocytes were differentiated in the organoids differentiated from the cells obtained by the stepwise enzymatic digestion method, the conventional mechanical separation and purification method, and the non-enzymatic picker purification method of the present invention. This shows that the cells isolated by the stepwise enzymatic digestion method of the present invention have the potential to differentiate into neurons and glial cells.

[0170] 4.4 Maestro Microelectrode Array (MEA) Electrophysiology

[0171] By allowing the obtained hNPCs to develop into organoids and culturing them on a dedicated transparent bottom well CytoView microelectrode array plate, real-time observation of the electrical activity of the organoids on living cells can be carried out in a Maestro microelectrode array (MEA) electrophysiological instrument with an internal CO 2 incubator, thereby judging the physiological function of the organoids and measuring the quality of hNPCs used to form the organoids. The MEA method can obtain the overall electrogram of the organoids, and the single cluster discharge (black) and network cluster discharge (blue) of the organoids can be displayed through the electrogram. For hNPCs, the organoids obtained from them should have more regular and more cluster discharges and synchronous discharges, which can prove that the organoids have mature electrophysiological activity. The specific operations are as follows.

[0172] The method for forming the organoids was the same as in Example 4.2. After 7 days of organoid culture, the three groups of cells were respectively seeded in a CytoView MEA 48-well plate (Cat#M768-tMEA-48B; Axion BioSystems) coated with polyethyleneimine (PEI) and Laminin as supports. The medium used was hNPC medium, and the medium was changed by half every 7 days. On the 30th day of organoid culture, data reading and analysis were carried out using a Maestro multi-well plate microelectrode array (MEA) and the accompanying software, and the results are as Figure 8 shown.

[0173] In Figure 8Among them, A, B, and C are the electrophysiological raster images of the organoids formed by the cells obtained from three examples in sequence. Each row of the raster image represents the action potentials received by one electrode. Each black vertical line indicates that an action potential has been received, and an electrode continuously and regularly receives action potentials.

[0174] The results of the raster images show that when comparing the three examples, the organoids formed by the cell cultures obtained in Example 1 (two-step enzyme digestion method) and Example 2 (manual selection method) both exhibited electrical activities. In the Example 1 group, the number of active electrodes was 8 after 30 days of culture, the weighted average discharge frequency (Hz) was 3.4, and the burst discharge frequency (Hz) was 0.17. For Example 2, the number of active electrodes was 4, the weighted average discharge frequency (Hz) was 3.0, and the burst discharge frequency (Hz) was 0.28. In contrast, the organoids formed by the cell cultures obtained in Example 3 (conventional dissociation method) hardly showed any electrical activity (the number of active electrodes was 0), indicating that although the cultures obtained by this method are also rich in hNPCs expressing the correct markers, they cannot have ideal neuroelectrophysiological functions after further differentiation.

[0175] Example 5. Effects of using different first digestive enzymes

[0176] In this example, the effects were tested when various reagents commonly used for dissociating stem cells or nerve cells were used to replace the first digestive enzyme of the present invention.

[0177] 5.1 Using STEMxyme 1 as the first digestive enzyme

[0178] STEMxyme 1 (Worthington#LS004106) is a combination of collagenase / neutral protease, which contains Clostridium histolyticum collagenase without animal origin and polymyxin neutral protease without animal origin, and can be used for the digestion of stem cells.

[0179] Under the condition that other conditions remained unchanged compared with Example 1, STEMxyme 1 was used as the first digestive enzyme to digest the rosette-like structure in the first step, with a concentration of 2 mg / mL, a volume of 1 mL, and treated at 37 °C for 8 min.

[0180] The results showed that the whole rosette-like structure detached from the well plate after digestion, and it was impossible to distinguish the heterogeneous cells from the hNPCs.

[0181] 5.2 Using Dispase as the first digestive enzyme

[0182] Dispase, Animal Component-Free (ACF) is a neutral endoaminopeptidase that can cleave the N-terminal peptide bond of non-polar amino acid residues. It is obtained from cultures without animal-derived materials and can be used to digest nerve tissue and stem cells.

[0183] Under the condition that other conditions remained unchanged compared with Example 1, Dispase, Animal Component-Free (ACF) was used as the first digestive enzyme to digest the rosette-like structure in the first step. The concentration was 5 U / mL, the volume was 1 mL, and it was treated at 37 °C for 8 min.

[0184] The results showed that the whole rosette-like structure detached from the well plate after digestion, and it was impossible to distinguish heterologous cells from hNPCs.

[0185] 5.3 Replace the first digestive enzyme with a non-enzymatic dissociation reagent

[0186] CTS TM Versene TM Solution (Gibco#A4239101) is a mild non-enzymatic cell dissociation reagent. Its main component is ethylenediaminetetraacetic acid (EDTA), which is obtained by preparing EDTA (about 0.5 mM) in calcium-free and magnesium-free D-PBS and does not contain animal-derived substances.

[0187] Under the condition that other conditions remained unchanged compared with Example 1, CTS TM Versene TM Solution was used to replace the first digestive enzyme to treat the rosette-like structure. The concentration was 0.48 mM, the volume was 1 mL, and it was treated at 37 °C for 30 min.

[0188] The results showed that the differentiation effect between heterologous cells and hNPCs after treatment was not as good as that in Example 1 when Collagenase was used as the first digestive enzyme, and the obtained cells could not be used for subsequent culture and differentiation.

[0189] 5.4 Replace the first digestive enzyme with a non-enzymatic dissociation and passage reagent

[0190] ReLeSR TM (Stemcell Technology#05872) is a cGMP-grade enzyme-free human pluripotent stem cell selection and passage reagent. ReLeSR TM can be used to digest human pluripotent stem cells.

