CN109260227A - It is a kind of for treating the autologous fat mesenchymal stem cell injection preparation method of Alzheimer's disease - Google Patents
It is a kind of for treating the autologous fat mesenchymal stem cell injection preparation method of Alzheimer's disease Download PDFInfo
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/42—Proteins; Polypeptides; Degradation products thereof; Derivatives thereof, e.g. albumin, gelatin or zein
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
- A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/11—Epidermal growth factor [EGF]
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- C—CHEMISTRY; METALLURGY
- 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
- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/13—Nerve growth factor [NGF]; Brain-derived neurotrophic factor [BDNF]; Cilliary neurotrophic factor [CNTF]; Glial-derived neurotrophic factor [GDNF]; Neurotrophins [NT]; Neuregulins
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- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a kind of for treating the autologous fat mesenchymal stem cell injection preparation method of Alzheimer's disease, fat mesenchymal stem cell is isolated from the adipose tissue acquired self, a large amount of fat mesenchymal stem cell is obtained through cultured and amplified in vitro, various cell factors in culture supernatant are obtained simultaneously, for treating the autologous fat mesenchymal stem cell injection of Alzheimer's disease made of the cell factor in cell and culture supernatant, there is unique advantage in therapeutic effect, brings hope for the better healing of Alzheimer's disease.
Description
Technical field
The present invention relates to biomedicine technical fields more particularly to a kind of for treating the autologous fat of Alzheimer's disease
Mesenchymal stem cell injection preparation method.
Background technique
Mescenchymal stem cell (Mesenchymal stem cells) is a kind of thin at soma with multi-lineage potential
Born of the same parents are primarily present in connective tissue and organ interstitial derived from the mesoderm of mesoderm growing early stage, can be from marrow, peripheral blood, fat
And it is obtained in the Various Tissues such as skin.Mescenchymal stem cell belongs to non-terminally differentiated cells, the feature of its existing interstitial cell, again
There is the feature of endothelial cell and epithelial cell；As a kind of multipotential stem cell, it is in vitro under specific inductive condition, Ke Yixiang
The directions such as bone, cartilage, muscle, tendon, liver, fat, nerve, endothelium and islet-like cells Proliferation, Differentiation.
It is raw that stem cell factor (SCF), epidermal growth factor (EGF), blood vessel endothelium can be secreted in mescenchymal stem cell incubation
The long factor (VEGF), basic fibroblast growth factor (bFGF), platelet derived growth factor A(PDGF-A), conversion life
It is the long factor (TGF-β), hepatocyte growth factor (HGF), interleukins (IL), insulin-like growth factor (IGF- II), huge
Phagocyte colony stimulating factor (GSFs), tumor necrosis factor (TNF-α), interferon (IFN), nerve growth factor (NGF), with
And other not yet by isolated molecular substance.This multiple cytokine can promote cell Proliferation, differentiation, again in human body
It is raw, reinforce metabolism, prevents cell ageing；Body cell signal transduction, activation equipment somatic stem cell are mediated, new blood capillary is promoted
Pipe is formed, and physiological reparation is carried out；Body inferior health is adjusted, the cell that immunity of organism removes damage, lesion, aging is improved.Have
Studies have shown that using stem cell secretion factor and excretion body and using stem cell effect having the same, therefore joined with stem cell
Close stem cell secretion factor treat with merely with stem cell compared with, it is with the obvious advantage.
It is domestic at present rarely found about the autologous fat mesenchymal stem cell injection preparation method in treatment Alzheimer's disease,
And this is very big for the meaning of the cell therapy of Alzheimer's disease, so and where the power of handwritten copy patent.
Summary of the invention
The present invention provides a kind of for treating the autologous fat mesenchymal stem cell injection preparation side of Alzheimer's disease
Method, this method operating process is simple and reliable, and application effect is preferable.
Specific steps are as follows:
1) aseptic collection autologous adipose tissue 20g
2) haemocyte on surface is washed away with physiological saline
3) tissue is cut into 0.3mm3 fritter below with aseptic operation knife, with brine and is centrifuged degreasing and part
Red blood cell.
4) lower liquid is collected with DMEMF12 termination digestion with the adipose tissue 5min of 0.25% pancreatin digestion chopping
5) with collagenase type I and neutral protein enzymatic digestion stage 4) remaining adipose tissue 30min, disappeared with physiological saline termination
Change, collects lower liquid
6) step 4) and step 5) are collected into the liquid of coming, after 200 mesh screens are filled into a 50ml centrifuge tube, 400g,
It is centrifuged 5min.
