CN107125240A - A kind of skin spermatogonium protection liquid and preparation method thereof - Google Patents
A kind of skin spermatogonium protection liquid and preparation method thereof Download PDFInfo
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- CN107125240A CN107125240A CN201610622369.5A CN201610622369A CN107125240A CN 107125240 A CN107125240 A CN 107125240A CN 201610622369 A CN201610622369 A CN 201610622369A CN 107125240 A CN107125240 A CN 107125240A
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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
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Abstract
The present invention provides a kind of skin stem spermatogonium protection liquid and preparation method thereof, the protection liquid mainly forms aminoglycoside antibiotics with DMEM culture medium aqueous dissolutions, the 120U of amino sugar glycoside antibiotic 80 described in DMEM culture medium aqueous dissolutions described in per mL, the concentration of the DMEM culture mediums aqueous solution is 13 25mg/mL, the skin stem spermatogonium protection liquid that the present invention is provided fully simulates human body vivo environment, it is more suitable for the existence of skin progenitor cell and stem spermatogonium, and the protection fluidity matter is stable, the survival rate of skin progenitor cell and stem spermatogonium can be significantly improved；Simultaneously while skin progenitor cell and stem spermatogonium is preserved, it can also further expand the culture of skin progenitor cell and stem spermatogonium.
Description
Technical field
The invention belongs to skin spermatogonium preservation field, more particularly to a kind of skin spermatogonium protection liquid and its preparation
Method.
Background technology
Spermatogonium belongs to the early development stage of male sex-cell, can constantly carry out cell division, increases cell
Quantity, and it is divided into sperm mother cell.Original blastocyte is through being differentiated to form spermatogonium, and spermatogonium is replicated to form primary essence
Mother cell, primary forms secondary spermatocyte through mother cell after initial meiosis, then by second meiotic division
Form spermatid.Study more for stem spermatogonium at present.
Skin is as the maximum organ of human body, with a variety of functions such as sensation, regulation body temperature, secretion and excretions, and its
The barrier for being also used as human body and external environment plays a part of maintaining homoiostasis, while it is also immune system
Important component.Skin has extremely strong reparation and power of regeneration, and the presence of this and skin progenitor cell has direct pass
System, study more has epidermal stem cells at present.
At present, the research for Skin Cell or spermatogonium is increasingly becoming a focus of cell field research, still
Understood based on prior art disclosure, greatest problem present in the research process to them is exactly that Skin Cell or essence are former
The storage and transport problem of cell, it includes all multi-steps, practical operation such as collection, transport, separation, culture in storing process
In need store longer time, once Skin Cell or spermatogonium collection after, departing from original vivo environment, its motility rate
Just influenceed by factors, time, temperature, osmotic pressure etc..Therefore it is former thin for the Skin Cell after collection or essence
The preservation or protection of born of the same parents is essential step in its storage or research process.The preservation of current Skin Cell or spermatogonium
Preserved more using directly bottling or adding the method for basal medium.It is small that the holding time directly bottled is generally less than 12
When, transport, handing-over and detection to Skin Cell or spermatogonium bring no small time pressure, also limit it and preserve industry
The scope of business development；In addition, Skin Cell or spermatogonium are preserved by adding basal medium, because basal medium is not
The other requirement of clinical grade can be reached, is that their preservation brings risk, has had a strong impact on epidermal stem cells or stem spermatogonium
Motility rate.In addition, prior art do not disclose also it is a kind of can be while preserving the stem cell protection of Skin Cell or spermatogonium
Liquid, and a kind of prior art CN105532641 placenta preserves liquid and its application method in preservation Skin Cell or smart former thin
During born of the same parents, preserve after 12h, the motility rate of epidermal stem cells or stem spermatogonium has just disappeared；Therefore being badly in need of offer one kind at present can
Enough preserving Skin Cell has the protection liquid that can preserve spermatogonium.
The content of the invention
In order to solve the above-mentioned technical problem, the present invention provides a kind of skin spermatogonium protection liquid, and the protection liquid is simulated
Human body vivo environment, is more suitable for the culture and preservation of Skin Cell or spermatogonium, and can significantly improve Skin Cell or
The activity of spermatogonium.
Concrete technical scheme of the present invention is as follows：
One aspect of the present invention provides a kind of skin spermatogonium protection liquid, and the protection liquid is mainly by aminoglycoside antibiosis
Element is formed with DMEM culture medium aqueous dissolutions, amino sugar glycoside antibiosis described in DMEM culture medium aqueous dissolutions described in per mL
Plain 80-120U, the concentration of the DMEM culture mediums aqueous solution is 13-25mg/mL.
