CN108192861A - A kind of preparation method of mescenchymal stem cell applied to climacteric syndrome - Google Patents
A kind of preparation method of mescenchymal stem cell applied to climacteric syndrome Download PDFInfo
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- CN108192861A CN108192861A CN201711449695.1A CN201711449695A CN108192861A CN 108192861 A CN108192861 A CN 108192861A CN 201711449695 A CN201711449695 A CN 201711449695A CN 108192861 A CN108192861 A CN 108192861A
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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
- 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
- C12N5/0668—Mesenchymal stem cells from other natural sources
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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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- 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
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The invention discloses a kind of preparation methods of the mescenchymal stem cell applied to climacteric syndrome：Step 1, separation；Step 2, just processing；Step 3, filtering；Step 4, inoculation and culture；Step 5, passage；Step 6, cell cryopreservation；Step 7；Cell recovery.The present invention intend since feedstock capture until cell prepare complete overall process carry out unification, system monitoring, since placenta tissue acquisition before screening up to cell be prepared into finished product centre product and finished product quality it is all controlled.
Description
Technical field
The present invention relates to cell biologies, are done carefully more particularly to a kind of mesenchyma applied to climacteric syndrome
The preparation method of born of the same parents.
Background technology
Stem cell (stem cell) is a kind of multipotential cell with the of self-replication capacity.Under certain condition, it can
To be divided into multiple functions cell.Stage of development according to residing for stem cell is divided into embryonic stem cell (embryonic stem
Cell, ES cell) and adult stem cell (somatic stem cell).It is divided into three classes according to the potentiality of development of stem cell：It is all-round
Stem cell (totipotent stem cell, TSC), multipotential stem cell (pluripotent stem cell) and single ability are thin
Born of the same parents (unipotent stem cell) (specially energy stem cell).Stem cell (Stem Cell) is a kind of inabundant differentiation, Shang Bucheng
Ripe cell, has the potential function for regenerating various histoorgans and human body, and medical field is known as " general-purpose cell ".Stem cell exists
In all multicellular tissues, a variety of specialized cells can be split into, and self can be utilized with differentiation via mitosis
It more newly arrives and more stem cells is provided.The source of stem cell has very much, including placenta, umbilical cord, Cord blood and marrow etc..
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) is that a kind of have multi-lineage potential, low exempt from
The adult stem cell of epidemic focus has adhesiveness, and in spindle-type, fibroblast sample form, middle embryo is derived from embryonic development
Layer has and is divided into the potential of a variety of mesoblastema systems, including osteocyte, adipocyte, cartilage cell, fibroblast,
Tenocyte cell etc..MSCs is universally present in the marrow of human body, fat, parodontium, neonatal umbilical cord, placenta, amnion, amniotic fluid
It waits in tissues, derive from a wealth of sources and is limited without ethics, is easily isolated and amplification in vitro, still kept after repeatedly dividing passage
Multi-lineage potential, clinical experimental study up to now do not find that MSCs has serious side effects, are a kind of ideal thin
Born of the same parents treat and the seed cell of regenerative medicine.
Premature ovarian failure refers to that women once had the natural menstrual cycle, and occurs ovarian atrophy before 40 years old and continue
Amenorrhoea.The cause of disease may be infection, iatrogenic premature ovarian failure, idiopathic premature ovarian failure and immune factor etc..
Meanwhile clinical signs secondary sex characters is shunk back, occur face warm, be vexed, the menopause symptoms such as irritability；Usually easily
Flu, the raising of blood follicular stimulating hormone level up to more than 40 units, are equivalent to postmenopausal women's level；And blood estradiol level is shown
It writes low.The apparent atrophy of gynecologial examination internal genitalia, vagina mucosa are thin and congested.It cuts open the belly and makees biopsy of ovary, it is seen that ovum
Nest atrophy is shown in ovary cortex to be fibr tissue under microscope, and the folliculus at different levels such as no primordium folliculus are visible.
