CN112980792B

CN112980792B - Chemical culture system and application thereof

Info

Publication number: CN112980792B
Application number: CN202110489193.1A
Authority: CN
Inventors: 魏君; 蔡萌
Original assignee: Wuhan Iregene Pharmaceutical Technology Co ltd
Current assignee: Wuhan Iregene Pharmaceutical Technology Co ltd
Priority date: 2020-10-16
Filing date: 2021-04-30
Publication date: 2022-06-21
Anticipated expiration: 2041-04-30
Also published as: CN112980792A; CN112226411A

Abstract

The invention discloses a chemical culture system and application thereof, wherein the chemical culture system comprises a basic culture medium and a small molecular compound to form a serum-free culture system with clear chemical components. The invention greatly improves the culture activity and in vitro survival time of the nerve cells. The invention does not adopt the currently widely used serum B27 substitute and GS21 substitute containing animal-derived components, but uses a pure chemical molecule activation signal path, uses non-animal-derived growth factors to replace animal-derived growth factors, has definite components, and avoids potential risks brought by the existence of serum and animal-derived components, thereby greatly expanding the clinical prospect of nerve cell transplantation.

Description

Chemical culture system and application thereof

Technical Field

The invention belongs to the field of cell biology, and particularly relates to a chemical culture system and application thereof.

Background

Neurodegenerative diseases are common aging diseases at present, the treatment and nursing costs are extremely high, and no specific medicine can be used for effective treatment on the market. Neurodegenerative diseases include Amyotrophic Lateral Sclerosis (ALS), Parkinson Disease (PD), Alzheimer Disease (AD), etc. The world health organization counts that in 2050, the number of patients with neurodegenerative diseases in China will exceed 3000 ten thousand, and the expected medical cost will exceed 1 trillion. At present, medicaments are mainly used for supplementing or stimulating levodopa, nerve nucleus destruction or deep brain electrical stimulation operation and other treatment methods which are insufficient in brain, but the treatment methods cannot achieve good curative effect and bring adverse reactions which seriously affect the life quality, such as 'dyskinesia' or 'drug effect fluctuation'.

In addition to neurodegenerative diseases, Spinal Cord Injury (SCI) is a common traumatic disease of the nervous system, and the treatment and rehabilitation cost of SCI patients for a lifetime is more than 75 ten thousand dollars on average according to foreign statistics, and the cost of SCI patients in the united states exceeds 60 million dollars per year. The prevalence rate of China is higher than that of developed countries in Europe and America, expensive treatment cost is high, and the huge influence on individuals and families caused by long-time rehabilitation and labor loss and the heavy burden on the society are also caused.

The treatment difficulty of neurodegenerative diseases and spinal cord injury is the non-regenerability of nerve cells of the central nervous system, and such diseases are caused by irreversible damage to the central nerve. Currently, transplantation of nerve cells is one of the most effective therapeutic modalities. The nerve cells include different nerve cell types such as nerve stem cells, mature neurons, astrocytes and oligodendrocytes. At present, the allotransplantation of the neural stem cells has achieved certain effects clinically. Part of clinical tests adopt a neural cell line transplanting method, but the existing neural cell line has limited resources, is mostly a neural tumor cell line, and has unstable factors brought by EB virus in the line establishing process; the clinical promotion of the above diseases is further hindered by the factors of difficult in vitro passage of the allogeneic neural cells and the ethical limitation and inconvenient obtaining technology of the allogeneic neural stem cells. In recent years, the development of embryonic stem cells and induced pluripotent stem Cell technology (iPSC) has been advanced greatly, and various types of adult cells can be obtained by utilizing such cells for directed differentiation (Cell, 2006, 124(4) pp.663-676; Cell, 2007, 131(5) pp.861-872). Therefore, the induction and differentiation of nerve cells by using totipotent stem cells or pluripotent stem cells including embryonic stem cells and iPSCs as raw materials can be used as a new idea of clinical treatment, and the application potential of the nerve cells in clinical medicine is greatly expanded.

From pluripotent stem cells to neural stem cells, there are two induction methods currently most widely used, the first being a two-step method: first, the cells aggregate to form embryoid bodies (embryoid bodies), and then the cells are further induced to become neuronal cells. The disadvantage of this method is that the cell differentiation is asynchronous and the purity of the neuronal cells is not high; in addition, the use of animal derived growth factors also limits the clinical use of neuronal cells. The second method is a double SMAD inhibition method (Nat Biotechnol, 2009, 05; 27 (5): 485), and the neural stem cells obtained by the method can be differentiated into other types of neuronal cells, and the principle is that the signal pathways in the early embryonic development stage are simulated by inhibiting BMP and TGFB pathways, so that the generation of the neural stem cells is induced. In this way, sensory neurons can be induced in the presence of a serous component (Knockout Serum Replacement) (Nat Biotechnol, 2009, 05; 27 (5): 485). Although the types of cells applicable to transplantation are greatly increased, the in vitro culture of nerve cells at present mostly depends on the existence of serum components, such as fetal bovine serum and B27 series (Thermo Fisher), GS21 (G0800, Sigma-Aldrich) which are widely used at present, and the serum components contain animal-derived components, so that a series of safety hazards exist, and the existence of the serum components greatly limits the clinical application of nerve cell transplantation. Therefore, the development of a nervous system basic medium containing no serum component is crucial to the development of nervous system regeneration medicine.

