CN108642002A - A kind of method of serum-free domestication culture human mesenchymal stem cell - Google Patents
A kind of method of serum-free domestication culture human mesenchymal stem cell Download PDFInfo
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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
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- 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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Abstract
The invention discloses a kind of methods that serum-free tames culture human mesenchymal stem cell.This method makes the mescenchymal stem cell in Human plactnta source adapt to external serum free culture system, and obtains cell density similar with there is serum free culture system and motility rate, more economical, more effective compared with existing human mesenchymal stem cell cultural method.Using the method, risen than not taming cell with the relevant bFGF, PDGF of angiogenic growth, vegf expression amount.It is prepared by the stem cell medicine that the cell after domestication can be used for being injected intravenously；The secretion activity factor component of tamed cell can be used as the component of facial mask, eye mask, hair film.
Description
Technical field
The present invention relates to a kind of cultural methods of human mesenchymal stem cell, it is more particularly related to using a kind of
Unique, safety domestication training method reaches the serum-free in vitro culture of human mesenchymal stem cell.
Background technology
Stem cell is the ancestors of human body cell, and all cells of ours in vivo are both from stem cell.When internal cell
When aging death or damage denaturation, stem cell, which will grow and transit out of, can substitute their cell.As seed cell,
Clinically it is mainly used for treating a variety of refractory diseases of histocyte and organ damage that body can not be repaired naturally；As exempting from
Epidemic disease adjusts cell, treats immunological rejection and autoimmune disease.Human mesenchymal stem cell（human mesenchymal stem
cells, HMSC）It is the important member of stem cell line, derives from the mesoderm of mesoderm growing early stage, belong to multipotential stem cell, MSC
Initially found in marrow, because its with multi-lineage potential, hematopoiesis support and promote stem cell implantation, immunoregulation and self
The features such as duplication and the concern for being increasingly subject to people.Initial clinical research is to be carried out by Lazarus et al. nineteen ninety-five, they
The self MSC of paracmasis patients with hematological tumor is collected, amplification cultivation 4 ~ 7 weeks, are then injected intravenously again into patient's body in vitro,
Patient is divided into 3 groups, gives the MSC of various dose respectively, and toxic side effect is not observed after injection, prompts MSC for transplanting
Treatment safety is reliable.Then the clinical report of self MSC gradually increases, and disease is related to hematopoietic reconstitution, transplanting after radiotherapy and chemotherapy
The anti-host disease of object（GVHD）, cardiac system disease etc., proved in these reports clinical safe and reliable through venoclysis.Between fill
The in vitro culture technique of matter stem cell is to be related to stem cell as drug or the committed step of beauty product safety in utilization.
The ingredient of especially culture medium is main affecting parameters in culture process.The cultural method generally used now has conventional add
Two kinds of the cultural method and culture method in serum-free of cow's serum.The method of serum free culture system is relatively simple, and cell growth state is preferable,
Main Quality Control object is serum, but because the serum proportion being added is between 10-20%, there are larger bovine serum albumin residuals
The problem of.In the patent retrieved（Including authorizing and disclosing unauthorized）In, the culture of mescenchymal stem cell mainly uses DMEM
Complete medium.Patent C12N5/0775 (2006.01) I mentions a kind of undifferentiated mescenchymal stem cell (MSC) of amplification
Method comprising：Undifferentiated MSC is cultivated in the culture medium comprising niacinamide and fibroblast growth factor 4 (FGF4),
In addition the wherein described culture medium does not add other growth factors and/or differentiation factor, thin to generate undifferentiated MSC amplifications
Born of the same parents.Although serum free medium ingredient is clear, with high costs, and asks there are the upgrowth situation of stem cell is undesirable etc.
Topic.It would therefore be highly desirable to which the method for improving Stem cells cultured in vitro makes cell obtain good growth while guaranteeing safety
State, while the economic cost of culture can also be reduced.
