CN109280640A - A kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder - Google Patents
A kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder Download PDFInfo
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- CN109280640A CN109280640A CN201811157862.XA CN201811157862A CN109280640A CN 109280640 A CN109280640 A CN 109280640A CN 201811157862 A CN201811157862 A CN 201811157862A CN 109280640 A CN109280640 A CN 109280640A
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0221—Freeze-process protecting agents, i.e. substances protecting cells from effects of the physical process, e.g. cryoprotectants, osmolarity regulators like oncotic agents
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Abstract
The present invention provides a kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder, and being separately cultured including umbilical cord mesenchymal stem cells is passed on and the preparation of stem cell freeze-dried powder.It is frozen after the cell culture supernatant of secondary culture is evacuated in centrifuge tube to solid-state like, solid-state like frost culture medium is placed in again and is frozen in bottle using what sealed membrane or preservative film tightly sealed, hole is pricked on sealed membrane or preservative film with syringe needle, it is then placed into freeze-drying on freeze drier and obtains umbilical cord mesenchymal stem cells freeze-dried powder to solid-like.Preparation method of the present invention is simple and effective, at low cost, and freeze-dried powder sample in freezing dry process can be prevented to be splashed to outside container, reduces that sample is especially a small amount of or the loss of relatively valuable sample, guarantees the yield of sample；The bubble for being evacuated generation in freezing dry process can be made to be smoothly discharged simultaneously, prevent bubble to be stained with container inner wall, ensure that the quality and appearance of sample.
Description
Technical field
The invention belongs to biomedical technical field, especially a kind of preparation sides of umbilical cord mesenchymal stem cells freeze-dried powder
Method.
Background technique
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.Currently, umbilical cord and Cord blood not will cause donor any with the acquisition pattern of its non-intrusion type
It injures and becomes a kind of ideal source for mesenchymal stem cells.And compared with Cord blood, the mescenchymal stem cell in umbilical cord source is more
It is easily obtained and cultivates.
Umbilical cord mesenchymal stem cells, which refer to, is present in one of neonatal umbilical cord tissue versatile stem cell, it can break up
At many kinds of histocytes, because having, acquisition is convenient, Proliferation, Differentiation ability is strong, immunogenicity is weak, many excellent without ethics problem etc.
Point, it is of increasing concern in fields such as organizational project, genetic engineerings.
Mescenchymal stem cell freeze-dried powder is extracted and is prepared, sample is carried out using various freeze driers in the market mostly
The method of physics distillation processing.During the freeze-drying process, vacuum can be pumped down in container, what this resulted in holding in container
Sample is largely overflowed due to pumping and out of container, and sample is caused to reduce, and since pumping causes to produce in freezing dry process
Raw a large amount of bubbles are stained with container inner wall, affect the quality, yield and appearance of sample.Particularly with umbilical cord mesenchymal stem cells
For the freeze-dried powder preparation of this small volume production, a kind of quality for effectively improving freeze-dried powder is studied, the side of the loss of freeze-dried powder is reduced
Method is of great significance.
Summary of the invention
In order to solve the problems existing in the prior art, the purpose of the present invention is to provide a kind of freeze-dryings of umbilical cord mesenchymal stem cells
The preparation method of powder, using the freeze drying technology for a small amount of or relatively valuable bioactivity sample, method is simple and effective, can subtract
Few sample loss, improves sample quality.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder, the difference is that, comprising the following steps:
Step a) umbilical cord mesenchymal stem cells are separately cultured: fresh umbilical cord taken, is shredded after the jelly of Wharton around separating blood vessel,
Digestive juice digestion abandons supernatant with being centrifuged after normal saline dilution, and culture dish is transferred to after adding culture medium to be suspended, and additive, double is added
Anti- and amphotericin B, is put into incubator and cultivates；
The passage of step b) umbilical cord mesenchymal stem cells: it after the cell of step a) culture covers with, is passed on；Step a) is taken to train
Partial medium in ware is supported physiological saline and pancreatin are added into centrifuge tube, mixes digestion, is added after digesting well to centrifuge tube
Culture medium terminates digestion, and cell is washed with the pipette piping and druming culture dish truth of a matter time, is then collected into centrifuge tube and is centrifuged, in abandoning
Clearly, the culture medium containing additive is added to be suspended, by culture dish is transferred to after the dilution of required cell density, is put into incubator and cultivates；
The preparation of step c) umbilical cord mesenchymal stem cells freeze-dried powder: by the resulting cell culture supernatant of step b) secondary culture
It is frozen after being evacuated in centrifuge tube to solid-state like, freezes culture medium completely solid；Solid-state like frost culture medium is placed in again
It is frozen in bottle using what sealed membrane or preservative film tightly sealed, then pricks hole on sealed membrane or preservative film with syringe needle, then put
It is placed in freeze-drying on freeze drier and obtains umbilical cord mesenchymal stem cells freeze-dried powder to solid-like.
