CN105056303A - Composition, preparation and application thereof - Google Patents
Composition, preparation and application thereof Download PDFInfo
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- CN105056303A CN105056303A CN201510602665.4A CN201510602665A CN105056303A CN 105056303 A CN105056303 A CN 105056303A CN 201510602665 A CN201510602665 A CN 201510602665A CN 105056303 A CN105056303 A CN 105056303A
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Abstract
The invention relates to the field of cells and in particular to a composition, a preparation and an application thereof. The composition comprises umbilical cord mesenchymal stem cells, growth factors and hyaluronic acid or a salt thereof. The invention also provides a stem cell preparation capable of quickly and efficiently repairing face defects. The proliferation differentiation ability of the stem cells, the moisture retention function of hyaluronic acid and sodium hyaluronate, and the effect of promoting cell growth of EGF are fully utilized, so that the problem of stably and efficiently repairing the face defects is solved.
Description
Technical field
The present invention relates to cell field, particularly a kind of compositions, preparation and uses thereof.
Background technology
From biologically, aging be biology As time goes on, spontaneous necessary process, it is complicated natural phenomena, and show as the degeneration of structure and the decline of function, adaptability and resistance go down.In a physiologically, aging is regarded as the ontogeny being performed until old age from germ cell.From pathology, aging be stress and strain, damage and infect, immunoreation fail, nutritional disorder, dysbolismus and carelessness and Drug abuse accumulate result.
Beauty treatment and defying age more and more become the means that people pursue beauty, also there is increasing beautifying and antisenility old product accordingly, in these products, based on the skin-protection product of external, play slow down aging and skin keeps elastic object, but these products cannot meet and have the crowd of the aspect problems such as face defect to the demand of beautifying and anti-aging.And the product of this patent can solve these face deletion problems, play the object of Facial defects reparation and face rejuvenation.
At present the normal high-concentration blood plasma concentrated (PRP) that adopts carries out Facial defects reparation, be utilize self-blood to make be rich in hematoblastic high-concentration blood plasma.But PRP preparation is acellular composition, mainly platelet, promote that the propagation of surrounding tissue cells reaches the function of tissue repair by all kinds of somatomedin of secretion of platelet, defect repair ability is low, and the time is long.The crowds such as platelet defect, fibrin dyssynthesis, hemodynamic are unstable can not use.PRP preparation needs to carry out platelet from autologous extraction peripheral blood and concentrates, and belongs to blood products, is not suitable for the institutional units not possessing medical qualification.
Therefore, a kind of stem cell medicine rapidly and efficiently revising face defect is provided all to have important realistic meaning.
Summary of the invention
In view of this, the invention provides a kind of compositions, preparation and uses thereof.Said preparation makes full use of the proliferation and differentiation ability of stem cell, hyaluronic acid and the moisturizing of hyaluronate sodium and the Promote cell's growth effect of EGF, reaches the repair surface hole defect problem of stability and high efficiency.
In order to realize foregoing invention object, the invention provides following technical scheme:
The invention provides a kind of compositions, comprise umbilical cord mesenchymal stem cells, somatomedin, hyaluronic acid or its salt.
In specific embodiments more of the present invention, compositions also comprises hyaluronic acid.
In specific embodiments more of the present invention, in mg/mL, described somatomedin mass volume ratio is in the composition 1.5 ~ 2.5:1.
In specific embodiments more of the present invention, in cell/mL, described umbilical cord mesenchymal stem cells density is in the composition 5 × 10
5.
In specific embodiments more of the present invention, described hyaluronate is hyaluronate sodium, and described hyaluronate sodium mass percentage in described compositions is 5% ~ 10%.
In specific embodiments more of the present invention, described hyaluronic acid or described hyaluronic acid mass percentage in described compositions is 10% ~ 20%.
In specific embodiments more of the present invention, the preparation method of described umbilical cord mesenchymal stem cells comprises the steps:
Step 1: after getting umbilical cord cleaning, peel off dynamic and static vascular and adventitia, shred into 1-2mm2 fritter, be inverted and cultivate;
Step 2: add complete medium and cultivate, the later half amount of 2 ~ 3d changes liquid, cultivates 6 ~ 8d, treats that cell climbs out of from piece of tissue, discards piece of tissue and continues to cultivate; Described complete medium comprises the EGF of X-VIVO15 serum-free medium and 10ng/ml;
Step 3: treat that Growth of Cells to 80% merges, discard culture fluid, cleaning, through 0.25% trypsinization 1-2min, stops digestion, the centrifugal 5min of 400g, collecting cell;
Step 4: add described complete medium resuspended in the cell that step 3 obtains, Secondary Culture, collects P2 or P3 for cell.