Invention content
The purpose of the present invention is being directed to deficiency in the prior art, a kind of unique cultural method is provided, is human mesenchyme
The in vitro culture of stem cell provides a kind of method of serum-free domestication.The purpose of the present invention gives reality by following technical proposals
It is existing.* unless otherwise indicated, the percentage employed in the present invention is weight percent.
The present invention provides a kind of methods of in vitro culture, which is characterized in that this method uses fresh serum-free basis
Culture medium, is added according to a certain percentage and that replaces external human mesenchymal stem cell has serum complete culture solution, is filled using
Matter stem cell, as supplement, makes mescenchymal stem cell complete without blood from the excretion body and micro-capsule ingredient secreted in incubation
The domestication cultivated clearly, finally realizes serum free culture system.The density and motility rate of mescenchymal stem cell after domestication, cell surface
Outstanding feature object and have a blood serum medium no significant difference, detect the component of the cell factor secreted by culture solution and excretion body
It finds, is risen than not taming cell with the relevant bFGF, PDGF of angiogenic growth, vegf expression amount, table 1 with abundance.Harvest
Cell can be used for venoclysis, culture supernatant, excretion body and the micro-capsule ingredient of harvest can be used for the preparation of beauty product.
Table 1：The human mesenchymal stem cell indices of serum-free domestication culture
| Detection project | Cell after serum-free domestication（P5） | Cell (P5) is not tamed |
| Density (a/ml) | More than 1 × 106/ml | More than 1 × 106/ml |
| Motility rate % | 90% | 90% |
| bFGF (pg/ml) | 25 | 17 |
| PDGF (pg/ml) | 18 | 12 |
| VEGF (pg/ml) | 9.7 | 8.5 |
Wherein, the human mesenchymal stem cell is mainly derived from but is not limited to Human plactnta affiliated group, such as amnion, umbilical cord.
The serum-free basal medium of addition refers to but is not limited to DMEM basal mediums.
The present invention provides the external serum-free domestication and culture methods of human mesenchymal stem cell, and identify using domestication training
The characteristic and growth kinetics feature for harvesting mescenchymal stem cell in different periods are supported, as a result, it has been found that, the growth cycle of cell is close
Degree and motility rate no apparent difference, mescenchymal stem cell of harvest compared with using complete medium can be used for being injected intravenously
The preparation of preparation；The mescenchymal stem cell excretion body and vesica of harvest suitable for medicinal stem cell medicine and cure grinding for U.S. product
System；With the mescenchymal stem cell of the method culture, density is up to 1x106/ ml, motility rate reach 90% or more, 109Mescenchymal stem cell
It cultivates 48 hours in vitro, the content of the excretion body in the culture supernatant of harvest is greater than or equal to 80mg.It is examined through Bio-Rad DC
It surveys（detergent compatible protein concentration assay）It analyzes, includes blood vessel life in excretion body
The concentration of long required VEGF, PDGF, bFGF are respectively 9.6pg/ml, 18pg/ml and 25pg/ml.
Compared with prior art, the invention has the advantages that：
The present invention by serum-free tame in the way of, the human mesenchymal stem cell of in vitro culture is tamed, it is made finally to fit
It should be in free serum culture.In the final harvest stage, the content of the bovine serum albumin(BSA) detected in culture medium supernatant is 20ng/
ML substantially increases the safety of final harvest cell less than or equal to the requirement for the residual content of ox in biological products regulation.
After mescenchymal stem cell adapts to free serum culture, other than the growth kinetics of cell change, the harvest of same generation is thin
The density and motility rate of born of the same parents, no difference compared with using complete medium.Angiogenesis class cell activity secreted by excretion body
The concentration of factor Ⅴ EGF, PDGF, bFGF are risen.With this method, it reduces and uses complete medium or nothing at present
The cost of serum free culture system based method.
