CN110447637A - Melanocyte frozen stock solution - Google Patents
Melanocyte frozen stock solution Download PDFInfo
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- CN110447637A CN110447637A CN201910788416.7A CN201910788416A CN110447637A CN 110447637 A CN110447637 A CN 110447637A CN 201910788416 A CN201910788416 A CN 201910788416A CN 110447637 A CN110447637 A CN 110447637A
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- melanocyte
- stock solution
- frozen stock
- cell
- phenylthiourea
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- 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
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N1/00—Preservation of bodies of humans or animals, or parts thereof
- A01N1/02—Preservation of living parts
- A01N1/0205—Chemical aspects
- A01N1/021—Preservation or perfusion media, liquids, solids or gases used in the preservation of cells, tissue, organs or bodily fluids
- A01N1/0226—Physiologically active agents, i.e. substances affecting physiological processes of cells and tissue to be preserved, e.g. anti-oxidants or nutrients
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- General Health & Medical Sciences (AREA)
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Abstract
The present invention provides a kind of melanocyte frozen stock solution, and the frozen stock solution includes cell culture fluid, fetal calf serum, dimethyl sulfoxide and N- phenylthiourea.After freezing and recovering, form is preferable for melanocyte frozen stock solution provided by the invention, safety and stability, favorable repeatability, and melanocyte, and survival rate is high, and proliferative capacity is good, and the ability that melanocyte synthesizes melanin granule is unaffected.
Description
Technical field
The invention belongs to cell and technical field of bioengineering, and in particular to a kind of melanocyte frozen stock solution.
Background technique
Melanocyte is located at the basal layer of epidermis, is the dentritic cell of synthesis and secretion melanocyte, in adult epidermal's substrate
Melanocyte sum in layer is about hundred million, and average out to accounts for about epidermal cell sum 3% ~ 5%.Under normal circumstances, melanocyte proliferation
Ability is very weak, and the melanocyte growth of in vitro culture is slow, is easy by fibroblast or the dominant growth of keratinocyte
It is covered.And primary melanocyte aging is very fast, therefore melanocyte originally culture freezes and the quality of melanocyte is risen
Very important effect.
Necessary a kind of solution when cells frozen storing liquid is cell cryopreservation.Mesh is precellular to be stored at room temperature technology and immature,
Cell saves the preserving type that cryogenic freezing is widely used.The cryo-conservation of cell needs to add cryoprotector, thin to reduce
Ice crystal physical injury caused by cell in born of the same parents' frozen storage process.Cryoprotector has two classes: osmosis type cryoprotector and non-
Osmosis type cryoprotector.Wherein osmosis type cryoprotector can pass through membrane permeability into the cell, be cell cryopreservation institute
Necessary, most common of them is exactly dimethyl sulfoxide (DMSO).
Since the difficult acquisition of melanocyte and the unit of domestic culture melanocyte are less, freezing for melanocyte does not have also
Have to form the system that freezes of standard, at present for the cell freezing method of melanocyte frozen or use wide spectrum: small ox blood
Clearly/90%+dimethyl sulfoxide of fetal calf serum 10%.
A large amount of practical biocytology experiment and a large amount of statistical data show the melanocyte frozen using this frozen stock solution
For survival rate generally in 30-50%, survival rate is relatively low after cell recovery.Freeze-stored cell is by recovery, general after culture 12 hours
Light passing microscopically observation, survival rate is generally 50% or so；When survival rate is less than 30%, cell growth and breeding is slow,
Cells Dendritic is formed slowly, and form is irregular, and cell is relatively fine, and growing way is poor, and recovery time is longer；Work as cell survival rate
At 50% or so, cell distribution is relatively uniform, and living cells is relatively more, and form is more normal, covers with single layer and needs 4-7 days.
Therefore, in cell and technical field of bioengineering, existing can overcome existing melanocyte frozen stock solution to make melanocyte
Cell survival rate declines after cultivating after cell recovery, influences the tight demand of the melanocyte frozen stock solution of melanocyte vigor.
Summary of the invention
In view of this, the purpose of the present invention is to provide a kind of cells that can be improved after cultivating after melanocyte is recovered to deposit
Motility rate improves the melanocyte frozen stock solution of melanocyte vigor.
For this purpose, the present invention provides following technical schemes.
