WO2018023828A1 - 一种皮肤精原细胞保护液及其制备方法 - Google Patents

一种皮肤精原细胞保护液及其制备方法 Download PDF

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WO2018023828A1
WO2018023828A1 PCT/CN2016/094676 CN2016094676W WO2018023828A1 WO 2018023828 A1 WO2018023828 A1 WO 2018023828A1 CN 2016094676 W CN2016094676 W CN 2016094676W WO 2018023828 A1 WO2018023828 A1 WO 2018023828A1
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dmem medium
skin
aqueous solution
solution
cell protection
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PCT/CN2016/094676
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English (en)
French (fr)
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曹毓琳
李霞云
白志惠
刘世红
卢承前
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北京世纪劲得生物技术有限公司
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Publication of WO2018023828A1 publication Critical patent/WO2018023828A1/zh

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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

Definitions

  • the invention belongs to the field of skin sperm cell preservation, and particularly relates to a skin sperm cell protection liquid and a preparation method thereof.
  • Spermatogonia belongs to the early developmental stage of male germ cells, which can continuously divide cells, increase the number of cells, and differentiate into spermatocytes.
  • the original embryonic cells are differentiated to form spermatogonia, and the spermatogonia is replicated to form primary spermatocytes.
  • the primary meristematic cells form secondary spermatocytes after the first meiosis, and then undergo a second meiosis.
  • Sperm cells At present, more research is done on spermatogonial stem cells.
  • the skin As the largest organ of the human body, the skin has many functions such as feeling, regulating body temperature, secretion and excretion, and it can also serve as a barrier between the human body and the external environment to maintain the stability of the internal environment. It is also an important component of the immune system. section.
  • the skin has a strong ability to repair and regenerate, which has a direct relationship with the existence of skin stem cells.
  • epidermal stem cells Currently, there are many epidermal stem cells.
  • the preservation of skin cells or spermatogonia is usually preserved by direct bottling or supplementation with basal medium.
  • Direct bottling usually takes less than 12 hours to give to the skin
  • the transportation, transfer and detection of cells or spermatogonia brings a lot of time pressure and limits the scope of its preservation business; in addition, by adding basal medium to preserve skin cells or spermatogonia, because the basal medium Failure to meet clinical grade requirements poses a risk to their preservation, seriously affecting the viability of epidermal stem cells or spermatogonial stem cells.
  • the prior art does not disclose a stem cell protection solution capable of simultaneously storing skin cells or spermatogonia, and a placental preservation solution of the prior art CN105532641 and a method of using the same are preserved when preserving skin cells or spermatogonia After 12 hours, the viability of epidermal stem cells or spermatogonial stem cells has disappeared; therefore, there is an urgent need to provide a protective solution capable of preserving skin cells and preserving spermatogonia cells.
  • the present invention provides a skin spermatogonium cell protection solution which simulates a human body environment, is more suitable for the cultivation and preservation of skin cells or spermatogonia, and can significantly increase skin cells or spermatogonia cells. Activity.
  • the present invention provides a skin spermatogonial cell protection solution, which is mainly prepared by dissolving an aminoglycoside antibiotic with an aqueous solution of DMEM medium, and dissolving the aminoglycoside antibiotic 80 per mL of the aqueous solution of DMEM medium.
  • a skin spermatogonial cell protection solution which is mainly prepared by dissolving an aminoglycoside antibiotic with an aqueous solution of DMEM medium, and dissolving the aminoglycoside antibiotic 80 per mL of the aqueous solution of DMEM medium.
  • concentration of the DMEM medium aqueous solution was 13-25 mg/mL.
  • DMEM Denscralose's modified eagle medisum
  • the components and contents of the DMEM medium are as follows: anhydrous calcium chloride 265 mg / L, iron nitrate non-ferrous nitrate 0.1 mg / L, potassium chloride 400 mg / L, anhydrous magnesium sulfate 97.6 mg / L 7, sodium chloride 6400 mg /L, anhydrous sodium dihydrogen phosphate 109mg / L, succinic acid 75mg / L, sodium succinate 100mg / L, L-arginine hydrochloride 84mg / L, L-hydrochloride cystine 63mg / L, glycine 30mg /L, L-histidine hydrochloride 42mg/L, L-isoleucine 105mg/L, L-serine 42mg/L, L-threonine 95mg/L,
  • the aminoglycoside antibiotic is selected from one or more of gentamicin, kanamycin, amikacin or tobramycin, preferably in a weight ratio of 5.5:1:1.2.
  • gentamicin kanamycin
  • amikacin tobramycin
  • the protective liquid provided by the invention is closer to the human body environment, and is suitable for the preservation of skin cells or spermatogonia, and the added aminoglycoside antibiotics can also have a bactericidal effect.
  • the DMEM medium in the aqueous solution of DMEM medium is a low-glucose DMEM medium.
  • the concentration of the aqueous DMEM medium solution is 16.9 mg.
  • the protective solution further comprises a supplement
  • the components and weight of the supplement dissolved in the mL of the DMEM medium solution are as follows: polyglutamic acid 15-20 mg, phenylalanine 5-10 mg, lysine 1-3 mg and glutamine 0.5-1 mg.
  • Supplements consisting of a mixture of polyglutamic acid, phenylalanine, lysine, and glutamine can provide more energy for the growth of skin cells or spermatogonia.
  • the protective solution further comprises a stimulating agent
