CN114099548B - Human neural stem cell preparation and preparation method thereof - Google Patents
Human neural stem cell preparation and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a human nerve stem cell preparation and a preparation method thereof, wherein the human nerve stem cell preparation consists of nerve stem cells and matrix solution, the matrix solution consists of dextran 40 glucose injection, 10-15% human serum albumin, 0.1-0.5ug/ml citicoline sodium injection, 20-50ug/ml ganglioside sodium and 1-10 uM coenzyme Q10. The stem cells in the preparation are high-purity and high-activity neural stem cells obtained by in-vitro induction and amplification of human embryonic stem cells, and the neural stem cell preparation prepared from the neural stem cells and a matrix solution can well maintain the activity, the dryness and the uniformity of the neural stem cells, has definite components, does not contain heterologous animal serum, has uniform cell morphology after being stored at 4-8 ℃ for 48h, has the survival rate of more than 90%, has positive specific surface markers Nestin of the neural stem cells, and has the characteristics of forming nerve balls and differentiating into neurons.
Description
Technical Field
The invention relates to a human neural stem cell preparation and a preparation method thereof, in particular to the technical field of stem cell culture.
Background
Neurodegenerative diseases are currently clinically common neurological diseases, mainly caused by the loss of neurons and/or their myelin sheaths, which worsen over time and become dysfunctional. It mainly comprises Cerebral Ischemia (CI), brain Injury (BI), and epilepsy; alzheimer's Disease (AD), parkinson's Disease (PD), huntington's Disease (HD), amyotrophic Lateral Sclerosis (ALS), and the like. The central nervous system is difficult to repair and regenerate after being damaged, and the main reasons are as follows: (1) neural cells such as neurons are highly differentiated terminal cells, and have small regeneration capacity per se; (2) the neurotrophic factor is not secreted enough, and the local microenvironment is not beneficial to the repair of a damaged nervous system; (3) after injury, the organism secretes inflammatory factors and various cytokines, inhibits synaptic regeneration and aggravates the occurrence of ischemia and hypoxia; (4) scar formation at the damaged part has physical and chemical barrier effect on nerve regeneration, and increases the difficulty of extending and growing nerve synapses.
Current treatments are drug therapy, exercise therapy and cell therapy. The medicine is generally a medicine with neuroprotection and has limited curative effect; exercise therapy has limitations for patients with severe loss of exercise capacity; in contrast, transplantation of neural stem cells is probably one of the most effective ways. Neural stem cells are a type of pluripotent stem cells that have self-renewal capacity and differentiate into neurons, astrocytes and oligodendrocytes under specific microenvironment. The neural stem cells can be differentiated into neurons or glial cells in the transplanting repair process, replace cells which are damaged and dead, express various neurotrophic factors, improve the microenvironment of the cells, inhibit the excessive proliferation of glial cells and the like.
Neural stem cells are mainly derived from fetal brain tissue or human embryonic stem cells or induced pluripotent stem cells (ipscs) to differentiate directionally. The limited sources of fetal brain tissue and ethical issues limit their large-scale clinical application. The safety of clinical application of IPSC is still to be further studied due to the embedding of foreign genes. Therefore, ESC is used as a raw material to qualitatively induce the neural stem cells, a new thought is provided for clinical treatment, the application potential of the neural stem cells in clinical medicine is greatly expanded, and clinical researches show that the neural precursor cells of human embryonic stem cell sources are safe and effective for treating the parkinsonism.
Neural stem cells are particularly important for exerting therapeutic effects, as a key component of clinical cell transplantation therapies, in terms of cell activity, stem cell uniformity and cell uniformity. As single components such as physiological saline, glucose injection and the like are used as infusion media of cells, the cell activity is reduced to below 90% in 12 h due to single nutrition and poor buffer capacity, the differentiation and regeneration capacity of the cells are insufficient, the uniformity is unstable, and potential safety hazards and poor curative effects exist in clinical research and application. Therefore, the invention has definite components, safe clinical infusion and effective long-time maintenance of the activity, dryness and uniformity of the infused stem cells, and becomes a problem to be solved in clinical treatment.
The human serum albumin provides nutrition for cells, maintains the activity of the cells, can enhance the stability of single cells, avoids aggregation and prevents embolism caused by the reinfusion process.
