CN106244652B - A kind of preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder and freeze-dried powder obtained - Google Patents
A kind of preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder and freeze-dried powder obtained Download PDFInfo
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Abstract
The invention discloses a kind of methods of induction human mesenchymal stem cell production stem cell factor；The invention also discloses a kind of preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder and freeze-dried powders obtained.The method induced efficiency of induction human mesenchymal stem cell production cell factor of the invention is high, stem cell factor yield is big, and a large amount of stem cell factor can be obtained in the short time；The preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder of the invention is simple, conveniently, compared to the method for non-low temperature induction, output increased 15%-20%；Freeze-dried powder prepared by the preparation method of invention human mesenchymal stem cell culture supernatant freeze-dried powder saves the various biologically active cytokine mixtures in human mesenchymal stem cell culture supernatant to the full extent, for human mesenchymal stem cell culture supernatant in terms of application lay a good foundation.
Description
Technical field
The invention belongs to human mesenchymal stem cell fields more particularly to a kind of human mesenchymal stem cell culture supernatant to be lyophilized
The preparation method of powder and freeze-dried powder obtained.
Background technique
Mescenchymal stem cell (Mesenchymal Stem Cells, MSCs) is a kind of important multipotential stem cell, mainly
It is distributed in mesoderm and ectoderm of the early stage with embryo.Wherein umbilical cord mesenchymal stem cells are that one kind is present in neonatal umbilical cord group
One of knit versatile stem cell.It is in vivo or in vitro under specific inductive condition, can be divided into bone, cartilage, tendon, tough
The Various Tissues cells such as band, nerve, liver, endothelium and cardiac muscle have wide potential applicability in clinical practice.Due to its immunogenicity compared with
Low, the features such as cell content is high, proliferative capacity is excellent are widely used in the fields such as beauty and shaping.But in the culture of stem cell
Exogenous albumen (fetal calf serum) also can be inevitably introduced in amplification procedure, easily causes immunological rejection.In cell culture
Clear liquid contains various biologically active cell factors, and immunological rejection when its application can be to avoid stem cell beauty is anti-
It answers.But when carrying out routine culture using the culture medium of no fetal calf serum, the activity of stem cell is lower, and the factor of secretion is seldom,
And a large amount of stem cell mortality in a short time.Using some conventional methods, for example, mechanical friction, ultraviolet light irradiation and
The schemes such as starvation culture, can not significantly improve the content of the factor in stem cell supernatant.In addition, the cell culture medium after concentration
In, the various factors can degrade and inactivate rapidly in a short time.
Summary of the invention
On the one hand, a kind of induction human world is provided it is an object of the invention to overcome the shortcomings of the prior art place
The method that mesenchymal stem cells produce cell factor, method induced efficiency of the invention is high, stem cell factor yield is big, can the short time
It is interior to obtain a large amount of stem cell factor.
The technical solution adopted by the present invention are as follows: a method of induction human mesenchymal stem cell produces cell factor, by people
Mescenchymal stem cell is cultivated in a low temperature of being placed in 2~10 DEG C.
Preferably, the culture medium used when culture is DMEM serum free medium.
On the other hand, the present invention also provides a kind of preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder,
The following steps are included:
1) human mesenchymal stem cell is placed in a low temperature of 2~10 DEG C and is cultivated；
2) culture supernatant is collected, to get human mesenchymal stem cell culture supernatant freeze-dried powder after concentration, freeze-drying.
Preferably, in the step 1), human mesenchymal stem cell is to pass on the low passage human mesenchyme that multiple was 1~10 generation
Stem cell.
Preferably, in the step 1), the culture medium that when culture uses is DMEM serum free medium.It is trained using serum-free
Feeding base is cultivated, and exogenous albumen (fetal calf serum) will not be introduced, thus is not easy to cause immunological rejection, jelly obtained
Dry powder uses safer.