Concrete, in other embodiments of the present invention, the preparation method of described umbilical cord mesenchymal stem cells comprises the steps:
1. get one, umbilical cord, the dual anti-solution of PBS+200U/m rinses 3 times;
2. umbilical cord is cut into the long segment of 2cm, peels off dynamic and static vascular and adventitia, and middle Fahrenheit glue shreds into 1-2mm
2fritter, is evenly attached at 10cm culture dish, and put upside down in 37 DEG C of 5%CO2 incubators 1-2h;
3. complete medium (EGF of X-VIVO15 serum-free medium+10ng/ml) 5ml, the 2-3 days later half amount that adds changes liquid, cultivates 6-8 days, treats that cell climbs out of from piece of tissue, discards piece of tissue and continues to cultivate;
4. band grows to 80% fusion, and remove culture fluid, PBS cleans one time, adds 0.25% trypsinization 1-2min, adds FBS and stops digestion, the centrifugal 5min of collecting cell 400g;
5. add complete medium resuspended, according to count results inoculation 2-3 ware;
6. treat that Growth of Cells to 80% fusion is follow-up and resume generation.Get P2 and P3 carries out preparation for cell preparation for cell conditioned medium liquid, P2 or P3.
In specific embodiments more of the present invention, the preparation method of described somatomedin comprises:
Step 1: after getting umbilical cord cleaning, peel off dynamic and static vascular and adventitia, shred into 1-2mm2 fritter, be inverted and cultivate;
Step 2: add complete medium and cultivate, the later half amount of 2 ~ 3d changes liquid, cultivates 6 ~ 8d, treats that cell climbs out of from piece of tissue, discards piece of tissue and continues to cultivate; Described complete medium comprises the EGF of X-VIVO15 serum-free medium and 10ng/ml;
Step 3: treat that Growth of Cells to 80% merges, discard culture fluid, cleaning, through 0.25% trypsinization 1-2min, stops digestion, the centrifugal 5min of 400g, collecting cell;
Step 4: add described complete medium resuspended in the cell that step 3 obtains, Secondary Culture, collects P2 or P3 for cell conditioned medium liquid;
Step 5: P2 or P3 getting step 4 acquisition is for cell conditioned medium liquid, and through 0.45 μm of membrane filtration removing large particulate matter, filtrate, again through 0.22 μm of membrane filtration, removes below 3KDa finely ground particle substance.
Concrete, the preparation method of described somatomedin comprises:
1. collect P2 and P3 for supernatant, through 0.45 μm of membrane filtration removing large particulate matter
2. filtrate is again through 0.22 μm of membrane filtration, removing below 3KDa finely ground particle substance
(concentration process adopts Slice200 slipstream device, and the time of circulating filtration is 3h), cocnentration factor: condensate precursor amasss: after concentrated, volume is 3 ~ 4:1.
3. lyophilized powder made by concentrated solution freeze dryer, for subsequent use.
In specific embodiments more of the present invention, the preparation method of described somatomedin also comprises the product getting step 5 acquisition and concentrates the step with lyophilizing.
Present invention also offers a kind of preparation, comprise described compositions and normal saline, the volumn concentration of described normal saline in described preparation is 70% ~ 90%.
Concrete, in whole preparation:
Somatomedin lyophilized powder concentration is: 1.5-2.5mg/ml; (being all take preparation as benchmark)
Umbilical cord mesenchymal stem cells density is: 5 × 10
5cell/ml
Hyaluronate sodium mass ratio is: 5%-10%
Hyaluronic acid (hyaluronic acid) volume ratio is: 10%-20%
Normal saline volume ratio is: 70-90%.See Fig. 1.
Present invention also offers described compositions or the application of described preparation in preparation beauty treatment and/or antidotal medicine, health product and/or cosmetics.
In specific embodiments more of the present invention, described beauty treatment is repair deficiency.Preferred, described beauty treatment is for repairing Facial defects.
The invention provides a kind of compositions, comprise umbilical cord mesenchymal stem cells, somatomedin, hyaluronic acid or its salt.Present invention also offers the stem cell medicine with rapidly and efficiently repair surface hole defect.Make full use of the moisturizing of the proliferation and differentiation ability of stem cell, hyaluronic acid and hyaluronate sodium and the Promote cell's growth effect of EGF, reach the repair surface hole defect problem of stability and high efficiency.