In the above-mentioned technical solutions, the digestive juice digestion in the step a), is that the umbilical cord shredded is first put into Type I collagen
In enzyme, uniformly mixed be placed in 37 DEG C of incubators of dual anti-and amphotericin B is added and stays overnight, pancreatin is added after taking-up, sets again
It is digested in 37 DEG C of incubators.
In the above-mentioned technical solutions, the extension rate of the physiological saline in the step a) is 4 ~ 6 times, and centrifugal condition is
3000rpm is centrifuged 15 ~ 20min.
In the above-mentioned technical solutions, the step b) is by 1:(3 ~ 5) it is passed on.
In the above-mentioned technical solutions, in the step b) pancreatin final concentration of 0.05%, centrifugal condition be 1800rpm from
Heart 5min.
In the above-mentioned technical solutions, conditions of cryopreservation is to be placed in low temperature refrigerator or liquid nitrogen container to freeze in the step c).
In the above-mentioned technical solutions, in the step c) on freeze drier sublimation drying be 12 ~ for 24 hours.
In the above-mentioned technical solutions, cell culture supernatant carries out umbilical cord mesenchyma before being evacuated to centrifuge tube in the step c)
The identification of stem cell.
In the above-mentioned technical solutions, the identification of the umbilical cord mesenchymal stem cells is disappeared with 0.1% trypsase/citric acid
Change liquid the cell dissociation in step c) in cell culture supernatant gets off, is suspended with PBS buffer solution, CD31- is then added
FITC, CD73-PE, DRPer CP and CD29-APC are protected from light incubation；After being cleaned again with PBS buffer solution, 1800rpm is centrifuged 5min,
After precipitating is resuspended with PBS, with flow cytometer Testing and appraisal cell surface antigen.
Compared with prior art, the invention has the following beneficial effects:
1) present invention covers sealed membrane or preservative film at the vessel port that freeze drier contains sample, can prevent from freezing
Freeze-dried powder sample is splashed to outside container in drying process, is reduced the loss of sample to greatest extent, be ensure that the yield of sample, right
It is particularly important in low-volume samples or relatively valuable sample；
2) present invention covers container using disposable sealed membrane or preservative film, had both improved the convenience of experimental implementation, and had also dropped
Low experimental cost；
3) present invention pricks hole on the sealed membrane or preservative film of covering container using syringe needle, can make to be freeze-dried
The bubble for being evacuated generation in the process is smoothly discharged, and bubble is prevented to be stained with container inner wall, ensure that the quality and appearance of sample.
Detailed description of the invention
Fig. 1 is 1 products therefrom sample of the embodiment of the present invention, and Fig. 2 is 1 products therefrom sample of comparative example.
Specific embodiment
Below with reference to embodiment, specific embodiments of the present invention will be described in further detail.Following embodiment is for saying
The bright present invention, but be not used in and limit the scope of the invention.
A kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder of the present invention, comprising the following steps:
Step a) umbilical cord mesenchymal stem cells are separately cultured: being taken fresh umbilical cord, shredded after the jelly of Wharton around separating blood vessel；
It is put into Type I collagen enzyme, dual anti-and amphotericin B is added, be uniformly mixed and be placed in 37 DEG C of incubator overnight；Add after taking-up
Enter pancreatin, the amount of pancreatin is added depending on collagenase digesting situation, is again placed in 37 DEG C of incubator and digests；Use physiology salt
3000rpm is centrifuged 15 ~ 20min after water dilutes 4 ~ 6 times, abandons supernatant, is transferred in culture dish after adding culture medium to be suspended, and addition is added
Agent, dual anti-and amphotericin B, are put into incubator and cultivate, and change liquid according to cultivation conditions timely and appropriate discovery；
Step b) umbilical cord mesenchymal stem cells passage: after the cell of step a) culture covers with, by 1:(3 ~ 5) it is passed on.It inhales
Take in step a) culture dish partial medium to centrifuge tube, remaining culture medium goes to waste liquid cylinder in culture dish.Into centrifuge tube
Physiological saline and pancreatin is added, wherein pancreatin preferably final concentration of 0.05%, mixes digestion amount of time, it is seen that cell floats up
Or culture medium is added after microscopic observation confirmation cell dissociation is good and terminates digestion；With the pipette piping and druming culture dish truth of a matter time cell
It washes, then be collected into centrifuge tube, 1800rpm is centrifuged 5min, abandons supernatant, and the culture medium containing additive is added and is suspended, presses
It is transferred to culture dish after required cell density dilution, is put into incubator and cultivates；
The preparation of step c) umbilical cord mesenchymal stem cells freeze-dried powder: by the resulting cell culture supernatant of step b) secondary culture
It is evacuated in centrifuge tube to place and be frozen in low temperature refrigerator or liquid nitrogen container to solid-state like, freeze culture medium completely solid；It will consolidate again
The frost culture medium of state shape is placed in be frozen in bottle using what sealed membrane or preservative film tightly sealed, then with syringe needle in sealed membrane
Or several holes are pricked on preservative film, it is then placed on freeze drier and is freeze-dried to solid-like, preferably freeze drying time
For 12 ~ for 24 hours, because the time not in place easily causes sample to melt to produce gas and dissolution produces gas loss, lead to the underproduction, freeze in freeze-drying process
Dry overlong time cost is high.Umbilical cord mesenchymal stem cells freeze-dried powder is obtained after freeze-drying.
Preferably, cell culture supernatant needs to carry out umbilical cord mesenchymal stem cells before being evacuated to centrifuge tube in the step c)
Identification then carries out the preparation of the step c) freeze-dried powder after the identified cell for proving culture is umbilical cord mesenchymal stem cells.
The identification of umbilical cord mesenchymal stem cells is with the following method: with 0.1% trypsase/citric acid digestive juice by step
C) the good cell dissociation of growth conditions in cell culture supernatant gets off, with 50 μ l PBS buffer solution be suspended, then plus
Enter 10 μ l CD31-FITC, 10 μ l CD73-PE, 10 μ l DRPer CP and 5 μ l CD29-APC, is protected from light and is incubated for 15min；It uses again
After PBS buffer solution cleaning, 1000rpm is centrifuged 5min, after precipitating is resuspended with 500 μ l PBS, with flow cytometer Testing and appraisal
Cell surface antigen.