Wherein, DMEM (Dulbecco ' s modified eagle medisum) culture medium contains various amino acid to be a kind of
With the culture medium of glucose.Each composition and content are as follows in the DMEM culture mediums：Anhydrous calcium chloride 265mg/L, nine water ferric nitrates
0.1mg/L, potassium chloride 400mg/L, anhydrous magnesium sulfate 97.6mg/L 7, sodium chloride 6400mg/L, AMSP
It is 109mg/L, succinic acid 75mg/L, sodium succinate 100mg/L, L- R-gene 84mg/L, L- hydrochloric acid cystine 63mg/L, sweet
Propylhomoserin 30mg/L, L- histidine monohydrochloride 42mg/L, ILE 105mg/L, Serine 42mg/L, L-threonine 95mg/
L, L-Trp 16mg/L, TYR 72mg/L, Valine 94mg/L, D-VB5 calcium 4mg/L, choline tartrate 7.2mg/
L, folic acid 4mg/L, inositol 7.2mg/L, niacinamide 4mg/L, riboflavin 0.4mg/L, thiamine hydrochloride 4mg/L, pyridoxine hydrochloride
4mg/L。
Further to improve, aminoglycoside antibiotics is selected from gentamicin, kanamycins, amikacin or TOB
In one or more, preferably ratio of weight and number be 5.5:1:The mixing of 1.2 gentamicin, kanamycins and TOB
Thing.
The protection liquid that the present invention is provided is adapted to the preservation of Skin Cell or spermatogonium, together closer to human body vivo environment
When the aminoglycoside antibiotics that adds may also function as the effect of sterilization.
Further to improve, the DMEM culture mediums in the DMEM culture medium aqueous solution are low-sugar type DMEM culture mediums.
Preferably, the concentration of the DMEM culture mediums aqueous solution is 16.9mg.
Further to improve, the protection liquid also includes replenishers, the supplement of DMEM culture medium aqueous dissolutions described in per mL
Each composition of agent and weight are as follows：Polyglutamic acid 15-20mg, phenylalanine 5-10mg, lysine 1-3mg and glutamine 0.5-
1mg.The replenishers for adding the mixture composition of polyglutamic acid, phenylalanine, lysine and glutamine can be Skin Cell
Or the growth of spermatogonium provides more energy.
Further to improve, protection liquid also includes activity increase agent, the solubilizer of DMEM culture medium aqueous dissolutions described in per mL
Each composition and weight are as follows：Deoxycholic acid 6.5-9 μ g, hydroxyethyl-β-cyclodextrin 15-20 μ g, casein sodium 1-3 μ g.
The mixture that the present invention passes through addition deoxycholic acid, hydroxyethyl-β-cyclodextrin and casein sodium in protection liquid
The activity increase agent of composition, can be deep into Skin Cell or spermatogonium internal structure, can be with Skin Cell or spermatogonium
Interior motility rate peptide formation inclusion compound, with reduction freezing point, reduces ice crystal formation, mitigates cell shrink degree and then play raising
The effect of Skin Cell or spermatogonium motility rate.
Further to improve, protection liquid also includes culture auxiliary agent, the culture of DMEM culture medium aqueous dissolutions described in per mL
Each composition of auxiliary agent and weight are as follows：Propiram 5-10 μ g, glutathione 6-10 μ g, aliphatic acid lactoyl ester 20-35 μ g, vitamin
E30-50 μ g, human serum albumins 1-3 μ g, ammonium nitrate 6-10 μ g, peptone 3-5 μ g and coconut monoethanolamide 15-20 μ
g。
By adding the culture auxiliary agent by above material composition in protection liquid, it is not only able to keep epidermal stem cells or essence
The motility rate of former stem cell, while amplification Skin Cell or spermatogonium number can be played together with the DMEM culture medium aqueous solution
Purpose.
Further to improve, protection liquid also includes delay agent, the delay agent of DMEM culture medium aqueous dissolutions described in per mL
Each composition and weight are as follows：Hamposyl L 7.5-10 μ g, hydrolecithin 35-50 μ g, monostearate calcium 1-3 μ g.
The present invention in protection liquid by adding by the mixture of Hamposyl L, hydrolecithin and monostearate calcium
The delay agent of composition, can significantly improve the holding time of Skin Cell or spermatogonium, and can improve the molten of each composition
Xie Du.
Further to improve, protection liquid also includes stabilizer, the stabilizer of DMEM culture medium aqueous dissolutions described in per mL
Each composition and weight are as follows：Sodium ascorbate 15-20 μ g, sodium starch phosphate 5-10 μ g and potassium citrate 3-6 μ g.
The present invention is made up of by being added in protection liquid the mixture of sodium ascorbate, sodium starch phosphate and potassium citrate
Stabilizer, primarily serve the osmotic equilibrium maintained in Skin Cell or spermatogonium, improve Skin Cell or spermatogonium
Adherent rate, while can increase the viscosity of protection liquid, improves the stability of the protection liquid.
Another aspect of the present invention provides the preparation method that a kind of skin spermatogonium protects liquid, and this method includes following step
Suddenly：
1) the DMEM culture medium aqueous solution of 1/3 volume is taken, the aminoglycoside antibiotics of 1/4 unit is added in 10 DEG C, is mixed
Close uniform, stand 5min；
2) the DMEM culture medium aqueous solution of 1/3 volume is added again, is mixed, and the aminoglycoside of 1/3 unit is added in 10 DEG C
Antibiotic, is well mixed, and stands 7min；
3) the DMEM culture medium aqueous solution of remaining 1/3 volume is added again, is mixed, and the ammonia of remaining 5/12 unit is added in 10 DEG C
Base glycoside antibiotic, is well mixed, and produces skin spermatogonium protection liquid.
The protection liquid prepared by the present invention is clarified, it is possible to increase the motility rate of epidermal stem cells or stem spermatogonium.
In protection liquid provided by the present invention, without special indicating preparation method, all components are exactly direct dissolving
Into the DMEM culture medium aqueous solution.