The human mesenchymal stem cell for the method culture tamed using serum-free can be developed into the injection of stem cell, use
In the exploitation of stem cell drugs, the excretion body and micro-capsule of harvest can also be used as the U.S. product of exploitation doctor, such as facial mask, eye mask, life
The important component of hair film etc..
Specific implementation mode
With reference to specific embodiment, the present invention is further clearly demonstrated, but they are not to be protected to the present invention
Protect the restriction of range.
Embodiment 1
--- the preparation method of human mesenchymal stem cell injection, using following steps：
Placenta source MSC cells include mainly umbilical cord, amnion-derived mescenchymal stem cell, and culture is carried out using following steps：
The processing of umbilical cord, amnion:Umbilical cord includes a vein and two arteries, is around magnificent Tong Shi glue（Wharton’s Jelly）,
Outer layer is wrapped up by amnion-derived epithelium, and for development angle, umbilical cord is that stem cell is formed and institute is through access, existing research table
The connective tissue of person of good sense's umbilical cord is abundant tissue-derived of mescenchymal stem cell.Processing method be first stripping umbilical cord arteriovenous and
Outer layer epithelium leaves gel tissue（Magnificent Tong Shi glue）, cut off the part of double-sided tape catcher mark and extravasated blood.
Amnion is fetal membrane innermost layer, and placenta wraps up one layer of thin and translucent film in fetus face, is sent out by embryo's amnion cyst wall
It educates, is affixed, is made of human amniotic mesenchymal cell and human amnion membrane, surface does not have with chorion in placenta face
The tissues such as nerve, blood vessel, muscle and lymph.
Disinfection:Placentas or amnion are taken out from liquid storage bottle（Transport liquid storage：Dual anti-the 50 of DMEM/F12+1%~
150ml;4 DEG C of shipping storage condition）, first rinsing liquid is then used to organizing the wiping that carries out disinfection with 75% alcohol（Not calcium-magnesium-containing but
Contain dual anti-sterile PBS）Tissue is rinsed repeatedly, until cleaning residual blood cells.
Tissue block processing:Umbilical cord tissue processing：It is washed 3 times with PBS, removes remaining haemocyte.Cutting navel cord at
The segment of 2.0 cm or so simultaneously rejects blood vessel（1 umbilical vein, 2 arteria umbilicalis）, inner membrance is left, inner membrance is China's Tong Shi glue, will
It is cut into the tissue block of about 1 mm3 of size（Fritter fine crushing；1mm3 sizes；In meat gruel shape, can be drawn as standard with suction pipe）.
Amnion tissue processing：Amnion is cut into big tissue block with the tissue shear and operating scissors for having done roasting sterilizing, on one side will
Big tissue block is cut into small pieces, and is held and is rinsed again with surgical forceps or brave tooth tweezer on one side, avoid overflowing, so weight
It is 3 times multiple, until rinsing liquid is limpid, rinsing liquid is abandoned, remaining PBS is exhausted with 1ml liquid-transfering guns or pipettor.This operation is sterile
It is operated in culture dish.It is added containing 1% dual anti-DMEM/F12 to sterile petri dish, continues to be shredded tissue with operating scissors
（Fritter fine crushing；1mm3 sizes；In meat gruel shape, can be drawn as standard with suction pipe）.
Tissue mass suspension is prepared and packing:It adds containing 1% dual anti-DMEM/F12 final volume 27ml, is added final concentration of
10% fetal calf serum 3ml blows and beats tissue mass suspension repeatedly with 5ml liquid-transfering guns or pipettor, then presses 10ml/T25 cells and trains
Foster bottle is dispensed.It is put into CO2 incubators by T25 bottles to cultivate, condition of culture is set：5%CO2 concentration, 37 DEG C；According to thin
Born of the same parents climb out of adherent situation and carry out replacing uterus tissue pieces liquid from 3 days, 5 days, 7 days（Original fluid more commutation is carefully drawn when changing liquid
The culture solution of same volume）, cell confluency degree situation is observed daily.
