CN108186682A - A kind of preparation method of placenta mesenchyma stem cell freeze-dried powder - Google Patents
A kind of preparation method of placenta mesenchyma stem cell freeze-dried powder Download PDFInfo
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Abstract
The present invention is prepared for placenta stem-cell using the fast method of tissue, and collects intracellular cell factor using ultrasonic fragmentation and mixed with the cell factor in supernatant, is lyophilized at low ambient temperatures, has fully ensured that the concentration and activity of the placenta stem-cell factor.Freeze-dried powder prepared by the preparation method of the present invention effectively saves the various biologically active cytokine mixtures in Human plactnta mescenchymal stem cell, storage life can extend to 2 years, the bottleneck that cell factor solution shelf-life is limited is efficiently solved, the commercial application for people's placenta mesenchyma stem cell cell factor is laid a good foundation.
Description
Technical field
The invention belongs to biotechnologies, are related to a kind of preparation method of placenta mesenchyma stem cell freeze-dried powder.
Background technology
Mescenchymal stem cell (mesenchymal stem cells, MSCs) is that a kind of have self-renewing, proliferation and more
To the adult stem cell of differentiation potential, tissue growth and reparation can be stimulated, enhances the power of regeneration of tissue, influences immunological regulation,
There is extremely wide application prospect in cell therapy field.MSCs can be from marrow, Cord blood, fat, synovial membrane, placenta, connective
The positions such as tissue obtain.
However, the abundance of MSCs in the tissue is extremely low, therefore, in vitro culture is to realize must walking for MSCs application values
Suddenly, research shows that, different from the MSCs of fresh separated, after cultured and amplified in vitro, biological characteristics can occur very big cell
Variation, and with the extension of incubation time, the ability of cell differentiation is gradually lost, this has with the environment residing for the cell of inside and outside
It closes, internal microenvironment composition is considerably complicated, it is impossible to these extrinsic factor institute moulds of temperature, pH value and the nutritional ingredient of stabilization
Intend.How the short time obtains a large amount of good MSCs of functional status, maintains the reset condition of MSCs and control MSCs differentiation sides
It is also difficult point to being the hot spot of current research.In addition, more and more evidences show to be injected intravenously or local injection,
After the MSCs of in vitro culture is entered in vivo, only minimal amount of cells survival, and the cell of this minority survival is big by secreting
The small-molecule substance of amount, for example, cell factor, growth factor, chemotactic factor (CF) etc. and play a role.
Placenta mesenchyma stem cell is a kind of multipotential stem cell, is the multipotential cell with the of self-replication capacity.One
Under fixed condition, it can be divided into multiple functions cell as APSC pluripotent cells.Compared to other stem cells, placenta mesenchyma is done
Cell has convenient sources, and cell quantity is sufficient, is easily isolated, cultivates, expands and purifies, and passage amplification still has after more than 30 generations
Stem cell properties.Placenta is the current best source for confirming stem cell.
Mesoderm is derived from embryonic development.In the normal tissue damage repair process of body, placenta mesenchyma is done
A kind of cell bank of important participation regeneration of cell.Under distinctive signal effect caused by tissue damage, placenta mesenchyma
Stem cell migration is proliferated in localized clusters, breaks up according to different damage signals along different approaches to damaged part.Between placenta
Mesenchymal stem cells are easily isolated amplification, and external multiplication capability is vigorous, even if 100,000,000 times of amplification remains to keep its Multidirectional Differentiation ability.
Therefore, placenta mesenchyma stem cell is a kind of tissue repair seed cell of practicality.Placenta stem-cell has immunoregulation effect,
By negative immune regulatory function, the immune response that inhibits body hyperfunction makes body's immunity restore balance, so as to
To treat the immunological rejection and the autoimmunities such as clone disease, lupus erythematosus, chorionitis after hematopoietic stem cell transplantation
Systemic disease.
Stem cell is widely used in beauty industry.Stem cell extract generally refer to stem cell medium supernatant and (or)
Cell pyrolysis liquid, cell culture supernatant liquid contain a large amount of protein, polypeptide and cell with multiple biological activities because
Son, they participate in cyto-architectural maintenance movable information exchange and tissue repair and regeneration, have preferable anti-light aging, resist
Oxidation, crease-resistant, skin whitening, wound healing, cell repair and other effects.Therefore, as the application value of stem cell extract is got over
It is more concerned by people, how to obtain stem cell extract a large amount of, stable and controllable for quality and endeavour to study as people
The problem of.Stem cell supernatant can be smeared directly, easy to operate.But the culture supernatant of liquid is when room temperature preserves
Between it is short, this just hinders its promotion and application.