Premature ovarian failure is commonly considered as autoimmune disease, it may be possible to which body is to itself ovary group caused by virus infection
The immunity oaritis knitted.All and see autoimmune phenomena caused by all immunity diseases or exist simultaneously more than one disease
Disease finds expression in polyadenous body exhaustion syndrome.Due in the serum of ovarian autoimmunity person there are a kind of anti-ovarian antibodies, thus ovum
The visible lymphocytic infiltration of nest biopsy.Have what is caused due to operation, radiotherapy influence ovary blood fortune.Have because ovarian follicle is congenital very few or promotees
Gonadin overstimulation accelerates follicle atresia and causes.Premature ovarian failure treatment is relatively difficult, and doctor trained in Western medicine clinically uses hormone replacement
Therapy needs to use estrogen and progestogen under the guidance of doctor；Research shows that when the estrogen and progestogen therapy, have certain
Effect, symptom can be improved, but unsatisfactory to the recovery of ovarian function, at the same be discontinued after symptom reappear, make ovum
Nest is in dependent status, and having leads to the worse possibility of ovarian function, that is to say, that hormone replacement therapy has functioned only as what is taken stopgap measures
Effect, and this method side effect is big, and exogenous hormones, which are used for a long time, has the danger for leading to endometrium canceration, and increase hat
The incidence of worry and cholecystopathy.Therefore, the effect of the hormone therapy is undesirable, exists simultaneously security risk.
The underlying cause that perimenopausal syndrome occurs is the ovary caused by physiological or pathologic or operation
Functional failure.Once ovarian function failure is removed and destroys, the estrogen of ovarian secretion will be reduced.Women whole body has
More than 400 kinds of estrogen receptor is distributed in almost women whole body all tissue and organ, receives the control and domination of estrogen,
Once estrogen is reduced, the degeneration variation of organ and tissue will be caused, a series of symptom occur.
In recent years, with gene therapy and the development of stem cells technology, the therapy of hormone is replaced using stem cells technology
More and more it is concerned；It has been currently, there are using venous re-transfusion mescenchymal stem cell/cell factor or using through invasive Jie of abdomen
Enter the method treatment premature ovarian failure of Laparoscopic Ovarian in-situ injection mescenchymal stem cell/cell factor, obtain the effect of certain,
But the curative effect is less desirable, and it is efficient low, it is in particular in：
First, venous re-transfusion mescenchymal stem cell is easily adhered agglomerating, and filtered input easily causes pulmonary vein embolism, table
Now for be short of breath, cyanosis, it is restless and faint from fear etc., also have the case for causing cardiovascular and cerebrovascular stroke hemiplegin；
Secondly, it although mescenchymal stem cell is low immunogenicity, does not express or low expression II class antigens, multiple vein is defeated
Enter mescenchymal stem cell, still there is certain proportion to generate leukocyte antibody, to inputting the amount of cell/time directly proportional, antigen-antibody is anti-
It should can lead to the reactions such as low-heat, tired, respiratory distress, vein input cell or the factor, the amount gone back to the nest is limited, also shadow to a certain degree
Curative effect is rung.
Interventional therapy will generally be open in abdomen, and interposing catheter is disposable to intervene to blood vessel or lesion part under laparoscope
Offer limited effectiveness, repeatedly intervention bring Iatrogenic injury to patient repeatedly, it is impossible to receive, be not easy to popularize.
Therefore, up to the present, there are no the function that definite effective method can restore ovary, so the premature ovarian failure cause of disease
It is complicated, treatment difficulty is larger, modern medicine not yet makes a breakthrough progress so far..
Invention content
In view of the drawbacks of the prior art, the present invention provides a kind of system of mescenchymal stem cell applied to climacteric syndrome
Preparation Method.
The application employs following technical scheme：
The present invention provides a kind of preparation method of mescenchymal stem cell applied to climacteric syndrome, the preparation method
Include the following steps：
Step 1：Separation：Qualified placenta is taken, after placenta is rinsed with Sterile Saline, careful separation placenta and amnion add
Enter Wall and lead to glue, shred into tissue fritter.
Step 2：Just processing：The small blood constituent in the block of tissue is washed away, rejects the vascular tissue on tissue fritter and nourishing
Layer tissue adds clostridiopetidase A, is placed in 37 DEG C of constant-temperature shaking incubator oscillation digestion.
Step 3：Filtering and centrifugation：The mixing liquid obtained after digestion is fallen into insoluble matter by strainer filtering, will be filtered
Cell suspension centrifuge washing.
Step 4：Inoculation and culture：UltraCULTURE serum-free mediums are added in cell mixing, with 1.0 × 105/
cm2Mixing liquid after filtering is inoculated in Tissue Culture Flask respectively, with the DF12 culture solutions containing fetal calf serum in 37 DEG C, 3
~8%CO2, saturated humidity incubator in cultivate.
Step 5：Passage：After cell is adherent, liquid is changed every 3~5d.When cell growth to 80~90% fusion, add
Enter trypsin digestion cell, passed on.
