CN108410805B - A kind of isolated culture method of human cord blood stem cell - Google Patents
A kind of isolated culture method of human cord blood stem cell Download PDFInfo
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Abstract
A kind of isolated culture method of human cord blood stem cell, comprising: (1) pretreatment of Cord blood whole blood cells separation and blood plasma；(2) Tissue Culture Dish coating 4 antibody of AntiCD3 McAb and anti-CD44 monoclonal antibody；(3) the Cord blood whole blood cells of step (1) preparation are added into Tissue Culture Dish, incline unbonded cellular layer；(4) pretreated blood plasma and cell culture fluid is added.The method of the invention can make full use of Cord blood, and a variety of stem cells are carried out a step and are separately cultured.
Description
Technical field
The invention belongs to field of biotechnology, especially Stem Cell Engineering field.It is dry thin more particularly to a kind of human cord blood
The isolated culture method of born of the same parents.
Background technique
Stem cell not only can produce or secrete a large amount of bioactie agent, and also life rich in stem cell
Active substances, these stem cell biological active factors or substance can Effective Regulation body cell signal transduction, activating human body it is dry
Cell, and then physiological reparation or substitution body injury, lesion and the cell of aging etc..Such as stem cell can produce stem cell
Growth factor (SCF), nerve growth factor (NGF), stroma cell source property growth factor (SDF), vascular endothelial cell growth because
Son (VEGF), basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF), epidermal growth factor
(EGF), interleukin-6 and IL-7 (IL-6 and IL-7), megakaryocyte colony stimulating factor (M-CSF), tumor necrosis factor
(TNF), the factors such as interferon (IFN), these cell factors have the function of to promote cell Proliferation, differentiation, anti-apoptotic etc.；Stem cell
Innate immunity albumen can also be generated, itself and extraneous factor can be resisted or be repaired to such as IgG, IgA, IgM, IgD, IgE etc.
It is damaged caused by body cell.
Therefore, the bioactive substance in the bioactie agent generated using stem cell secretion or stem cell activates body
Stem cell, and then body injury, lesion and the cell of aging are repaired or substituted by autologous stem cells physiological, in disease prevention and cure
It has broad application prospects with health and beauty field.
The blood being retained in placenta and umbilical cord after baby due is the important sources of stem cell.Numerous studies have shown that navel
It can be used to treat a variety of diseases in the blood system and disease of immune system with the candidate stem cell in blood, including hematological system is pernicious
It is tumour (such as: acute leukemia, chronic leukemia, Huppert's disease, marrow abnormality proliferation syndrome and lymthoma), blood red
Albumen disease (such as: Thalassemia), marrow hematopoiesis function failure (such as: alpastic anemia), congenital metabolic disease, elder generation
Nature immunodeficiency disorders, autoimmune conditions, certain entity tumors are (such as: Small Cell Lung Cancer, neuroblastoma, ovary
Cancer and progressive muscular dystrophy etc.).From navel blood stem cell in 1988 be just used to treat root up to syndrome, it is prosperous up to syndrome and
Draw many children diseases such as syndrome and acute lymphatic leukemia.Currently, Cord blood can not only effectively treat it is several
Ten kinds of refractory diseases and a variety of incurable diseases, and the kinds of Diseases that it can be treated also are being constantly increasing.Self storage
Cord blood once needing in use, being not required to distribution type, cell activity is strong, the danger of no immunological rejection, and transplanting success is high, cures
Rate is high, and medical expense is low.More and more medical research institutes pay attention to and carry out the teiology and Mechanism Study of breast cancer, and
Research is also concentrated mainly on Antineoplastic combined chemotherapy protocols, neoplastic hematologic disorder marker etc., however research achievement is rarely applied to face at present
Bed test, limited tumor markers such as CA15.3 and BR27.29 sensibility is lower, therefore they are used for breast cancer not yet
Screening, therefore there is an urgent need to a kind of invasive methods to screen effective standard items for the early diagnosis of malignant galactophore lesion and pre-
Anti- even Index for diagnosis.
It is newest also to contain a large amount of mescenchymal stem cells studies have shown that not only contain candidate stem cell in Cord blood, however
Current Cord blood is separately cultured mainly for candidate stem cell, for non-hematopoietic stem cell using less.In addition, though navel
With in blood candidate stem cell to be separately cultured technology comparatively mature, but how as far as possible main Research Thinking is at present
It is conveniently separated candidate stem cell, and promotes umbilical cord blood hematopoietic dry thin by adding various cell factors in synthetic media
The proliferation of born of the same parents carries out passage amplification, and the cell factor of addition is more, at high cost, complicated for operation, effect is also bad.
Summary of the invention
Aiming at the shortcomings in the prior art, technical problem to be solved by the invention is to provide one kind can make full use of navel
The isolated culture method of cord blood stem cell with blood resource, the method can not only be by the candidate stem cells in Cord blood point
From culture, and mescenchymal stem cell can be separated and be cultivated.In addition, isolated umbilical cord blood be used to prepare it is dry
Cell culture medium avoids high cost caused by single-factor is added one by one and complex operations.