Based on the technical problems in the prior art, there is a need to develop a novel serum-free culture medium for nervous system, which does not adopt the currently widely used serum-substitutes B27 and GS21 containing animal-derived components. The pure chemical molecule is used for activating a signal path, and the non-animal-derived growth factor is used for replacing the animal-derived growth factor, so that the components are clear, and the clinical prospect of the nerve cell transplantation is expanded.

Disclosure of Invention

In view of the above, the present invention provides a chemical culture system, which uses pure chemical molecules to activate signal pathways, and uses non-animal growth factors to replace animal growth factors, thereby avoiding potential risks caused by the presence of serum and animal-derived components.

In order to solve the above problems, the present invention provides a chemical culture system, comprising a basic culture medium and an additive, wherein the additive comprises a small molecule compound, and the small molecule compound is: one or more of piperidinol oxide, luteolin, D (+) -galactose and recombinant human transferrin.

Further, the small molecular compound is at least two of piperidinol oxide, luteolin, D (+) -galactose and recombinant human transferrin.

Further, the small molecular compound is piperidinol oxide, luteolin, D (+) -galactose and recombinant human transferrin.

Further, the dosage of the piperidinol oxide is 10uM-200 uM; preferably 1uM, 5uM, 10uM, 20uM, 50uM, 100uM, 150uM or 200 uM.

Further, the dosage of the luteolin is 5uM-150 uM; preferably 5uM, 10uM, 50uM, 75uM, 100uM or 150 uM.

Further, the dosage of the D (+) -galactose is 5-25 ug/ml; preferably 5.5 ug/ml, 7.5ug/ml, 10 ug/ml, 12.5ug/ml, 15 ug/ml, 17.5 ug/ml, 20 ug/ml, 22.5ug/ml or 25 ug/ml.

Further, the dosage of the recombinant human transferrin is 50-200 ng/ml; preferably 55 ng/ml, 75ng/ml, 100ng/ml, 125 ng/ml, 150ng/ml, 175 ng/ml or 200 ng/ml.

Further, the basal medium is a DMEM medium or a DMEM/F12 medium, and the basal medium includes minimal essential medium non-essential amino acids.

Furthermore, the additive in the chemical culture system provided by the invention also comprises a growth factor and an inorganic salt.

Further, the growth factors include vitamins, progesterone, putrescine, and insulin; the inorganic salt includes sodium chloride and sodium selenite. Further, the vitamins include 1ug/ml vitamin E, 1.2uM vitamin B12, 64mg/L vitamin C; the progesterone is 6.3ng/ml, the putrescine is 23ug/ml, and the insulin is 22 ug/ml; the sodium chloride is 0.5g/L, and the sodium selenite is 13.6 ug/L.

Another object of the present invention is to provide the use of the chemical culture system in primary neural cell culture and neural cell line culture.

The invention also aims to provide application of the chemical culture system in the induction of the pluripotent stem cells to the neural stem cells.

The invention also aims to provide the application of the chemical culture system in inducing the neural directional differentiation of the stem cells and the culture of differentiated cells. Further, the application comprises astrocyte differentiation, DRG neuron differentiation culture or cortical neuron differentiation culture; furthermore, cytokines are added in the differentiation and culture process of the DRG neurons; and a chemical small molecule inhibitor is added in the differentiation culture process of the cortical neurons.

It is still another object of the present invention to provide the use of said chemical culture system in organoid induction.

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

1. the invention uses one or more novel chemical small molecules, combines with a basic culture medium and the combination of inorganic salt and growth factors, and can support the in vitro induction of a plurality of neurons, thereby establishing a serum-free and animal-derived substance-free sensory neuron cell induction culture system and showing better induction culture effect.

2. The neural stem cells obtained by the culture medium have complete biological functions, are stable in state among a plurality of batches and high in purity, and solve the problems of low purity and long period in the production process of cell medicines.

3. The invention also solves the problems of animal-derived culture method and trophoblast pollution for a long time, and can be used for in vitro screening of nervous system disease drugs, thereby having great economic and social effects.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a Cyquant curve of primary cells and human neural stem cell line ReNcell VM cultured in NouvNeu.

FIG. 2 is a graph showing the results of core component screening in NouvNeu using the human neural cell line ReNcell VM.

FIG. 3 is a diagram showing transcriptional analysis of neural rosette structures and markers obtained after the directional induction of neural stem cells using the NouvNeu system.

FIG. 4 is a schematic diagram showing the identification of the expression of a sensory neuron-specific molecular marker by immunofluorescence after induction of neurons and glial cells using the NouvNeu system.

FIG. 5 is a graph showing the electrophysiological activity of DRG neural cells using the NouvNeu system.

FIG. 6 is a diagram showing the cell activity during the induction of the differentiation of cortical neuronal cells using the NouvNeu system.

FIG. 7 is a schematic representation of the brain-like organ induction using the NouvNeu system, and the analysis of brain-like organ markers and electrophysiological activity.

Detailed Description

The following examples are intended to illustrate the invention without limiting its scope. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.

In a particular embodiment of the invention, serum-free medium means that it does not contain serum isolated directly from blood. Serum is a clear liquid portion of plasma that is free of fibrinogen or blood cells and remains a liquid after blood coagulation. The serum-free medium may contain a serum substitute, and examples of the serum substitute include purified substances such as serum albumin, transferrin, fatty acids, and the like, which are well known in the art as substances that can substitute for serum.

