CN114107207A - Composition for inducing neural stem cell differentiation, method and application - Google Patents
Composition for inducing neural stem cell differentiation, method and application Download PDFInfo
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- CN114107207A CN114107207A CN202111420544.XA CN202111420544A CN114107207A CN 114107207 A CN114107207 A CN 114107207A CN 202111420544 A CN202111420544 A CN 202111420544A CN 114107207 A CN114107207 A CN 114107207A
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Abstract
The invention discloses a composition for inducing neural stem cell differentiation, a method and application thereof. The composition comprises an induction culture solution and a laminin solution with the concentration of 3-20 mu g/ml, wherein the induction culture solution contains a KnockOut serum substitute. The composition has low cost and high induction rate for inducing neural stem cells to differentiate into astrocytes.
Description
Technical Field
The invention relates to a composition for inducing neural stem cell differentiation, a method and application thereof, in particular to application of a laminin solution and application of poly-L-lysine.
Background
Astrocytes are important glial cells of the central nervous system and are also one of the three types of cells that make up the blood-brain barrier. Astrocytes are important glial cells that help central nervous system neurons acquire nutrients, aiding in the excretion of metabolic waste products. Astrocytes also provide important support for the barrier function of the blood brain barrier. Research shows that astrocytes have close relation with diseases such as sleep regulation disorder, epilepsy, cognitive dysfunction, cerebral apoplexy, cerebral hemorrhage and the like. Therefore, astrocytes play an important role in scientific research. At present, in neuroscience research, astrocytes and reactive astrocytes are found in the central nervous system by observing and detecting various aspects of molecular expression, functional morphology, and the like of astrocytes under normal or different disease conditions. Reactive astrocytes are astrocytes that have been altered in shape, molecule, and function in response to central nervous system injury, disease, or infection. The up-regulation of GFAP protein is considered as a marker of reactive astrocytes, and can be detected in various nervous system diseases such as Alzheimer's disease, cerebral apoplexy, Parkinson's disease and the like.
The in vitro induced astrocytes and reactive astrocytes can be applied to experimental research in the aspects of central nervous lesion mechanism, drug screening, clinical treatment and the like, and have important significance.
CN111235110A discloses an in vitro culture method of neural stem cells, which comprises (1) isolating neural stem cells from hippocampus of newborn rat; (2) placing the separated neural stem cells in a culture medium for culture and amplification; (3) the effect of bFGF and EGF at a concentration of 20ng/ml on the differentiation of neural stem cells into neurons, astrocytes and oligodendrocytes was observed. The method has low induction rate of inducing the neural stem cells into the astrocytes, and requires the use of growth factors. The growth factor has the disadvantages of high preparation cost, complex preparation and storage processes, low yield and the like.
CN112912495A discloses a method for producing astrocytes, comprising: (1) dissociating the embryoid into single cells, and performing suspension culture in a serum-free culture dish containing basic fibroblast growth factor and epidermal growth factor to obtain a neural stem cell mass; and (2) dissociating the neural stem cell mass into single cells and performing adherent culture in a serum-free medium containing BDNF and GDNF in the serum, so as to obtain a cell population containing the astrocytes. The method needs to add BDNF and GDNF into the culture medium, has higher cost and complex preparation and storage processes of BDNF and GDNF.
Disclosure of Invention
In view of the above, an object of the present invention is to provide a composition for inducing differentiation of neural stem cells, which is low in cost and has a high rate of inducing differentiation of neural stem cells into astrocytes.
Another object of the present invention is to provide a use of laminin for directionally inducing differentiation of neural stem cells into astrocytes.
It is still another object of the present invention to provide a method for inducing differentiation of neural stem cells, which has a high induction rate of inducing differentiation of neural stem cells into astrocytes and a low cost.
Still another object of the present invention is to provide a use of poly-L-lysine, which can directionally induce differentiation of neural stem cells into reactive astrocytes.
The technical purpose is realized by the following technical scheme.
In one aspect, the invention provides a composition for inducing neural stem cell differentiation, which comprises an induction culture solution and a laminin solution with the concentration of 3-20 μ g/ml, wherein the induction culture solution contains a KnockOut serum substitute.
According to the composition of the invention, preferably, the volume content of the KnockOut serum substitute in the induction culture solution is 3-25 vol%.
Preferably, the induction medium comprises KnockOut serum replacement and DMEM/F12 liquid medium.
According to the composition of the present invention, preferably, the KnockOut serum replacement and DMEM/F12 liquid medium have a volume ratio of 1: (6-9), wherein the volume ratio of a DMEM medium to a F12 medium in the DMEM/F12 liquid medium is 1: 1.
Preferably, the composition according to the present invention further comprises B27 cell culture additives and a penicillin-streptomycin-glutamine solution in the induction medium.
On the other hand, the invention provides an application of a laminin solution in directionally inducing neural stem cells to differentiate into astrocytes, wherein the concentration of the laminin solution is 3-20 mug/ml.
According to the use of the present invention, preferably, the process of directionally inducing differentiation of neural stem cells into astrocytes is performed in the presence of a KnockOut serum replacement.
In another aspect, the present invention provides a method for inducing neural stem cell differentiation using the above composition, comprising the steps of:
and (3) placing the neural stem cells to be differentiated and the induction culture solution containing the KnockOut serum substitute into a culture container coated with laminin with the concentration of 3-20 mu g/ml for culture.