In a first aspect, the present invention provides a kind of melanocyte frozen stock solutions, wherein the frozen stock solution includes cell culture
Liquid, fetal calf serum, dimethyl sulfoxide and N- phenylthiourea.
In a preferred embodiment, wherein include the cell culture fluid of 500-800mL, example in frozen stock solution described in every 1L
Such as, may include 500 in frozen stock solution described in every 1L, 510,520,530,540,550,560,570,580,590,600,610,
620,630,640,650,660,670,680,690,700,710,720,730,740,750,760,770,780,790 or
The cell culture fluid of 800mL.
In preferred embodiment, comprising the cell culture fluid of 600-750mL in frozen stock solution described in every 1L, for example,
May include 600 in frozen stock solution described in every 1L, 610,620,630,640,650,660,670,680,690,700,710,720,
730, the cell culture fluid of 740 or 750mL.
In a preferred embodiment, the cell culture fluid uses the culture solution of melanocyte.
In preferred embodiment, the culture solution of the melanocyte can be Medium 254 or MelM-2.
It in a preferred embodiment, include the fetal calf serum of 100-200mL in frozen stock solution described in every 1L, for example, every 1L
It may include the fetal calf serum of 100,110,120,130,140,150,160,170,180,190 or 200mL in the frozen stock solution.
In preferred embodiment, comprising the fetal calf serum of 120-160mL in frozen stock solution described in every 1L, for example, often
May include 120 in frozen stock solution described in 1L, 122,125,127,130,132,135,137,140,141,143,146,150,
152, the fetal calf serum of 155,158 or 160mL.
It in a preferred embodiment, include the dimethyl sulfoxide of 30-150mL in frozen stock solution described in every 1L, for example, every 1L
The dimethyl that may include 30,40,50,60,70,80,90,100,110,120,130,140 or 150mL in the frozen stock solution is sub-
Sulfone.
In preferred embodiment, comprising the dimethyl sulfoxide of 50-100mL in frozen stock solution described in every 1L, for example, often
It may include the dimethyl sulfoxide of 50,55,60,65,70,75,80,85,90,95 or 100mL in frozen stock solution described in 1L.
It in a preferred embodiment, include the N- phenylthiourea of 500-800 μm of ol/L, example in frozen stock solution described in every 1L
Such as, may include 500 in frozen stock solution described in every 1L, 520,550,580,600,650,660,680,690,700,710,720,
730, the N- phenylthiourea of 750,760,770,780,790 or 800 μm of ol/L.
In preferred embodiment, the N- phenylthiourea of 500-600 μm of ol/L, example are included in frozen stock solution described in every 1L
Such as, 500,510,520,530,540,550,560,570,580,590 or 600 μm of ol/L be may include in frozen stock solution described in every 1L
N- phenylthiourea.
Second aspect provides a kind of melanocyte and freezes reagent, and the reagent includes frozen stock solution of the present invention.
Further it is provided that purposes of the melanocyte frozen stock solution of the present invention in melanocyte freezes.
Each component in melanocyte frozen stock solution of the present invention, can buy from market, wherein
Medium 254 is purchased from Gibco company, article No. M-254-500；It can be supplied for the cells with nutrient of dormant period
It gives and maintains.
MelM-2 is purchased from Promocell company, article No. C-39420；It can be the cells with nutrient of dormant period
Supply and maintenance.
Fetal calf serum is purchased from Hangzhou Sijiqing Biological Engineering Material Co., Ltd., article No. 1101-8611；It can be mentioned
For there is no or measuring seldom nutrients and main low molecule nutrients in cell culture fluid；It can also identify vitamin, rouge
Class, metal and other hormones etc. can combine or change the substance vitality that they are combined.
Dimethyl sulfoxide is purchased from Sigma-Aldrich company, article No. D4540-1L；It is a kind of permeability protection
Agent, can reduce cell freezing point, reduce the formation of ice crystal during cell cryopreservation, mitigate free radical to cell damage,
Change biomembrane to the permeability of electrolyte, drug, poisonous substance and metabolite.Prevent during cryopreservation ice crystal shape in cell
At and influence of the products of cellular metabolism to cell.
N- phenylthiourea is purchased from Sigma-Aldrich company, article No. P7629；It can reduce melanocyte and freeze
The secretion of melanin granule during depositing guarantees the vigor of melanocyte synthesis melanocyte.