In a specific embodiment of the invention, a culture medium for induction of a plurality of cells is provided, comprising a basal medium and an additive; the additive comprises a small molecule compound, common inorganic salt and growth factors;

in a specific embodiment of the invention, the small molecule compound is one or more of piperidinol oxide, luteolin, D (+) -galactose and recombinant human transferrin; preferably two, more preferably four.

In a particular embodiment of the invention, the basal medium is DMEM medium, minimal essential medium non-essential amino acids are added, and other media comprising minimal essential medium non-essential amino acids may be selected.

In particular embodiments of the invention, the growth factors include vitamins, progesterone, putrescine, and insulin; other growth factors commonly used may also be selected for substitution or addition.

In particular embodiments of the invention, the inorganic salts include sodium chloride, sodium selenite; other inorganic salts commonly used can also be selected to replace or be added.

Example 1 media formulation

The formulation of the NouvNeu medium is hereinafter referred to as NouvNeu:

duke's modified eagle's Medium (DMEM Medium, serum-free DMEM-F12 may also be selected), minimal Essential Medium Non-Essential Amino Acids (minimal Essential Medium Non-Essential Amino Acids, cat # 11140076, Thermo Fisher), piperidinol oxide (4-Hydroxy-TEMPO, 10nM-200uM, optimal concentration of 50uM), D (+) -galactose (D (+) -galactonase, 5-25ug/ml, optimal concentration of 12.5 ug/ml), vitamin E (1 ug/ml), vitamin B12 (1.2 uM), vitamin C (L-ascobic, 64 mg/L), Progesterone (Progesterone, 6.3 ng/ml), Putrescine (Putrescine, 23 ug/ml), Luteolin (Luteolin, 5uM-150uM, optimal concentration of 60 Sodium Chloride, 0.5 ug/ml), sodium selenite (13.6 ug/L), insulin (22 ug/ml), and recombinant human transferrin (50-100 ng/ml, with an optimal concentration of 100 ng/ml).

Meanwhile, the NouvNeu medium was prepared according to the standard of Table 1 under the condition that other components were not changed.

Table 1: NouvNeu component analysis Medium component Table

The reference culture medium I is N2B27 culture medium, hereinafter referred to as N2B27, and the formula thereof is as follows: 50% Duchenne modified eagle's F12 medium (DMEM-F12), 50% neural basal medium (Neurobasal), 0.1% N2 additive (N2 supplement), 2% B27 additive (B27 supplement).

Control medium II was a modified eagle's medium (DMEM medium) containing 10% FBS, hereinafter abbreviated as FBS.

Example 2 culture of Primary neural cells and neural cell lines and Compound concentration screening

Coating a 6-well culture plate with 50ug/ml polylysine (SIGMA, P6407), laying the plate, and incubating in a 37 ℃ incubator for more than 3 hours; then further coated with 5ug/ml Laminin (Laminin, I2020, SIGMA ALDRICH), plated and incubated in a 37 ℃ incubator for more than 3 hours. Mouse primary cells and the human neural cell line ReNcell VM were cultured, wherein the isolation of the mouse primary cells was performed according to the method described in Nature protocols, Volume 7, pages 1741-1754 (2012). The cell culture conditions were 37 ℃ and 5% carbon dioxide (Panasonic, MCO-18 AC), cell adherent culture. Comparisons were made using the inventive medium, Neuralbasal +10% FBS and N2B27 medium, respectively. Cell passage was digested with 0.05% trypsin/EDTA at 37 ℃ for 5 min, and cell digestion was stopped with DMEM +10% FBS. After washing and centrifuging the cells, the ratio of the cells to the total volume of the cells is 2X 10⁵The ratio of each flask was re-inoculated in a T25 plate. Cyquant experiments were performed after passage 2 of the cells.

The adherent culture described in steps 1 to 3 in this example is preferably carried out in the presence of a basement membrane. The basement membrane can form a layer of membrane consisting of extracellular matrix molecules on the surface of a culture vessel, and can provide support similar to the in-vivo environment for the parameters of the morphology, growth, differentiation, movement and the like of cells. In particular embodiments, the basement membrane is a combination of one or more of a substrate gel (Matrigel) (stem cell Technologies), a Laminin (lamin), and a vitronectin.

The Cyquant test is used for quantitative detection of cell viability, so that the influence of different culture medium components on the physiological state of cells in the long-term culture process of the cells is compared. Coating a 96-hole opaque cell culture plate, and after coating, arranging the cells according to the 5 multiplied by 10⁴The number of cells per well was seeded separately and three replicates were set (the average of the three sets was calculated as data). The culture conditions were 37 ℃ and 5% carbon dioxide, half-changes were made every 2 days, using the medium in the known method as a control. Samples were taken on

days

1, 5, 15 and 30, respectively, and cell viability assays were performed using the CyQuant Kit (Invitrogen, X12223) and data reads were performed using the SpectraMax i3 Multi-Mode Microplate Reader (VWR, model ID 3-STD) according to the instructions. The results are shown in fig. 1, and indicate that Cyquant experiments show that both mouse primary cells (fig. 1 a) and human neural stem cell lines (fig. 1B) exhibit better cell viability in the novvneu system compared to FBS control and N2B27 control.