7) visual cell precipitating in red blood cell number decide whether splitting erythrocyte, if red blood cell is less, remove supernatant, use
3ml erythrocyte cracked liquid splitting erythrocyte 5min adds physiological saline to 45ml, 400g, is centrifuged 5min.If red blood cell is more,
Cell precipitation can be then resuspended with physiological saline, be added to the enterprising line density gradient centrifugation of Percoll of 1.070g/ml, 800g,
It is centrifuged 15min, has been centrifuged and has been followed successively by physiological saline from top to bottom, fat mesenchymal stem cell layer, Percoll layers, lymphocyte
Layer, red blood cell layer in aspirating adipose mescenchymal stem cell layer and Percoll layers to one new 50ml centrifuge tubes, add physiology
Salt water is centrifuged 5min to 45ml, 500g.
8) with 10% serum substitute is contained, the MEM culture medium of 10ng/mlEGF, 6.408ng/ml mercaptoethanol press 6000/cm2
Density cell culture is got up.It carries out changing liquid for the first time after 24 hours, mainly removes extra red blood cell and cell fragment.
It changes the liquid once within every 3 days, when cell reaches 80-90% fusion, is passed on, the inoculum density of passage is 8000/cm2. later
9) when cell culture is to predetermined quantity, cell is harvested, streaming, endotoxin, sterile and detection of mycoplasma is qualified
Afterwards, cell normally freezes spare.
10) culture supernatant is poured into centrifuge tube, after 0.22um membrane filtration, endotoxin, sterile and detection of mycoplasma qualification
Afterwards, -80 DEG C of freezen protectives are spare.
11) by 39 DEG C of the cell recoveries that step 9) obtains and 37 DEG C of the supernatant thawings that step 10) obtains, with the 10% white egg of people's blood
The white autologous fat mesenchymal stem cell injection being mixed for treating Alzheimer's disease.
It is a kind of for treating the autologous fat mesenchymal stem cell injection preparation method of Alzheimer's disease as the present invention
A kind of preferred embodiment, the concentration of collagenase type I described in step 5) are 0.2%.
Compared with prior art, the beneficial effects of the present invention are: being digested respectively with three kinds of enzymes, obtained primary cell quantity
It is considerable, it may separate out 30*10 in every 1ml adipose tissue6；The fat stem cell activity separated is good, and amplification rate is fast, purity
It is high.
In order to better illustrate the present invention, specific embodiment is:
1 aseptic collection fat: microbiologic inhibition tests, HIV, HBV, HCV, TP inspection first are carried out to fat mesenchymal stem cell depositary
It surveys, then aseptic collection fat；Collection point is preferably the abundanter position of body fat, generally abdomen, femoribus internus etc.；
It is manually cut off under conditions of local anaesthesia or liposuction procedures, manually cuts off obtained fat, preferably equivalent at least 150g, liposuction side
The liquid aliphatic that method obtains is preferably in 200ml.
The removal of 2 greases and some red blood cells: taking adipose tissue, is first shredded with sterile surgical scissors or pocket knife, length is wanted
It asks no more than 3mm, is then collected in 50mL centrifuge tube, isometric physiological saline is added, 500g is centrifuged 5min, is divided into three layers
After suck upper layer grease, careful collection middle layer part (includes mescenchymal stem cell, fat cell, physiological saline and red thin
Born of the same parents), remove the red blood cell of lower layer, the middle layer part of part physiological saline, collection uses brine 3 times.
The digestion of 3 pancreatin: with the adipose tissue 5min of 0.25% pancreatin digestion chopping, being terminated with DMEMF12 and digested, 400g centrifugation
5min collects lower liquid.
The digestion of 4 clostridiopetidase As: being added 0.2% isometric clostridiopetidase A I for the remaining upper-layer fat tissue of step 3 and digest, and 37 DEG C
Water-bath 30min；Isometric physiological saline is added and terminates digestion, 10min layering is stored at room temperature after being sufficiently mixed；Remove upper layer
(lipid/fragment), 20 DEG C, 400g is centrifuged 5min and collects underclad portion (stromal vascular part, stem cell, red blood cell).
The cell precipitation for the underclad portion that 5 steps 3 and step 4 are collected is resuspended with physiological saline, after the filtering of 200 mesh filter screens,
400g is centrifuged 5min.