The beneficial effects of the invention are as follows：The skin spermatogonium protection liquid that the present invention is provided fully simulates body inner ring
Border, is more suitable for the existence of Skin Cell or spermatogonium, and the protection fluidity matter is stable, can significantly improve epidermal stem cells
Or the motility rate of stem spermatogonium；Simultaneously while Skin Cell or spermatogonium is preserved, skin can also be further expanded thin
Born of the same parents or the number of spermatogonium.
Embodiment
Embodiment 1
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin DMEM culture mediums aqueous dissolution
Into per gentamicin 80U described in DMEM culture medium aqueous dissolutions described in mL, the concentration of the DMEM culture mediums aqueous solution is
13mg/mL。
Embodiment 2
A kind of skin spermatogonium protects liquid, and the protection liquid is by kanamycins DMEM culture mediums aqueous dissolution
Into per gentamicin 120U described in DMEM culture medium aqueous dissolutions described in mL, the concentration of the DMEM culture mediums aqueous solution is
25mg/mL, the preparation method of the protection liquid comprises the following steps：
1) the DMEM culture medium aqueous solution of 1/3 volume is taken, the kanamycins of 1/4 unit is added in 10 DEG C, is well mixed, it is quiet
Put 5min；
2) the DMEM culture medium aqueous solution of 1/3 volume is added again, is mixed, the kanamycins of 1/3 unit is added in 10 DEG C, is mixed
Close uniform, stand 7min；
3) the DMEM culture medium aqueous solution of remaining 1/3 volume is added again, is mixed, and the card of remaining 5/12 unit is added in 10 DEG C
That mycin, is well mixed, and produces skin spermatogonium protection liquid.
Embodiment 3
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin, kanamycins and TOB DMEM
Culture medium aqueous dissolution is formed, per DMEM culture medium aqueous dissolution gentamicins 82.5U described in mL, kanamycins 15U, appropriate
Obramycin 18U, the concentration of the DMEM culture mediums aqueous solution is 16.9mg/mL.
Embodiment 4
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin DMEM culture mediums aqueous dissolution
Into per DMEM culture medium aqueous dissolution gentamicin 90U described in mL, the concentration of the DMEM culture mediums aqueous solution is
DMEM culture mediums in 16.9mg/mL, DMEM the culture medium aqueous solution are low-sugar type DMEM culture mediums.
Embodiment 5
A kind of skin spermatogonium protects liquid, and the protection liquid is by TOB and the replenishers DMEM culture medium aqueous solution
Dissolving is formed, DMEM culture medium aqueous dissolution TOB 90U described in per mL, and the concentration of the DMEM culture mediums aqueous solution is
21mg/mL, each composition of replenishers and weight of DMEM culture medium aqueous dissolutions described in per mL is as follows：Polyglutamic acid 15mg,
Phenylalanine 5mg, lysine 1mg and glutamine 0.5mg.
Embodiment 6
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin and the replenishers DMEM culture medium aqueous solution
Dissolving is formed, DMEM culture medium aqueous dissolution gentamicin 100U, the concentration of the DMEM culture mediums aqueous solution described in per mL
For 20mg/mL, the DMEM culture mediums in the DMEM culture medium aqueous solution are low-sugar type DMEM culture mediums, DMEM culture mediums described in per mL
Each composition of replenishers and weight of aqueous dissolution are as follows：Polyglutamic acid 20mg, phenylalanine 10mg, lysine 3mg and paddy
Glutamine 1mg.
Embodiment 7
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin and the activity increase agent DMEM culture medium aqueous solution
Dissolving is formed, DMEM culture medium aqueous dissolution gentamicin 95U described in per mL, and the concentration of the DMEM culture mediums aqueous solution is
20mg/mL, each composition of activity increase agent and weight of DMEM culture medium aqueous dissolutions described in per mL is as follows：The μ g of deoxycholic acid 6.5, hydroxyl
The μ g of the second group-beta-cyclodextrin 15 and μ g of casein sodium 1.
Embodiment 8
A kind of skin spermatogonium protects liquid, and the protection liquid is to cultivate gentamicin, replenishers and activity increase agent with DMEM
Base aqueous dissolution is formed, and DMEM culture medium aqueous dissolution gentamicin 105U described in per mL, the DMEM culture mediums are water-soluble
The concentration of liquid is 15mg/mL, and each composition of replenishers and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：Poly paddy
Propylhomoserin 16mg, phenylalanine 8mg, lysine 2mg and glutamine 0.6mg, DMEM culture medium aqueous dissolutions described in per mL
Each composition of activity increase agent and weight are as follows：The μ g of deoxycholic acid 9, the μ g of hydroxyethyl-β-cyclodextrin 20 and the μ g of casein sodium 3, wherein
DMEM culture mediums in the DMEM culture medium aqueous solution are low-sugar type DMEM culture mediums.
Embodiment 9
A kind of skin spermatogonium protects liquid, and the protection liquid is that gentamicin and culture auxiliary agent DMEM culture mediums is water-soluble
Liquid dissolving is formed, per DMEM culture medium aqueous dissolution gentamicin 100U described in mL, the DMEM culture mediums aqueous solution it is dense
Spend for 18mg/mL, each composition of culture auxiliary agent and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：The μ g of Propiram 5,
The μ g of glutathione 6, the μ g of aliphatic acid lactoyl ester 20, the μ g of vitamin E 30, the μ g of human serum albumins 1, the μ g of ammonium nitrate 6, the μ g of peptone 3
With the μ g of coconut monoethanolamide 15.