Compared with the existing technology, the beneficial effects of the present invention are:
Melanocyte frozen stock solution provided by the invention, safety and stability, favorable repeatability, and melanocyte are freezing and are recovering
Afterwards, survival rate is high, and proliferative capacity is good, and the ability that melanocyte synthesizes melanin granule is unaffected.
Using melanocyte frozen stock solution provided by the invention, cell survival rate can reach after freezing melanocyte recovery culture
80% or more, it is significantly improved than the survival rate after the recovery culture of regular growth frozen stock solution institute's freeze-stored cell.
The cell culture fluid for the certain content that the present invention uses can supply for the cells with nutrient of dormant period.This
The fetal calf serum of the certain content used is invented, being capable of providing in cell culture fluid does not have or measures seldom nutrients, and
Main low molecule nutrients can also identify vitamin, lipid, metal and other hormones, can combine or change their institutes
In conjunction with substance vitality；Fetal calf serum too high levels cause wastage of material, increase cost；Fetal calf serum content is too low, cannot mention
For enough nutrients.The dimethyl sulfoxide for the certain content that the present invention uses can prevent cell during cryopreservation
The influence of middle ice crystal formation and products of cellular metabolism to cell；Dimethyl sulfoxide too high levels are increased to black in melanocyte
The destruction of crude granule secretion；Dimethyl sulfoxide content is too low, cannot reduce ice crystal during cell cryopreservation well
Formation, not can be well protected cell from the damage of free radical.The N- phenylthiourea for the certain content that the present invention uses, energy
The secretion of melanin granule of the melanocyte in frozen storage process is reduced, guarantees the vigor of melanocyte synthesis melanocyte；N- phenyl sulphur
Urea too high levels will cause certain cytotoxicity；N- phenylthiourea content is too low to spit outside melanin granule in improving cell
Effect is not significant.
Detailed description of the invention
It is thin after the recovery for 24 hours of trimestral melanocyte that Fig. 1 is that the melanocyte frozen stock solution prepared using embodiment 1 is frozen
Born of the same parents' form schematic diagram；
Fig. 2 is that the melanocyte frozen stock solution prepared using embodiment 2 freezes the cell shape after trimestral melanocyte recovery for 24 hours
State schematic diagram；
Fig. 3 is the cell shape for freezing melanocyte frozen stock solution prepared with embodiment 3 after trimestral melanocyte recovery for 24 hours
State schematic diagram；
Fig. 4 is that the melanocyte frozen stock solution prepared using comparative example 1 freezes the cell shape after trimestral melanocyte recovery for 24 hours
State schematic diagram；
Fig. 5 is that the melanocyte frozen stock solution prepared using comparative example 2 freezes the cell shape after trimestral melanocyte recovery for 24 hours
State schematic diagram；
Fig. 6 is that the melanocyte frozen stock solution prepared using comparative example 3 freezes the cell shape after trimestral melanocyte recovery for 24 hours
State schematic diagram；
Fig. 7 is that the melanocyte frozen stock solution prepared using comparative example 4 freezes the cell shape after trimestral melanocyte recovery for 24 hours
State schematic diagram；
Fig. 8 is that the melanocyte frozen stock solution prepared using comparative example 5 freezes the cell shape after trimestral melanocyte recovery for 24 hours
State schematic diagram；
Fig. 9 is that the melanocyte frozen stock solution prepared using comparative example 6 freezes the cell shape after trimestral melanocyte recovery for 24 hours
State schematic diagram；
Figure 10 is after freezing trimestral melanocyte recovery for 24 hours using melanocyte frozen stock solution prepared by 7 embodiment 3 of comparative example
Cellular morphology schematic diagram；
Figure 11 is that the melanocyte frozen stock solution prepared using comparative example 8 freezes the cell after trimestral melanocyte recovery for 24 hours
Form schematic diagram；
Figure 12 is thin to freeze trimestral melanocyte using melanocyte frozen stock solution prepared by embodiment 3, comparative example 1 and comparative example 8
Cell survival rate comparison diagram after born of the same parents' recovery.
Specific embodiment
Below in conjunction with specific embodiment and embodiment, it is specifically described the present invention, advantages of the present invention and various effects
It thus more will clearly present.It will be understood by those skilled in the art that these specific embodiments and embodiment are for illustrating
The present invention is not intended to limit the present invention.