6 erythrocyte splittings: visual cell precipitating in red blood cell number decide whether splitting erythrocyte, if red blood cell is less,
Remove supernatant, with the erythrocyte cracked liquid splitting erythrocyte 5min of 2 times of volumes, adds physiological saline to 45ml, 400g, centrifugation
5min.If red blood cell is more, cell precipitation can be resuspended with physiological saline, the Percoll for being added to 1.070g/ml is enterprising
Line density gradient centrifugation, 800g are centrifuged 15min, have been centrifuged and have been followed successively by physiological saline from top to bottom, fat mesenchymal stem cell
Layer, Percoll layer, buffy coat, red blood cell layer, aspirating adipose mescenchymal stem cell layer and Percoll layers to one are newly
In 50ml centrifuge tube, physiological saline is added to 45ml, 500g, is centrifuged 5min.Supernatant is removed, MEM culture solution is added into gained cell,
It is collected by centrifugation cell, and in containing 10% serum substitute, 10ng/mlEGF is trained in the MEM culture medium of 6.408ng/ml mercaptoethanol
It supports, changing the liquid time for the first time is 24 hours, is changed the liquid once within every 3 days later, when cell reaches 80-90% fusion, is passed on, is passed
The inoculum density in generation is 8000/cm2.
7 when cell culture is to predetermined quantity, harvests cell, streaming, endotoxin, and sterile and detection of mycoplasma is qualified
Afterwards, cell normally freezes spare.
8 pour into culture supernatant in centrifuge tube, after 0.22um membrane filtration, endotoxin, and sterile and detection of mycoplasma qualification
Afterwards, -80 DEG C of freezen protectives are spare.
9 37 DEG C of supernatant thawings for obtaining 39 DEG C of the cell recoveries that step 6 obtains with step 7, mix with 10% human serum albumin
The autologous fat mesenchymal stem cell injection for treating Alzheimer's disease is made.
The biology and fluidic cell of fat mesenchymal stem cell are identified.
1, cell growth and its Morphological Characteristics: digestion method of the invention is used, mixture is applied on culture dish and is carried out
Adhere-wall culture, through inverted microscope observation (as shown in Figure 1) when culture was to the 3-5 days, it is possible to find there is more adherent cell growth；
When culture was to 10 days, cell confluency reaches 70%；When organizing fritter secondary use, cell progress has been observed in culture afterwards for 24 hours
Adherent growth, cell confluency reaches 70% or so after 9 days.
Detailed description of the invention Fig. 1 is the photo under the fat mesenchymal stem cell microscope for cultivating third day.
2, fluidic cell identifies mescenchymal stem cell surface marker.
Second generation cell is collected, phosphate buffered saline solution is washed 2 times (1000rpm is centrifuged 5min), adjustment cell concentration to 5 ×
106A/ml；It is incubated for 30min after the corresponding antibody of cell surface marker is added, be available on the machine after washing gravity treatment (streaming again
Cell instrument FACSCalibur, BD company) detection.Antibody used includes the antibody of following cell surface molecule: CD90, CD29,
CD105, CD73 are positive mark, and CD45, CD34, CD79a, CD14, HLA-DR are negative marker.Concrete outcome is referring to table 4.
Table 4: fat mesenchymal stem cell surface marker testing result.
As shown in Table 4: in the present invention the primary umbilical cord mesenchymal stem cells that harvest of tissue fritter after secondary culture twice,
Still conform to standard of the international cell therapy association about mescenchymal stem cell: positive expression rate >=95%, negative expression rate≤2%；
Cell obtained can be used as the seed cell of clinical research and application.
Claims (4)
1. a kind of for treating the autologous fat mesenchymal stem cell injection preparation method of Alzheimer's disease, this method operation
Process is simple and reliable, and application effect is preferable.
Specific steps are as follows:
1) aseptic collection autologous adipose tissue 20g
2) haemocyte on surface is washed away with physiological saline
3) tissue is cut into 0.3mm3 fritter below with aseptic operation knife, with brine and is centrifuged degreasing and part
Red blood cell.
4) lower liquid is collected with DMEMF12 termination digestion with the adipose tissue 5min of 0.25% pancreatin digestion chopping
5) with collagenase type I and neutral protein enzymatic digestion stage 4) remaining adipose tissue 30min, disappeared with physiological saline termination
Change, collects lower liquid
6) step 4) and step 5) are collected into the liquid of coming, after 200 mesh screens are filled into a 50ml centrifuge tube, 400g,
It is centrifuged 5min.
7) visual cell precipitating in red blood cell number decide whether splitting erythrocyte, if red blood cell is less, remove supernatant, use
3ml erythrocyte cracked liquid splitting erythrocyte 5min adds physiological saline to 45ml, 400g, is centrifuged 5min.If red blood cell is more,
Cell precipitation can be then resuspended with physiological saline, be added to the enterprising line density gradient centrifugation of Percoll of 1.070g/ml, 800g,
It is centrifuged 15min, has been centrifuged and has been followed successively by physiological saline from top to bottom, fat mesenchymal stem cell layer, Percoll layers, lymphocyte
Layer, red blood cell layer in aspirating adipose mescenchymal stem cell layer and Percoll layers to one new 50ml centrifuge tubes, add physiology
Salt water is centrifuged 5min to 45ml, 500g.