Embodiment 10
A kind of skin spermatogonium protects liquid, and the protection liquid is to train gentamicin, replenishers and culture auxiliary agent with DMEM
Support base aqueous dissolution to form, DMEM culture medium aqueous dissolution gentamicin 100U, the DMEM culture mediums water described in per mL
The concentration of solution is 19mg/mL, and each composition of replenishers and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：Poly
Glutamic acid 17mg, phenylalanine 6mg, lysine 1.5mg and glutamine 0.7mg, the DMEM culture medium aqueous solution described in per mL are molten
Each composition of culture auxiliary agent and weight of solution are as follows：The μ g of Propiram 10, the μ g of glutathione 10, the μ g of aliphatic acid lactoyl ester 35, vitamin
E50 μ g, the μ g of human serum albumins 3, the μ g of ammonium nitrate 10, the μ g of peptone 5 and coconut monoethanolamide 20 μ g, wherein DMEM training
It is low-sugar type DMEM culture mediums to support the DMEM culture mediums in the base aqueous solution.
Embodiment 11
A kind of skin spermatogonium protects liquid, and the protection liquid is to train gentamicin, activity increase agent and culture auxiliary agent with DMEM
Support base aqueous dissolution to form, DMEM culture medium aqueous dissolution gentamicin 100U, the DMEM culture mediums water described in per mL
The concentration of solution is 20mg/mL, and each composition of activity increase agent and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：Deoxidation
The μ g of cholic acid 7, the μ g of hydroxyethyl-β-cyclodextrin 17, the μ g of casein sodium 2, the culture of DMEM culture medium aqueous dissolutions described in per mL
Each composition of auxiliary agent and weight are as follows：The μ g of Propiram 7.5, the μ g of glutathione 8, the μ g of aliphatic acid lactoyl ester 30, the μ g of vitamin E 40, people
The μ g of seralbumin 2, the μ g of ammonium nitrate 7.5, the μ g of peptone 4 and the μ g of coconut monoethanolamide 17.
Embodiment 12
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin and the delay agent DMEM culture medium aqueous solution
Dissolving is formed, DMEM culture medium aqueous dissolution gentamicin 110U, the concentration of the DMEM culture mediums aqueous solution described in per mL
For 25mg/mL, often each composition of delay agent and weight of DMEM culture medium aqueous dissolutions described in mL are as follows：Hamposyl L
7.5 μ g, the μ g of hydrolecithin 50 and the μ g of monostearate calcium 1.
Embodiment 13
A kind of skin spermatogonium protects liquid, and the protection liquid is to cultivate gentamicin, activity increase agent and delay agent with DMEM
Base aqueous dissolution is formed, and DMEM culture medium aqueous dissolution gentamicin 110U described in per mL, the DMEM culture mediums are water-soluble
The concentration of liquid is 13mg/mL, and each composition of activity increase agent and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：Deoxidation courage
Acid 8 μ g, the μ g of the hydroxyethyl-β-cyclodextrin 18.5 and μ g of casein sodium 2.5, DMEM culture medium aqueous dissolutions prolongs described in per mL
When each composition of agent and weight it is as follows：The μ g of Hamposyl L 10, the μ g of hydrolecithin 35 and the μ g of monostearate calcium 3.
Embodiment 14
A kind of skin spermatogonium protects liquid, and the protection liquid is to train gentamicin, culture auxiliary agent and delay agent with DMEM
Support base aqueous dissolution to form, DMEM culture medium aqueous dissolution gentamicin 110U, the DMEM culture mediums water described in per mL
The concentration of solution is 18mg/mL, and each composition of culture auxiliary agent and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：It is general
Shandong 8 μ g of orchid, the μ g of glutathione 7.5, the μ g of aliphatic acid lactoyl ester 25, the μ g of vitamin E 37.5, the μ g of human serum albumins 2.5, ammonium nitrate
9 μ g, the μ g of peptone 4.5 and the μ g of coconut monoethanolamide 17.5, the delay of DMEM culture medium aqueous dissolutions described in per mL
Each composition of agent and weight are as follows：The μ g of Hamposyl L 9, the μ g of hydrolecithin 45 and the μ g of monostearate calcium 2.
Embodiment 15
A kind of skin spermatogonium protects liquid, and the protection liquid is to use gentamicin, activity increase agent, culture auxiliary agent and delay agent
DMEM culture medium aqueous dissolutions are formed, DMEM culture medium aqueous dissolution gentamicins 110U described in per mL, the DMEM trainings
The concentration for supporting the base aqueous solution is 16.9mg/mL, each composition of activity increase agent and weight of DMEM culture medium aqueous dissolutions described in per mL
It is as follows：The μ g of deoxycholic acid 7.2, the μ g of hydroxyethyl-β-cyclodextrin 18.5 and the μ g of casein sodium 2.2, DMEM culture mediums described in per mL
Each composition of culture auxiliary agent and weight of aqueous dissolution are as follows：The μ g of Propiram 9, the μ g of glutathione 6.5, aliphatic acid lactoyl ester 22.5
μ g, the μ g of vitamin E 35, the μ g of human serum albumins 2.6, the μ g of ammonium nitrate 6.5, the μ g of peptone 4.5 and coconut monoethanolamide
16.5 μ g, each composition of delay agent and weight of DMEM culture medium aqueous dissolutions described in per mL is as follows：The μ g of Hamposyl L 9,
The μ g of the hydrolecithin 45 and μ g of monostearate calcium 1.5.