MSC secondary cultures:After cell converges up to 80% or more, each T25 bottles is added 2~3ml, 0.25% Trypsin-
EDTA pancreatin digestion (be added pancreatin after rotate T25 bottle, allow pancreatin tile infiltrating cells face 10s, incline pancreatin immediately, placement
30s gently pats T25 bottles, and 10ml uterus tissue pieces liquid is added immediately and terminates digestion), it is passed on to T75 bottles in 1: 2 ratio
（Fast culture solution is organized containing 50ml）, continue amplification cultivation.Observation cell growth and degree of converging situation daily.
When cell confluency degree reaches 50%, be sucked out 25% volume culture solution, and be added same volume be free of ox blood
Cleer and peaceful phenol red DMEM/F12 culture mediums, continue to cultivate in incubator.
When cell confluency degree reaches 80%, be sucked out 50% volume culture solution, and be added same volume be free of ox blood
Cleer and peaceful phenol red DMEM/F12 culture mediums, continue to cultivate in incubator.
When cell confluency degree is higher than 85%, manner described above carries out had digestive transfer culture.
50% complete medium and 50% is added after passage and is free of cow's serum and phenol red DMEM/F12 culture mediums, in incubator
In continue to cultivate.
When cell confluency degree reaches 50%, be sucked out 75% volume culture solution, and be added same volume be free of ox blood
Cleer and peaceful phenol red DMEM/F12 culture mediums.
When cell confluency degree reaches 80%, be sucked out 25% volume culture solution, and be added same volume be free of ox blood
Cleer and peaceful phenol red DMEM/F12 culture mediums.
Continue incubation time to reach 40 hours, carries out cell harvest.Cell is cleaned with physiological saline 3 times, manner described above
Cell, is then resuspended in the injection stage physiological saline containing 2% human serum albumin by digestion.
Embodiment 2
--- human mesenchymal stem cell secretes the preparation of component concentrated essence liquid or freeze-dried powder, using processing step below：
The existing micro-capsule of ingredient of human mesenchymal stem cell secretion, diameter are more than 200nm, also there is an excretion body, a diameter of 30-150nm,
These vesicas have double-layer of lipoid membrane structure.
The culture of MSC cells is cultivated using one method of above-described embodiment.It is clear with PBS when cell confluency degree reaches 80%
Cell is washed, serum free medium, including but not limited to MesenGro hMSC medium (StemRD, San is added
Francisco, CA, USA), then cell continues at 37 DEG C, and 5%CO2 is cultivated 48 hours, this CMC model is based on 4 DEG C, 500g
Centrifugation 10 minutes takes supernatant, and 4 DEG C, 1000g is centrifuged 10 minutes.
0.22- μm of (Steritop of supernatant;Millipore) filter, with molecular cut off be 50KD ultrafiltration membrane into
5~10 times of row concentration, collects trapped fluid, and it with changing solvent is 20mM PBS or physiological saline that the method for ultrafiltration, which is used in combination, in room temperature
With -20 DEG C of multigelations 3 times, frozen in -80 DEG C after packing.The Essence of preparation is directly applied to skin surface and uses or be added to
It is used in conjunction in cosmetics.
Multiple cytokine activity liquid or cell factor concentrate with 2ml/ bottles, it is filling in cillin bottle under vacuum degree
It is lyophilized（- 70 DEG C, for 24 hours）, that is, obtain mescenchymal stem cell and close cytokine activity liquid or concentrate freeze-dried powder.It will freeze-drying
1 bottle of powder after the phosphate buffer of 2ml 20mM or normal saline dilution with being coated directly on skin surface.
This Essence ingredient can also arrange in pairs or groups from different nursing facial masks and use.