Embodiment 1
A kind of umbilical cord mesenchymal stem cells freeze-dried powder is prepared, method is as follows:
Step a) umbilical cord mesenchymal stem cells are separately cultured: fetching fresh umbilical cord with aseptic bottle, the Wo Dun around separating blood vessel
It is shredded after glue；It is put into 2mg/ml Type I collagen enzyme 15ml, is added that 200 μ l are dual anti-and 200 μ l amphotericin Bs, uniformly mixed postposition
In 37 DEG C of incubator overnight；Pancreatin 10ml is added after taking-up, is again placed in 37 DEG C of incubator and digests 30min；With life
It manages 3000rpm after salt water dilutes 5 times and is centrifuged 20min, abandon supernatant, be transferred in 3 culture dishes after adding culture medium to be suspended, every ware is added
1ml additive, 200 μ l are dual anti-and 200 μ l amphotericin Bs, are put into incubator and cultivate, are changed according to cultivation conditions timely and appropriate discovery
Liquid；
The passage of step b) umbilical cord mesenchymal stem cells: it after the cell of step a) culture covers with, is passed on by 1:3.Every ware is inhaled
Take 3ml culture medium in step a) culture dish remaining to go to waste liquid cylinder to 50ml centrifuge tube.Physiological saline is added into centrifuge tube
And pancreatin, pancreatin final concentration of 0.05%, mix digestion 1min, it is seen that cell floats up or confirms cell dissociation in microscopic observation
Every ware is added 3ml culture medium and terminates digestion after good；6 times cell is washed with pipette piping and druming culture dish ware bottom, then collect
Into 50ml centrifuge tube, 1800rpm is centrifuged 5min, abandons supernatant, every ware is added culture medium of the 18ml containing additive and is suspended, by required
It is transferred to culture dish after cell density dilution, is put into incubator and cultivates；
The preparation of step c) umbilical cord mesenchymal stem cells freeze-dried powder: it first takes step b) 3 to be commissioned to train and supports resulting cell culture supernatant
The identification of umbilical cord mesenchymal stem cells is carried out, qualification result proves the cell of culture as after human mesenchymal stem cell, cell is trained
Feeding supernatant is evacuated in centrifuge tube to place and be frozen in low temperature refrigerator or liquid nitrogen container to solid-state like, makes the complete ice freeze of culture medium
It is real；Solid-state like frost culture medium is placed in again and is frozen in bottle using what sealed membrane or preservative film tightly sealed, then uses syringe needle
Head pricks several holes on sealed membrane or preservative film, is then placed on freeze drier and is freeze-dried for 24 hours to solid-like, freeze-drying
After obtain umbilical cord mesenchymal stem cells freeze-dried powder.
Comparative example 1
Comparative example 1 prepares umbilical cord mesenchymal stem cells using normal freeze-drying method.Except being freeze-dried in step c)
Bottle is frozen in journey and tightly seals bottleneck without using sealed membrane or preservative film, if pricking on sealed membrane or preservative film without using syringe needle
Outside dry hole, other preparation steps are same as Example 1.
As seen from Figure 1, the umbilical cord mesenchymal stem cells freeze-dried powder appearance that 1 preparation method of the embodiment of the present invention obtains is white
It is powdered, bottle inner wall bubble-free is lyophilized.From Figure 2 it can be seen that umbilical cord mesenchymal stem cells freeze-dried powder and embodiment prepared by comparative example 1
1 compared to having lost 60%, and sample almost remains little, and freeze-drying bottle inner wall has a large amount of blisterings, has seriously affected the appearance of freeze-dried powder
And yield.This is primarily due to the freeze-drying bottle opening of the invention for containing sample in freeze drier and uses sealed membrane or fresh-keeping
Film tightly seals bottleneck, and freeze-dried powder sample in freezing dry process can be prevented to be splashed to outside container, thus can be reduced the loss of sample,
It ensure that the yield of sample.Hole is pricked on sealed membrane or preservative film using syringe needle, freezing dry process can be made
The bubble that middle pumping generates is smoothly discharged, and prevents bubble to be stained with container inner wall, to ensure that the quality and appearance of sample.
It is obtained prepared by hole it can be seen that freeze-drying container applies sealed membrane and pricked on sealed membrane with syringe needle
To umbilical cord mesenchymal stem cells freeze-dried powder no matter have obvious optimization from yield and quality or in appearance.The present invention is cold
Lyophilizer, which contains, covers sealed membrane or preservative film at the vessel port of sample, can prevent freeze-dried powder in freezing dry process
Sample is splashed to outside container, is reduced the loss of sample to greatest extent, be ensure that sample especially low-volume samples or somewhat expensive
The yield of same product；Hole is pricked on the sealed membrane or preservative film of covering container with syringe needle, can make to be freeze-dried
The bubble for being evacuated generation in the process is smoothly discharged, and bubble is prevented to be stained with container inner wall, ensure that the quality and appearance of sample.