Specifically, the present invention provides a kind of isolated culture methods of human cord blood stem cell, comprising:
(1) pretreatment of Cord blood whole blood cells separation and blood plasma；
(2) Tissue Culture Dish coating 4 antibody of AntiCD3 McAb and anti-CD44 monoclonal antibody；
(3) the Cord blood whole blood cells of step (1) preparation are added into Tissue Culture Dish, remove unbonded cell；
(4) pretreated blood plasma and cell culture fluid is added.
The isolated culture method of a kind of human cord blood stem cell of the present invention, it is characterised in that: the step (1) includes
Anticoagulant Cord blood is taken, 2000rpm is centrifuged 10min, takes upper plasma, and it is standby that the resuspension of HANK ' S liquid is added into the haemocyte of lower layer
With.
A kind of isolated culture method of human cord blood stem cell of the present invention, it is characterised in that: blood in the step (1)
Slurry pretreatment includes 56 DEG C of heat preservation 30min.
The isolated culture method of a kind of human cord blood stem cell of the present invention, which is characterized in that the step (2) includes
Take 4 antibody of AntiCD3 McAb and anti-CD44 monoclonal antibody with 0.01M respectively, the buffer of pH9.6 is diluted to 10 μ g/L, into Tissue Culture Dish
4 antibody diluent of AntiCD3 McAb and anti-CD44 monoclonal antibody dilution of equivalent is added, 4 DEG C overnight, and incline extra liquid.
The isolated culture method of a kind of human cord blood stem cell of the present invention, which is characterized in that the culture dish is
Polystyrene culture dish.
The isolated culture method of a kind of human cord blood stem cell of the present invention, which is characterized in that the step (3) includes
It is 1 × 10 that concentration is added into culture dish4-1×106The Cord blood whole blood cells of a/mL, 37 DEG C of standing 2h, jog remove not
In conjunction with cell.
The isolated culture method of a kind of human cord blood stem cell of the present invention, which is characterized in that the step (4) includes
By pretreated blood plasma in step (1), 1640 cell culture fluids are added in 1:1 by volume, and the cell training of step (3) is then added
It supports in ware, 37 DEG C, is cultivated in 5%CO2.
A kind of isolated culture method of human cord blood stem cell of the present invention, which is characterized in that the 1640 cell training
Nutrient solution contains 10% inactivation human serum.
The isolated culture method of a kind of human cord blood stem cell of the present invention, it is further characterized in that in cell culture fluid also
Added with fibronectin, the additive amount of the fibronectin is relative to 2 μ g/cm2 of culture dish floor space.
Human cord blood stem cell isolated culture method of the present invention, it is characterised in that further include step (5) whole blood cells
After overnight incubation or culture 8h, liquid is changed with 1640 complete culture solutions added with fibrin, fibronectin, per changing liquid one for 24 hours
It is secondary, had digestive transfer culture after three days.
Compared with the prior art, the advantages of the present invention are as follows:
Firstly, the present invention takes full advantage of candidate stem cell and mescenchymal stem cell in Cord blood, it is coated with by antibody
Mode by two kinds of stem cell separated in synchronization cultures in Cord blood whole blood cells, mescenchymal stem cell being capable of continuous release cell
The factor stimulates the growth of candidate stem cell, and can also provide more differentiation directions.
Secondly, the present invention separates and be utilized the anticoagulant blood plasma of Cord blood, by itself and synthetic media collectively as stem cell
The medium component of culture, the components such as fibrin, fibronectin, cell factor naturally contained in blood plasma can be Cord blood
The growth of stem cell provides better nutrition and growing environment, promotes the adherent proliferation of cell.
In addition, operation of the present invention is simple, without the expensive instrument such as flow cytometer；It can also even if remaining a small amount of red blood cell
It is only to generate haemolysis during follow-up cultivation.
Detailed description of the invention
Cord blood stem cell culture figure after Fig. 1 passage
Fig. 1 shows that the cord blood stem cell of the method for the invention preparation had both contained adherent cell mescenchymal stem cell
(polygonal or spindle), also containing being attached on attached cell or half adherent candidate stem cell (subcircular or ellipse).
The cord blood stem cell fluidic cell figure of Fig. 2 culture
Abscissa is anti-CD44 monoclonal antibody, and ordinate is 4 antibody of AntiCD3 McAb.The result shows that the method for the invention preparation is dry
Both contain CD44+ mescenchymal stem cell in cell, also contain CD34+ candidate stem cell.
Specific embodiment
Below in conjunction with specific embodiment, the present invention is further explained.These embodiments are merely to illustrate the present invention and do not have to
In limiting the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition or presses
According to condition proposed by manufacturer.Unless otherwise defined, all professional and scientific terms as used herein and this field are ripe
It is identical to practice meaning known to personnel.In addition, any method similar to or equal to what is recorded and material all can be applied to
In the method for the present invention.The preferred methods and materials described herein are for illustrative purposes only.