The citicoline is a nucleoside derivative, can improve the recovery of nerve cell function, has a certain effect on promoting the recovery of brain function, can be absorbed into nerve cell membranes under the cerebral ischemia state, promotes the biosynthesis of phospholipid, improves the metabolism of phospholipid, and improves the cell activity and the dryness of cells during in-vitro and in-vivo transplantation.
Dextran 40 glucose injection: the blood volume expander can improve the colloid osmotic pressure of blood plasma after intravenous injection, absorb the water outside blood vessels to increase the blood volume, and raise and maintain the blood pressure. It can depolymerize the aggregated red blood cells and platelets, reduce blood viscosity, improve microcirculation, and prevent thrombosis; glucose is the energy source of living cells and an intermediate product of metabolism, the primary energy supply of organisms.
Gangliosides can promote functional recovery of central nervous system injury caused by various reasons. The mechanism of action is to promote "neural remodeling" including neuronal cell survival, axonal growth and synaptic growth. Has protecting effect on nerve cells, and can improve nerve cell activity and nerve differentiation ability.
Coenzyme Q10 (Coenzyme Q10) is one of the electron transfer chains and involved in aerobic respiration in cell mitochondria, and can significantly improve cell activity by protecting cells from oxidative damage by free radicals and reducing apoptosis, thereby playing an important role in the antioxidant system of cells.
Disclosure of Invention
The invention aims to provide a neural stem cell preparation which can effectively maintain the activity, the dryness and the uniformity of the neural stem cells, has definite components, does not contain heterologous animal serum and has high safety performance, and aims to overcome the defects of the prior art.
In order to achieve the above purpose, the present invention provides the following technical solutions: the human neural stem cell preparation consists of neural stem cells and matrix solution, wherein the matrix solution consists of dextran 40 glucose injection, 10-15% human serum albumin, 0.1-0.5ug/ml citicoline sodium injection, 20-50ug/ml ganglioside sodium and 1-10 uM coenzyme Q10.
The neural stem cells are obtained by in vitro induced differentiation and amplification of human embryonic stem cells, and the concentration of the neural stem cells in the neural stem cell preparation is 1-10 multiplied by 10 6 /ml。
The concentration of the dextran in the dextran 40 glucose injection is 6%, and the concentration of the glucose is 5%.
The human embryonic stem cells are H9 human embryonic stem cells, the cells are provided by the national academy of sciences of China, and the H9 human embryonic stem cell line is originally derived from WICell company in the United states;
the preparation method of the human neural stem cell preparation comprises the following specific steps:
human embryonic stem cell H9 culture: culturing human embryonic Stem cells in a T25 culture bottle pre-coated with VITRONECTUIN XF (Stem cell), adding TeSRTM-E8 culture medium, changing liquid every day, incubating with separating enzyme at 37 deg.C for 3-5min for digestion when clone grows to about 80% confluence, and freezing, passaging or induced differentiation of digested ESC;
induction of neural stem cells: suspending the digested ESC in a culture flask or a culture dish which is not subjected to tissue treatment, adding a nerve induction culture medium, culturing for 7-10 days, and changing the liquid every 2 days; culturing suspended cell spheres in a culture flask or a culture dish which is pre-coated with matrigel, adding a nerve induction medium without SB431542 and DMH1, and culturing for 10-12 days, wherein a large amount of neural stem cells are observed; the culture condition of the neural stem cells is 37 ℃ and 5% CO 2 A concentration incubator;
expansion of neural stem cells: adding separating enzyme into nerve stem cells, incubating at 37 ℃ for 3-5min, adding 2 times of culture medium to stop digestion, lightly blowing down the cells, centrifuging at 1400rpm for 3-5min, suspending the bottom cell sediment in the nerve stem cell amplification culture medium to amplify, mechanically cutting nerve balls into small balls every 3 days, and supplementing 2 times of culture medium to continuously culture and amplify, thus obtaining a large number of nerve stem cells;
neural stem cell identification: absorbing the culture medium, washing 3 times with PBS, incubating for 10 min at room temperature with 4% paraformaldehyde, washing 3 times with PBS, blocking for 1 h at room temperature with blocking solution, washing 3 times with PBS, adding Nestin antibody, and incubating overnight at 4 ℃; washing 3 times by using PBS, adding a secondary antibody, incubating for 2 hours at room temperature in a dark place, rinsing 3 times by using PBS, and detecting the expression condition of each marker by using an EVOSFL auto full-automatic fluorescence microscopic imaging system and photographing in dark place;
preparation of neural stem cell preparation: 100 Preparing matrix solution ml, 10-15 ml human serum albumin, 0.2-1 ul citicoline sodium injection (100 mg/2 ml), 40-100 ul ganglioside sodium (100 mg/2 ml), 32-320 ul coenzyme Q10 (5 mg/2 ml), supplementing dextran 40 glucose injection to 100 ml, placing in a miniature oscillator (VORTEX GENIE), shaking at 180rpm for 30s-1 min, mixing, and storing at 4deg.C;
pouring the amplified neural stem cells into 50 ml centrifuge tube, centrifuging at 1400rpm for 3-5min, adding 3-5 ml separating enzyme into the bottom precipitate, incubating at 37deg.C for 3-5min, shaking every 1 min, stopping digestion when cell ball becomes significantly smaller, adding 2 times of culture medium, repeatedly gently beating, centrifuging at 1400rpm for 3-5min, washing cells with physiological saline for 2-3 times, filtering with 100 mesh sieve, counting, and collecting cells at 1-10×10 6 The density of the solution is/ml suspended in a matrix solution, and the neural stem cell preparation is prepared.