In still another aspect, the present invention provides a use of poly-L-lysine for directionally inducing differentiation of neural stem cells into reactive astrocytes.
According to the use of the present invention, preferably, the process of directionally inducing differentiation of neural stem cells into reactive astrocytes is carried out in the presence of a KnockOut serum replacement.
The composition comprises a KnockOut serum substitute and a laminin solution with the concentration of 3-20 mu g/ml. The combination of the culture medium and the extracellular matrix can be used for improving the induction rate of inducing the neural stem cells to differentiate into the astrocytes. The composition has low cost and convenient use, and has high induction rate for inducing the neural stem cells to differentiate into the astrocytes. The present inventors have unexpectedly found that poly-L-lysine is used as an extracellular matrix to induce neural stem cells in a targeted manner into reactive astrocytes.
Drawings
FIG. 1 is a graph showing the expression levels of GFAP protein in differentiated cells obtained in examples 1 to 2 and comparative example 3.
FIG. 2 is an immunofluorescence map of the differentiated cells obtained in example 1.
FIG. 3 is an immunofluorescence chart of the differentiated cells obtained in comparative example 1.
FIG. 4 is an immunofluorescence map of the differentiated cells obtained in example 2.
Detailed Description
In the present invention,% means vol%, that is, volume percentage, unless otherwise specified.
< composition for inducing differentiation of neural Stem cell >
The composition for inducing differentiation of neural stem cells of the present invention comprises an induction culture solution and a laminin solution. In certain embodiments, an anticoagulant is also included. The compositions of the present invention do not contain cytokines. According to one embodiment of the invention, the composition of the invention consists of an induction broth, an anticoagulant and a laminin solution.
The induction medium of the present invention contains a KnockOut serum replacement. The volume content of the KnockOut serum substitute in the induction culture solution can be 3-25 vol%; preferably 5 to 20 vol%; more preferably 8 to 15 vol%. According to one embodiment of the invention, the KnockOut serum replacement is present in the induction broth in an amount of 10 vol%. The KnockOut serum substitute and the laminin solution are combined for use, so that the neural stem cells can be induced to differentiate into astrocytes, and the induction rate is high. Thus, cytokines are not needed, the cost of the composition is reduced, and the induction rate can be kept high.
The induction culture solution of the invention can also contain one or more of DMEM/F12 liquid medium, B27 cell culture additives and penicillin-streptomycin-glutamine solution. According to one embodiment of the invention, the induction medium consists of KnockOut serum replacement, DMEM/F12 liquid medium, B27 cell culture additives and penicillin-streptomycin-glutamine solution.
The volume content of the DMEM/F12 liquid culture medium in the induction culture solution can be 60-95 vol%; preferably 75-90 vol%; more preferably 85 to 90 vol%. According to one embodiment of the present invention, the content of DMEM/F12 liquid medium in the induction culture solution is 87 vol%. The volume ratio of the DMEM medium to the F12 medium in the DMEM/F12 liquid medium may be 1: 1. According to one embodiment of the present invention, the DMEM/F12 liquid medium is DMEM/F12(1:1) liquid medium (1X).
In the present invention, the ratio of KnockOut serum replacement to DMEM/F12 liquid medium may be 1: (6-9); preferably 1: (7-9); more preferably 1: (8-9).
The induction culture medium of the present invention may further contain B27 cell culture additives. The concentration of the B27 cell culture additive in the induction medium was the working concentration. For example, using B27 cell culture supplement (50 ×), the volume content of B27 cell culture supplement in the induction medium was 2 vol%.
The inducing solution of the present invention may further contain a penicillin-streptomycin-glutamine solution. The concentration of the penicillin-streptomycin-glutamine solution in the induction culture solution is the working concentration. For example, when a penicillin-streptomycin-glutamine solution (100 ×) was used, the volume content of the penicillin-streptomycin-glutamine solution in the induction culture solution was 1 vol%.
The composition of the present invention may contain an anticoagulant. Taking the induction culture solution as a reference, wherein the dosage of the anticoagulant is 5-40 mu g/ml; preferably 10-30 mu g/ml; more preferably 15 to 25. mu.g/ml. The anticoagulant may be heparin sodium.
In the composition, the concentration of the laminin solution is 3-20 mug/ml; preferably 5-15 mu g/ml; more preferably 8 to 12. mu.g/ml. The culture dish coated by the laminin solution is more suitable for inducing and differentiating the neural stem cells into the astrocytes, so that the induction rate is improved. Concentrations of laminin solution outside the above range result in deterioration of the induction effect. The specific reason is not clear, and it is possible that only a laminin solution in a suitable concentration range forms a laminin layer suitable for induction on the surface of the coated culture dish. The concentration of the laminin solution is too low to completely coat the culture dish; too high concentration of laminin solution results in too thick laminin layer coated by the culture dish, which is not favorable for inducing differentiation of neural stem cells into astrocytes.
According to one embodiment of the invention, the composition comprises an induction broth, an anticoagulant and a laminin solution. Induction medium included 87 vol% DMEM/F12(1:1) liquid medium (1X), 2 vol% B27 cell culture supplement (50X), 1 vol% penicillin-streptomycin-glutamine solution (100X) and 10 vol% KnockOut serum replacement. The amount of anticoagulant added was 20. mu.g/ml based on the induction culture medium. The concentration of the laminin solution was 9.6 μ g/ml.