Embodiment 1
The present invention provides a kind of melanocyte frozen stock solution, wherein includes in frozen stock solution described in every 1L: 650mL Medium 254 is black
Plain cell culture fluid, 200mL fetal calf serum, 50mL dimethyl sulfoxide and 500 μm of ol/L N- phenylthioureas.
The present embodiment melanocyte frozen stock solution the preparation method is as follows:
It weighs N- phenylthiourea to be dissolved in 254 melanocyte culture solution of Medium, constant volume is configured to 500 μm of ol/L at 100mL
N- phenylthiourea solution.
The dimethyl of the 254 melanocyte culture solution of Medium of measurement 650mL, the fetal calf serum of 200mL, 50mL respectively
The N- phenylthiourea solution mixing system of 500 μm of ol/L of sulfoxide and 100mL obtain.
Embodiment 2
The present invention provides a kind of melanocyte frozen stock solution, wherein includes in frozen stock solution described in every 1L: 700mL Medium 254 is black
Plain cell culture fluid, 100mL fetal calf serum, 100mL dimethyl sulfoxide and 500 μm of ol/L N- phenylthioureas.
The present embodiment melanocyte frozen stock solution the preparation method is as follows:
It weighs N- phenylthiourea to be dissolved in 254 melanocyte culture solution of Medium, constant volume is configured to 500 μm of ol/L at 100mL
N- phenylthiourea solution.
The dimethyl of the 254 melanocyte culture solution of Medium of measurement 700mL, the fetal calf serum of 100mL, 100mL respectively
The N- phenylthiourea solution mixing system of 500 μm of ol/L of sulfoxide and 100mL obtain.
Embodiment 3
The present invention provides a kind of melanocyte frozen stock solution, wherein includes in frozen stock solution described in every 1L: 690mL Medium 254 is black
Plain cell culture fluid, 160mL fetal calf serum, 150mL dimethyl sulfoxide and 120 μm of ol/L N- phenylthioureas.
The present embodiment melanocyte frozen stock solution the preparation method is as follows:
It weighs N- phenylthiourea to be dissolved in 254 melanocyte culture solution of Medium, constant volume is configured to 500 μm of ol/L at 100mL
N- phenylthiourea solution.
The dimethyl of the 254 melanocyte culture solution of Medium of measurement 590mL, the fetal calf serum of 160mL, 150mL respectively
The N- phenylthiourea solution mixing system of 500 μm of ol/L of sulfoxide and 100mL obtain.
Comparative example 1
Beneficial effect in order to further illustrate the present invention provides comparative example 1 and compares with embodiment 3, the difference of the comparative example 1
It is: does not add N- phenylthiourea.
The comparative example melanocyte frozen stock solution the preparation method is as follows:
The dimethyl sulfoxide of the 254 melanocyte culture solution of Medium of measurement 690mL, the fetal calf serum of 160mL, 150mL respectively
It is mixed to prepare.
Comparative example 2
Beneficial effect in order to further illustrate the present invention provides comparative example 2 and compares with embodiment 3, the difference of the comparative example 2
Be: the content of N- phenylthiourea is adjusted to 460 μm of ol/L.
The comparative example melanocyte frozen stock solution the preparation method is as follows:
It weighs N- phenylthiourea to be dissolved in 254 melanocyte culture solution of Medium, constant volume is configured to 460 μm of ol/L at 100mL
N- phenylthiourea solution.
The dimethyl of the 254 melanocyte culture solution of Medium of measurement 590mL, the fetal calf serum of 160mL, 150mL respectively
The N- phenylthiourea solution mixing system of 460 μm of ol/L of sulfoxide and 100mL obtain.
Comparative example 3
Beneficial effect in order to further illustrate the present invention provides comparative example 3 and compares with embodiment 3, the difference of the comparative example 3
Be: the content of N- phenylthiourea is adjusted to 810 μm of ol/L.
The comparative example melanocyte frozen stock solution the preparation method is as follows:
It weighs N- phenylthiourea to be dissolved in 254 melanocyte culture solution of Medium, constant volume is configured to 500 μm of ol/L at 100mL
N- phenylthiourea solution.
The dimethyl of the 254 melanocyte culture solution of Medium of measurement 590mL, the fetal calf serum of 160mL, 150mL respectively
The N- phenylthiourea solution mixing system of 810 μm of ol/L of sulfoxide and 100mL obtain.
Comparative example 4
Beneficial effect in order to further illustrate the present invention provides comparative example 4 and compares with embodiment 3, the difference of the comparative example 4
Be: the content of fetal calf serum is adjusted to 95mL.