The compounds screened in the Cyquant single-product experiment are respectively piperidinol oxide, D (+) -galactose, luteolin and recombinant human transferrin. When concentration screening is carried out, only the piperidinol oxide, the D (+) -galactose, the recombinant human transferrin and the luteolin in the culture medium formula are respectively changed in concentration, and the concentrations of the other components are not changed.

The screening results are shown in FIGS. 2a-2d, and FIG. 2a shows that luteolin is used in a range of 5uM to 150 uM; it is not difficult to derive one of 1uM, 10uM, 50uM, 75uM, 100uM and 150uM that may be preferable.

FIG. 2b shows the use of D (+) -galactose ranging from 5-25 ug/ml; it will be appreciated that 5.5 ug/ml, 7.5ug/ml, 10 ug/ml, 12.5ug/ml, 15 ug/ml, 17.5 ug/ml, 20 ug/ml, 22.5ug/ml and 25ug/ml can be selected as preferred amounts.

FIG. 2c shows the range of use of piperidinol oxide from 10uM to 200 uM; it will be appreciated that 1uM, 5uM, 10uM, 20uM, 50uM, 100uM, 150uM or 200uM may be selected as preferred amounts.

FIG. 2d shows recombinant human transferrin is used in the range of 50-200 ng/ml; it will be appreciated that 55 ng/ml, 75ng/ml, 100ng/ml, 125 ng/ml, 150ng/ml, 175 ng/ml or 200ng/ml may be selected as preferred amounts.

Cyquant screened the single product combinations using the NouvNeu assay medium according to Table 1, with the screening results shown in 2 e. The results showed that combining luteolin, D (+) -galactose, piperidinol oxide and human recombinant transferrin in novnenu showed that novnenu containing four components had the best cell proliferation activity in all combinations.

From the above screening results (fig. 2a-2 e), the increase of the concentration of each individual component in NouvNeu can play a role in cell proliferation, so it can be determined that the composition contains one or more of luteolin, D (+) -galactose, piperidinol oxide and human recombinant transferrin, which can realize cell proliferation.

Table 2: cyquant enumeration of primary cell cultures with four NouvNeu Components combinations (FIG. 2 e)

Example 3 Induction culture of neural Stem cells

Human pluripotent stem cells include embryonic pluripotent stem cells such as the H9 cell line and human induced pluripotent stem cells. Wherein the human-induced pluripotent stem cells are obtained by reprogramming CD34+ cells in accordance with "a reprogramming medium and a method for culturing the reprogramming-induced pluripotent stem cells" (patent publication No. CN 109628383A).

Human pluripotent stem cells T25 cell culture flasks were coated with matrigel (STEMCELL technologies), plated, and incubated in a 37 ℃ incubator for more than one hour. According to 1 × 10⁶Cells were seeded in T25 flasks for expansion and passaging.

When performing nerve induction, coating a 6-hole culture plate with 50ug/ml polylysine (SIGMA, P6407), laying the plate, and incubating in a 37 deg.C incubator for more than 3 hr; then further coated with 5ug/ml Laminin (Laminin, I2020, SIGMA ALDRICH), plated and incubated in a 37 ℃ incubator for more than 3 hours. When the pluripotent stem cells reached 70% coverage, they were digested with EDTA at 37 ℃ for 5 minutes, and cell digestion was stopped with DMEM. After washing and centrifuging the cells, the ratio of the cells to the total volume of the cells is 2X 10⁵The ratio of each flask was re-inoculated in a T25 plate. Neural induction was carried out using the medium of the present invention and a combination of neural inducing compounds of 100nM LDN-193189 HCl (Selleck, cat # S7507) and 10uM SB431542 (Selleck, cat # S1067) at 37 ℃ with 5% CO₂(Panasonic, MCO-18 AC), the medium was changed daily until the formation of rosette structure of neural stem cells.

Neural stem cells were digested with EDTA at 37 ℃ for 5 minutes, and cell digestion was stopped using DMEM. After washing and centrifuging the cells, the ratio of the cells to the total volume of the cells is 2X 10⁵The culture medium was inoculated into T25 culture plates coated with 50ug/ml polylysine and 5ug/ml laminin at a ratio of each flask for amplification culture. The culture conditions were 37 ℃ and 5% CO₂（Panasonic，MCO-18AC）。

And (3) performing immunofluorescence staining identification on the neural stem cells which are subjected to passage after the induction of the N2B27 system and the NouvNeu system is completed respectively: fixing the cells with 4% paraformaldehyde at room temperature for 40 minutes, and washing twice with DPBS buffer solution; then permeabilizing with 0.1% Triton X-100 for 5 minutes, and washing twice with DPBS buffer solution; cells were then incubated overnight at 4 ℃ with DPBS buffer containing 10% horse serum and 0.1% Triton X-100; then, the antibody diluted with the DPBS buffer was added, incubated at 37 ℃ for 2 hours, washed three times with the DPBS buffer, photographed, and photographed using a Leica DMi 8. Details of antibody use are shown in table 3.