Step 6：Cell cryopreservation：Collection cell and counting and motility rate, by 3 × 107/ ml/ pipes are put into cell cryopreservation tube, add in
Frozen solution is put into liquid nitrogen container and carries out freezen protective；
Step 7：Cell recovery：Cell cryopreservation tube from liquid nitrogen container is taken out, is put into rapidly in 37 DEG C of water-baths, constantly shakes
It is dynamic, in 2min within thaw, centrifuge washing abandons supernatant, obtains mescenchymal stem cell, as desired use.
The mescenchymal stem cell prepared by placenta has powerful immunoregulation effect, can apply and be controlled in preparation
It treats in autoimmune disease drug.
The autoimmune disease refers to include systemic loupus erythematosus, systemic sclerosis, polymyositis and skin
A major class disease including myositis, rheumatoid arthritis, multiple sclerosis, type 1 diabetes etc..
The autoimmune disease especially systemic loupus erythematosus, multiple sclerosis, type 1 diabetes, rheumatoid
Arthritis.
The method of the preparation can also include after detecting step.Such as cell surface molecule Mark Detection.It collects
107Cell suspension is made in the 3rd, the 4 generation fibroblast-like cells of 3~4 d of a culture, is separately added into antihuman CD 34-PE, CD45-
FITC、CD105-PE、CD90-FITC、CD73-PE、CD29-PE、CD44-FITC、CD14-PE、CD19-FITC、HLA-
Each 5 μ l, mouse IgG1 such as Drpercp-A are negative control.4 DEG C of 30 min of reaction, use flow cytomery.
The also detection of ability of cell proliferation takes primary, 2,4,6 generation fibroblast-like cells respectively, adds in
Cell suspension is made in UItraCULTURE serum-free mediums, with 1 × 105The density of/ml is inoculated in 6 orifice plates.3d rises, often
Every 1d digestion and cell count, curve is drawn according to cell quantity.
Preferably, in step 1, the size for shredding into tissue fritter is 0.6~1.0mm3。
Preferably, in step 2, the clostridiopetidase A of the addition is IV type, a concentration of 0.8~1.2g/L.
Preferably, in step 2, the time for being put into constant-temperature shaking incubator oscillation digestion is 2~4h, it is highly preferred that for 3h.
Preferably, the strainer in step 3 is 80 mesh filter screens.
Preferably, the condition of the centrifugation in step 3 be 800~1500r/min, 3~10min, more preferably 1000r/
min,5min。
Preferably, in step 4, the content of fetal calf serum is 6~15%, more preferably 10%.
Preferably, in step 4, a concentration of the 4~6% of CO2, more preferably 5%.
Preferably, in step 5, the opportunity for adding in pancreatin is that cell is produced to 80~90%, more preferably 85%.
Preferably, in step 5, the ratio passed on is 1:3 or 1:2.
Preferably, in step 6, the cell freezing protection liquid used in freezen protective is matched by complete medium and dimethyl sulfoxide (DMSO)
It makes, wherein dimethyl sulfoxide (DMSO) final concentration 8%.
Compared with prior art, the present invention has following advantageous effect：
1. overall process total quality monitor, since placenta acquisition before screening until cell be prepared into finished product centre product and
The quality of finished product is all controlled.
2. it collects to obtain the time-consuming short, at low cost of mescenchymal stem cell, and placenta group is effectively utilized by this method
It knits.
3. serum free medium, without using antibiotic, disease or allergy caused by avoiding heterologous protein and antibiotic possible
Risk.
4. low concentration freezing protective agent keeps the activity of cell and the dryness of stem cell, makes the cell after recovery as far as possible
Ground ensures that various biological characteristics are injury-free.
Specific embodiment
With reference to specific embodiment, the present invention is described in detail.Following embodiment will be helpful to the technology of this field
Personnel further understand the present invention, but the invention is not limited in any way.It should be pointed out that the ordinary skill to this field
For personnel, without departing from the inventive concept of the premise, various modifications and improvements can be made.These belong to the present invention
Protection domain.
Embodiment 1
A kind of preparation method of mescenchymal stem cell applied to climacteric syndrome of the present embodiment, specifically includes following step
Suddenly：
Step 1：Separation：Qualified placenta is taken, after placenta is rinsed with Sterile Saline, careful separation placenta and amnion add
Enter Wall and lead to glue, shred into 0.6~1.0mm3Organize fritter.
Step 2：Just processing：The small blood constituent in the block of tissue is washed away, rejects the vascular tissue on tissue fritter and nourishing
Layer tissue adds type Ⅳ collagenase (a concentration of 1.0g/L), is placed in 37 DEG C of constant-temperature shaking incubator oscillation digestion 3h.