The comparative example melanocyte frozen stock solution the preparation method is as follows:
It weighs N- phenylthiourea to be dissolved in 254 melanocyte culture solution of Medium, constant volume is configured to 500 μm of ol/L at 100mL
N- phenylthiourea solution.
The dimethyl of the 254 melanocyte culture solution of Medium of measurement 655mL, the fetal calf serum of 95mL, 150mL respectively
The N- phenylthiourea solution mixing system of 500 μm of ol/L of sulfoxide and 100mL obtain.
Comparative example 5
Beneficial effect in order to further illustrate the present invention provides comparative example 5 and compares with embodiment 3, the difference of the comparative example 5
Be: the content of fetal calf serum is adjusted to 202mL.
The comparative example melanocyte frozen stock solution the preparation method is as follows:
It weighs N- phenylthiourea to be dissolved in 254 melanocyte culture solution of Medium, constant volume is configured to 500 μm of ol/L at 100mL
N- phenylthiourea solution.
The dimethyl of the 254 melanocyte culture solution of Medium of measurement 548mL, the fetal calf serum of 202mL, 150mL respectively
The N- phenylthiourea solution mixing system of 500 μm of ol/L of sulfoxide and 100mL obtain.
Comparative example 6
Beneficial effect in order to further illustrate the present invention provides comparative example 5 and compares with embodiment 3, the difference of the comparative example 5
Be: the content of dimethyl sulfoxide is adjusted to 28mL.
The comparative example melanocyte frozen stock solution the preparation method is as follows:
It weighs N- phenylthiourea to be dissolved in 254 melanocyte culture solution of Medium, constant volume is configured to 500 μm of ol/L at 100mL
N- phenylthiourea solution.
The dimethyl of the 254 melanocyte culture solution of Medium of measurement 712mL, the fetal calf serum of 160mL, 28mL respectively
The N- phenylthiourea solution mixing system of 500 μm of ol/L of sulfoxide and 100mL obtain.
Comparative example 7
Beneficial effect in order to further illustrate the present invention provides comparative example 5 and compares with embodiment 3, the difference of the comparative example 5
Be: the content of dimethyl sulfoxide is adjusted to 155mL.
The comparative example melanocyte frozen stock solution the preparation method is as follows:
It weighs N- phenylthiourea to be dissolved in 254 melanocyte culture solution of Medium, constant volume is configured to 500 μm of ol/L at 100mL
N- phenylthiourea solution.
The dimethyl of the 254 melanocyte culture solution of Medium of measurement 585mL, the fetal calf serum of 160mL, 155mL respectively
The N- phenylthiourea solution mixing system of 500 μm of ol/L of sulfoxide and 100mL obtain.
Comparative example 8
Beneficial effect in order to further illustrate the present invention, provides a kind of common melanocyte frozen stock solution, which freezes
Liquid storage includes 90%+DMSO 10% of fetal calf serum.
The comparative example melanocyte frozen stock solution the preparation method is as follows:
The dimethyl sulfoxide of the fetal calf serum and 100mL that measure 900mL respectively is mixed to prepare.
Experiment 1: compliance test result
Given the test agent: the sample that 1-3 of the embodiment of the present invention, comparative example 1-8 are prepared.
Experimental method:
When the melanocyte of culture rate to be fused reaches 80%, the culture solution of melanocyte is discarded, using 0.25% pancreatin, 37 DEG C
Melanocyte 1-3min is digested, digestion is terminated with the culture solution of 10% fetal calf serum, the digestion suspension of cell is moved into centrifuge tube
In, 1000g is centrifuged 5-10min, and density is made in melanocyte using cells frozen storing liquid prepared by embodiment 1-3, comparative example 1-8
For 1-5 × 106 The cell suspension of cells/mL moves to cell suspension in cryopreservation tube according to each cryopreservation tube 1mL.Cryopreservation tube is close
It is honored as a queen and places in cell cryopreservation box, after -80 DEG C are placed for 24 hours, go in liquid nitrogen pipe and freeze for a long time.
After freezing three months from liquid nitrogen, 37 DEG C of water-bath dissolutions are placed in, 1000r/min is centrifuged 10min, discards supernatant, press
Bed board is carried out to cell according to cell concentration when freezing.Plating cells density is 1 ~ 3 × 105 cells/mL.37 DEG C of cultures are set, are taken pictures
Observe cell recovery situation.Cell recovery the result is shown in Figure 1-11.