Invention content
Of the existing technology to solve the problems, such as, the purpose of the present invention is to provide a kind of freeze-dryings of placenta mesenchyma stem cell
The preparation method of powder.
In order to achieve the above objects and other related objects, the present invention provides a kind of system of placenta mesenchyma stem cell freeze-dried powder
Preparation Method includes the following steps：
(1), the separation and culture of placenta stem-cell：Aseptically, the placenta of health is collected, in GMP laboratory biologicals
Safety cabinet shreds placenta to 1.5-2.5mm3The tissue block of size places it in the DMEM-LG cells that T175 culture bottles are contained
In culture solution, 37 DEG C are put, 5%CO2It is cultivated in incubator；Liquid was changed every 2-3 days, until placenta stem-cell is precipitated, discards tissue
Block；After cell covers with, the first generation is denoted as, had digestive transfer culture is carried out with 0.28% pancreatin；Use antibiotic-free DMEM- after the second generation instead
LG culture mediums, and carry out secondary culture；
(2), the collection of cell factor：After the third generation, cell length to 90% or so can start to collect cell factor；By cell
Cell in good condition collects, and carries out ultrasonication, it is made to come out the cytokine release not discharged completely into the cell；
And 1500rpm is centrifuged 10 minutes, removes pellet cell debris；
(3), the ultralow temperature freeze-drying of stem cell extract：The placenta stem-cell culture stoste for removing pellet cell debris is added
Enter 10%BSA as excipient, fully after dissolving, with the sterilizing filter filtration sterilization of 0.45um, and dispense to 2ml XiLin
In bottle, every bottle of 1ml；Cillin bottle is placed in -80 DEG C of ultra low temperature freezers after packing and is lyophilized in advance, is taken out after pre-freeze 12h, then put
In being freeze-dried in freeze dryer；Obtain placenta mesenchyma stem cell freeze-dried powder.
(4) the Activity determination of stem cell factor before and after, being lyophilized：Prepare freeze-dried powder solvent；Using the growth curve of 3T3 cells,
Simulate growing state of the fibroblast under cell factor promotion；After 3T3 is cultivated, digested using pancreatin, it, will be thin after counting
Born of the same parents are spread to four piece of six orifice plate, every two pieces one group, and every piece of four holes, wherein per hole 1 × 103A cell；First group is set as control
Group adds the cell of step (2) middle collection in the control group；Second group is set as experimental group, is added in the experimental group
The cell factor dissolved after freeze-drying with solvent；It cultivates respectively for 24 hours, after 48h, 72h, 96h, 1 hole is taken to count per plate, it is bent to draw growth
Line.
In said program, related content is explained as follows：
1st, in said program, step (1) described in DMEM-LG cell culture fluids containing 10%FBS, bFGF 5ng/mL, L-
Glutamine 2mM, 1 μ g/mL of penicillin 100U/mL, 100 μ g/mL of streptomysin and amphotericin B.
2nd, in said program, step (1) described in antibiotic-free DMEM-LG culture mediums containing 10%FBS, bFGF 5ng/mL,
L-Glutamine 2mM.
3rd, in said program, step (3) described in freeze dryer be vacuum freeze drier.
4th, in said program, step (4) described in freeze-dried powder solvent be 2% hyaluronic acid, 10% glycerine and distilled water.
5th, in said program, the principle of stem cells factor beauty is to stimulate skin fibroblasts by cell factor
It is newborn and reach from solving the problems, such as ageing skin at all；Therefore the activity of test freeze-dried powder cell factor is most important.
Compared with prior art, advantage of the invention is that：
(1), the present invention is prepared for placenta stem-cell using the fast method of tissue, and is collected into the cell using ultrasonic fragmentation
Cell factor is mixed with the cell factor in supernatant, is lyophilized at low ambient temperatures, has fully ensured that the dense of the placenta stem-cell factor
Degree and activity.Freeze-dried powder prepared by the preparation method of the present invention effectively saves various in Human plactnta mescenchymal stem cell
Biologically active cytokine mixture, storage life can extend to 2 years, and efficiently solving cell factor solution shelf-life has
The bottleneck that limit, the commercial application for people's placenta mesenchyma stem cell cell factor are laid a good foundation.
(2), freeze-dried powder is made in placenta mesenchyma stem cell extract by the present invention, preferably placenta mesenchyma can be kept to do
The bioactivity of cell extract, it is not degradable in prolonged storage and easy to use.