The neural induction culture medium in the induction step of the neural stem cells is a DMEM/F12-based culture medium, and contains 1% of N2, 20-50 ng/mlbFGF,2-10uM SB431542 and 2-10uM DMH1;
the neural stem cell expansion culture medium in the neural stem cell expansion step uses DMEM/F12 as a basic culture medium, contains 0.5-2% of platelet lysate, 0.8-1.5 xITS and 0.5-1% of N2;1-2% B27;20-50 ng/ml human basic fibroblast growth factor (bFGF), 10-20 ng/ml IGF-II,0.1-0.2 ml beta-mercaptoethanol (BME).
Compared with the prior art, the invention has the beneficial effects that: the invention can effectively induce and amplify a large number of high-purity and high-activity neural stem cells from human embryonic stem cells, the neural stem cell preparation prepared from the neural stem cells and matrix solution can well maintain the activity, the dryness and the uniformity of the neural stem cells, the invention has definite components, does not contain heterologous animal serum, has uniform cell morphology after 48-h is preserved at 4-8 ℃, the survival rate is still maintained to be more than 90%, and the specific surface mark Nestin of the neural stem cells is positive.
Drawings
FIG. 1 is a morphology of human embryonic stem cells of example 1;
FIG. 2 is a diagram of suspension Embryoid Bodies (EBs) in example 1;
FIG. 3 is a graph of neural stem cells cultured by adherence in example 1;
FIG. 4 is a graph of neural stem transistors in example 1;
FIG. 5 is a fluorescence staining chart of rosettes structure in example 1;
FIG. 6 is a neurosphere diagram of example 1;
FIG. 7 is an immunofluorescence of the differentiation of neurospheres into neurons in example 1;
FIG. 8 is an immunofluorescence of a single neural stem cell of example 1;
FIG. 9 is an immunofluorescence of neural stem cell preparation differentiated into neurons after storage 48 h;
FIG. 10 shows the neurospheres formed after 48h of the neural stem cell preparation;
FIG. 11 is a neuro-sphere immunofluorescence map;
FIG. 12 shows the preparation of neural stem cells (5X 10) of example 3 6 Density/ml) store 12 h, 24 h, 48h cell morphology maps.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
Culturing human embryonic stem cells without a feeder layer; and (3) coating a culture bottle: coating T25 or T75 culture flasks with 1-5 ug/ml VITRONECTUIN XF (Stem cell), incubating at 37deg.C for 1-h or 4deg.C overnight, inoculating human embryonic Stem cells into pre-coated culture flasks, adding TeSRTM-E8 medium, changing liquid every day, incubating with an isolating enzyme at 37deg.C for 3-5min for digestion, and subjecting digested ESCs to 1:3-5, or inducing differentiation.
Induction of neural stem cells: the digested ESCs were suspended in non-tissue-treated flasks or petri dishes, nerve induction medium (DMEM/F12+1% N2+25 ng/ml bFGF+3 uM SB431542+3 uM DMH1) was added, incubated for 8 days, and the cells were suspended as Embryoid Bodies (EBs) every 2 days, as shown in FIG. 2. The suspension embryoid bodies were cultivated in a culture flask or a culture dish which was coated with matrigel in advance, and a neural induction medium without SB431542 and DMH1 was added for cultivation for 12 days, and a large number of neural stem cells were observed to proliferate as shown in FIG. 3, and a typical "rosettes" structure was partially observed as shown in FIG. 4, and cells were positive as identified by Nestin staining as shown in FIG. 5.