Step 3：Filtering and centrifugation：The mixing liquid obtained after digestion is filtered out into insoluble matter by 80 mesh filter screens, it will
The cell suspension centrifuge washing of filtration, centrifugal condition 1000r/min, 5min.
Step 4：Inoculation and culture：UltraCULTURE serum-free mediums are added in cell mixing, with 1.0 × 105/
cm2Mixing liquid after filtering is inoculated in Tissue Culture Flask respectively, with the DF12 culture solutions containing 10% fetal calf serum in 37
DEG C, 5%CO2, saturated humidity incubator in cultivate.
Step 5：Passage：After cell is adherent, liquid is changed every 3~5d.When cell growth to 80~90% fusion, add
Enter trypsin digestion cell, according to 1:3 ratio is passed on.
Step 6：Cell cryopreservation：Collection cell and counting and motility rate, by 3 × 107/ ml/ pipes are put into cell cryopreservation tube, add in
Frozen solution is prepared by complete medium and dimethyl sulfoxide (DMSO), is put into liquid nitrogen container and carries out freezen protective；
Step 7：Cell recovery：Cell cryopreservation tube from liquid nitrogen container is taken out, is put into rapidly in 37 DEG C of water-baths, constantly shakes
It is dynamic, in 2min within thaw, centrifuge washing abandons supernatant, obtains mescenchymal stem cell, as desired use.
Embodiment 2
A kind of preparation method of mescenchymal stem cell applied to climacteric syndrome of the present embodiment, specifically includes following step
Suddenly：
Step 1：Separation：Qualified placenta is taken, after placenta is rinsed with Sterile Saline, careful separation placenta and amnion add
Enter Wall and lead to glue, shred into 0.6~1.0mm3Organize fritter.
Step 2：Just processing：The small blood constituent in the block of tissue is washed away, rejects the vascular tissue on tissue fritter and nourishing
Layer tissue adds type Ⅳ collagenase (a concentration of 1.2g/L), is placed in 37 DEG C of constant-temperature shaking incubator oscillation digestion 4h.
Step 3：Filtering and centrifugation：The mixing liquid obtained after digestion is filtered out into insoluble matter by 80 mesh filter screens, it will
The cell suspension centrifuge washing of filtration, centrifugal condition 1200r/min, 5min.
Step 4：Inoculation and culture：UltraCULTURE serum-free mediums are added in cell mixing, with 1.0 × 105/
cm2Mixing liquid after filtering is inoculated in Tissue Culture Flask respectively, with the DF12 culture solutions containing 12% fetal calf serum in 37
DEG C, 5%CO2, saturated humidity incubator in cultivate.
Step 5：Passage：After cell is adherent, liquid is changed every 3~5d.When cell growth to 80~90% fusion, add
Enter trypsin digestion cell, according to 1:2 ratio is passed on.
Step 6：Cell cryopreservation：Collection cell and counting and motility rate, by 3 × 107/ ml/ pipes are put into cell cryopreservation tube, add in
Frozen solution is prepared by complete medium and dimethyl sulfoxide (DMSO), is put into liquid nitrogen container and carries out freezen protective；
Step 7：Cell recovery：Cell cryopreservation tube from liquid nitrogen container is taken out, is put into rapidly in 37 DEG C of water-baths, constantly shakes
It is dynamic, in 2min within thaw, centrifuge washing abandons supernatant, obtains mescenchymal stem cell, as desired use.
Embodiment 3
A kind of preparation method of mescenchymal stem cell applied to climacteric syndrome of the present embodiment, specifically includes following step
Suddenly：
Step 1：Separation：Qualified placenta is taken, after placenta is rinsed with Sterile Saline, careful separation placenta and amnion add
Enter Wall and lead to glue, shred into 0.6~1.0mm3Organize fritter.
Step 2：Just processing：The small blood constituent in the block of tissue is washed away, rejects the vascular tissue on tissue fritter and nourishing
Layer tissue adds type Ⅳ collagenase (a concentration of 0.9g/L), is placed in 37 DEG C of constant-temperature shaking incubator oscillation digestion 4h.
Step 3：Filtering and centrifugation：The mixing liquid obtained after digestion is filtered out into insoluble matter by 80 mesh filter screens, it will
The cell suspension centrifuge washing of filtration, centrifugal condition 900r/min, 6min.