Table 3: antibodies for cellular immunofluorescence staining

Q-PCR was used to detect transcriptional changes of different marker genes during induction from pluripotent stem cells to neural stem cells. The induced pluripotent stem cells used in example 3, the induced neural stem cells obtained using the NouvNeu and N2B27 systems, and the cells after passage of 3 generations using the NouvNeu component analysis medium were subjected to total RNA extraction using RNeasy Mini or Micro Kit (QIAGEN), and 1 mg of RNA was synthesized into cDNA using SuperScript III First-Strand Synthesis System (Invitrogen), respectively. The labeling and reaction of the Quan-reactive PCR were performed using SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa), and beta-Actin was used as an internal reference. All data were analyzed using delta-Ct method. Each set of experiments was performed using three replicates and variance statistics were performed. The primer sequences used to identify the genes encoding the different cellular markers are shown in table 4.

Table 4: different marker gene primers in process of inducing pluripotent stem cells into neural stem cells

FIG. 3 is a graph showing the results of induction of neural stem cells using NouvNeu and a control group. In contrast to the control N2B27 system (fig. 3a, fig. 3 c), NouvNeu was able to form a uniformly tiled aggregation of nerval rings (rossette) during the monolayer induction phase (fig. 3B), and cells were able to form further nerval ring structures after passaging (fig. 3 d); the neuvneu (fig. 3 f) and N2B27 (fig. 3e) control formed neural rosette structures expressing structural protein ZO1 and nuclear transcription factor SOX2 at the central position of the rosette; Q-PCR analysis of RNA expression in neural stem cells, NouvNeu induced neural stem cells were able to express Pax6 (FIG. 3g), Sox2 (FIG. 3i) and Nestin (FIG. 3 j). The combination of luteolin, D (+) -galactose, piperidinol oxide and human recombinant transferrin in NouvNeu shows that all the combinations can induce the expression of the neural stem cell specific marker Pax6 in the neural stem cell induction process. In the process of maintaining and inducing the growth of the neural stem cells, the medium containing one or more of luteolin, D (+) -galactose, piperidinol oxide and human recombinant transferrin can support the passage and growth of the neural stem cells, but can cause the expression of Caspase3 (figure 3k) under the condition of ultra-long-term culture and passage, and the result shows that the NouvNeu containing four compounds can maintain the passage and growth of the neural stem cells at a lower expression level of Caspase 3.

Example 4 induced differentiation and culture of neural Stem cells

Neural stem cells obtained from the NouvNeu system in example 3 were subjected to committed differentiation in NouvNeu medium.

4.1 astrocyte differentiation

Human induced astrocytes Using the neural stem cells obtained in example 3, 10ng/mL PDGFAA (R) was added to NouvNeu basal medium&D Systems), 20 ng/mL fibroplast growth factor 2 (Heyu life), 20 ng/mL epidermal growth factor (epidermal growth factor, Peprotech), Human LIF (10 ng/mL, Alomone Labs), 10% fetal bovine serum (Jiety life), with fresh medium changed every 3 days until day 21. The culture conditions were 37 ℃ and 5% CO₂(Panasonic, MCO-18 AC). Control experiments were performed with N2B27 basal medium.

FIGS. 4a-4B show astrocytes induced using the N2B27 control, which are flat star-shaped in morphology and express the astrocyte marker GFAP; FIGS. 4c-4d show that astrocytes induced using NouvNeu have the same cell morphology and marker expression as compared to the N2B27 control. The experimental results show that both the NouvNeu and N2B27 systems can differentiate to obtain astrocytes, and the obtained astrocytes have typical morphology and express the astrocyte marker MAP2 (FIGS. 4a-4 d).

4.2 DRG neuronal differentiation

Human induced DRG neurons Using the nerves obtained in example 3Stem cells were cultured in NouvNeu basal medium with 3uM CHIR99021 (Selleck, cat # S2924), 10uM SU5402 (Tocris, cat # 3300/1), 10uM DAPT (Selleck, cat # S2215), and fresh medium was changed every 3 days until day 21. The culture conditions were 37 ℃ and 5% CO₂（Panasonic，MCO-18AC）。

The control experiment (CK hereinafter) was carried out according to a published method (Nat Biotechnol., 2013, 30(7): 715-720) as follows: 820ml of knock-out Dulbert's modified eagle's Medium (Knockout DMEM Medium, cat # 10829018, Thermo Fisher), 150ml of knock-out Serum Replacement (Knockout Serum Replacement, cat # 10828028, Thermo Fisher), 1mM L-Glutamine (L-Glutamine, cat # 25030081, Thermo Fisher), 100 mM minimal Essential Medium Non-Essential Amino Acids (Minium Essential Medium Non-Essential Amino Acids, cat # 11140076, Thermo Fisher) and 0.1mM beta-Mercaptoethanol (beta-Mercaptoethanol, cat # 21985023, Thermo Fisher) were used to prepare a basal Medium, and 100nM LDN-193189 and 10 mM SB431542 were added to the basal Medium from day 0 to day 5. 1% (v/v) of N2 additive (N2 supplement, cat # 17502045, Thermo Fisher) was added to a neural basal medium (Neurobasal medium, cat # 21103049, Thermo Fisher) to form N2 medium (hereinafter referred to as N2 system). N2 medium was added in 25% increments every other day starting on day 4 (100% N2 on day 10). Three inhibitors, 3uM CHIR99021 (Selleck, cat # S2924), 10uM SU5402 (Tocris, cat # 3300/1), 10uM DAPT (Selleck, cat # S2215), were added on day 10, and fresh medium was changed every 3 days until day 31. The culture conditions were 37 ℃ and 5% CO2 (Panasonic, MCO-18 AC).