< method for inducing differentiation of neural Stem cell >
Culturing the neural stem cells to be differentiated in the presence of an induction culture solution containing a KnockOut serum substitute and a laminin solution with the concentration of 3-20 mu g/ml. Preferably, the neural stem cells to be differentiated are cultured in the presence of an induction culture solution, an anticoagulant and a laminin solution with the concentration of 3-20 mu g/ml. The selection and amounts of induction medium, laminin solution, and anticoagulant are as described above and will not be described further herein.
Specifically, neural stem cells to be differentiated and an induction culture solution are placed in a culture container coated with laminin for culture. Preferably, the neural stem cells to be differentiated, the induction culture solution and the anticoagulant are cultured in a laminin-coated culture vessel. Optionally, a step of preparing a fibronectin-coated culture vessel may also be included. "culture vessel" refers to a laboratory vessel in which neural stem cells can be cultured and differentiated. For example: petri dishes, culture plates, and the like. The neural stem cells to be differentiated, the induction culture solution and the optional anticoagulant can form single cell suspension, and the single cell suspension is placed into a culture container coated by the laminin solution for culture so as to differentiate the neural stem cells.
The culture container coated with laminin can be prepared by the following method: coating a laminin solution with the concentration of 3-20 mug/ml on a culture container, and forming the laminin coated culture container. Specifically, a laminin solution was added to a culture vessel, the culture vessel with laminin was incubated, and then washed with PBS buffer to obtain a culture vessel coated with a laminin solution. The concentration of the laminin solution is specifically as described previously. The culture vessel with laminin was incubated at room temperature (20-38 ℃). The incubation time is 40-90 min; preferably 50-70 min. The washing may be performed multiple times, e.g., 3 times, with PBS buffer. The laminin solution can be added in an amount to completely cover the bottom of the culture container.
At 3X 105Taking the individual neural stem cells as a reference, and using 0.5-5 ml of induction culture solution; preferably 1-4 ml; more preferably 1.5 to 3 ml.
The neural stem cell to be differentiated according to the present invention may be derived from a mammal such as a mouse, a human, or a monkey. The neural stem cells to be differentiated can be obtained by screening the extracted neural stem cells by adopting an Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF) dual-factor screening method and culturing the neural stem cells in a culture container coated by gelatin for 3-7 generations.
According to one embodiment of the present invention, the neural stem cell to be differentiated is prepared by the following steps:
(1) a step of isolating neural stem cells;
(2) purifying and expanding the neural stem cells.
The step of isolating the neural stem cell may include the steps of: and placing the separated neural stem cells and the neural stem cell culture solution into a gelatin-coated culture container to culture single separated single cells into a first-generation neural stem cell clone group. Specifically, the method comprises the following steps: digesting the striatum obtained by separation with Trypsin (Trypsin) enzyme, stopping digestion with neural stem cell culture solution, centrifuging, discarding supernatant, adding neural stem cell culture solution, and blowing into single cell suspension; and (3) planting the single cell suspension in a culture container coated by gelatin, removing 50% of supernatant every 1-3 days, and replacing the neural stem cell culture solution until single isolated single cells become a first generation neural stem cell clone group.
The digestion temperature of Trypsin (Trypsin) enzyme can be 34-40 ℃; preferably 36 to 38 ℃. The digestion time can be 5-12 min; preferably 8-10 min.
The single cell suspension can be planted in a gelatin-coated culture container at a concentration of 150000-250000 cells/ml. Preferably, the single cell suspension is planted in a gelatin-coated culture vessel at a concentration of 180000-220000 cells/ml. More preferably, the single cell suspension is planted in a gelatin-coated culture vessel at a concentration of 195000-205000 cells/ml.
The step of purifying and expanding neural stem cells may include the steps of: placing the first generation neural stem cell clone group and neural stem cell culture solution into a culture container coated by gelatin to culture a single first generation neural stem cell single cell into a second generation neural stem cell clone group. Specifically, the method comprises the following steps: blowing and beating the first generation neural stem cell clone group and centrifuging, removing supernatant, adding neural stem cell culture solution and blowing and beating to obtain first generation neural stem cell single cell suspension. Planting the first generation neural stem cell suspension in a culture container coated by gelatin, removing 50% of supernatant every 1-3 days, and replacing the neural stem cell culture solution until a single first generation single cell becomes a second generation neural stem cell clone group. Repeating the steps for 1-5 times, and correspondingly obtaining three-to seven-generation neural stem cell clone groups. Blowing and centrifuging the clone groups of the neural stem cells of any generation from three to seven generations, and removing supernate to obtain the neural stem cells to be differentiated.
The primary single cell suspension can be planted in a gelatin-coated culture container at a concentration of 150000-250000 cells/ml. Preferably, the first generation single cell suspension is seeded in a gelatin coated culture vessel at a concentration of 180000-220000 cells/ml. More preferably, the primary single cell suspension is seeded in a gelatin-coated culture vessel at a concentration of 195000-205000 cells/ml.
The single cell suspension of neural stem cells can be counted by a counting plate to determine the content of neural stem cells in the unit volume of single cell suspension of neural stem cells.