Accompanying drawing explanation
In order to be illustrated more clearly in the embodiment of the present invention or technical scheme of the prior art, be briefly described to the accompanying drawing used required in embodiment or description of the prior art below.
Fig. 1 shows flow chart prepared by preparation;
Fig. 2 shows that preparation prepared by embodiment 4 uses front and back design sketch; Wherein Fig. 2 (A) shows that preparation prepared by embodiment 4 uses front design sketch; Fig. 2 (B) shows that preparation prepared by embodiment 4 uses front design sketch;
Fig. 3 shows cell proliferation curve in embodiment 5.
Detailed description of the invention
The invention discloses a kind of compositions, preparation and uses thereof, those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Method of the present invention and application are described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope methods and applications as herein described are changed or suitably change with combination, realize and apply the technology of the present invention.
Terminological interpretation:
Hyaluronate sodium: hyaluronate sodium (SODIUMHYALURONATE) is the material extracted from cockscomb; also obtain by Lactococcus fermentation; for white or off-white color granule or powder; odorless; time dry; nitrogen content is 2.8%-4.0%, and glucuronic acid content is 37.0%-51.0%.Use more in cosmetic field, have moisture-keeping function.
Hyaluronic acid: the straight chain polymer polysaccharide that hyaluronic acid is made up of dissacharide units (glucuronic acid-N-second sulfur aminoglucose) is the material naturally existed in a kind of tissue.In the connective tissue that hyaluronic acid is present in human body in a large number and skin corium, be a kind of transparent colloid substance, have powerful moisture-keeping functions, the hyaluronic acid of 1 g can absorb the moisture of 500C.C, is equivalent to glycerol 500 times of water absorbing capacities.In the skin corium of human skin, play the key player of substrate, be that the transport between maintenance overall in organizational structure or cell all has very important function.Hyaluronic acid not only has the function keeping skin elasticity, large quantity of moisture can also be pinned, to tissue, there is moisturizing lubrication, make the full youth of skin flexible, but with advancing age, when the speed that hyaluronic acid runs off is than fast growth, skin also will become shortage moisture gradually, tarnish elasticity, gets off for a long time and just occur the catabiosis such as wrinkle.
Epithelical cell growth factor: EGF is a kind of somatomedin existed in human body, and its major function is the division promoting Skin Cell.Research shows: the EGF of denier can intense stimulus Growth of Cells, suppresses aging gene to be expressed, stops skin aging, make skin respectively form part and keep best physiological status.In addition, it can also the synthesis of the outer macromole (as hyaluronic acid and collagen protein etc.) of irritation cell and secretion, and skin care determines skin vitality and healthy key factor.Research finds, EGF can promote epithelial cell, fibroblastic increment; Strengthen the vigor of epidermis cell; Delay the aging of epidermis cell, make each composition of skin keep best physiological status.
Umbilical cord mesenchymal stem cells: umbilical cord mesenchymal stem cells (MesnchymalStemCells, MSCs) a kind of versatile stem cell be present in neonatal umbilical cord tissue is referred to, it can be divided into many kinds of histiocytes, has wide potential applicability in clinical practice.Umbilical cord mesenchymal stem cells (MSCs) has higher differentiation potential, can break up to multiple directions.It has wide potential applicability in clinical practice in the organizational projects such as bone, cartilage, muscle, tendon, ligament, nerve, liver, endothelium and cardiac muscle.
In compositions provided by the invention, preparation and uses thereof, raw materials used and reagent all can be buied by market.
Below in conjunction with embodiment, set forth the present invention further:
The cultivation of embodiment 1 umbilical cord mesenchymal stem cells:
1. get one, healthy pregnant women umbilical cord, the dual anti-solution of PBS+200U/m rinses 3 times
2. umbilical cord is cut into the long segment of 2cm, peels off dynamic and static vascular and adventitia, and middle Fahrenheit glue shreds into 1-2mm
2fritter, is evenly attached at 10cm culture dish, and put upside down in 37 DEG C of 5%CO2 incubators 1-2h
3. complete medium (EGF of X-VIVO15 serum-free medium+10ng/ml) 5ml, the 2-3 days later half amount that adds changes liquid, cultivates 6-8 days, treats that cell climbs out of from piece of tissue, discards piece of tissue and continues to cultivate.