Step 4：Inoculation and culture：UltraCULTURE serum-free mediums are added in cell mixing, with 1.0 × 105/
cm2Mixing liquid after filtering is inoculated in Tissue Culture Flask respectively, with the DF12 culture solutions containing 10% fetal calf serum in 37
DEG C, 5%CO2, saturated humidity incubator in cultivate.
Step 5：Passage：After cell is adherent, liquid is changed every 3~5d.When cell growth to 80~90% fusion, add
Enter trypsin digestion cell, according to 1:3 ratio is passed on.
Step 6：Cell cryopreservation：Collection cell and counting and motility rate, by 3 × 107/ ml/ pipes are put into cell cryopreservation tube, add in
Frozen solution is prepared by complete medium and dimethyl sulfoxide (DMSO), is put into liquid nitrogen container and carries out freezen protective；
Step 7：Cell recovery：Cell cryopreservation tube from liquid nitrogen container is taken out, is put into rapidly in 37 DEG C of water-baths, constantly shakes
It is dynamic, in 2min within thaw, centrifuge washing abandons supernatant, obtains mescenchymal stem cell, as desired use.
Performance test
Clinical criteria detection, testing result such as Tables 1 and 2 institute are carried out to mescenchymal stem cell prepared by Examples 1 to 3
It states：
Table 1 freezes preceding testing result
Testing result after 2 Cryopreservation of table
Claims (10)
1. a kind of preparation method of mescenchymal stem cell applied to climacteric syndrome, the preparation method includes following step
Suddenly：
Step 1：Separation：Qualified placenta is taken, after placenta is rinsed with Sterile Saline, careful separation placenta and amnion add in China
You lead to glue, shred into tissue fritter.
Step 2：Just processing：The small blood constituent in the block of tissue is washed away, rejects the vascular tissue on tissue fritter and trophoderm group
It knits, adds clostridiopetidase A, be placed in 37 DEG C of constant-temperature shaking incubator oscillation digestion.
Step 3：Filtering and centrifugation：The mixing liquid obtained after digestion is fallen into insoluble matter by strainer filtering, by the thin of filtration
Born of the same parents' suspension centrifuge washing.
Step 4：Inoculation and culture：UltraCULTURE serum-free mediums are added in cell mixing, with 1.0 × 105/cm2It will
Mixing liquid after filtering is inoculated in Tissue Culture Flask respectively, with the DF12 culture solutions containing fetal calf serum in 37 DEG C, 3~8%
CO2, saturated humidity incubator in cultivate.
Step 5：Passage：After cell is adherent, liquid is changed every 3~5d.When cell growth to 80~90% fusion, pancreas is added in
Enzymic digestion cell, is passed on.
Step 6：Cell cryopreservation：Collection cell and counting and motility rate, by 3 × 107/ ml/ pipes are put into cell cryopreservation tube, add in freezing
Liquid is protected, is put into liquid nitrogen container and carries out freezen protective；
Step 7：Cell recovery：Cell cryopreservation tube from liquid nitrogen container is taken out, is put into rapidly in 37 DEG C of water-baths, is constantly shaken,
It thaws in 2min, centrifuge washing abandons supernatant, obtains mescenchymal stem cell, as desired use.
2. preparation method according to claim 1, which is characterized in that the size that tissue fritter is shredded into step 1 is 0.6
~1.0mm3。
3. preparation method according to claim 1, which is characterized in that the clostridiopetidase A added in described in step 2 is IV type, dense
It spends for 0.8~1.2g/L.
4. preparation method according to claim 1, which is characterized in that constant-temperature shaking incubator oscillation is put into step 2 and is disappeared
The time 3h of change.
5. preparation method according to claim 1, which is characterized in that the strainer in step 3 is 80 mesh filter screens.
6. preparation method according to claim 1, which is characterized in that the condition of the centrifugation in step 3 is 800~1500r/
Min, 3~10min.
7. preparation method according to claim 1, which is characterized in that in step 4 content of fetal calf serum for 6~
15%.
8. preparation method according to claim 1, which is characterized in that the opportunity that pancreatin is added in step 5 is cell life
It produces to 85%.
9. preparation method according to claim 1, which is characterized in that in step 5, the ratio passed on is 1:3 or
Person 1:2.
10. preparation method according to claim 1, which is characterized in that in step 6, the cell used in freezen protective is cold
Freeze protection liquid to be formulated by complete medium and dimethyl sulfoxide (DMSO), wherein dimethyl sulfoxide (DMSO) final concentration 8%.
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| CN104232573A (en) * | 2014-09-11 | 2014-12-24 | 安沂华 | Growth medium and use thereof, and method for cultivating umbilical cord mesenchymal stem cells |
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