CN105838673A - Extraction and separation method for mesenchymal stem cells (MSCs) - Google Patents
Extraction and separation method for mesenchymal stem cells (MSCs) Download PDFInfo
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- CN105838673A CN105838673A CN201610398252.3A CN201610398252A CN105838673A CN 105838673 A CN105838673 A CN 105838673A CN 201610398252 A CN201610398252 A CN 201610398252A CN 105838673 A CN105838673 A CN 105838673A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
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Abstract
The invention discloses an extraction and separation method for mesenchymal stem cells (MSCs), and the extraction and separation method is used for local-anesthesia downlink bone marrow puncture. The method comprises the following steps: totally extracting 130ml of bone marrow from two sides by use of a 20ml dry and sterile syringe, injecting the bone marrow into a heparin blood bag which is prepared in advance, and rapidly delivering the heparin blood bag to a laboratory; adding sodium citrate used as an anticoagulant into a special reagent for stem cell extraction, and extracting about 0.8ml stem cell suspension by use of a stem cell separation and extraction technology; and adding the stem cell suspension into a gentamicin brine so as to dilute the stem cell suspension to 5ml. According to the method, firstly the MSCs can be extracted from bone marrow of a patient; secondly, the MSCs can be rapidly cultured and amplified in vitro, and can be subjected to directional differentiation towards nerve cells; furthermore, the MSCs can be input back into the body of the patient, and can be integrated in a host central nervous system and exist for a long time; the risks of the method are less than those of allotransplantation, the ethical issues are rarely involved, and BMSCs have favorable prospect and development potential in the aspect of clinic treatment.
Description
Technical field
The invention belongs to medicine technology field, particularly relate to a kind of mesenchymal stem cells MSCs MSCs and extract separation
Method.
Background technology
For a long time, medical circle is thought always, and nerve cell belongs to a kind of permanent cell, lacks power of regeneration.
Therefore, traditional view is thought: when causing a large amount of neuron loss after central nervous system is impaired, because not producing
Raw new neuron, sets up new synaptic contact, and causes the function after damage and be difficult to recover.Therefore, maincenter
The nervous system disease is always the global difficult problem that clinical treatment faces.Progress and doctor along with science and technology
The development for the treatment of technology, it has been found that the tremendous potential in the damage of stem cell pivot nervous system in the treatment, dry
The discovery of cell changes thinks the unrenewable understanding of Adult Mammals central nerve neuronal in the past, because of
This stem cell transplantation becomes the focus that current international medical community is paid close attention to, and the treatment for human neurological diseases brings
Wish.
Mesenchymal stem cells MSCs (MSCs) is mesoderma origin cell, and MSCs is a kind of pluripotent stem cell,
Its treatment to the neurological dysfunction that central nervous system ischemic, amyloidogenic, demyelinating diseases alter
Effect is more significant.After transplanting, MSCs migrates to the nerve fiber Regional Differentiation of pathology and becomes all kinds maincenter god
Through system cells.MSCs has vast prospect in infrastest and clinical research, especially in gene therapy,
Nervous function is recovered and in hematopoietic reconstitution.
Along with the arrival of social senilization, the nervous system disease has become as and causes human death and maimed master
Want one of reason.Causing the big cardiovascular disease of the cause of death of the mankind four, tumour, cerebrovascular disease and old age
In degenerative disease, the nervous system disease account for two, the nervous system disease has that the incidence of disease is high, case fatality rate high,
The feature that disability rate is high and recurrence rate is high, along with stem cells technology is constantly promoted in China, it is applied to maincenter
There is many dramatization, beyond thought good curative effect in nervous system disease, improves nervous system disease
Sick cure rate, decreases disability rate.The discovery of stem cell changes thinks Adult Mammals maincenter in the past
The neural unrenewable understanding of neuron, therefore stem cell transplantation becomes the heat that current international medical community is paid close attention to
Point, the treatment for human neurological diseases brings hope.Autologous bone marrow mesenchymal stem cells transplantation treatment has
Without the advantage that rejection, security and health giving quality are high.
Summary of the invention
It is an object of the invention to provide a kind of mesenchymal stem cells MSCs MSCs extraction separation method, it is intended to solve
When causing a large amount of neuron loss after certainly central nervous system is impaired, because new neuron can not be produced, set up
New synaptic contact, and cause the problem that the function after damage is difficult to recover.