Preferably, the step 1) specifically: human mesenchymal stem cell is cultivated in a low temperature of 2~10 DEG C, when the human world is filled
When matter stem cell growth is to 70-90% convergence degree, culture supernatant is collected, and mix with DMEM serum free medium, continue to use
In the expansion culture of human mesenchymal stem cell, it was passaged to for 10~15 generations to cell repeatedly.
It is highly preferred that the culture supernatant mixes in equal volume with DMEM serum free medium.
Preferably, the low temperature is 4 DEG C.The stem cell factor yield induced at this temperature is bigger.
Preferably, the time of the culture is 48~96h, more preferably 72h.
Preferably, the human mesenchymal stem cell is human umbilical cord mesenchymal stem cells.
Preferably, in the step 1), the acquisition methods of human mesenchymal stem cell are as follows: (1) training of human mesenchymal stem cell
It supports: with the mescenchymal stem cell culture medium subculture human mesenchymal stem cell containing fetal calf serum；(2) human mesenchymal stem cell
Collection: collect low passage human mesenchymal stem cell and simultaneously eluted for several times with physiological saline, is then resuspended in physiological saline,
Wherein low passage human mesenchymal stem cell refers to that passage multiple is the human mesenchymal stem cell in 0~10 generation.
Preferably, the step 2) specifically: culture supernatant is 1. collected, in an aseptic environment by human mesenchymal stem cell
Culture supernatant is placed in 3.5KDa ultra-filtration centrifuge tube, centrifugation；2. opening centrifuge tube in an aseptic environment, it is sucked out with sterile pipette tips
Supernatant in centrifuge tube is into 3.5KDa bag filter, under the conditions of 4 DEG C, bag filter is embedded in PEG20000 and is dehydrated 4 hours；
3. bag filter is vacuumized, lyophilization, ice crystal is removed, freeze-dried powder is finally made.
In another aspect, the present invention also provides the preparations according to the human mesenchymal stem cell culture supernatant freeze-dried powder
Human mesenchymal stem cell culture supernatant freeze-dried powder made from method.
Compared with the existing technology, the invention has the benefit that
The method induced efficiency height of induction human mesenchymal stem cell production cell factor of the invention, stem cell factor yield
Greatly, a large amount of stem cell factor can be obtained in the short time；
The preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder of the invention is simply, conveniently, low compared to not
The method of temperature induction, output increased 15%-20%, and the product protein content obtained are between 97-99%.
Freeze-dried powder prepared by the preparation method of invention human mesenchymal stem cell culture supernatant freeze-dried powder is to the full extent
The various biologically active cytokine mixtures in human mesenchymal stem cell culture supernatant are saved, it is dry for human mesenchyme
Cell culture supernatant in terms of application lay a good foundation.
Detailed description of the invention
Fig. 1 shows human mesenchymal stem cell supernatant that different Fiber differentiation modes obtains to fibroblasts of adult human dermis
The facilitation of growth.
Specific embodiment
To better illustrate the object, technical solutions and advantages of the present invention, below in conjunction with specific embodiment to the present invention
It is described further.
Embodiment 1
The acquisition methods of human mesenchymal stem cell
The acquisition methods of human mesenchymal stem cell, comprising the following steps:
(1) cell origin used in takes fully the infant umbilical cord 5-10cm of monthly output, physiological saline wash clean to group in aseptic condition
No color is knitted, removal epidermis, two arteries and a vein blood vessel shred tissue, with 37 DEG C of 0.1%I Collagenase Type
Digestion 1 hour, 400g are centrifuged 5min, inhale and abandon supernatant, mix 200 mesh net filtrations, cell suspension 1200r/ obtained
Min, is centrifuged 8min, and the DMEN/F12 culture medium (containing dual anti-) containing 10%FBS, 37 DEG C, saturated humidity, 5% is added in cell precipitation
CO2: in about one week of culture, primary human umbilical cord mesenchymal stem cells can be obtained.When cell fusion degree 70%-80%,
0.25% trypsase (containing 0.02%EDTA) vitellophag secondary culture.