FIGS. 4e-4f show DRG neurons induced using N2B27, expressing the DRG neuron-specific marker sodium channel 1.8 and the neural cell marker Tuj; FIGS. 4g-4h show that DRG neurons induced using NouvNeu have the same cell morphology and marker expression compared to the N2B27 control.

Experimental results show that both NouvNeu and N2 systems can be differentiated to obtain DRG neurons, and the obtained DRG neurons have axon structures and express DRG neuron markers KCa2.2 and nerve cell markers Nestin (FIGS. 4e-4 h).

Cell self-emission signal detection was performed on the induced DRG neuronal cells using a multichannel electrode, and the cells were cultured according to the DRG neuronal differentiation method using a NouvNeu component analysis series medium, a NouvNeu medium, and an N2B27 control medium, respectively. Coating a 96-well MEA system multi-channel electrode plate (AXION Biosystem, US) by using 100ng/ml polylysine (Poly-L-lysine, Sigma-Aldrich, P4707), and coating in a 5% carbon dioxide cell culture box (Panasonic, MCO-18 AC) at 37 ℃ for 12 hours; the polylysine-coated MEA multi-channel electrode plate was taken out, the polylysine was aspirated, and after washing with sterilized water for 3 times, a PBS solution containing 3ug/ml gelatin (Laminin, I2020, SIGMA ALDRICH) was used as a nerve cell coating substrate, and added to the MEA multi-channel electrode plate, and the MEA multi-channel electrode plate was placed in a 5% carbon dioxide cell incubator at 37 ℃ and coated for 3 hours (Panasonic, MCO-18 AC). After the MEA multi-channel electrode plate is coated, the thickness of the MEA multi-channel electrode plate is 5 multiplied by 10⁵The number of each cell per well was seeded with cells that induced neuronal cells. And placing the inoculated MEA multichannel electrode plate in an MEA chamber, adjusting in Axis Navigator 2.0.2 software, and operating for 10 minutes until the chamber environment is stable under the cell culture condition of 37 ℃ and 5% of carbon dioxide. The cell self-emission signals were recorded using the Axis Navigator 2.0.2 software (AXION Biosystem, US).

The experimental result shows that DRG neurons induced by the NouNeu system show good electrophysiological activity. Fig. 5a-5k show the growth patterns of the drug neurons induced by the NouNeu system and the N2B27 control in MEA electrode plates, and the NouvNeu system was more active than the N2B27 control by comparing the number of discharges per unit time (fig. 5 i) and the average discharge rate (fig. 5 m) of the cells when the MEA electrode plates were further used to detect the spontaneous discharge of the cells in a multichannel electrode system. FIGS. 5n-5o show that the combination of luteolin, D (+) -galactose, piperidinol oxide and human recombinant transferrin in NouvNeu shows that NouvNeu containing four components has the best cellular electrophysiological activity in this combination.

4.3 cortical neuron differentiation

When cortical neuron differentiation is carried out, coating a 6-hole culture plate with 50ug/ml polylysine (SIGMA, P6407), laying the plate, and incubating in a 37 ℃ incubator for more than 3 hours; then further coated with 3ug/ml Laminin (Laminin, I2020, SIGMA ALDRICH), plated and incubated in a 37 ℃ incubator for more than 3 hours. Induced nerve trunk obtained in example 3 was as 1X 10⁵The ratio of each flask was re-inoculated in a T25 plate. Culturing neural stem cells in NouvNeu medium at 37 deg.C and 5% CO₂(Panasonic, MCO-18 AC), fresh medium was changed every three days until neuronal formation on day 30. The control experiment was performed using N2B27 medium for neuronal induction, using the same procedures as above.

FIGS. 4i-4j show cortical neurons induced using N2B27, expressing the cortical neuron specific markers MAP2 and NeuN; FIGS. 4k-4l show that cortical neurons induced using NouvNeu have the same cell morphology and marker expression compared to the N2B27 control.

Experimental results show that both the NouvNeu and N2B27 systems can differentiate to obtain cortical neurons, which have axon structures and express the cortical neuron markers NeuN and MAP2 (FIGS. 4i-4 l).

And taking two control materials of each group after induction is finished to carry out immunofluorescence staining identification: fixing the cells with 4% paraformaldehyde at room temperature for 40 minutes, and washing twice with DPBS buffer solution; then permeabilizing with 0.1% Triton X-100 for 5 minutes, and washing twice with DPBS buffer solution; cells were then incubated overnight at 4 ℃ with DPBS buffer containing 10% horse serum and 0.1% Triton X-100; then, the antibody diluted with DPBS buffer was added, incubated at 37 ℃ for 2 hours, washed three times with DPBS buffer, and photographed. Details of antibody use are shown in table 5.