4. band grows to 80% fusion, and remove culture fluid, PBS cleans one time, adds 0.25% trypsinization 1-2min, adds FBS and stops digestion, the centrifugal 5min of collecting cell 400g
5. add complete medium resuspended, according to count results inoculation 2-3 ware.
6. treat that Growth of Cells to 80% fusion is follow-up and resume generation.Get P2 and P3 carries out preparation for cell preparation for cell conditioned medium liquid, P2 or P3.
The preparation of embodiment 2 somatomedin lyophilized powder
1. collect P2 and P3 for supernatant, through 0.45 μm of membrane filtration removing large particulate matter
2. filtrate is again through 0.22 μm of membrane filtration, removing below 3KDa finely ground particle substance
(concentration process adopts Slice200 slipstream device, and the time of circulating filtration is 3h)
3. lyophilized powder made by concentrated solution freeze dryer, for subsequent use.
The preparation of embodiment 3 compositions
1. get P2 or P3 for umbilical cord mesenchymal stem cells one ware, remove supernatant, add PBS and clean 1-2 time, add 0.25% trypsinization 1-2min, digest complete, add FBS and stop digestion, collecting cell, the centrifugal 5min of 400g, obtains lower confluent monolayer cells;
2. get 4-5ml sodium hyaluronate solution: 1-2ml re-suspended cell, 3-4ml dissolves stem cell factor lyophilized powder
3. mix cell suspension, stem cell lyophilized powder solution and 0.5-1ml hyaluronic acid with tee T, make the compositions of 4.5-6ml.
The preparation (being all take preparation as benchmark) of embodiment 4 preparation
Somatomedin lyophilized powder concentration prepared by embodiment 2 is: 1.5-2.5mg/ml;
Umbilical cord mesenchymal stem cells density prepared by embodiment 1 is: 5 × 10
5cell/ml;
Hyaluronate sodium mass ratio is: 5%-10%;
Hyaluronic acid (hyaluronic acid) volume ratio is: 10%-20%;
Normal saline volume ratio is: 70-90%.
The contrast experiment of embodiment 5 and PRP
Face reparation is the process filling up defect after an epidermin blast cell cell proliferation, needs the stimulation of somatomedin; This simulation experiment is by adding PRP and this patent composition cultivation epidermin blast cell respectively, and observation of cell proliferative conditions contrasts face repair ability and effect.
Carry out 5 groups of experiments, the mice P1 getting 5 groups of equivalent respectively represents hide fiber blast cell 1 × 10
5, be inoculated in 10cm ware,
Group A adds culture medium (DMEM high glucose medium+10%FBS) 5ml,
Group B1 adds culture medium (DMEM high glucose medium+10%FBS) 5ml, and adds 10%PRP composition;
Group C1 adds culture medium (DMEM high glucose medium+10%FBS) 5ml, the preparation in 10% embodiment 4, and formula is:
Somatomedin lyophilized powder concentration is: 1.5mg/ml;
Umbilical cord mesenchymal stem cells density is: 5 × 10
5cell/ml;
Hyaluronate sodium mass ratio is: 5%;
Hyaluronic acid (hyaluronic acid) volume ratio is: 10%;
Remaining as normal saline;
Group C2 adds culture medium (DMEM high glucose medium+10%FBS) 5ml, the preparation in 20% embodiment 4, and formula is:
Somatomedin lyophilized powder concentration is: 2.5mg/ml;
Umbilical cord mesenchymal stem cells density is: 5 × 10
5cell/ml;
Hyaluronate sodium mass ratio is: 10%;
Hyaluronic acid (hyaluronic acid) volume ratio is: 20%;
Remaining as normal saline;
Group C3 adds culture medium (DMEM high glucose medium+10%FBS) 5ml, the preparation in 15% embodiment 4, and formula is:
Somatomedin lyophilized powder concentration is: 2.0mg/ml;
Umbilical cord mesenchymal stem cells density is: 5 × 10
5cell/ml;
Hyaluronate sodium mass ratio is: 8%;
Hyaluronic acid (hyaluronic acid) volume ratio is: 15%;
Remaining as normal saline;
In incubation, cell reaches 80% fusion and goes down to posterity.
The results are shown in Table 1:
Table 1 result of the test
Shown by table 1, Fig. 3 result:
B and C1, C2, C3 growth rate are obviously better than A (P < 0.05); C1 growth rate is also obviously better than B (P < 0.05), has statistical significance.So the cultivation effect of the promotion epidermin blast cell of the preparation of the embodiment of the present invention 4 preparation is obviously better than PRP (P < 0.05).