(2) collection of stem cell: human umbilical cord mesenchymal stem cells are cultivated in 10cm culture dish, when growing into 70%-
When 80% degrees of fusion, culture stem cell is collected, three times with normal saline flushing cell.
Embodiment 2
The preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder
The preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder, comprising the following steps:
(1) the low algebra cell collected in embodiment 1 secretion of low temperature induction stem cell factor: is resuspended in physiology salt
Water, in 4 DEG C, saturated humidity, 5%CO2Under the conditions of, it cultivates 72 hours；Therebetween, when human mesenchymal stem cell grows into 70-90%
When convergence degree, culture supernatant is collected, and mix in equal volume with DMEM serum free medium, continue on for human mesenchymal stem cell
Expansion culture, be passaged to for 10~15 generations to cell repeatedly, collect whole cell culture supernatants, be used for subsequent freeze-dried powder
Preparation.
(2) it the preparation of freeze-dried powder: by the culture supernatant of collection into 3.5KDa bag filter, under the conditions of 4 DEG C, will dialyse
It is dehydrated 4 hours in bag embedment PEG20000；Bag filter is set and is vacuumized in a round bottom flask, lyophilization removes ice crystal, finally
Freeze-dried powder, -20 DEG C of preservations are made.
Embodiment 3
It is specific as follows using the stem cell in the 5th generation collected in different method Fiber differentiation embodiments 1:
1.1 blank control
The stem cell for taking for the 5th generation three times with brine cell is added DMEM serum free medium and continues culture 72
Hour, collect supernatant.Stem cell culture supernatant is taken to measure it to fibroblasts of adult human dermis proliferation activity.
1.2 ultraviolet induction
Three times with brine cell DMEM serum free medium is added in the stem cell for taking for the 5th generation, and UVA irradiation is thin
Born of the same parents 2h (interval 22 hours, irradiation is three times), cultivates 72 hours, collects supernatant.Stem cell culture supernatant is taken to measure it to human dermis
Proliferative activity of fibroblasts.
1.3 mechanical friction
The stem cell for taking for the 5th generation three times with brine cell is added DMEM serum free medium, uses sterile needle
Head is cultivated 72 hours cell surface frictionally damage cell (friction 10 minutes every time are done primary for every 24 hours), collects supernatant.
Stem cell culture supernatant is taken to measure it to fibroblasts of adult human dermis proliferation activity.
1.4 low nutrition methods
The stem cell for taking for the 5th generation, three times with brine cell, old DMEM serum free medium, which is added, (will wash
Cell culture medium before washing is collected, and Tissue Culture Flask is added after cell washing, supports cell with old culture medium), continue to train
It supports 72 hours, collects supernatant.Stem cell culture supernatant is taken to measure it to fibroblasts of adult human dermis proliferation activity.
1.5 low temperature
The 5th generation stem cell is taken, three times with brine cell, DMEM serum free medium is added, cell is distinguished
Be placed in 4 DEG C, 8 DEG C, in 10 DEG C of constant temperature refrigerator, continue culture 72 hours, collect supernatant.Stem cell culture supernatant is taken to measure its right
Fibroblasts of adult human dermis proliferation activity.
The detection of 2.1 different inductive condition stem cell secretion cell factor amounts
Using the amount of the cell factor of Coomassie Brilliant Blue detection stem cell secretion.Determination of protein concentration is obtained first
Standard curve, then measure the light absorption value of sample, by comparing with standard curve, obtain the concentration of albumen in sample, as a result
As shown in the table.
As seen from the above table: 1) stem cell can secrete a large amount of cell factor and other cellular products, in the condition not induced
Lower cell can secrete a large amount of cell factor (blank control, 753 μ g/ml)；2) it under different inductive conditions, can stimulate dry
Cell-secretion factor.Wherein inducing effect are as follows: low temperature induction > ultraviolet induction > low nutrition induces > friction induction.Wherein, ultraviolet
The method of line induction and low temperature induction has the effect of more obvious, and low temperature induction is optimal set, at especially 4 DEG C；3) low temperature
Induction is used as optimal set, and possible reason is under other inductive conditions, and cell has a large amount of death, and in cryogenic conditions
Under, cell death quantity is few.