Embodiment 16
A kind of skin spermatogonium protects liquid, and the protection liquid is to use gentamicin, replenishers, activity increase agent and delay agent
DMEM culture medium aqueous dissolutions are formed, DMEM culture medium aqueous dissolution gentamicins 110U described in per mL, the DMEM trainings
The concentration for supporting the base aqueous solution is 23mg/mL, and the replenishers composition and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：
Polyglutamic acid 17mg, phenylalanine 6mg, lysine 1.5mg and glutamine 0.7mg, DMEM culture mediums described in per mL are water-soluble
Each composition of activity increase agent and weight of liquid dissolving are as follows：The μ g of deoxycholic acid 7.2, the μ g of hydroxyethyl-β-cyclodextrin 18.5 and caseinic acid
The μ g of sodium 2.2, each composition of culture auxiliary agent and weight of DMEM culture medium aqueous dissolutions described in per mL is as follows：The μ g of Propiram 9, paddy Guang
The sweet μ g of peptide 6.5, the μ g of aliphatic acid lactoyl ester 22.5, the μ g of vitamin E 35, the μ g of human serum albumins 2.6, the μ g of ammonium nitrate 6.5, peptone
The 4.5 μ g and μ g of coconut monoethanolamide 16.5, each composition of delay agent per DMEM culture medium aqueous dissolutions described in mL and
Weight is as follows：The μ g of Hamposyl L 9, the μ g of hydrolecithin 45 and the μ g of monostearate calcium 1.5, wherein the DMEM culture mediums aqueous solution
In DMEM culture mediums be low-sugar type DMEM culture mediums.
Embodiment 17
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin and the stabilizer DMEM culture medium aqueous solution
Dissolving is formed, DMEM culture medium aqueous dissolution gentamicin 100U, the concentration of the DMEM culture mediums aqueous solution described in per mL
For 17.5mg/mL, often each composition of stabilizer and weight of DMEM culture medium aqueous dissolutions described in mL are as follows：Sodium ascorbate 15
μ g, the μ g of sodium starch phosphate 10 and the μ g of potassium citrate 6.
Embodiment 18
A kind of skin spermatogonium protects liquid, and the protection liquid is to cultivate gentamicin, activity increase agent and stabilizer with DMEM
Base aqueous dissolution is formed, and DMEM culture medium aqueous dissolution gentamicin 100U described in per mL, the DMEM culture mediums are water-soluble
The concentration of liquid is 15mg/mL, and each composition of activity increase agent and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：Deoxidation courage
Acid 7 μ g, the μ g of the hydroxyethyl-β-cyclodextrin 17 and μ g of casein sodium 2, the stabilizer of DMEM culture medium aqueous dissolutions described in per mL
Each composition and weight are as follows：The μ g of sodium ascorbate 20, the μ g of sodium starch phosphate 5 and the μ g of potassium citrate 3.
Embodiment 19
A kind of skin spermatogonium protects liquid, and the protection liquid is to train gentamicin, culture auxiliary agent and stabilizer with DMEM
Support base aqueous dissolution to form, DMEM culture medium aqueous dissolution gentamicin 100U, the DMEM culture mediums water described in per mL
The concentration of solution is 22mg/mL, and each composition of culture auxiliary agent and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：It is general
Shandong 6 μ g of orchid, the μ g of glutathione 7, the μ g of aliphatic acid lactoyl ester 30, the μ g of vitamin E 40, the μ g of human serum albumins 2, the μ g of ammonium nitrate 6, egg
The white μ g of the peptone 5 and μ g of coconut monoethanolamide 17, each composition of stabilizer of DMEM culture medium aqueous dissolutions described in per mL and
Weight is as follows：The μ g of sodium ascorbate 17.5, the μ g of sodium starch phosphate 7.5 and the μ g of potassium citrate 5.
Embodiment 20
A kind of skin spermatogonium protects liquid, the protection liquid be by gentamicin, replenishers, culture auxiliary agent, activity increase agent and
Stabilizer is formed with DMEM culture medium aqueous dissolutions, DMEM culture medium aqueous dissolution gentamicin 100U, institute described in per mL
The concentration for stating the DMEM culture medium aqueous solution is 16.9mg/mL, the replenishers per DMEM culture medium aqueous dissolutions described in mL it is each into
Divide and weight is as follows：Polyglutamic acid 17mg, phenylalanine 6mg, lysine 1.5mg and glutamine 0.7mg, described in per mL
Each composition of activity increase agent and weight of DMEM culture medium aqueous dissolutions are as follows：The μ g of deoxycholic acid 7, the μ g of hydroxyethyl-β-cyclodextrin 17
With the μ g of casein sodium 2, often each composition of culture auxiliary agent and weight of DMEM culture medium aqueous dissolutions described in mL are as follows：Propiram
6 μ g, the μ g of glutathione 7, the μ g of aliphatic acid lactoyl ester 30, the μ g of vitamin E 40, the μ g of human serum albumins 2, the μ g of ammonium nitrate 6, peptone
The 5 μ g and μ g of coconut monoethanolamide 17, each composition of delay agent and weight of DMEM culture medium aqueous dissolutions described in per mL
It is as follows：The μ g of Hamposyl L 9, the μ g of hydrolecithin 40, the μ g of monostearate calcium 2, the DMEM culture medium aqueous solution described in per mL are molten
Each composition of stabilizer and weight of solution are as follows：The μ g of sodium ascorbate 18, the μ g of sodium starch phosphate 8 and potassium citrate 4 μ g, wherein DMEM
DMEM culture mediums in the culture medium aqueous solution are low-sugar type DMEM culture mediums.