Group C2 and C3 growth rate close, no difference of science of statistics (P > 0.05), and C2, C3 growth rate is obviously better than C1 (P < 0.05), so cell proliferation rate is the fastest when patent composition reaches 15%, concentration increases again, does not have promotion cultivation effect.
Compliance test result is tested:
Random collection 20 has the volunteer of facial SOL in various degree and hollow, respectively at the same day, carries out a defect proprietary preparation injection, and carry out Contrast on effect after 1 one months after 1 month, 80% and above repair and improve have 18 examples.Wherein the improvement effect of a patient is shown in Fig. 2.Wherein Fig. 2 (A) is before the preparation using the embodiment of the present invention 4 to prepare, and Fig. 2 (B) is after the preparation using the embodiment of the present invention 4 to prepare.As apparent from Fig. 2 can, preparation provided by the invention has the effect of significantly treatment Facial defects.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a compositions, is characterized in that, comprises umbilical cord mesenchymal stem cells, somatomedin, hyaluronic acid or its salt.
2. compositions according to claim 1, is characterized in that, also comprises hyaluronic acid.
3. compositions according to claim 1 and 2, is characterized in that, in mg/mL, described somatomedin mass volume ratio is in the composition 1.5 ~ 2.5:1.
4. the compositions according to any one of claims 1 to 3, is characterized in that, in cell/mL, described umbilical cord mesenchymal stem cells density is in the composition 5 × 10
5.
5. the compositions according to any one of Claims 1-4, is characterized in that, described hyaluronate is hyaluronate sodium, and described hyaluronate sodium mass percentage in described compositions is 5% ~ 10%.
6. the compositions according to any one of claim 1 to 5, is characterized in that, described hyaluronic acid or described hyaluronic acid mass percentage in described compositions is 10% ~ 20%.
7. the compositions according to any one of claim 1 to 6, is characterized in that, the preparation method of described umbilical cord mesenchymal stem cells comprises the steps:
Step 1: after getting umbilical cord cleaning, peel off dynamic and static vascular and adventitia, shred into 1-2mm
3fritter, is inverted and cultivates;
Step 2: add complete medium and cultivate, the later half amount of 2 ~ 3d changes liquid, cultivates 6 ~ 8d, treats that cell climbs out of from piece of tissue, discards piece of tissue and continues to cultivate; Described complete medium comprises the EGF of X-VIVO15 serum-free medium and 10ng/ml;
Step 3: treat that Growth of Cells to 80% merges, discard culture fluid, cleaning, through 0.25% trypsinization 1-2min, stops digestion, the centrifugal 5min of 400g, collecting cell;
Step 4: add described complete medium resuspended in the cell that step 3 obtains, Secondary Culture, collects P2 or P3 for cell.
8. the compositions according to any one of claim 1 to 7, is characterized in that, the preparation method of described somatomedin comprises:
Step 1: after getting umbilical cord cleaning, peel off dynamic and static vascular and adventitia, shred into 1-2mm
3fritter, is inverted and cultivates;
Step 2: add complete medium and cultivate, the later half amount of 2 ~ 3d changes liquid, cultivates 6 ~ 8d, treats that cell climbs out of from piece of tissue, discards piece of tissue and continues to cultivate; Described complete medium comprises the EGF of X-VIVO15 serum-free medium and 10ng/ml;
Step 3: treat that Growth of Cells to 80% merges, discard culture fluid, cleaning, through 0.25% trypsinization 1-2min, stops digestion, the centrifugal 5min of 400g, collecting cell;
Step 4: add described complete medium resuspended in the cell that step 3 obtains, Secondary Culture, collects P2 or P3 for cell conditioned medium liquid;
Step 5: P2 or P3 getting step 4 acquisition is for cell conditioned medium liquid, and through 0.45 μm of membrane filtration removing large particulate matter, filtrate, again through 0.22 μm of membrane filtration, removes below 3KDa finely ground particle substance.
9. a preparation, is characterized in that, comprises the compositions as described in any one of claim 1 to 6 and normal saline, and the volumn concentration of described normal saline in described preparation is 70% ~ 90%.
10. the compositions according to any one of claim 1 to 8 or the application of preparation as claimed in claim 9 in preparation beauty treatment and/or antidotal medicine, health product and/or cosmetics.
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