The present invention is achieved in that a kind of mesenchymal stem cells MSCs MSCs extraction separation method, this marrow
Mescenchymal stem cell MSCs extraction separation method comprises the following steps:
In the descending bone marrow puncture of local anaesthesia；
Preprepared with the bone marrow extraction 130 milliliters injection altogether of 20 milliliters of dry asepsis injector both sides
In heparin blood bag, deliver to rapidly laboratory；
Use sodium citrate anti-freezing, add to stem cell extraction special agent, through stem cell separation and Extraction skill
Art, extracts stem cell suspension about 0.8ml；
Stem cell suspension addition after extracting is diluted to 5ml to gentamicin salt solution.
The stem cell transplantation technology individual event that the present invention provides charges about 20,000 yuan, and patient has the ability to assume charge,
Relatively other stem cell transplantations are without the fee of material of supplementary payments allosome stem cell, and economic benefit is higher.
Mesenchymal stem cells MSCs is otherwise known as marrow stromal cell (Bone marrow stromal
Cells, BMSCs), it is a kind of population mixture, mesenchymal stem cells MSCs can break up that to become nervous system thin
Born of the same parents, including neuronal cell, neural endothelial cells and Deiter's cells etc..Medulla mesenchyma is done
Cell compares by relatively other types stem cell, and it has following several advantage, and first it can be from the marrow of patient own
Extract；Secondly it can cultivate rapidly amplification in vitro, it is possible to neuralward cell directional breaks up；And it
Can be fed back in the patient after can integrate in host's central nervous system and long-term existence；And
BMSCs has the advantages that immunogenicity is low, so its danger is lower than the danger of heteroplastic transplantation, seldom
Relate to ethics problem.The most several advantages, BMSCs has preferably in terms of the clinical treatment of cerebral apoplexy
Prospect and development potentiality.
Accompanying drawing explanation
Fig. 1 is a kind of mesenchymal stem cells MSCs MSCs extraction separation method flow process that the embodiment of the present invention provides
Figure,
Detailed description of the invention
In order to make the purpose of the present invention, technical scheme and advantage clearer, below in conjunction with embodiment,
The present invention is further elaborated.Should be appreciated that specific embodiment described herein only in order to
Explain the present invention, be not intended to limit the present invention.
Below in conjunction with the accompanying drawings and the application principle of the present invention is further described by specific embodiment.
A kind of mesenchymal stem cells MSCs MSCs extraction separation method, this mesenchymal stem cells MSCs MSCs carries
Take separation method to comprise the following steps:
S101: in the descending bone marrow puncture of local anaesthesia；
S102: prepare in advance with the 130 milliliters of injections of bone marrow extraction altogether of 20 milliliters of dry asepsis injector both sides
In good heparin blood bag, deliver to rapidly laboratory；
S103: use sodium citrate anti-freezing, adds and extracts in special agent to stem cell, separate through stem cell
Extractive technique, extracts stem cell suspension about 0.8ml；
S104: the stem cell suspension addition after extracting is diluted to 5ml to gentamicin salt solution.
Below in conjunction with embodiment, the present invention is further described.
Case inclusion criteria: 1. age < 50 years old.2. clinical symptoms and combine imaging data (Cranial Computed Tomography or
Head mri) it is diagnosed as Patients With Central Nervous Diseases.The most lasting neurologic impairment.The most unconscious
Obstacle.5. patient and family members agree to.6. without epilepsy.7. be not in the mood for, liver, kidney and other organs serious
Function injures.After improving preoperative coherence check,
Determine operation plan.In the descending bone marrow puncture of local anaesthesia, with 20 milliliters of dry asepsis injector both sides
During bone marrow extraction 130 milliliters injects preprepared heparin blood bag altogether, deliver to rapidly laboratory, use citron
Acid sodium anti-freezing, adds and extracts in special agent to stem cell, through stem cell separation and extraction technology, extract dry
Cell suspension about 0.8ml, is added into being diluted to 5ml to gentamicin salt solution.Bone marrow aspiration is postoperative sternly
The close observation every vital sign of patient.
The mode row autologous bone marrow mesenchymal stem cells of postoperative employing lumbar puncture subarachnoid injection is transplanted and is controlled
Treat.