8) with 10% serum substitute is contained, the MEM culture medium of 10ng/mlEGF, 6.408ng/ml mercaptoethanol press 6000/cm2
Density cell culture is got up.It carries out changing liquid for the first time after 24 hours, mainly removes extra red blood cell and cell fragment.
It changing the liquid once within every 3 days, when cell reaches 80-90% fusion, is passed on later, the inoculum density of passage is 8000/
cm2.
9) when cell culture is to predetermined quantity, cell is harvested, streaming, endotoxin, sterile and detection of mycoplasma is qualified
Afterwards, cell normally freezes spare.
10) culture supernatant is poured into centrifuge tube, after 0.22um membrane filtration, endotoxin, sterile and detection of mycoplasma qualification
Afterwards, -80 DEG C of freezen protectives are spare.
11) by 39 DEG C of the cell recoveries that step 9) obtains and 37 DEG C of the supernatant thawings that step 10) obtains, with the 10% white egg of people's blood
The white autologous fat mesenchymal stem cell injection being mixed for treating Alzheimer's disease.
2. as described in claim 1, it is characterised in that digestive ferment used in step 4) and step 5) is three kinds and according to enzyme
Digestion condition difference and avoid mutual influence, the sequencing of digestion is first to use pancreatin, afterwards with clostridiopetidase A and neutral egg
White enzyme.
3. as described in claim 1, it is characterised in that culture solution used in culture fat mesenchymal stem cell is free serum culture
Liquid adds 10% serum substitute, and EGF, mercaptoethanol is conducive to the secretion of neurotrophic factor, and is input to people
The differentiation of body fat mesenchymal stem cells into nerve stem cell.
4. as described in claim 1, it is characterised in that the autologous fat mescenchymal stem cell for treating Alzheimer's disease is infused
Liquid is penetrated to be mixed by autologous fat mescenchymal stem cell, cell culture supernatant and 10% human serum albumin.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109954002A (en) * | 2019-03-11 | 2019-07-02 | 北京诺兰药谷科技有限公司 | Application of the human umbilical cord mesenchymal stem cells in preparation prevention Parkinson's disease or the drug for treating Early Parkinson's disease |
| CN111346051A (en) * | 2020-03-19 | 2020-06-30 | 瑞太干细胞中心(沈阳)有限公司 | Preparation method of umbilical cord mesenchymal stem cell injection for treating cerebral infarction |
| CN111690597A (en) * | 2020-06-29 | 2020-09-22 | 海口健康岛生物科技有限公司 | Extraction reagent of human autologous fat vascular stroma component SVF and extraction, amplification and culture method |
| CN111733128A (en) * | 2020-05-14 | 2020-10-02 | 厚朴生物科技（苏州）有限公司 | Preparation method of human adipose-derived mesenchymal stem cells and in-vitro differentiation capacity identification method |
| CN113181216A (en) * | 2021-06-17 | 2021-07-30 | 上海市同济医院 | Mesenchymal stem cell exosome-AM 1241 complex and application thereof in treating Alzheimer disease |
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| CN108315297A (en) * | 2018-02-26 | 2018-07-24 | 福建省银丰干细胞工程有限公司 | A method of it detached from adipose tissue, purify fat stem cell |
Non-Patent Citations (4)
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109954002A (en) * | 2019-03-11 | 2019-07-02 | 北京诺兰药谷科技有限公司 | Application of the human umbilical cord mesenchymal stem cells in preparation prevention Parkinson's disease or the drug for treating Early Parkinson's disease |
| CN111346051A (en) * | 2020-03-19 | 2020-06-30 | 瑞太干细胞中心(沈阳)有限公司 | Preparation method of umbilical cord mesenchymal stem cell injection for treating cerebral infarction |
| CN111733128A (en) * | 2020-05-14 | 2020-10-02 | 厚朴生物科技（苏州）有限公司 | Preparation method of human adipose-derived mesenchymal stem cells and in-vitro differentiation capacity identification method |
| CN111690597A (en) * | 2020-06-29 | 2020-09-22 | 海口健康岛生物科技有限公司 | Extraction reagent of human autologous fat vascular stroma component SVF and extraction, amplification and culture method |
| CN111690597B (en) * | 2020-06-29 | 2023-12-29 | 广东康盾创新产业集团股份公司 | Extraction reagent and extraction and amplification culture method for human autologous adipose-derived vascular stroma component SVF |
| CN113181216A (en) * | 2021-06-17 | 2021-07-30 | 上海市同济医院 | Mesenchymal stem cell exosome-AM 1241 complex and application thereof in treating Alzheimer disease |
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