Expansion of neural stem cells: adding separating enzyme into neural stem cells, incubating at 37deg.C for 3-5min, adding 2 times of culture medium to terminate digestion, gently blowing down the cells, centrifuging at 1400rpm for 3-5min, suspending the bottom cell pellet in neural stem cell amplification culture medium (DMEM/F12, 1% platelet lysis, 1× ITS,1%N2,2%B27,25 ng/ml bFGF,15 ng/ml IGF-II,0.15 ml beta-mercaptoethanol (BME) for amplification, mechanically cutting the neural sphere into pellets every 3 days, and supplementing 2 times of culture medium for continuous culture amplification, thus obtaining a large number of neural spheres, as shown in FIG. 6.
Neuron differentiation: the nerve balls or the digested nerve stem cells are inoculated into matrigel coated culture flasks or culture plates, a neuron culture medium is added for culture, the liquid is changed for 1 time every 3-5 days, the nerve stem cells are differentiated into the nerve cells in the third day, a large number of nerve filaments climb out of the nerve balls radially along with the extension of the culture time, the digested nerve stem cells are differentiated into typical nerve cells, and the nerve stem cells are subjected to immunofluorescent staining to show that Tuj-1 is positive, as shown in figures 7 and 8, the obtained nerve stem cells have the capacity of differentiating into the nerve cells.
Example 2
Preparing a neural stem cell preparation; 100 Preparation of matrix solution ml comprises adding 10 ml human serum albumin, 0.2 ul citicoline sodium injection (100 mg/2 ml), 40 ul ganglioside sodium (100 mg/2 ml), 32 ul coenzyme Q10 (5 mg/2 ml), supplementing 100 ml with dextran 40 glucose injection, placing in micro oscillator (VORTEX GENIE), shaking thoroughly, 180rpm, shaking for 30s-1 min, mixing, and storing at 4deg.C.
Preparation of neural stem cell preparation: transferring the amplified neurospheres into 50 ml centrifuge tube, centrifuging at 1400rpm for 3-5min, adding 3-5 ml separating enzyme into the bottom precipitate, incubating at 37deg.C for 3-5min, shaking every 1 min, adding 2 times when the cytopheres become significantly smallerStopping digestion of the culture medium, repeatedly gently blowing at 1400rpm, centrifuging for 3-5min, washing cells with physiological saline for 2-3 times, filtering with 100 mesh sieve, counting, and collecting cells at 1-10X10 6 The density of the solution is/ml suspended in a matrix solution, and the neural stem cell preparation is prepared.
Example 3
Preparing a neural stem cell preparation; 100 Preparation of ml matrix solution comprises adding 12 ml human serum albumin, 0.6 ul citicoline sodium injection (100 mg/2 ml), 70 ul ganglioside sodium (100 mg/2 ml), 160 ul coenzyme Q10 (5 mg/2 ml), supplementing 100 ml with dextran 40 glucose injection, placing in a micro oscillator (VORTEX GENIE), shaking at 180rpm for 30s-1 min, mixing, and preserving at 4deg.C. The remaining steps are identical to those of example 2, neural stem cell preparation (5X 10 6 Density/ml) cells morphology of 12 h, 24 h, 48h are shown in fig. 12.
Example 4
Preparing a neural stem cell preparation; 100 Preparation of matrix solution by adding 15ml of human serum albumin, 1 ul cytidine diphosphate sodium injection (100 mg/2 ml), 100 ul ganglioside sodium (100 mg/2 ml), 320 ul coenzyme Q10 (5 mg/2 ml), supplementing 100 ml with dextran 40 glucose injection, shaking in a miniature shaker (VORTEX GENIE), shaking at 180rpm for 30s-1 min, mixing, and storing at 4deg.C. The remaining steps are identical to those of example 2.