Description of the drawings
Fig. 1 is the growth curve that 3T3 cells are utilized in the present invention.
Specific embodiment
The present invention is further described, but the present invention is not limited only to following embodiment below by way of specific embodiment.
Embodiment 1：
A kind of preparation method of placenta mesenchyma stem cell freeze-dried powder, includes the following steps：
(1), the separation and culture of placenta stem-cell：Aseptically, the placenta of health is collected, in GMP laboratory biologicals
Safety cabinet shreds placenta to 1.5-2.5mm3The tissue block of size places it in the DMEM-LG cells that T175 culture bottles are contained
In culture solution, 37 DEG C are put, 5%CO2It is cultivated in incubator；Liquid was changed every 2-3 days, until placenta stem-cell is precipitated, discards tissue
Block；After cell covers with, the first generation is denoted as, had digestive transfer culture is carried out with 0.28% pancreatin；Use antibiotic-free DMEM- after the second generation instead
LG culture mediums, and carry out secondary culture；Containing 10%FBS, bFGF 5ng/mL, L- paddy ammonia in the DMEM-LG cell culture fluids
Amide 2mM, 1 μ g/mL of penicillin 100U/mL, 100 μ g/mL of streptomysin and amphotericin B；The antibiotic-free DMEM-LG trainings
Base is supported containing 10%FBS, bFGF 5ng/mL, L-Glutamine 2mM.
(2), the collection of cell factor：After the third generation, cell length to 90% or so can start to collect cell factor；By cell
Cell in good condition collects, and carries out ultrasonication, it is made to come out the cytokine release not discharged completely into the cell；
And 1500rpm is centrifuged 10 minutes, removes pellet cell debris；
(3), the ultralow temperature freeze-drying of stem cell extract：The placenta stem-cell culture stoste for removing pellet cell debris is added
Enter 10%BSA as excipient, fully after dissolving, with the sterilizing filter filtration sterilization of 0.45um, and dispense to 2ml cillin bottles
In, every bottle of 1ml；Cillin bottle is placed in -80 DEG C of ultra low temperature freezers after packing and is lyophilized in advance, is taken out after pre-freeze 12h, then be placed in
It is freeze-dried in freeze dryer；Obtain placenta mesenchyma stem cell freeze-dried powder.The freeze dryer is vacuum freeze drier.
Freeze dryer sets freeze-drying curve such as table：
| Temperature (DEG C) | Time (hour) |
| -30 | 5 |
| -20 | 5 |
| -5 | 10 |
| 5 | 20 |
| 4 | It preserves to taking-up |
(4) the Activity determination of stem cell factor before and after, being lyophilized：Prepare freeze-dried powder solvent；The original of stem cells factor beauty
Reason is to stimulate skin fibroblasts newborn by cell factor to reach from solving the problems, such as ageing skin at all；Therefore it tests
The activity of freeze-dried powder cell factor is most important.
As shown in Figure 1, using the growth curve of 3T3 cells, growth of the simulation fibroblast under cell factor promotion
Situation；It after 3T3 is cultivated, is digested using pancreatin, after counting, cell is spread to four piece of six orifice plate, every two pieces one group, and every piece
Four holes, wherein per 1*103, hole cell；First group is set as control group, step (2) middle collection is added in the control group
Cell；Second group is set as experimental group, the cell factor dissolved after addition freeze-drying with solvent in the experimental group；It trains respectively
Support for 24 hours, after 48h, 72h, 96h, per plate take 1 hole count, draw growth curve.The freeze-dried powder solvent for 2% hyaluronic acid,
10% glycerine and distilled water.
The present invention is prepared for placenta stem-cell using the fast method of tissue, and intracellular cell is collected using ultrasonic fragmentation
The factor is mixed with the cell factor in supernatant, is lyophilized at low ambient temperatures, fully ensured that the placenta stem-cell factor concentration and
Activity.What the freeze-dried powder prepared by the preparation method of the present invention effectively saved in Human plactnta mescenchymal stem cell various has
The cytokine mixture of bioactivity, storage life can extend to 2 years, efficiently solve cell factor solution shelf-life it is limited this
One bottleneck, the commercial application for people's placenta mesenchyma stem cell cell factor are laid a good foundation.
Freeze-dried powder is made in placenta mesenchyma stem cell extract by the present invention, can preferably keep placenta mesenchyma stem cell
The bioactivity of extract, it is not degradable in prolonged storage and easy to use.