2.2 pairs of fibroblasts of adult human dermis proliferation activity detections
With 1*104The amount of the fibroblasts of adult human dermis in/hole is added 96 orifice plates, 37 DEG C, 5%CO2, saturated humidity cell
Incubator culture 24 hours, supernatant is abandoned, is separately added into blank control, ultraviolet light lures, rub, low nutrition, low temperature induction method obtain
Stem cell supernatant, while fetal calf serum (10%) is added, 37 DEG C, the cell incubator culture 48 of 5%CO2, saturated humidity
Hour, 20 hole μ l/ MTT is added and abandons supernatant after culture 4 hours, 10020 hole μ l/ dimethyl sulfoxide (DMSO), concussion is added
15min, microplate reader colorimetric (wavelength 570nm, reference wavelength 630nm) survey absorbance A value.Stem cell supernatant is calculated to human dermis
The facilitation of fibroblastic growth.Cell growth promotion rate=sample-adding group A570/1*104Fibroblasts of adult human dermis A570,
As a result as shown in Figure 1, wherein low temperature is the result at 10 DEG C.
To sum up by comparing influence of the different physical stimulations for the cell factor of stem cell secretion, it can be seen that low
Temperature has more significant effect for the secretion of cell factor.
Human mesenchymal stem cell can be secreted many cell factors (protein) during the cultivation process, therefore cells and supernatant
Liquid contains the biologically active cell factor of various secretions, mainly has: epidermal growth factor (EGF), and basic fibroblast is raw
The long factor (BFGF), insulin-like growth factor-i (IGF-1), transforming growth factor-β (TGF-β), platelet derived growth because
Sub (PDGF), keratinocyte growth factor (KGF) etc..Epidermal growth factor can promote epidermal cell metabolism, tachymetabolism
Aging cutin repairs acne print, refines wrinkle, enhances the water conservation and water lock ability of itself skin, has enhancing elasticity of skin, reduces
The effects of wrinkle prevents skin aging and infiltration nourishing, promotes the synthesis of collagen.Basic Fibroblast Growth Factor promotes
The proliferation of dermal cell, differentiation, improve cell life microenvironment, promote reparation and the refinement wrinkle of damaged skin, restore cell
Vigor, endogenous adjust collagen and fill by skin flexible, shine healthy glow, the deep layer that can be used for problem skin is repaired
Multiple and smoothing wrinkle, while starting skin wound healing process, reduce the formation of cicatricial tissue in wound healing process.Insulin-Like is raw
Long -1 Human Umbilical Vein Endothelial Cells of the factor play chemotaxis, quickly repair impaired skin, accelerate the division and proliferation of different type cell,
The synthesis for increasing collagen, removes wrinkle.Transforming growth factor-β, adjusts the proliferation and differentiation of cell, it can promote collagen
The formation of fiber, reticular fibre and elastomer adjusts collagen and is formed, reduces wrinkle, adjust the secretion of skin oil and fat, make
Skin-tightening is glossy.Platelet derived growth factor assembles cell to skin lesion sites, and breaks up there, is proliferated,
Repair damaged skin tissue；The dissolution for also inhibiting phoirocyte simultaneously, extends its time-to-live, to selectively stimulate
Phoirocyte growth, reparation and healing after promoting dermal tissue insult.Keratinocyte growth factor be keratinocyte and
Most important influence factor in hair follicle forming process, stimulates the synthesis of DNA, promote to maintain human keratinocyte, epidermal cell and on
The growth of chrotoplast, to adjust various skin problems on the whole.