Transfer autologous bone marrow stem cells has been injected by lumbar puncture.Patient's left lateral position on flat bed,
Head, as far as possible to front flexing, takes hip knee position in the wrong, and back is vertical with bed surface, takes 3-4 spinous process of lumbar vertebra gap for wearing
Thorn point, routine disinfection, spread aseptic hole-towel, tough to intervertebral with 2% lidocaine 3ml local and infiltration anesthesia
Band, thrusts from point of puncture with direction, vertical back with No. 9 puncture needles, and after thrusting about about 5cm, pin cutting edge of a knife or a sword supports
Anti-sense disappears, and slowly extracts nook closing member.
Treat that water white cerebrospinal fluid flows out, BMSCs 5 milliliters is slowly injected into cavum subarachnoidale, penetrates pin
Core, extracts puncture needle, immobilization with adhesive tape, advises patient to remove pillow flat sleeping 6 hours.
Postoperative application antibiotic 6 days is in case infecting, and close observation change of illness state.Before patient's stem-cell therapy
Blindness, right eye only has light sensation, double lower-limb muscular strength 4-levels, can't take care of oneself, follow up a case by regular visits to patient after treatment
Eyes all have partial vision to recover, and about double lower-limb muscular strength 5-levels, can walk, and life is basic takes care of oneself, and
Without any postoperative complication.
In present invention operation, key issue is: 1, the extraction amount of marrow is relatively big, needs consummate bone marrow aspiration skill
Art.2, the separation of mesenchymal stem cells MSCs, needs high-grade marrow separation equipment and good reagent.3、
Lumbar puncture approach stem cell is injected: cerebrospinal fluid is mainly secreted by choroid plexus of lateral ventricle, wherein comprises
Some growth factors and neurotrophic factor.
The present invention uses, by lumbar puncture approach, BMSCs is implanted into cavum subarachnoidale.With other ways
Footpath is compared, and substantial amounts of stem cell can be converged to the focal area generation effect of damage by cerebrospinal fluid approach,
And normal central nervous system will not be produced detrimental effect in early days.
The foregoing is only presently preferred embodiments of the present invention, not in order to limit the present invention, all at this
Any amendment, equivalent and the improvement etc. made within bright spirit and principle, should be included in the present invention
Protection domain within.
Claims (3)
1. a mesenchymal stem cells MSCs MSCs extraction separation method, it is characterised in that fill between this marrow
Matter stem cell MSCs extraction separation method comprises the following steps:
In the descending bone marrow puncture of local anaesthesia；
With in dry asepsis injector the both sides altogether preprepared heparin blood bag of bone marrow extraction injection, send rapidly
To laboratory；
Use sodium citrate anti-freezing, add to stem cell extraction special agent, through stem cell separation and Extraction skill
Art, extracts stem cell suspension；
Stem cell suspension addition after extracting is diluted to 5ml to gentamicin salt solution.
2. mesenchymal stem cells MSCs MSCs extraction separation method as claimed in claim 1, its feature
It is, injects accurate in advance with 20 milliliters of dry asepsis injector both sides bone marrow extraction 130 milliliters altogether
In the heparin blood bag got ready.
3. mesenchymal stem cells MSCs MSCs extraction separation method as claimed in claim 1, its feature
It is, extracts stem cell suspension 0.8ml.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610398252.3A CN105838673A (en) | 2016-06-07 | 2016-06-07 | Extraction and separation method for mesenchymal stem cells (MSCs) |
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| CN201610398252.3A CN105838673A (en) | 2016-06-07 | 2016-06-07 | Extraction and separation method for mesenchymal stem cells (MSCs) |
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| CN201610398252.3A Pending CN105838673A (en) | 2016-06-07 | 2016-06-07 | Extraction and separation method for mesenchymal stem cells (MSCs) |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114886921A (en) * | 2022-05-13 | 2022-08-12 | 深圳中检联新药检测有限责任公司 | Novel stem cell preparation and application thereof in treating cerebral hemorrhage |
-
2016
- 2016-06-07 CN CN201610398252.3A patent/CN105838673A/en active Pending
Non-Patent Citations (3)
| Title |
|---|
| 关勇等: "兔骨髓间充质干细胞的分离、培养、鉴定及DiI荧光标记", 《天津医药》 * |
| 王旭等: "自体骨髓间充质干细胞移植治疗帕金森病的疗效", 《中国老年学杂志》 * |
| 郭珈宜著: "《中西医结合治疗股骨头坏死》", 30 April 2014, 湖北科学急速出版社 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114886921A (en) * | 2022-05-13 | 2022-08-12 | 深圳中检联新药检测有限责任公司 | Novel stem cell preparation and application thereof in treating cerebral hemorrhage |
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