Finally, it should be noted that above embodiments are merely to illustrate the protection scope that the present invention is not intended to limit the present invention.
In addition, after reading the technical contents of the present invention, those skilled in the art the present invention can be made various changes, modification or
Modification, all these equivalent forms also belong within the required protection scope limited of the application.
Claims (9)
1. a kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder, it is characterised in that: the following steps are included:
Step a) umbilical cord mesenchymal stem cells are separately cultured: fresh umbilical cord taken, is shredded after the jelly of Wharton around separating blood vessel,
Digestive juice digestion abandons supernatant with being centrifuged after normal saline dilution, and culture dish is transferred to after adding culture medium to be suspended, and additive, double is added
Anti- and amphotericin B, is put into incubator and cultivates；
The passage of step b) umbilical cord mesenchymal stem cells: it after the cell of step a) culture covers with, is passed on；Step a) is taken to train
Partial medium in ware is supported physiological saline and pancreatin are added into centrifuge tube, mixes digestion, is added after digesting well to centrifuge tube
Culture medium terminates digestion, and cell is washed with the pipette piping and druming culture dish truth of a matter time, is then collected into centrifuge tube and is centrifuged, in abandoning
Clearly, the culture medium containing additive is added to be suspended, by culture dish is transferred to after the dilution of required cell density, is put into incubator and cultivates；
The preparation of step c) umbilical cord mesenchymal stem cells freeze-dried powder: by the resulting cell culture supernatant of step b) secondary culture
It is frozen after being evacuated in centrifuge tube to solid-state like, freezes culture medium completely solid；Solid-state like frost culture medium is placed in again
It is frozen in bottle using what sealed membrane or preservative film tightly sealed, then pricks hole on sealed membrane or preservative film with syringe needle, then put
It is placed in freeze-drying on freeze drier and obtains umbilical cord mesenchymal stem cells freeze-dried powder to solid-like.
2. a kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder according to claim 1, it is characterised in that: described
Digestive juice digestion in step a), is that first the umbilical cord shredded is put into Type I collagen enzyme, and the mixing of dual anti-and amphotericin B is added
It is uniformly placed in 37 DEG C of incubators overnight, pancreatin is added after taking-up, is again placed in 37 DEG C of incubators and digests.
3. a kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder according to claim 1, it is characterised in that: described
The extension rate of physiological saline in step a) is 4 ~ 6 times, and centrifugal condition is that 3000rpm is centrifuged 15 ~ 20min.
4. a kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder according to claim 1, it is characterised in that: described
Step b) is by 1:(3 ~ 5) it is passed on.
5. a kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder according to claim 1, it is characterised in that: described
Final concentration of the 0.05% of pancreatin in step b), centrifugal condition are that 1800rpm is centrifuged 5min.
6. a kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder according to claim 1, it is characterised in that: described
Conditions of cryopreservation is to be placed in low temperature refrigerator or liquid nitrogen container to freeze in step c).
7. a kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder according to claim 1, it is characterised in that: described
In step c) on freeze drier sublimation drying be 12 ~ for 24 hours.
8. a kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder according to claim 1, it is characterised in that: described
Cell culture supernatant is evacuated to the identification of progress umbilical cord mesenchymal stem cells before centrifuge tube in step c).
9. a kind of preparation method of umbilical cord mesenchymal stem cells freeze-dried powder according to claim 8, it is characterised in that: described
The identification of umbilical cord mesenchymal stem cells is will be in step c) in cell culture supernatant with 0.1% trypsase/citric acid digestive juice
Cell dissociation get off, be suspended with PBS buffer solution, CD31-FITC, CD73-PE, DRPer CP and CD29-APC be then added,
It is protected from light incubation；After being cleaned again with PBS buffer solution, 1800rpm is centrifuged 5min and is examined after precipitating is resuspended with PBS with flow cytometer
Survey identification of cell surface antigen.
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Application publication date: 20190129 |
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