[0191] Under the condition that other conditions remained unchanged compared with Example 1, 1 mL of ReLeSR was used in the first step of digestion TMThe stock solution was used to replace the first digestive enzyme to treat the rosette-like structure, and the treatment was carried out at 37 °C for 8 min, with other operations remaining unchanged.

[0192] The results showed that ReLeSR TM After digestion, the rosette-like structure detached from the well plate as a whole, and it was impossible to distinguish the contaminating cells from hNPCs.

[0193] 5.5 Use another collagenase to replace the first digestive enzyme

[0194] Collagenase NB 6GMP Grade (Amsbio#N0002779) is a mixture of type I and type II collagenases and is often used for tissue dissociation, such as dissociating human stem cells, neurons, and endothelial cells.

[0195] Under the condition that other conditions remained unchanged as in Example 1, Collagenase NB 6GMP Grade was used instead of the first digestive enzyme to treat the rosette-like structure in the first digestion, with a concentration of 0.5 PZU / mL and a volume of 1 mL. The treatment was carried out at 37 °C for 8 min, and other operations remained unchanged.

[0196] The results showed that after digestion with Collagenase NB 6GMP Grade, the contaminating cells detached, but the target cells still adhered to the well plate, and it was possible to distinguish the contaminating cells from hNPCs, indicating that Collagenase NB 6GMP Grade can be used to replace Collagenase (Gibco#17104019).

[0197] Example 6. Influence of digestion time

[0198] In this example, the influence of the treatment time of the first digestive enzyme on the purification result was explored. Under the condition that other conditions remained unchanged, it was found that when the treatment time of the first digestive enzyme was less than 5 min, the contaminating cells could not detach; when the treatment time of the first digestive enzyme was greater than 15 min, a small part of the target cells (such as hNPCs) detached, which meant that the yield of the target cells would be lost.

[0199] Example 7. Effects of different second digestive enzymes

[0200] In this example, the influence of different second digestive enzymes on the purification result was explored. On the premise that other conditions were the same, Collagenase (Gibco#17104019), a type IV collagenase, was used as the second digestive enzyme. The results showed that after treatment for 30 min, the cells detached in flakes and the detachment was incomplete, and finally they could not be blown into single cells.

Claims

1. A method for preparing a purified human neural progenitor cell culture, the method comprises: (a) treating rosette-like structures with a first digestive enzyme; and (b) treating the rosette-like structures treated in step (a) with a second digestive enzyme to obtain a purified human neural progenitor cell culture, wherein the first digestive enzyme is collagenase.

2. The method according to claim 1, wherein the collagenase is selected from type I collagenase, type II collagenase, type III collagenase, type IV collagenase, or a combination of one or more thereof.

3. The method according to claim 2, wherein the collagenase is type IV collagenase.

4. The method according to claim 2, wherein the collagenase is a combination of type I collagenase and type II collagenase.

5. The method according to any one of claims 1 to 4, wherein the treatment with collagenase in step (a) causes the non-target cells at the edge of the rosette-like structures to detach, and keeps the central part of the rosette-like structures from detaching.

6. The method according to any one of claims 1 to 5, wherein in step (a), the rosette-like structures are treated with the collagenase for 5 - 20 minutes, preferably 5 - 15 minutes, more preferably 10 - 15 minutes.

7. The method according to any one of claims 1 to 6, wherein the second digestive enzyme is trypsin.

8. The method according to any one of claims 1 to 7, wherein the treatment in step (b) causes all of the rosette-like structures to detach from the culture surface and optionally disperse, thereby obtaining a purified human neural progenitor cell culture.

9. The method according to any one of claims 1 to 8, the method further comprises: (c) culturing the purified human neural progenitor cell culture obtained in step (b) into neurospheres under suitable conditions; and (d) digesting the neurospheres obtained in step (c) with a third digestive enzyme to obtain a further purified culture containing hNPCs.

10. The method according to claim 9, wherein the third digestive enzyme is the same as or different from the second digestive enzyme, preferably the same.

11. The method according to any one of claims 1 to 10, wherein during the process from rosette-like structures to a purified human neural progenitor cell culture, the method does not include any sorting steps for human neural progenitor cells, such as manual picking steps, density gradient centrifugation steps, and / or screening steps based on cell type markers.

12. A purified human neural progenitor cell culture obtained by the method of any one of claims 1 to 11, wherein the proportion of hNPCs expressing PAX6 + FOXG1 + SOX2 + is at least 80%, preferably at least 85%, of the total number of all cells in the culture.

13. A method for obtaining neural cells or brain organoids by differentiation, which comprises using the purified human neural progenitor cell culture according to claim 12.

14. A combination of enzymes, the combination comprising a first digestive enzyme and a second digestive enzyme, wherein the first digestive enzyme is collagenase and the second digestive enzyme is trypsin, and the first digestive enzyme and the second digestive enzyme are used sequentially to treat rosette-like structures to obtain a purified human neural progenitor cell culture.

15. A kit, the kit comprising a first digestive enzyme and a second digestive enzyme, wherein the first digestive enzyme is collagenase, the second digestive enzyme is trypsin, and the first digestive enzyme and the second digestive enzyme are contained in separate containers, and the first digestive enzyme and the second digestive enzyme are used sequentially to process rosette-like structures to obtain a purified human neural progenitor cell culture.

16. Use of collagenase for processing rosette-like structures in a method for culturing human neural progenitor cell cultures, which is used to detach the heterogeneous cells at the edge of the rosette-like structures and keep the central part of the rosette-like structures from detaching, so as to increase the proportion of human neural progenitor cells in the cell population.

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