Reference examples 1
A kind of skin spermatogonium protects liquid, and the protection liquid is to form penicillin with DMEM culture medium aqueous dissolutions,
Penicillin 80U described in DMEM culture medium aqueous dissolutions described in per mL, the concentration of the DMEM culture mediums aqueous solution is 13mg/
mL。
Reference examples 2
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin and the replenishers DMEM culture medium aqueous solution
Dissolving is formed, gentamicin 115U described in DMEM culture medium aqueous dissolutions described in per mL, the DMEM culture mediums aqueous solution
Concentration is 21mg/mL, and each composition of replenishers and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：Polyglutamic acid
20mg, phenylalanine 6mg, lysine 1.5mg.
Reference examples 3
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin and the activity increase agent DMEM culture medium aqueous solution
Dissolving is formed, DMEM culture medium aqueous dissolution gentamicin 95U described in per mL, and the concentration of the DMEM culture mediums aqueous solution is
20mg/mL, each composition of activity increase agent and weight of DMEM culture medium aqueous dissolutions described in per mL is as follows：The μ g of deoxycholic acid 6.5, hydroxyl
The μ g of the second group-beta-cyclodextrin 15 and μ g of trisodium citrate 1.
Reference examples 4
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin and the activity increase agent DMEM culture medium aqueous solution
Dissolving is formed, DMEM culture medium aqueous dissolution gentamicin 95U described in per mL, and the concentration of the DMEM culture mediums aqueous solution is
20mg/mL, each composition of activity increase agent and weight of DMEM culture medium aqueous dissolutions described in per mL is as follows：The μ g of deoxycholic acid 6.5 and
The μ g of casein sodium 1.
Reference examples 5
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin and the delay agent DMEM culture medium aqueous solution
Dissolving is formed, DMEM culture medium aqueous dissolution gentamicin 110U, the concentration of the DMEM culture mediums aqueous solution described in per mL
For 25mg/mL, often each composition of delay agent and weight of DMEM culture medium aqueous dissolutions described in mL are as follows：Lauroyl lysine
7.5 μ g, the μ g of lecithin 50 and the μ g of monostearate calcium 1.
Reference examples 6
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin and the delay agent DMEM culture medium aqueous solution
Dissolving is formed, DMEM culture medium aqueous dissolution gentamicin 110U, the concentration of the DMEM culture mediums aqueous solution described in per mL
For 25mg/mL, often each composition of delay agent and weight of DMEM culture medium aqueous dissolutions described in mL are as follows：Hamposyl L
The 7.5 μ g and μ g of hydrolecithin 50.
Reference examples 7
A kind of skin spermatogonium protects liquid, and the protection liquid is that gentamicin and culture auxiliary agent DMEM culture mediums is water-soluble
Liquid dissolving is formed, per DMEM culture medium aqueous dissolution gentamicin 100U described in mL, the DMEM culture mediums aqueous solution it is dense
Spend for 18mg/mL, each composition of culture auxiliary agent and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：The μ g of Propiram 5,
The μ g of glutathione 6, the μ g of aliphatic acid lactoyl ester 20, the μ g of vitamin C 30, the μ g of human serum albumins 1, the μ g of ammonium nitrate 6, the μ of Sodium Pyruvate 3
The g and μ g of coconut monoethanolamide 15.
Reference examples 8
A kind of skin spermatogonium protects liquid, and the protection liquid is that gentamicin and culture auxiliary agent DMEM culture mediums is water-soluble
Liquid dissolving is formed, per DMEM culture medium aqueous dissolution gentamicin 100U described in mL, the DMEM culture mediums aqueous solution it is dense
Spend for 18mg/mL, each composition of culture auxiliary agent and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：The μ of glutathione 6
G, the μ g of aliphatic acid lactoyl ester 20, the μ g of vitamin E 30, the μ g of ammonium nitrate 6, the μ g of peptone 3 and the μ g of coconut monoethanolamide 15.
Reference examples 9
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin and the stabilizer DMEM culture medium aqueous solution
Dissolving is formed, DMEM culture medium aqueous dissolution gentamicin 100U, the concentration of the DMEM culture mediums aqueous solution described in per mL
For 17.5mg/mL, often each composition of stabilizer and weight of DMEM culture medium aqueous dissolutions described in mL are as follows：The μ of ascorbic acid 15
G, the μ g of sodium starch phosphate 10 and the μ g of citric acid 6.