Table 5: antibodies for directed differentiation immunofluorescence staining

Use ofThe Cyquant test is used for quantitatively detecting the cell activity, so that the influence of different culture medium components on the physiological state of the cells in the long-term culture process of the cells is compared. Coating a 96-hole opaque cell culture plate, and after coating, arranging the cells according to the 5 multiplied by 10⁴The number of cells per well was seeded separately and three sets of replicates were set up (the average of the three sets was calculated as data). The culture conditions were 37 ℃ and 5% carbon dioxide, half-changes were made every 2 days, using the medium in the known method as a control. Samples were taken on

days

1, 10, 15 and 35, respectively, and cell viability assays were performed using the CyQuant Kit (Invitrogen, X12223) and data reads were performed using the SpectraMax i3 Multi-Mode Microplate Reader (VWR, model ID 3-STD) according to the instructions. The results are shown in fig. 6, fig. 6a to 6k show that the growth morphology of cortical neurons induced by the NouNeu system and N2B27 control under the same cell initial concentration, and the Cyquant experiment shows that the NouvNeu can better maintain the cell activity in the process of inducing the directional differentiation of cortical neuron cells.

FIG. 6l shows the range of use of piperidinol oxide from 10uM to 200uM, and FIG. 6m shows the range of use of luteolin from 5uM to 150 uM; FIG. 6n shows the use of D (+) -galactose ranging from 5-25 ug/ml; FIG. 6o shows the use range of recombinant human transferrin is 50ng/ml to 200 ng/ml; FIG. 6p (data shown in Table 6) shows that the combination of luteolin, D (+) -galactose, piperidinol oxide and human recombinant transferrin in NouvNeu indicates that NouvNeu containing four components has the best cell proliferation activity in this combination.

Table 6: cyquant counts in cortical neuron induction by four-component combinations

EXAMPLE 53 Induction culture of class D organs

The brain-like organ culture is carried out by adopting human induced pluripotent stem cells, and the formula of the brain-like organ induction culture medium is as follows: the NouvNeu basal medium is added with 10 mu M SB-431542 and 100nM LDN-193189, and other inhibitors with similar effect can be selected.

Induced pluripotent stem cells were digested into single cells using Accutase, and seeded into U-bottom-ultra-low adherence 96-well plates at a ratio of 9000 cells per well per 150ml brain-like induction medium. And 50. mu.M ROCK inhibitor Y27632 and 5% (v/v) heat-inactivated FBS (Life Technologies) were added and incubated at 37 ℃ in a 5% carbon dioxide cell incubator for hours (Panasonic, MCO-18 AC). The next day, the fresh brain-like organ induction medium was changed until day 10. On day 10, the brain-like organs were transferred to an ultra-low-attachmen 6-well plate, replaced with NouvNeu medium, and spun-cultured at 80 rpm on a horizontal spinner in a 5% carbon dioxide cell incubator at 37 ℃ until day 30.

The control experiments were carried out according to published methods (Nat methods, 2019 Nov;16(11): 1169-: the basic culture medium of N2B27 was supplemented with 10uM SB-431542, 100nM LDN-193189, 2uM XAV-939. The rest of the operations are the same as above.

And detecting the cell spontaneous emission signals of the induced brain-like organ by using a multi-channel electrode. The brain-like organs induced for 30 days were digested with 10% pancreatin/EDTA at 37 ℃ for 5-8 minutes, coated with 100ng/ml polylysine (Poly-L-lysine, Sigma-Aldrich, P4707) in a 96-well MEA-system multichannel electrode plate (AXION Biosystem, US), placed in a 5% carbon dioxide cell incubator (Panasonic, MCO-18 AC) at 37 ℃ for 12 hours; the polylysine-coated MEA multi-channel electrode plate was taken out, the polylysine was aspirated, and after washing with sterilized water for 3 times, a PBS solution containing 3ug/ml gelatin (Laminin, I2020, SIGMA ALDRICH) was used as a nerve cell coating substrate, and added to the MEA multi-channel electrode plate, and the MEA multi-channel electrode plate was placed in a 5% carbon dioxide cell incubator at 37 ℃ and coated for 3 hours (Panasonic, MCO-18 AC). After the MEA multi-channel electrode plate is coated, the thickness of the MEA multi-channel electrode plate is 5 multiplied by 10⁵The number of each cell per well was seeded with the digested brain-like organ mixed cells. The inoculated MEA multichannel electrode plate is placed in an MEA chamber and is regulated in Axis Navigator 2.0.2 software, the cell culture condition is 37 ℃, 5% carbon dioxide,run for 10 minutes until the chamber environment stabilized. The cell self-emission signals were recorded using the Axis Navigator 2.0.2 software (AXION Biosystem, US).

Transcriptional changes of different marker genes following organogenesis were detected using Q-PCR: total RNA of 18-day-old brain-like organs cultured by different methods and human induced pluripotent stem cell controls were extracted using RNeasy Mini or Micro Kit (QIAGEN), and 1 mg of RNA was synthesized into cDNA using SuperScript III First-Strand Synthesis System (Invitrogen). The labeling and reaction of the Quan-reactive PCR were performed using SYBR Premix Ex Taq (TaKaRa) and Thermal Cycler Dice Real Time System (TaKaRa), and beta-Actin was used as an internal reference. All data were analyzed using delta-Ct method. Each set of experiments was performed using three replicates and variance statistics were performed. The primer sequences used to identify the genes encoding the different cellular markers are shown in table 7. The results showed that combining luteolin, D (+) -galactose, piperidinol oxide and human recombinant transferrin in novneu showed that novneu containing four components had the best cell proliferation activity in all combinations (fig. 7 c); the brain-like organs formed by the NouvNeu system and the N2B27 system were able to generate glial cells, neural progenitor cells and neurons (FIGS. 7d-7 e).