Embodiment one
(1) sterile to take Cord blood, anticoagulant heparin, 2000rpm is centrifuged 10min, takes upper plasma, 56 DEG C of heat preservation 30min.To
It is spare that the resuspension of HANK ' S liquid is added in the haemocyte of lower layer.
(2) take 4 antibody of AntiCD3 McAb and anti-CD44 monoclonal antibody with 0.01M respectively, the buffer of pH9.6 is diluted to 10 μ g/L, to 6
4 antibody diluent of AntiCD3 McAb and anti-CD44 monoclonal antibody dilution of equivalent are added in the Tissue Culture Dish of hole, 4 DEG C overnight, and it is extra to incline
Liquid.
(3) it is 1 × 10 that concentration is added into culture dish4-1×106The Cord blood whole blood cells of a/mL, 37 DEG C of standing 2h,
Jog, incline unbonded cell.
(4) by pretreated blood plasma in step (1), the inactivation human serum containing 10%, fibronectin is added in 1:1 by volume
1640 cell culture fluids, then be added step (3) Tissue Culture Dish in, 37 DEG C, cultivated in 5%CO2.
(5) after overnight incubation, liquid is changed with 1640 complete culture solutions added with fibrin, fibronectin, it is every to change liquid for 24 hours
Once, had digestive transfer culture after three days.
Embodiment two
By the plating cells after had digestive transfer culture, microscopy observes cellular morphology under inverted microscope after covering with culture plate.
Show that the cord blood stem cell of the method for the invention preparation had both contained adherent cell mescenchymal stem cell (polygonal or spindle
Shape), also contain and is largely attached to attached cell or half adherent candidate stem cell (subcircular or ellipse).
4 antibody of AntiCD3 McAb of the different fluorescent markers of cell of third time passage and anti-CD44 monoclonal antibody are detected.As a result
Show both containing CD44+ mescenchymal stem cell in the stem cell of the method for the invention preparation, it is also thin containing CD34+ Hematopoietic Stem
Born of the same parents.
Above description is not limitation of the present invention, and the present invention is also not limited to the example above.The art it is common
Within the essential scope of the present invention, the variations, modifications, additions or substitutions made also should belong to protection of the invention to technical staff
Range, protection scope of the present invention are subject to claims.
Claims (7)
1. a kind of isolated culture method of human cord blood stem cell, comprising:
(1) pretreatment of Cord blood whole blood cells separation and blood plasma；
(2) Tissue Culture Dish coating 4 antibody of AntiCD3 McAb and anti-CD44 monoclonal antibody, including 4 antibody of AntiCD3 McAb and anti-CD44 monoclonal antibody are taken respectively
With 0.01M, the buffer of pH9.6 is diluted to 10 μ g/L, into Tissue Culture Dish be added equivalent 4 antibody diluent of AntiCD3 McAb and
Anti-CD44 monoclonal antibody dilution, 4 DEG C overnight, and incline extra liquid；
(3) the Cord blood whole blood cells of step (1) preparation are added into Tissue Culture Dish, remove unbonded cell；
(4) pretreated blood plasma and cell culture fluid is added；
Wherein, the step (1) includes taking anticoagulant Cord blood, and 2000rpm is centrifuged 10min, takes upper plasma, the blood to lower layer is thin
It is spare that the resuspension of HANK ' S liquid is added in born of the same parents, blood plasma pretreatment includes 56 DEG C of heat preservation 30min in the step (1).
2. a kind of isolated culture method of human cord blood stem cell as described in claim 1, which is characterized in that the culture
Ware is polystyrene culture dish.
3. a kind of isolated culture method of human cord blood stem cell as described in claim 1, which is characterized in that the step
(3) include into culture dish be added concentration be 1 × 104-1×106The Cord blood whole blood cells of a/mL, 37 DEG C of standing 2h, jog,
Remove unbonded cell.
4. a kind of isolated culture method of human cord blood stem cell as described in claim 1, which is characterized in that the step
(4) include that 1640 cell culture fluids are added in 1:1 by volume by pretreated blood plasma in step (1), be then added step (3)
In Tissue Culture Dish, 37 DEG C, 5%CO2Middle culture.
5. a kind of isolated culture method of human cord blood stem cell as claimed in claim 4, which is characterized in that described 1640 is thin
Born of the same parents' culture solution contains 10% inactivation human serum.
6. a kind of isolated culture method of human cord blood stem cell as described in claim 1, it is further characterized in that cell culture
Fibronectin is also added in liquid, the additive amount of the fibronectin is relative to 2 μ g/cm of culture dish floor space2。
7. a kind of isolated culture method of human cord blood stem cell as described in claim 1, it is characterised in that further include step
(5) whole blood culture changes liquid with 1640 complete culture solutions added with fibrin, fibronectin overnight or after culture 8h,
It is every to change the liquid once for 24 hours, had digestive transfer culture after three days.
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