It is described herein and claimed invention is not limited to the ranges of particular aspects disclosed here, because of these sides
Face is intended as illustrations of several aspects of the invention.It is expected that any equivalent aspect is all in the scope of the present invention.In fact, it removes
Shown here and description except those, different modifications of the invention are for those of ordinary skills from foregoing description
It will be apparent.Such modification, which is also intended to, to be fallen within the scope of the appended claims.In case of conflict, it is fixed to include
Subject to the present disclosure of justice.
Claims (5)
1. a kind of preparation method of placenta mesenchyma stem cell freeze-dried powder, it is characterised in that：Include the following steps：
(1), the separation and culture of placenta stem-cell：Aseptically, the placenta of health is collected, in GMP Laboratory biosafeties
Cabinet shreds placenta to 1.5-2.5mm3The tissue block of size places it in the DMEM-LG cell culture that T175 culture bottles are contained
In liquid, 37 DEG C are put, 5%CO2It is cultivated in incubator；Liquid was changed every 2-3 days, until placenta stem-cell is precipitated, discards tissue block；Carefully
After born of the same parents cover with, the first generation is denoted as, had digestive transfer culture is carried out with 0.28% pancreatin；Use antibiotic-free DMEM-LG cultures after the second generation instead
Base, and carry out secondary culture；
(2), the collection of cell factor：After the third generation, cell length to 90% or so can start to collect cell factor；By cell state
Good cell collects, and carries out ultrasonication, it is made to come out the cytokine release not discharged completely into the cell；And
1500rpm is centrifuged 10 minutes, removes pellet cell debris；
(3), the ultralow temperature freeze-drying of stem cell extract：The placenta stem-cell culture stoste for removing pellet cell debris is added in
10%BSA is as excipient, fully after dissolving, with the sterilizing filter filtration sterilization of 0.45um, and dispenses to 2ml cillin bottles
In, every bottle of 1ml；Cillin bottle is placed in -80 DEG C of ultra low temperature freezers after packing and is lyophilized in advance, is taken out after pre-freeze 12h, then be placed in
It is freeze-dried in freeze dryer；Obtain placenta mesenchyma stem cell freeze-dried powder.
(4) the Activity determination of stem cell factor before and after, being lyophilized：Prepare freeze-dried powder solvent；Utilize the growth curve of 3T3 cells, simulation
Growing state of the fibroblast under cell factor promotion；It after 3T3 is cultivated, is digested using pancreatin, after counting, cell is spread
To four piece of six orifice plate, every two pieces one group, and every piece of four holes, wherein per 1*103, hole cell；First group is set as control group,
The cell of step (2) middle collection is added in the control group；Second group is set as experimental group, freeze-drying is added in the experimental group
The cell factor dissolved afterwards with solvent；Cultivate respectively for 24 hours, after 48h, 72h, 96h, per plate take 1 hole count, draw growth curve.
2. the preparation method of placenta mesenchyma stem cell freeze-dried powder according to claim 1, it is characterised in that：Step (1) in
Containing 10%FBS, bFGF 5ng/mL, L-Glutamine 2mM, penicillin 100U/mL, strepto- in the DMEM-LG cell culture fluids
1 μ g/mL of element 100 μ g/mL and amphotericin B.
3. the preparation method of placenta mesenchyma stem cell freeze-dried powder according to claim 1, it is characterised in that：Step (1) in
The antibiotic-free DMEM-LG culture mediums are containing 10%FBS, bFGF 5ng/mL, L-Glutamine 2mM.
4. the preparation method of placenta mesenchyma stem cell freeze-dried powder according to claim 1, it is characterised in that：Step (3) in
The freeze dryer is vacuum freeze drier.
5. the preparation method of placenta mesenchyma stem cell freeze-dried powder according to claim 1, it is characterised in that：Step (4) in
The freeze-dried powder solvent is 2% hyaluronic acid, 10% glycerine and distilled water.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108771638A (en) * | 2018-07-22 | 2018-11-09 | 张晓南 | A kind of biomass placenta freeze-dried powder preparation and preparation method thereof |
| CN111602651A (en) * | 2020-06-20 | 2020-09-01 | 河南和泽干细胞基因工程有限公司 | Placental cell freezing device and freezing method thereof |
| CN112353817A (en) * | 2020-11-30 | 2021-02-12 | 湖南科诺康美生命科技有限公司 | Medicament formula for treating female vaginal relaxation syndrome |
| CN112831466A (en) * | 2021-02-03 | 2021-05-25 | 北京益华生物科技有限公司 | Method for improving content of basic fibroblast growth factor in mesenchymal stem cell culture solution |
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