Comparative example 5
Adding 12 ml human serum albumin into 88 ml sodium chloride injection, and fully mixing to prepare a matrix solution. Placing the prepared matrix solution in a 100 ml sterile culture medium bottle, and preserving at 4 ℃;
comparative example 6
Adding 12 ml human serum albumin into 88 ml glucose injection, and fully mixing to prepare a matrix solution. Placing the prepared matrix solution in a 100 ml sterile culture medium bottle, and preserving at 4 ℃;
comparative example 7
Adding 12 ml human serum albumin into 88 ml dextran 40 glucose injection, and mixing completely to obtain matrix solution. Placing the prepared matrix solutions into 100 ml sterile culture medium bottles respectively, and preserving at 4 ℃;
the neural stem cells obtained in example 1 were resuspended in the matrix solutions of examples 2 to 4 and comparative examples 5 to 7, and the cell density was adjusted to 1 to 10X 10 after filtration through a 100-mesh screen 6 Each ml of the system was 50 ml and stored at 4 ℃.
Cell and preparation quality evaluation, specific examples of which are shown in the following table:
analyzing through the table;
cell morphology: a+: round or elliptic, uniformly distributed, diameter between 18-25 and um, and good refractive index of 2-3 cells; after standing, the particles are slightly agglomerated, and after shaking, the particles are dispersed.
A: round or oval, even distribution, good refraction, and darkening of a small number of cells; a small amount of agglomerations are left to stand for agglomerations, and small agglomerations remain after shaking; b: individual cells become larger in volume, refractive index decreases, cells under the mirror darken, and a small amount of flocculent dead mass appears on standing for a long time.
-: indicating no detection.
After 48 and h are prepared, the cell morphology is uniform, the refractive index is strong, the cell survival rate is higher than 90 percent, the cell survival rate is obviously higher than that of a control group, and the cell density is 5 multiplied by 10 in the preparation prepared in the examples 2 to 4 6 The effect is particularly obvious when the dosage is per ml; bacteria and fungi are negative, and endotoxin detection is less than 0.1EU/ml.
Cell re-culture, differentiation and immunofluorescence identification after 48h 6 methods were used to preserve 48h stem cells at 1X 10 respectively 5 Inoculating into matrigel coated 6-well plate and suspending in 6-well plate, culturing, adding nerve differentiation culture medium and nerve stem cell amplification culture medium, adding 3 ml culture medium into each well, standing at 37deg.C, saturated humidity, and CO 2 Culturing in an incubator with a volume fraction of 5%, and continuously culturing for 7-10 days to observe cell differentiation and proliferation.The results show that the cells after the cell preparation of the invention is preserved by 48h still have good adherence and are differentiated into neurons, and the neurons are positive after being identified by Tuj-1 immunofluorescence staining, as shown in figure 9, the neural stem cells can continue to be suspended and proliferated to form neurospheres, as shown in figure 10, and the neurospheres are cultured for 1 day in adherence and are positive after being identified by Nestin immunofluorescence staining, as shown in figure 11.
Therefore, the neural stem cell preparation of the embodiment 2-4 can be verified to be capable of effectively maintaining the activity, the stem property and the differentiation capability of the neural stem cells, the cell morphology is uniform, the survival rate is still kept above 90% after 48h is stored at the temperature of 4-8 ℃, and the neural stem cell specific marker Nestin is positive.
Although the present invention has been described with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications may be made to the embodiments described, or equivalents may be substituted for elements thereof, and any modifications, equivalents, improvements and changes may be made without departing from the spirit and principles of the present invention.
Claims (4)
1. A human neural stem cell preparation, characterized in that: the human neural stem cell preparation consists of neural stem cells and a matrix solution, wherein the matrix solution consists of dextran 40 glucose injection, 10-15% human serum albumin, 0.1-0.5ug/ml citicoline sodium injection, 20-50ug/ml ganglioside sodium and 1-10 uM coenzyme Q10.
2. A human neural stem cell preparation according to claim 1, wherein: the neural stem cells are obtained by in vitro induced differentiation and amplification of human embryonic stem cells, and the concentration of the neural stem cells in the neural stem cell preparation is 1-10 multiplied by 10 6 /ml。
3. A human neural stem cell preparation according to claim 1, wherein: the concentration of the dextran in the dextran 40 glucose injection is 6%, and the concentration of the glucose is 5%.