The gelatin-coated culture vessel can be prepared by the following method: adding the gelatin solution into the culture container, incubating, and washing with PBS buffer solution to obtain the gelatin-coated culture container.
The incubation can be carried out at 35-39 ℃ and 5% CO2Under the condition of the reaction. The incubation time can be 15-50 min; preferably 20-40 min.
The neural stem cell culture solution comprises a basic culture solution, an Epidermal Growth Factor (EGF) and a basic fibroblast growth factor (bFGF). In certain embodiments, the neural stem cell culture fluid consists of basal medium, Epidermal Growth Factor (EGF), and basic fibroblast growth factor (bFGF).
The basic culture solution contains DMEM/F12 liquid medium, N-2 additive and penicillin-streptomycin-glutamine solution. The volume ratio of DMEM to F12 in DMEM/F12 was 1: 1. The DMEM/F12 liquid medium according to one embodiment of the present invention was DMEM/F12(1:1) liquid medium (1 ×). The concentration of the N-2 supplement in the basal medium was the working concentration. For example, if the N-2 supplement is N-2 supplement (100X), the volume content thereof in the basic culture solution is 1 vol%. The concentration of the penicillin-streptomycin-glutamine solution in the basal medium is the working concentration. For example, if the penicillin-streptomycin-glutamine solution is penicillin-streptomycin-glutamine solution (100 ×), the volume content thereof in the basic culture solution is 1 vol%. According to one embodiment of the present invention, the basal medium consists of 98 vol% DMEM/F12(1:1) liquid medium (1X), 1 vol% N-2 supplement (100X) and 1 vol% penicillin-streptomycin-glutamine solution (100X).
Based on the basic culture solution, the addition amount of the epidermal growth factor EGF can be 5-35 ng/ml; preferably 10-30 ng/ml; more preferably 15 to 25 ng/ml. According to one embodiment of the present invention, the epidermal growth factor EGF is added in an amount of 20 ng/ml.
Based on the basic culture solution, the addition amount of basic fibroblast growth factor (bFGF) can be 5-35 ng/ml; preferably 10-30 ng/ml; more preferably 15 to 25 ng/ml. According to one embodiment of the present invention, basic fibroblast growth factor, bFGF, is added in an amount of 20 ng/ml.
< use of laminin solution >
The neural stem cells to be differentiated and the induction culture solution containing the KnockOut serum substitute are placed in a multilayer adherent protein coated culture container for culture, and the neural stem cells can be directionally induced and differentiated into astrocytes. Therefore, the invention provides the application of the laminin solution in directionally inducing the differentiation of the neural stem cells into the astrocytes. Preferably, the process of directionally inducing differentiation of neural stem cells into astrocytes is performed in the presence of a KnockOut serum replacement. More preferably, the process of directionally inducing differentiation of neural stem cells into astrocytes is performed in the presence of KnockOut serum replacement, DMEM/F12 liquid medium, B27 cell culture additives, penicillin-streptomycin-glutamine solution and anticoagulant. The concentration of the laminin solution is 3-20 mug/ml; preferably 5-15 mu g/ml; more preferably 8 to 12. mu.g/ml. The astrocytes may be type 1 astrocytes (fibrous astrocytes). The selection and amounts of the various materials are as indicated above and will not be described further herein.
The application of the laminin solution in directionally inducing the neural stem cells to differentiate into the astrocytes specifically comprises the following steps: culturing the neural stem cells to be differentiated in the presence of an induction culture solution, an anticoagulant and a laminin solution with the concentration of 3-20 mug/ml. According to one embodiment of the present invention, neural stem cells to be differentiated, an induction culture solution and an anticoagulant are cultured in a laminin-coated culture vessel. Optionally, a step of preparing a fibronectin-coated culture vessel may also be included. The specific steps and parameters are as described above, and are not described herein again.
< use of poly-L-lysine >
The present inventors have unexpectedly found that poly-L-lysine as an extracellular matrix can directionally induce differentiation of neural stem cells into reactive astrocytes. The compound has important effects on pathological mechanism of the central nervous system, drug screening, clinical research and the like. Thus, the present invention provides a use of poly-L-lysine for the directed induction of differentiation of neural stem cells into reactive astrocytes. Preferably, the process of directionally inducing differentiation of neural stem cells into reactive astrocytes is performed in the presence of a KnockOut serum replacement. More preferably, the process of directionally inducing differentiation of neural stem cells into reactive astrocytes is performed in the presence of KnockOut serum replacement, DMEM/F12 liquid medium, B27 cell culture additive, penicillin-streptomycin-glutamine solution and anticoagulant. In some embodiments, neural stem cells to be differentiated and an induction culture solution containing a KnockOut serum substitute are cultured in a culture vessel coated with a poly-L-lysine solution having a concentration of 0.001 to 0.05 wt%. Reactive astrocytes are astrocytes that have been altered in shape, molecule, and function in response to central nervous system injury, disease, or infection. Upregulation of GFAP protein expression is considered a marker of reactive astrocytes.
Poly-L-lysine can be used in the form of a solution. The concentration of the poly-L-lysine solution can be 0.001-0.05 wt%; preferably 0.005-0.03 wt%; more preferably 0.008 to 0.015 wt%. According to one embodiment of the invention, the concentration of poly-L-lysine solution is 0.01 wt%.