TABLE 7 primers for the peripheral neuron and pluripotent stem cell marker Q-PCR:

FIG. 7 shows: both NouvNeu (7B) and N2B27 controls (7 a) produced organoids when cultured for 30 days; when spontaneous cell discharge was detected using the multichannel electrode system, the novneu system was more active than the N2B27 control by comparing the number of discharges per unit time of the cells (fig. 7 c) and the average discharge rate (fig. 7 d). Q-PCR analysis of cell RNA expression in organoid cells, combining luteolin, D (+) -galactose, piperidinol oxide and human recombinant transferrin in NouvNeu, revealed that NouvNeu containing four components had the best cell proliferation activity in all combinations, while some combinations resulted in massive apoptosis (FIG. 7 e). Both novrneu-induced brain-like organs were able to express EN1 (fig. 7 f), Sox2 (fig. 7 g) and Nestin (fig. 7 h) compared to the N2B27 control. The above results demonstrate that NouvNeu is able to maintain the growth of brain-like organs and the electrophysiological activity of brain-like organs.

The one or more novel chemical small molecules used in the above embodiment of the invention, in combination with the basal medium and the combination of inorganic salts and growth factors, can support the in vitro induction of multiple neurons, thereby establishing a serum-free and animal-derived substance-free sensory neuron cell induction culture system, and having better inducer culture effect compared with the existing culture medium containing animal serum.

The neural stem cells obtained by the culture system have complete biological functions, stable test state after repeated times, and shortened period compared with the culture medium in the prior art, and have great application prospect.

The invention does not adopt the serum B27 and GS21 containing animal-derived components, uses a pure chemical molecule activation signal path, and uses non-animal-derived growth factors to replace animal-derived growth factors, and the components are clear. Solves the problems of animal-derived culture method and trophoblast pollution for a long time, can be used for in vitro screening of nervous system disease drugs, and has great economic and social effects.

The invention is not limited solely to that described in the specification and embodiments, and additional advantages and modifications will readily occur to those skilled in the art, so that the invention is not limited to the specific details, representative embodiments, and illustrative examples shown and described herein, without departing from the spirit and scope of the general concept as defined by the appended claims and their equivalents.

Claims

1. A chemical culture system, which is characterized by comprising a basic culture medium and an additive; the additive comprises a small molecule compound which is:

piperidinol oxide and luteolin; or the like, or, alternatively,

at least one of piperidinol oxide, luteolin, at least one of D (+) -galactose and recombinant human transferrin;

the dosage of the piperidinol oxide is 10uM-200 uM;

the dosage of luteolin is 5uM-150 uM;

the dosage of the D (+) -galactose is 5-25 ug/ml;

the dosage of the recombinant human transferrin is 50-200 ng/ml.

2. The chemical culture system of claim 1, wherein the small molecule compound is piperidinol oxide, luteolin, D (+) -galactose, recombinant human transferrin.

3. The chemical culture system of claim 1, wherein the piperidinol oxide is used in an amount of 10uM, 20uM, 50uM, 100uM, 150uM, or 200 uM.

4. The chemical culture system of claim 1, wherein the luteolin is present in an amount of 5uM, 10uM, 50uM, 75uM, 100uM, or 150 uM.

5. The chemical culture system of claim 1, wherein the amount of D (+) -galactose is 5.5 ug/ml, 7.5ug/ml, 10 ug/ml, 12.5ug/ml, 15 ug/ml, 17.5 ug/ml, 20 ug/ml, 22.5ug/ml or 25 ug/ml.

6. The chemical culture system of claim 1, wherein the recombinant human transferrin is administered in an amount of 55 ng/ml, 75ng/ml, 100ng/ml, 125 ng/ml, 150ng/ml, 175 ng/ml or 200 ng/ml.

7. The chemical culture system of claim 1, wherein the basal medium is DMEM medium or DMEM-F12 medium, and the basal medium is supplemented with minimal essential medium non-essential amino acids.

8. The chemical cultivation system as claimed in claim 1, wherein the additive further comprises a growth factor and an inorganic salt.

9. The chemical culture system of claim 8, wherein the growth factors comprise vitamins, progesterone, putrescine, and insulin; the inorganic salt includes sodium chloride and sodium selenite.

10. The chemical culture system of claim 9, wherein the vitamins include 1ug/ml vitamin E, 1.2uM vitamin B12, 64mg/L vitamin C; the progesterone is 6.3ng/ml, the putrescine is 23ug/ml, and the insulin is 22 ug/ml; the sodium chloride is 0.5g/L, and the sodium selenite is 13.6 ug/L.

11. Use of a chemical culture system according to any of claims 1 to 10 in primary neural cell culture and neural cell line culture.

12. Use of a chemical culture system according to any one of claims 1 to 10 for the induction of pluripotent stem cells into neural stem cells.

13. Use of a chemical culture system according to any of claims 1 to 10 for inducing neural directed differentiation of stem cells and culture of differentiated cells.

14. The use of claim 13, wherein said use comprises astrocyte differentiation, DRG neuron differentiation culture or cortical neuron differentiation culture.

15. The use of claim 14, wherein cytokines are added during the differentiation and culture of DRG neurons.

16. The use of claim 14, wherein a small chemical molecule inhibitor is added during the differentiation culture of the cortical neurons.

17. Use of a chemical culture system according to any of claims 1 to 10 for organoid induction.