4. A method for preparing a human neural stem cell preparation, which is characterized by comprising the following steps: human embryonic stem cell H9 culture: culturing human embryonic stem cells in a T25 culture bottle pre-coated with VITRONECTUIN XF, adding TeSRTM-E8 culture medium, changing liquid every day, incubating with separating enzyme at 37 deg.C for 3-5min for digestion when the clone grows to about 80% confluence, and freezing, passaging or induced differentiation of the digested ESC;
induction of neural stem cells: suspending the digested ESC in a culture flask or a culture dish which is not subjected to tissue treatment, adding a nerve induction culture medium, culturing for 7-10 days, and changing the liquid every 2 days; culturing the suspended cell ball in a culture bottle or a culture dish which is pre-coated with matrigel, adding a nerve induction culture medium without SB431542 and DMH1, culturing for 10-12 days, and observing a large amount of neural stem cells; the culture condition of the neural stem cells is 37 ℃ and 5% CO 2 A concentration incubator;
expansion of neural stem cells: adding separating enzyme into nerve stem cells, incubating at 37 ℃ for 3-5min, adding 2 times of culture medium to stop digestion, lightly blowing down the cells, centrifuging at 1400rpm for 3-5min, suspending the bottom cell sediment in the nerve stem cell amplification culture medium to amplify, mechanically cutting nerve balls into small balls every 3 days, and supplementing 2 times of culture medium to continuously culture and amplify, thus obtaining a large number of nerve stem cells;
neural stem cell identification: absorbing the culture medium, washing 3 times with PBS, incubating for 10 min at room temperature with 4% paraformaldehyde, washing 3 times with PBS, blocking for 1 h at room temperature with blocking solution, washing 3 times with PBS, adding Nestin antibody, and incubating overnight at 4 ℃; washing 3 times by using PBS, adding a secondary antibody, incubating for 2 hours at room temperature in a dark place, rinsing 3 times by using PBS, and detecting the expression condition of each marker by using an EVOSFL auto full-automatic fluorescence microscopic imaging system and photographing in dark place;
preparation of neural stem cell preparation: 100 Preparing matrix solution, 10-15 ml human serum albumin, 0.2-1 ul 100mg/2ml citicoline sodium injection, 40-100 ul 100mg/2ml ganglioside sodium, 32-320 ul 5mg/2 ml coenzyme Q10, supplementing 100 ml with dextran 40 glucose injection, placing in a micro oscillator for full oscillation, shaking at 180rpm for 30s-1 min, mixing, and storing at 4deg.C;
pouring the amplified neural stem cells into 50 ml centrifuge tube, centrifuging at 1400rpm for 3-5min, adding 3-5 ml separating enzyme into the bottom precipitate, incubating at 37deg.C for 3-5min, shaking every 1 min, stopping digestion when cell ball becomes significantly smaller, adding 2 times of culture medium, repeatedly gently beating, centrifuging at 1400rpm for 3-5min, washing cells with physiological saline for 2-3 times, filtering with 100 mesh sieve, counting, and collecting cells at 1-10×10 6 Suspending the density of/ml in matrix solution to prepare the neural stem cell preparation;
the neural induction culture medium in the induction step of the neural stem cells is a DMEM/F12-based culture medium, and contains 1% of N2, 20-50 ng/mlbFGF,2-10uM SB431542 and 2-10uM DMH1;
the neural stem cell expansion culture medium in the neural stem cell expansion step uses DMEM/F12 as a basic culture medium, contains 0.5-2% of platelet lysate, 0.8-1.5 xITS and 0.5-1% of N2;1-2% B27;20-50 ng/ml human basic fibroblast growth factor, 10-20 ng/ml IGF-II, beta-mercaptoethanol.
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| CN113633663A (en) * | 2021-10-12 | 2021-11-12 | 呈诺再生医学科技(北京)有限公司 | Application of EPC derived from induced pluripotent stem cell differentiation in preparation of cerebral apoplexy therapeutic agent |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN108184818A (en) * | 2018-01-23 | 2018-06-22 | 领航干细胞再生医学工程有限公司 | A kind of Human plactnta mesenchyma stem cell suspension protective agent |
| CN113633663A (en) * | 2021-10-12 | 2021-11-12 | 呈诺再生医学科技(北京)有限公司 | Application of EPC derived from induced pluripotent stem cell differentiation in preparation of cerebral apoplexy therapeutic agent |
Non-Patent Citations (2)
| Title |
|---|
| Coenzyme Q10 protects neural stem cells against hypoxia by enhancing survival signals;Jinse Park等;Brain Research;第1478卷;64-73页 * |
| Effects of storage media, supplements and cryopreservation methods on quality of stem cells;Ozgur Dogus Erol等;World Journal of Stem Cells;第13卷(第9期);1197-1214页 * |
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