Specifically, the method comprises the following steps: adding poly-L-lysine solution into a culture container, then incubating, and washing with PBS buffer solution to obtain a poly-L-lysine coated culture container; and (3) placing the neural stem cells to be differentiated and the induction culture solution containing the KnockOut serum substitute into a poly-L-lysine coated culture container for culture to obtain the reactive astrocytes. Preferably, the method comprises the following steps: adding poly-L-lysine solution into a culture container, then incubating, and washing with PBS buffer solution to obtain a poly-L-lysine coated culture container; and (3) placing the neural stem cells to be differentiated, the induction culture solution containing the KnockOut serum substitute and the anticoagulant in a poly-L-lysine coated culture container for culture to obtain the reactive astrocytes. "culture vessel" refers to a laboratory vessel in which neural stem cells can be cultured and differentiated. For example: petri dishes, culture plates, and the like.
The concentration of poly-L-lysine solution is as described above. The incubation temperature can be 20-35 ℃. The incubation time can be 2-30 min; preferably 3-10 min.
The volume content of the KnockOut serum substitute in the induction culture solution can be 3-25 vol%; preferably 5 to 20 vol%; more preferably 8 to 15 vol%. According to one embodiment of the invention, the KnockOut serum replacement is present in the induction broth in an amount of 10 vol%. The combination of KnockOut serum replacement and poly-L-lysine can induce differentiation of neural stem cells into reactive astrocytes.
The induction culture solution of the invention can also contain one or more of DMEM/F12 liquid medium, B27 cell culture additives and penicillin-streptomycin-glutamine solution. According to one embodiment of the invention, the induction medium consists of KnockOut serum replacement, DMEM/F12 liquid medium, B27 cell culture additives and penicillin-streptomycin-glutamine solution.
The volume content of the DMEM/F12 liquid culture medium in the induction culture solution can be 60-95 vol%; preferably 75-90 vol%; more preferably 85 to 90 vol%. According to one embodiment of the present invention, the content of DMEM/F12 liquid medium in the induction culture solution is 87 vol%. The volume ratio of the DMEM medium to the F12 medium in the DMEM/F12 liquid medium may be 1: 1. According to one embodiment of the present invention, the DMEM/F12 liquid medium is DMEM/F12(1:1) liquid medium (1X).
In the present invention, the ratio of KnockOut serum replacement to DMEM/F12 liquid medium may be 1: (6-9); preferably 1: (7-9); more preferably 1: (8-9).
The induction culture medium of the present invention may further contain B27 cell culture additives. The concentration of the B27 cell culture additive in the induction medium was the working concentration. For example, using B27 cell culture supplement (50 ×), the volume content of B27 cell culture supplement in the induction medium was 2 vol%.
The induction culture solution of the present invention may further contain a penicillin-streptomycin-glutamine solution. The concentration of the penicillin-streptomycin-glutamine solution in the induction culture solution is the working concentration. For example, when a penicillin-streptomycin-glutamine solution (100 ×) was used, the volume content of the penicillin-streptomycin-glutamine solution in the induction culture solution was 1 vol%.
Taking the induction culture solution as a reference, wherein the dosage of the anticoagulant is 5-40 mu g/ml; preferably 10-30 mu g/ml; more preferably 15 to 25. mu.g/ml. The anticoagulant may be heparin sodium.
At 3X 105Taking the individual neural stem cells as a reference, and using 0.5-5 ml of induction culture solution; preferably 1-4 ml; more preferably 1.5 to 3 ml.
The neural stem cell to be differentiated according to the present invention may be derived from a mammal such as a mouse, a human, or a monkey. The neural stem cells to be differentiated can be obtained by screening the extracted neural stem cells by adopting an Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF) dual-factor screening method and culturing the neural stem cells in a culture container coated by gelatin for 3-7 generations.
According to one embodiment of the present invention, the neural stem cell to be differentiated is prepared by the following steps:
(1) a step of isolating neural stem cells;
(2) purifying and expanding the neural stem cells.
The step of isolating the neural stem cell may include the steps of: and placing the separated neural stem cells and the neural stem cell culture solution into a gelatin-coated culture container to culture single separated single cells into a first-generation neural stem cell clone group. Specifically, the method comprises the following steps: digesting the striatum obtained by separation with Trypsin (Trypsin) enzyme, stopping digestion with neural stem cell culture solution, centrifuging, discarding supernatant, adding neural stem cell culture solution, and blowing into single cell suspension; and (3) planting the single cell suspension in a culture container coated by gelatin, removing 50% of supernatant every 1-3 days, and replacing the neural stem cell culture solution until single isolated single cells become a first generation neural stem cell clone group.
The digestion temperature of Trypsin (Trypsin) enzyme can be 34-40 ℃; preferably 36 to 38 ℃. The digestion time can be 5-12 min; preferably 8-10 min.
The single cell suspension can be planted in a gelatin-coated culture container at a concentration of 150000-250000 cells/ml. Preferably, the single cell suspension is planted in a gelatin-coated culture vessel at a concentration of 180000-220000 cells/ml. More preferably, the single cell suspension is planted in a gelatin-coated culture vessel at a concentration of 195000-205000 cells/ml.