Take freeze-dried powder 10mg uniform dissolutions in 1% colloidal fluid of hyaluronic acid（Hyaluronic acid 1%, purified water 99%）In；Then
It is added in daily nursing facial mask, such as, but not limited to, 12% polyvinyl alcohol, 10% glycerine, 1% imidazolidinyl urea, 5% ethyl alcohol, 7%
Ethoxylated dodecyl alcohol, preservative；The ingredients such as sodium hyaluronate, snake venom.Long-term face application can play skin-whitening, reparation,
The effect of anti-aging.
Embodiment 3
--- the preparation of human mesenchymal stem cell facial mask, using processing step below：
The culture of MSC cells is cultivated using 1 method of above-described embodiment.
When cell confluency degree reaches 80%, after incubation time reaches 40 hours in serum free medium, in harvest culture
Clearly, by culture supernatant in 1000g, 4 DEG C centrifuge 5 minutes, remove cell relic.0.22- μm of (Steritop of supernatant;
Millipore it) filters, the ultrafiltration membrane for being 50KD with molecular cut off carries out 5~10 times of concentration, collects trapped fluid, ultrafiltration is used in combination
Method with changing solvent be 20mM PBS or physiological saline, in room temperature and -20 DEG C of multigelations 3 times, frozen in -80 after packing
℃.Be added to according to a certain percentage in facial mask base solvent when use, such as but be not limited to+90% water for injection of 1% hyaluronic acid+
10% glycerine, slight concussion dissolving mixing.
Daily nursing facial mask ingredient is：Mixing in facial mask liquid is added in cell factor and is prepared as facial mask, long-term face application
It can play the role of skin-whitening, reparation, anti-aging.Specific ingredient is as follows：12% polyvinyl alcohol, 10% glycerine, 1% imidazolidine
Base urea, 5% ethyl alcohol, 7% ethoxylated dodecyl alcohol, 0.1% cell factor liquid, appropriate amount of essence, preservative supplement purified water are extremely
100%。
4 DEG C are adsorbed in mask matrix, are used as cotton pads, silk are prepared into facial mask.
Embodiment 4
--- the preparation of human mesenchymal stem cell excretion body eye mask, using processing step below：
Human mesenchymal stem cell excretion body component Essence or freeze-dried powder are prepared using 2 method of above-described embodiment.
Human mesenchymal stem cell excretion body component Essence or freeze-dried powder 10mg are added in eye mask base solvent, such as but not
It is limited to+10% Aloe Vera Gel of+90% water for injection of 1% hyaluronic acid, slight concussion dissolving mixing.
It is filtered with 0.22 μm of disposable sterilized filter.
Filtered fluid is fitted into aseptic bottle, is sealed for use in 2-8 DEG C.
4 DEG C are adsorbed in eye mask matrix, are used as cotton pads, silk are prepared into facial mask.
Embodiment 5
--- human mesenchymal stem cell secretes the preparation of essence hair film, using processing step below：
Human mesenchymal stem cell excretion body component freeze-dried powder is prepared using 2 method of above-described embodiment.
Human mesenchymal stem cell excretion body freeze-dried powder such as but is not limited to 10mg according to a certain percentage, and it is molten to be added to hair film basis
In agent, such as but it is not limited to+99% water for injection of 1% olive oil, slight concussion dissolving mixing.
It is packed into sterile chamber, is sealed for use in 2-8 DEG C
Embodiment 6
--- human mesenchymal stem cell secretes the preparation of surfactant, using processing step below：
Human mesenchymal stem cell excretion body component harvest liquid is prepared using 2 method of above-described embodiment.
Human mesenchymal stem cell secretes essence freeze-dried powder 2mg, is dissolved in 1ml physiological saline, slightly shakes molten before use
Mixing is solved, face is applied to.
Embodiment 7
--- it issues with reference to Products in China regulation and newly《Stem cell General Requirement》Carry out mescenchymal stem cell harvest
Liquid is examined and determine, the preferred free from infection of placenta tissue, chronic disease, hereditary disease, while without smoking, excessive drinking, age in pregnancy period moderate parent,
With complete ethics formality, relevant mother's clinical detection result data archive is put on record, with the mescenchymal stem cell after culture
Detection is compared.