Reference examples 10
A kind of skin spermatogonium protects liquid, and the protection liquid is by gentamicin and the stabilizer DMEM culture medium aqueous solution
Dissolving is formed, DMEM culture medium aqueous dissolution gentamicin 100U, the concentration of the DMEM culture mediums aqueous solution described in per mL
For 17.5mg/mL, often each composition of stabilizer and weight of DMEM culture medium aqueous dissolutions described in mL are as follows：Sodium ascorbate 15
The μ g and μ g of potassium citrate 6.
The survival rate test of test example 1
The Skin Cell and spermatogonium preserved respectively using embodiment 1,3,5,7-8 and 12-13 protection liquid is experiment 1-7
Group, the Skin Cell and spermatogonium preserved respectively using reference examples 1-6 protection liquid is control 1-6 group.Each experimental group and control
The Skin Cell and spermatogonium of group enter test procedure in 24h, 48h, 96h, 240h and 480h respectively, adjustment Skin Cell and
The density of spermatogonium is 1 × 106cells/mL.By cell suspension：0.4% trypan blue=3:1(v:V) fully mix, take
20 μ L cell suspensions are added in cell counting count board, detect that each experimental group and control group protect liquid with Countstar cell counters
Endepidermis stem cell and the motility rate of stem spermatogonium, as a result as shown in table 1.
The experimental group of table 1 and cellular control unit motility rate result
Note：" -- " represents no motility rate.
As shown in Table 1, the Skin Cell that preserves respectively of protection liquid of experiment 1-2 groups and the Cell viability of spermatogonium than
The Skin Cell and the Cell viability of spermatogonium preserved respectively using 1 group of protection liquid used of control is high；Experiment 3-5 groups are used
The Skin Cell that preserves respectively of protection liquid and spermatogonium Cell viability than the protection liquid point that is used using control 2-4 groups
The Skin Cell and the Cell viability of spermatogonium not preserved are high；And test the skin that the protection liquid that 6-7 groups use is preserved respectively
The time of skin cell and spermatogonium is longer than the time of control 5-6 groups and experiment 1-4 groups, can reach 480h, its motility rate still reaches
To more than 50%.
It follows that the aminoglycoside antibiotics in the protection liquid that the present invention is provided has changed the antibiotic such as penicillin into
Afterwards, the motility rate of Skin Cell or spermatogonium is significantly reduced.Added when protecting in liquid by polyglutamic acid, phenylalanine, bad ammonia
Replenishers or add deoxycholic acid, hydroxyethyl-β-cyclodextrin and casein sodium in protection liquid that acid and glutamine are constituted
After the activity increase agent of mixture composition, the motility rate that protection liquid preserves Skin Cell or spermatogonium can be significantly improved, when poly paddy
In the composition of propylhomoserin, phenylalanine, lysine and glutamine and deoxycholic acid, hydroxyethyl-β-cyclodextrin and casein sodium
After lacking one or being substituted by other composition, the motility rate of Skin Cell or spermatogonium decreases；And in protection liquid
Protection liquid can also be improved to Skin Cell or essence original by adding the mixture of Hamposyl L, hydrolecithin and monostearate calcium
The holding time of cell, the holding time can be brought up to 480h, when Hamposyl L, hydrolecithin and monostearate calcium
After three kinds of compositions lack one or substituted by other composition, the holding time of Skin Cell or spermatogonium also can be only sustained at
In 96h.
The expansion of stem cells culture of test example 2 is detected
The Skin Cell and spermatogonium preserved respectively using the protection liquid of embodiment 7, embodiment 9 and embodiment 11 is experiment
1-3 groups, a kind of fowl disclosed in the Skin Cell and spermatogonium and CN1935984 preserved respectively with reference examples 7-8 protection liquid
The spermatogonium of the extracorporeal culturing method culture of class spermatogonium is control 1-3 groups.The Skin Cell of each experimental group and control group
Respectively preserve and sampled after 2 days with spermatogonium, carry out cell count (trypan blue staining), to protection liquid endepidermis stem cell and essence
The motility rate of former stem cell carries out the measure of TCS, amplification times, Cell viability respectively, the results are shown in Table 2.
The amplification cultivation result of the experimental group of table 2 and control group
As can be seen from the table, the protection liquid that the embodiment of the present invention 9 and embodiment 11 are provided is remarkably improved stem cell
Amplification times, its amplification times are significantly higher than embodiment 7, control 1-3 groups, and this protects the mentioned component added in liquid not shadow
Ring the motility rate of stem cell, it follows that, added in the protection liquid that the present invention is provided after the mixtures such as Propiram, glutathione,
It can significantly improve the amplification times of stem cell, but when above composition changes, or omit after a certain composition, the protection liquid
Amplification times to stem cell are significantly reduced.
The adherent rate of test example 3 is detected
The Skin Cell and spermatogonium preserved respectively using the protection liquid of embodiment 17 and embodiment 20 as experiment 1-2 groups,
The Skin Cell and spermatogonium preserved respectively using reference examples 9-10 protection liquid is control 1-2 group.Each experimental group and control group
Skin Cell and spermatogonium enter test procedure in 24h, 48h, 96h, 240h and 480h respectively, adjustment Skin Cell and essence
The density of archaeocyte is 5 × 105In cells/mL, the plate for being inoculated with 8mL to a diameter of 9cm, its natural adherent growth 48 is treated
After hour, remove after not adherent cell, digested with 0.25m/v% trypsase, calculate adherent epidermal stem cells
With the quantity of stem spermatogonium, adherent rate is drawn.As a result it is as shown in table 3.