The step of purifying and expanding neural stem cells may include the steps of: placing the first generation neural stem cell clone group and neural stem cell culture solution into a culture container coated by gelatin to culture a single first generation neural stem cell single cell into a second generation neural stem cell clone group. Specifically, the method comprises the following steps: blowing and beating the first generation neural stem cell clone group and centrifuging, removing supernatant, adding neural stem cell culture solution and blowing and beating to obtain first generation neural stem cell single cell suspension. Planting the first generation neural stem cell suspension in a culture container coated by gelatin, removing 50% of supernatant every 1-3 days, and replacing the neural stem cell culture solution until a single first generation single cell becomes a second generation neural stem cell clone group. Repeating the steps for 1-5 times, and correspondingly obtaining three-to seven-generation neural stem cell clone groups. Blowing and centrifuging the clone groups of the neural stem cells of any generation from three to seven generations, and removing supernate to obtain the neural stem cells to be differentiated.
The primary single cell suspension can be planted in a gelatin-coated culture container at a concentration of 150000-250000 cells/ml. Preferably, the first generation single cell suspension is seeded in a gelatin coated culture vessel at a concentration of 180000-220000 cells/ml. More preferably, the primary single cell suspension is seeded in a gelatin-coated culture vessel at a concentration of 195000-205000 cells/ml.
The single cell suspension of neural stem cells can be counted by a counting plate to determine the content of neural stem cells in the unit volume of single cell suspension of neural stem cells.
The gelatin-coated culture vessel can be prepared by the following method: adding the gelatin solution into the culture container, incubating, and washing with PBS buffer solution to obtain the gelatin-coated culture container.
The incubation can be carried out at 35-39 ℃ and 5% CO2Under the condition of the reaction. The incubation time can be 15-50 min; preferably 20-40 min.
The neural stem cell culture solution comprises a basic culture solution, an Epidermal Growth Factor (EGF) and a basic fibroblast growth factor (bFGF). In certain embodiments, the neural stem cell culture fluid consists of basal medium, Epidermal Growth Factor (EGF), and basic fibroblast growth factor (bFGF).
The basic culture solution contains DMEM/F12 liquid medium, N-2 additive and penicillin-streptomycin-glutamine solution. The volume ratio of DMEM to F12 in DMEM/F12 was 1: 1. The DMEM/F12 liquid medium according to one embodiment of the present invention was DMEM/F12(1:1) liquid medium (1 ×). The concentration of the N-2 supplement in the basal medium was the working concentration. For example, if the N-2 supplement is N-2 supplement (100X), the volume content thereof in the basic culture solution is 1 vol%. The concentration of the penicillin-streptomycin-glutamine solution in the basal medium is the working concentration. For example, if the penicillin-streptomycin-glutamine solution is penicillin-streptomycin-glutamine solution (100 ×), the volume content thereof in the basic culture solution is 1 vol%. According to one embodiment of the present invention, the basal medium consists of 98 vol% DMEM/F12(1:1) liquid medium (1X), 1 vol% N-2 supplement (100X) and 1 vol% penicillin-streptomycin-glutamine solution (100X).
Based on the basic culture solution, the addition amount of the epidermal growth factor EGF can be 5-35 ng/ml; preferably 10-30 ng/ml; more preferably 15 to 25 ng/ml. According to one embodiment of the present invention, the epidermal growth factor EGF is added in an amount of 20 ng/ml.
Based on the basic culture solution, the addition amount of basic fibroblast growth factor (bFGF) can be 5-35 ng/ml; preferably 10-30 ng/ml; more preferably 15 to 25 ng/ml. According to one embodiment of the present invention, basic fibroblast growth factor, bFGF, is added in an amount of 20 ng/ml.
The experimental materials are presented below:
ICR mice were purchased from the experimental animals research center of university of inner mongolia;
trypsin (Trypsin) enzyme was purchased from Japan biotechnology company (TAKARA, Japan);
DMEM/F12(1:1) liquid medium (1X), branded Gibco, purchased from Saimer Feishel technologies, Inc.;
n-2 supplement (100 x) with the brand Gibco, purchased from Saimer Feishell science, Inc.;
penicillin-streptomycin-glutamine solution (100 ×), branded Gibco, purchased from siemmer feishel technologies ltd;
epidermal Growth Factor (EGF) was purchased from petotack (Pepro Tech Inc.);
basic fibroblast growth factor (bFGF) was purchased from petotack (Pepro Tech Inc.);
gelatin solution, brand Sigma-Aldrich, purchased from Merck group;
laminin solutions were purchased from Corning (Corning);
KnockOut serum replacement, Gibco, purchased from Saimer Feishel technologies, Inc.;
fetal bovine serum, branded Gibco, purchased from Saimer Feishell science Inc.;
b27 cell culture additive (50 ×), branded Gibco, purchased from siemer feishel technologies ltd;
the neural stem cell culture solution consists of a basic culture solution, Epidermal Growth Factor (EGF) and basic fibroblast growth factor (bFGF). The basal medium consisted of 98 vol% DMEM/F12(1:1) liquid medium (1X), 1 vol% N-2 supplement (100X), 1 vol% penicillin-streptomycin-glutamine solution (100X). Based on the basic culture solution, the addition amount of the epidermal growth factor EGF is 20ng/ml, and the addition amount of the basic fibroblast growth factor bFGF is 20 ng/ml.