Biological characteristics
Biological characteristics detection includes marker, the detection of cellular morphology and metabolic enzyme hypotype spectrum.The positive of mescenchymal stem cell
Marker is CD19, CD34, CD44, CD45, CD73, CD90, CD105 and CD106.The main mark of excretion body be CD63 and
CD81 is measured with flow cytometer.After testing, the table of the mescenchymal stem cell of method culture described in embodiment one
Face molecular labeling meets description；The main mark of excretion body meets description.The cell cultivated form under the microscope and
Mesenchymal stem cells are consistent.Metabolic enzyme hypotype is detected using isodynamic enzyme method.
Safety detection
Safety detection includes sterility test（Bacterium, fungi）, mycoplasma, intracellular foreign aid's virulence factor endotoxin, abnormal exempts from
Epidemiology reaction, oncogenicity detection, culture medium and related component residue etc..After testing, the cell sterility test cultivated, Zhi Yuan
Body, intracellular foreign aid's virulence factor, endotoxin, abnormal immune reaction, oncogenicity are detected as feminine gender, are examined with enzyme-linked immunization
It surveys, bovine serum protein residual content should be not higher than 50ng/mL.The average value of testing result is 20ng/mL.
Stability
The detection of density, purity, survival rate has been carried out to the mescenchymal stem cell of separation.In the 3rd generation of harvest, mesenchyma is dry thin
The density of born of the same parents reaches 1X106/ ml, motility rate 90%.
Validity
The relevant cell factor that detection is expressed with the method culture Human plactnta mescenchymal stem cell：VEGF, PDGF, bFGF's is dense
Degree is respectively 9.6pg/ml, 18pg/ml and 25pg/ml.
Mescenchymal stem cell secretes many cell factors, growth factor.But the composition of these active factors and
Concentration is variant, and is the growth microenvironment residing for mescenchymal stem cell and the dynamic change of condition of culture generation.Cause
It is the pass for influencing product quality that this, which keeps the condition of culture of cell and state and the extraction process of secretion activity ingredient to stablize all,
Key factor.
Claims (5)
1. the method for serum-free domestication culture human mesenchymal stem cell, which is characterized in that this method makes Human plactnta derived mesenchymal
The method that stem cell changes liquid by gradient, is adapted to the culture system in vitro of serum-free, and obtains effective amplification.
2. the Human plactnta mescenchymal stem cell of domestication is used to be injected intravenously the preparation of stem cell medicine.
3. the secretion activity factor component of tamed cell can be used as the component of facial mask, eye mask, hair film.
4. preparation method according to claim 1, which is characterized in that the method for domestication is that gradient changes liquid method.
5. the preparation method described according to claim 1, which is characterized in that the motility rate of the human mesenchymal stem cell after domestication reaches
To 90%, density reaches 1 × 106The expression quantity of/ml or more, angiogenesis relevant cell factor bFGF, PDGF, VEGF is respectively：
25pg/ml、18pg/ml、9.7pg/ml。
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111206017A (en) * | 2019-04-30 | 2020-05-29 | 浙江大学 | Serum-free culture medium for stem cells and application thereof |
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| CN112029717A (en) * | 2020-09-17 | 2020-12-04 | 英科博雅基因科技（天津）有限公司 | Serum-free in vitro domestication culture of human mesenchymal stem cells |
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| CN112680414A (en) * | 2021-01-29 | 2021-04-20 | 宏之俊生物科技(上海)有限公司 | Preparation method of aging-reversing mesenchymal stem cells |
| CN113073081A (en) * | 2021-03-19 | 2021-07-06 | 广州远想生物科技有限公司 | bFGF mesenchymal stem cell exosome and preparation method and application thereof |
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