The experimental group of table 3 and cellular control unit adherent rate result
As shown in Table 3, experiment 1-2 groups are after 96h is preserved, and adherence rate remains at more than 80%, and adherent effect is bright
It is aobvious to be better than control 1-2 groups, and 2 groups of experiment adherence rate after 480h is preserved also is maintained at more than 50%, it follows that, resist
The mixture of bad hematic acid sodium, sodium starch phosphate and potassium citrate can improve the adherent rate of Skin Cell or spermatogonium, when wherein
After one composition transfer or omission, the adherent rate of Skin Cell or spermatogonium is significantly reduced.
Embodiment described above only expresses the several embodiments of the present invention, and it describes more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention can not be interpreted as.It should be pointed out that for one of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (10)
1. a kind of skin spermatogonium protects liquid, it is characterised in that the protection liquid mainly uses aminoglycoside antibiotics
DMEM culture medium aqueous dissolutions are formed, amino sugar glycoside antibiotic described in DMEM culture medium aqueous dissolutions described in per mL
80-120U, the concentration of the DMEM culture mediums aqueous solution is 13-25mg/mL.
2. skin spermatogonium as claimed in claim 1 protects liquid, it is characterised in that the aminoglycoside antibiotics is selected from
One or more in gentamicin, kanamycins, amikacin or TOB.
3. skin spermatogonium as claimed in claim 1 protects liquid, it is characterised in that in the DMEM culture mediums aqueous solution
DMEM culture mediums are low-sugar type DMEM culture mediums.
4. skin spermatogonium as claimed in claim 1 protects liquid, it is characterised in that the concentration of the DMEM culture mediums aqueous solution
For 16.9mg/mL.
5. skin spermatogonium as claimed in claim 3 protects liquid, it is characterised in that the protection liquid also includes replenishers,
Each composition of replenishers and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：Polyglutamic acid 15-20mg, phenylpropyl alcohol ammonia
Sour 5-10mg, lysine 1-3mg and glutamine 0.5-1mg.
6. skin spermatogonium as claimed in claim 1 protects liquid, it is characterised in that the protection liquid also includes activity increase agent,
Each composition of activity increase agent and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：Deoxycholic acid 6.5-9 μ g, ethoxy-
Beta-schardinger dextrin 15-20 μ g, casein sodium 1-3 μ g.
7. skin spermatogonium as claimed in claim 1 protects liquid, it is characterised in that the protection liquid is also helped including culture
Agent, each composition of culture auxiliary agent and weight of DMEM culture medium aqueous dissolutions described in per mL is as follows：Propiram 5-10 μ g, gluathione
Peptide 6-10 μ g, aliphatic acid lactoyl ester 20-35 μ g, vitamin E 30-50 μ g, human serum albumins 1-3 μ g, ammonium nitrate 6-10 μ g, egg
White peptone 3-5 μ g and coconut monoethanolamide 15-20 μ g.
8. skin spermatogonium as claimed in claim 1 protects liquid, it is characterised in that the protection liquid also includes delay agent,
Each composition of delay agent and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：Hamposyl L 7.5-10 μ g, hydrogen
Change lecithin 35-50 μ g, monostearate calcium 1-3 μ g.
9. skin spermatogonium as claimed in claim 1 protects liquid, it is characterised in that the protection liquid also includes stabilizer,
Each composition of stabilizer and weight of DMEM culture medium aqueous dissolutions described in per mL are as follows：Sodium ascorbate 15-20 μ g, phosphoric acid form sediment
Powder sodium 5-10 μ g and potassium citrate 3-6 μ g.
10. the skin spermatogonium described in a kind of claim 1 protects the preparation method of liquid, it is characterised in that methods described bag
Include following steps：
1) the DMEM culture medium aqueous solution of 1/3 volume is taken, the aminoglycoside antibiotics of 1/4 unit is added in 10 DEG C, mixing is equal
It is even, stand 5min；
2) the DMEM culture medium aqueous solution of 1/3 volume is added again, is mixed, and the aminoglycoside antibiosis of 1/3 unit is added in 10 DEG C
Element, is well mixed, and stands 7min；
3) the DMEM culture medium aqueous solution of remaining 1/3 volume is added again, is mixed, and the amino sugar of remaining 5/12 unit is added in 10 DEG C
Tobramycin antibiotic, is well mixed, and produces skin spermatogonium protection liquid.
Priority Applications (2)
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| CN201610622369.5A CN107125240A (en) | 2016-08-01 | 2016-08-01 | A kind of skin spermatogonium protection liquid and preparation method thereof |
| PCT/CN2016/094676 WO2018023828A1 (en) | 2016-08-01 | 2016-08-11 | Protective fluid for skin and spermatogonial cells and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610622369.5A CN107125240A (en) | 2016-08-01 | 2016-08-01 | A kind of skin spermatogonium protection liquid and preparation method thereof |
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| CN111838138A (en) * | 2020-08-31 | 2020-10-30 | 海南济民博鳌国际医院有限公司 | Stem cell cryopreservation liquid and preparation and use methods thereof |
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| WO2018023828A1 (en) | 2018-02-08 |
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