Survivaling cell is counted after freezing recovery in three months.Melanocyte survival results are shown in Figure 12.
It is by Fig. 1-3 it is found that thin after freezing trimestral melanocyte recovery for 24 hours using melanocyte frozen stock solution of the present invention
Born of the same parents' form is good, and there are many living cells, and the dendron of melanocyte is average at 2-3, and karyon is larger.Illustrate that melanocyte of the present invention is thin
Born of the same parents' frozen stock solution freeze that effect is apparently higher than traditional melanocyte frozen stock solution freeze effect.
By Fig. 4-11 it is found that after formula system by adjusting melanocyte frozen stock solution of the present invention, melanocyte is recovered for 24 hours
Cellular morphology afterwards is most preferably, and living cells significantly reduces.
As shown in Figure 12, it is deposited using the cell that melanocyte frozen stock solution of the present invention freezes after trimestral melanocyte recovery
Motility rate is higher, and is significantly higher than the cell using the melanocyte frozen stock solution commonly comprising 90%+DMSO 10% of fetal calf serum
Survival rate.
It should be understood that the present invention disclosed is not limited only to specific method, scheme and the substance of description, because these
It is alterable.It will also be understood that purpose of the terminology used here just for the sake of the specific embodiment scheme of description, rather than
It is intended to limit the scope of the invention, the scope of the present invention is limited solely by the attached claims.
Those skilled in the art, which will also be appreciated that or be able to confirm that, uses no more than routine experiment, institute herein
The many equivalents for the specific embodiment of the invention stated.These equivalents are also contained in the attached claims.
Claims (10)
1. a kind of melanocyte frozen stock solution, wherein the frozen stock solution include cell culture fluid, fetal calf serum, dimethyl sulfoxide and
N- phenylthiourea.
2. frozen stock solution according to claim 1, wherein include the cell culture of 500-800mL in frozen stock solution described in every 1L
Liquid.
3. frozen stock solution according to claim 2, wherein include the cell culture of 600-750mL in frozen stock solution described in every 1L
Liquid.
4. frozen stock solution according to claim 1, wherein include the fetal calf serum of 100-200mL in frozen stock solution described in every 1L.
5. frozen stock solution according to claim 4, wherein include the fetal calf serum of 120-160mL in frozen stock solution described in every 1L.
6. frozen stock solution according to claim 1, wherein the dimethyl in frozen stock solution described in every 1L comprising 30-150mL is sub-
Sulfone.
7. frozen stock solution according to claim 6, wherein the dimethyl in frozen stock solution described in every 1L comprising 50-100mL is sub-
Sulfone.
8. frozen stock solution according to claim 1, wherein include the N- benzene of 500-800 μm of ol/L in frozen stock solution described in every 1L
Base thiocarbamide.
9. frozen stock solution according to claim 8, wherein include the N- benzene of 500-600 μm of ol/L in frozen stock solution described in every 1L
Base thiocarbamide.
10. a kind of melanocyte freezes reagent, wherein the reagent includes the described in any item frozen stock solutions of claim 1-8.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111139217A (en) * | 2020-01-20 | 2020-05-12 | 杭州协合医疗用品有限公司 | Epidermal melanocyte separation culture and cryopreservation method based on clinical application and cryopreservation liquid of melanocytes |
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| US20060110373A1 (en) * | 2004-11-10 | 2006-05-25 | L'oreal | PTU compounds for promoting the in vitro culture of (highly pigmented) melanocytes |
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2019
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20060110373A1 (en) * | 2004-11-10 | 2006-05-25 | L'oreal | PTU compounds for promoting the in vitro culture of (highly pigmented) melanocytes |
| TW201129394A (en) * | 2010-02-26 | 2011-09-01 | Univ Kaohsiung Medical | Composition for inhibiting melanogenesis and use thereof |
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| LAUREN S.GODWIN ET AL.: ""Isolation,culture, and transfection of Melanocytes"", 《PROTOCOLS IN CELL BIOLOGY》 * |
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| CN111139217A (en) * | 2020-01-20 | 2020-05-12 | 杭州协合医疗用品有限公司 | Epidermal melanocyte separation culture and cryopreservation method based on clinical application and cryopreservation liquid of melanocytes |
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Application publication date: 20191115 |