The gelatin-coated culture dish of preparation example 1 was prepared as follows: 2ml of 1 wt% gelatin solution was placed in a petri dish (60 mm diameter) in 5% CO2And incubated in an incubator at 37 ℃ for 30min, and then washed 3 times with PBS buffer to obtain a gelatin-coated petri dish.
Preparation example 1
Step of isolating neural stem cells
A. ICR mice pregnant for 14.5 days were sacrificed by cervical dislocation, the abdomen was dissected, and the uterus and fetal mice were removed.
B. And (3) washing the uterus and the fetal rat for 2-3 times by using an ice PBS buffer solution, and separating the fetal rat from other tissues.
C. Fetal rat heads were isolated with forceps under a microscope, washed with ice PBS buffer and dissected out of fetal rat brains.
D. Separating striatum from the brain of a fetal rat, shearing the striatum in an ice PBS buffer solution, and then digesting the striatum in a water bath kettle at 37 ℃ for 8-10 min by Trypsin (Trypsin) enzyme to obtain the separated neural stem cells.
F. Adding neural stem cell culture solution into the separated neural stem cells to terminate digestion, and then centrifuging in a centrifuge (with the rotation speed of 1000rpm) for 5min at 4 ℃; centrifuging, removing supernatant, adding neural stem cell culture solution, blowing and separating to obtain single cell suspension.
G. And (3) planting the single cell suspension in a gelatin-coated culture dish at the concentration of 200000 cells/ml for culture, replacing 50% of supernatant every two days, and replacing the neural stem cell culture solution until single isolated single cells become a generation of neural stem cell clone masses.
Step of purifying and expanding neural stem cells
a. Collecting the first generation neural stem cell clone group in a centrifuge tube, blowing and beating for 20 times, and then centrifuging for 5min under the conditions of 4 ℃ and 1000rpm to obtain a first generation neural stem cell suspension.
b. Taking the first generation of neural stem cell suspension out of the centrifuge, discarding the supernatant, adding 1ml of neural stem cell culture solution, and blowing to obtain the first generation of neural stem cell single cell suspension.
c. Planting the first generation neural stem cell single cell suspension in a gelatin-coated culture dish at the concentration of 200000 cells/mL, removing 50% of supernatant every two days, and replacing the neural stem cell culture solution until the single first generation neural stem cell single cell becomes a second generation neural stem cell clone group.
d. Repeating the steps (a) to (c) until a third-generation to seventh-generation neural stem cell clone group is obtained.
e. And centrifuging the neural stem cell clone groups from the third generation to the seventh generation for 5 minutes at 25 ℃ and 1000rpm, and removing supernatant to obtain the neural stem cells to be differentiated.
Preparation example 2
Adding 1ml of laminin solution with the concentration of 9.6 mug/ml into a culture dish with the diameter of 35 mm; the culture dish with the laminin solution was incubated at 25 ℃ for 60min, and then washed 3 times with PBS buffer to obtain a laminin-coated culture dish.
Preparation example 3
1ml of a 1 wt% strength gelatin solution was added to a 35mm diameter petri dish; the petri dish with the gelatin solution was incubated at 37 ℃ for 30min, and then washed 3 times with PBS buffer to obtain a gelatin-coated petri dish.
Preparation example 4
1ml of poly-L-lysine solution with a concentration of 0.01 wt% was added to a petri dish with a diameter of 35 mm; the petri dish with the poly-L-lysine solution was incubated at 25 ℃ for 5min, and then washed 3 times with PBS buffer to obtain a petri dish coated with the poly-L-lysine solution.
Examples 1 to 2 and comparative examples 1 to 4
The neural stem cell to be differentiated obtained in preparation example 1, an induction culture solution and heparin sodium are formed into a neural stem cell suspension to be differentiated. Induction medium consisted of 10 vol% serum or its replacement, 87 vol% DMEM/F12(1:1) liquid medium (1 ×), 2 vol% B27 cell culture additive (50 ×), and 1 vol% penicillin-streptomycin-glutamine solution (100 ×). Taking the induction culture solution as a reference, wherein the dosage of the heparin sodium is 20 mu g/ml; every 3X 105Each neural stem cell to be differentiated was suspended in 2ml of induction medium. Inoculating 2ml of neural stem cell suspension to be differentiated into a culture dish, removing supernatant every two days, replacing induction culture solution and heparin sodium, and culturing for 13 days to obtain differentiated cells. The selection of serum or its substitutes and the culture dish is specifically shown in table 1.
TABLE 1
Experimental example 1
The differentiated cells obtained in examples 1 to 2 and comparative examples 1 to 4, which were cultured up to day 11, were collected to obtain 6 groups of cells. And (3) respectively loading each group of cells into two centrifuge tubes, wherein one centrifuge tube is added with the fluorescent antibody of the ACAS-2 protein, and the other centrifuge tube is not added with any antibody as a blank control. The astrocyte content of 6 groups of cells was analyzed by flow cytometry, and the results are shown in Table 2.