But storage life is only several weeks after conventional cytokine is dissolved in liquid, be unfavorable for stem cell in terms of push away
Wide application.And freeze-dried powder prepared by the preparation method of the present inventor's umbilical cord mesenchymal stem cells culture supernatant freeze-dried powder is effective
Ground saves the various biologically active cytokine mixtures in human mesenchymal stem cell culture supernatant, and storage life can
It extends to 2 years, efficiently solves the bottleneck that supernatant storage life is limited, be the industrialization of human mesenchymal stem cell culture supernatant
Using laying a good foundation.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than protects to the present invention
The limitation of range is protected, although the invention is described in detail with reference to the preferred embodiments, those skilled in the art should
Understand, it can be with modification or equivalent replacement of the technical solution of the present invention are made, without departing from the essence of technical solution of the present invention
And range.
Claims (9)
1. a kind of preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder, it is characterised in that: the following steps are included:
(1) it obtains human mesenchymal stem cell: umbilical cord tissue is shredded, digested with 0.1%I Collagenase Type, prepare cell suspending liquid,
And be centrifuged, it is cultivated with the cell precipitation of acquisition, 37 DEG C, saturated humidity, 5%CO2Under the conditions of, it cultivates 5~7 days, obtains
To primary human umbilical cord mesenchymal stem cells；Primary cell uses low propagating method secondary culture human mesenchymal stem cell, is passing on
Cell fusion degree collects human mesenchymal stem cell when being 70%~80%；
(2) human mesenchymal stem cell prepared by step (1) is resuspended in physiological saline, in a low temperature of 2~10 DEG C, saturated humidity
5%CO2Under the conditions of carry out proliferative culture；
(3) step (2) culture supernatant is collected, to get the freeze-drying of human mesenchymal stem cell culture supernatant after concentration, freeze-drying
Powder.
2. the preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder according to claim 1, it is characterised in that:
In the step (2), human mesenchymal stem cell is to pass on the low passage human mesenchymal stem cell that multiple was 1~10 generation.
3. the preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder according to claim 1, it is characterised in that:
In the step (2), the culture medium that when culture uses is DMEM serum free medium.
4. the preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder according to claim 1, it is characterised in that:
The step (2) specifically: human mesenchymal stem cell is cultivated in a low temperature of 2~10 DEG C, when human mesenchymal stem cell is grown into
When 70-90% convergence degree, culture supernatant is collected, and mix with DMEM serum free medium, it is dry thin to continue on for human mesenchyme
The expansion culture of born of the same parents, was passaged to for 10~15 generations to cell repeatedly.
5. the preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder according to claim 1, it is characterised in that:
The time cultivated in the step (2) is 48~96h.
6. the preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder according to claim 1, it is characterised in that:
The culture of human mesenchymal stem cell uses the mescenchymal stem cell culture medium subculture containing fetal calf serum in the step (1)
Human mesenchymal stem cell；The low passage human mesenchymal stem cell refers to that passage multiple is the human mesenchymal stem cell in 0~10 generation.
7. the preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder according to claim 6, it is characterised in that:
Fetal calf serum content in the mescenchymal stem cell culture medium is less than 1%.
8. the preparation method of human mesenchymal stem cell culture supernatant freeze-dried powder according to claim 1, it is characterised in that:
The step (3) specifically: 1. collect culture supernatant, be in an aseptic environment placed in human mesenchymal stem cell culture supernatant
In 3.5KDa ultra-filtration centrifuge tube, centrifugation；2. opening centrifuge tube in an aseptic environment, the supernatant in centrifuge tube is sucked out with sterile pipette tips
Liquid is into 3.5KDa bag filter, under the conditions of 4 DEG C, bag filter is embedded in PEG20000 and is dehydrated 4 hours；3. bag filter is taken out true
Sky, lyophilization remove ice crystal, and freeze-dried powder is finally made.
9. the preparation method system of described in any item human mesenchymal stem cell culture supernatant freeze-dried powders according to claim 1~8
The human mesenchymal stem cell culture supernatant freeze-dried powder obtained.
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