TABLE 2
| Serial number | Astrocyte induction rate (%) |
| Example 1 | 94.2 |
| Example 2 | 86.8 |
| Comparative example 1 | 30.4 |
| Comparative example 2 | 36.2 |
| Comparative example 3 | 29.9 |
| Comparative example 4 | 52.2 |
As can be seen from table 2, the composition of the present invention can effectively induce neural stem cells into astrocytes, and has a high induction rate, wherein the content of astrocytes reaches 94.2%. Comparative example 1 substitution of KnockOut serum replacement with fetal bovine serum, the induction rate was significantly reduced. Comparative example 3 is different from the present invention only in that the extracellular matrix coating the culture dish is changed into gelatin, and the induction rate thereof is also significantly lower than that of the present invention. Comparative example 2 the induction rate was also significantly lower than that of the present invention using a combination of fetal bovine serum and gelatin-coated culture dishes. Example 2 replacement of extracellular matrix coating the culture dish with poly-L-lysine reduced the induction rate by 7.4%. The extracellular matrix of the coated culture dish of comparative example 4 is poly-L-lysine, and fetal bovine serum is used as an inducing solution, and the inducing rate is significantly lower than that of the present invention. This indicates a synergistic effect between the compositions of the present invention, which are not a routine choice.
Experimental example 2
The differentiated cells of example 1, example 2 and comparative example 3 were collected to obtain 3 groups of cells. After each group of cells are cracked, GFAP protein and beta-actin protein are marked through a protein immunoblotting method, and the relative expression quantity of the GFAP protein of 3 groups of cells is analyzed.
As can be seen from FIG. 2, the relative expression amount of the GFAP protein of example 2 is higher than that of example 1; as can be seen from table 2, the induction rate of example 2 is lower than that of example 1. Therefore, it is inferred that the expression level of the GFAP protein in a single cell was significantly increased in the astrocytes induced in example 2, the astrocytes induced in example 2 were reactive astrocytes, and the astrocytes induced in example 1 were non-reactive astrocytes.
Experimental example 3
The differentiated cells of example 1, example 2 and comparative example 1 were collected to obtain 3 groups of cells. And (3) cracking each group of cells, dyeing by an immunofluorescence staining method, and judging the result by using a fluorescence microscope.
As is clear from FIGS. 2 to 4, the immunofluorescent staining results of example 1 showed that the astrocytes induced by the staining were thin and long in foot, indicating that type 1 astrocytes (fibrous astrocytes) were induced. The immunofluorescent staining result of comparative example 1 showed that the astrocyte foot processes induced by the staining were wide and short, indicating that type 2 astrocytes (protoplasmic astrocytes) were induced. The immunofluorescent staining result of example 2 shows that the induced astrocytes have a glial scar structure specific to reactive astrocytes, indicating that the induced astrocytes have reactive astrocytes.
The present invention is not limited to the above-described embodiments, and any variations, modifications, and substitutions which may occur to those skilled in the art may be made without departing from the spirit of the invention.
Claims (10)
1. A composition for inducing neural stem cell differentiation is characterized by comprising an induction culture solution and a laminin solution with the concentration of 3-20 mu g/ml, wherein the induction culture solution contains a KnockOut serum substitute.
2. The composition of claim 1, wherein the KnockOut serum replacement is present in the induction medium in an amount of 3 to 25 vol%.
3. The composition of claim 1, wherein said induction medium comprises KnockOut serum replacement and DMEM/F12 liquid medium.
4. The composition of claim 3, wherein the KnockOut serum replacement and DMEM/F12 liquid media are present in a volume ratio of 1: (6-9), wherein the volume ratio of a DMEM medium to a F12 medium in the DMEM/F12 liquid medium is 1: 1.
5. The composition of claim 3, wherein the induction medium further comprises B27 cell culture additives and a penicillin-streptomycin-glutamine solution.
6. The application of the laminin solution in directionally inducing the neural stem cells to differentiate into astrocytes is characterized in that the concentration of the laminin solution is 3-20 mug/ml.
7. The use according to claim 6, wherein the process of directionally inducing differentiation of neural stem cells into astrocytes is carried out in the presence of KnockOut serum replacement.
8. A method for inducing differentiation of neural stem cells using the composition according to any one of claims 1 to 5, comprising the steps of:
and (3) placing the neural stem cells to be differentiated and the induction culture solution containing the KnockOut serum substitute into a culture container coated with laminin with the concentration of 3-20 mu g/ml for culture.
9. Use of poly-L-lysine for the directed induction of differentiation of neural stem cells into reactive astrocytes.
10. The use according to claim 9, characterized in that the process of directed induction of differentiation of neural stem cells into reactive astrocytes is carried out in the presence of a KnockOut serum replacement.
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| JPWO2020054647A1 (en) * | 2018-09-14 | 2021-11-11 | 学校法人慶應義塾 | How to make astrocytes |
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| CN1852971A (en) * | 2002-07-11 | 2006-10-25 | 加利福尼亚大学董事会 | Oligodendrocytes derived from human embryonic stem cells for remyelination and treatment of spinal cord injury |
| US20080096273A1 (en) * | 2004-09-06 | 2008-04-24 | Yasushi Kondo | Method for Preparing Conditioned Medium of Astrocyte-Like Cells |
| US20110201113A1 (en) * | 2010-02-17 | 2011-08-18 | Kaia Palm | Ex Vivo Progenitor and Stem Cell Expansion and Differentiation for Use in the Treatment of Disease of the Nervous System |
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