  • the components and weights of the solubilizing agent dissolved per mL of the DMEM medium aqueous solution are as follows: deoxycholic acid 6.5-9 ⁇ g, hydroxyethyl- ⁇ -cyclodextrin 15-20 ⁇ g, Sodium caseinate 1-3 ⁇ g.
  • the invention can penetrate deep into the internal structure of skin cells or spermatogonia by adding a stimulating agent composed of a mixture of deoxycholic acid, hydroxyethyl- ⁇ -cyclodextrin and sodium caseinate in the protective liquid, and can be combined with the skin.
  • a stimulating agent composed of a mixture of deoxycholic acid, hydroxyethyl- ⁇ -cyclodextrin and sodium caseinate in the protective liquid, and can be combined with the skin.
  • the cell or the live peptide in the spermatogonium forms an inclusion complex, which has the effect of lowering the freezing point, reducing the formation of ice crystals, reducing the degree of cell shrinkage, and thereby increasing the viability of skin cells or spermatogonial cells.
  • the protective solution further comprises a culture aid, and the DMEM medium per mL
  • the components and weights of the culture aid dissolved in the aqueous solution are as follows: 5-10 ⁇ g of pullulan, 6-10 ⁇ g of glutathione, 20-35 ⁇ g of fatty acid lactyl ester, 50-35 ⁇ g of vitamin E30, 1-3 ⁇ g of human serum albumin, ammonium nitrate 6-10 ⁇ g, peptone 3-5 ⁇ g and coconut oleic acid monoethanolamide 15-20 ⁇ g.
  • a culture aid composed of the above substances By adding a culture aid composed of the above substances to the protective solution, not only can the activity of the epidermal stem cells or the spermatogonial stem cells be maintained, but also the purpose of expanding the number of skin cells or spermatogonia together with the DMEM medium aqueous solution. .
  • the protective solution further comprises a delaying agent, and the components and weight of the retarding agent dissolved per mL of the aqueous solution of DMEM medium are as follows: 7.5-10 ⁇ g of lauroyl sarcosine, 35-50 ⁇ g of hydrogenated lecithin, and monostearyl Calcium acid 1-3 ⁇ g.
  • the present invention can significantly increase the preservation time of skin cells or spermatogonia by adding a delay agent composed of a mixture of lauroyl sarcosine, hydrogenated lecithin and calcium monostearate to the protective liquid, and can improve the components. Solubility.
  • the protective solution further includes a stabilizer, and the components and weights of the stabilizer dissolved per mL of the DMEM medium aqueous solution are as follows: 15-20 ⁇ g of sodium ascorbate, 5-10 ⁇ g of sodium starch phosphate, and 3-6 ⁇ g of potassium citrate.
  • the invention adds a stabilizer composed of a mixture of sodium ascorbate, sodium starch phosphate and potassium citrate to the protective liquid, mainly to maintain the osmotic balance in the skin cells or spermatogonia, and to improve the skin cells or spermatogonia.
  • the adhesion rate can increase the viscosity of the protective liquid and improve the stability of the protective liquid.
  • Another aspect of the invention provides a method for preparing a skin spermatogonial cell protection solution, the method comprising the steps of:
  • the clarification of the protective solution prepared by the present invention can increase the viability of epidermal stem cells or spermatogonial stem cells.
  • the beneficial effects of the invention are: the skin spermatogonial cell protection solution provided by the invention fully simulates the human body environment, is more suitable for the survival of skin cells or spermatogonia, and the protective liquid has stable properties and can significantly improve epidermal stem cells or spermatogonium.
  • the survival rate of stem cells at the same time, while preserving skin cells or spermatogonia, the number of skin cells or spermatogonia can be further expanded.
  • a skin spermatogonium cell protection solution which is prepared by dissolving gentamicin in an aqueous solution of DMEM medium, and dissolving the gentamicin 80 U per mL of the DMEM medium solution, the DMEM medium solution The concentration was 13 mg/mL.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving kanamycin in a DMEM medium aqueous solution, and dissolving the gentamicin 120 U per mL of the DMEM medium aqueous solution, the DMEM medium solution.
  • concentration of the protective solution is 25 mg/mL
  • the preparation method of the protective solution comprises the following steps:
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin, kanamycin and tobramycin in an aqueous solution of DMEM medium, and dissolving gentamicin per mL of the aqueous solution of DMEM medium. 82.5 U, kanamycin 15 U, tobramycin 18 U, and the concentration of the DMEM medium aqueous solution was 16.9 mg/mL.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin in an aqueous solution of DMEM medium, and dissolving gentamicin 90 U per mL of the aqueous solution of DMEM medium, the concentration of the aqueous solution of DMEM medium.
  • the DMEM medium in the DMEM medium aqueous solution was 16.9 mg/mL, which was a low glucose type DMEM medium.
  • the concentration of the aqueous solution was 21 mg/mL, and the components and weights of the supplement dissolved per mL of the DMEM medium solution were as follows: polyglutamic acid 15 mg, phenylalanine 5 mg, lysine 1 mg, and glutamine 0.5 mg.
  • a skin sperm cell protection solution for gentamicin and supplements DMEM medium solution is dissolved, 100 U of gentamicin is dissolved per mL of the DMEM medium solution, the concentration of the DMEM medium solution is 20 mg/mL, and the DMEM medium in the DMEM medium is cultured in low glucose type DMEM.
  • the components and weights of the supplement dissolved in each mL of the DMEM medium aqueous solution are as follows: polyglutamic acid 20 mg, phenylalanine 10 mg, lysine 3 mg, and glutamine 1 mg.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin and a stimulating agent in an aqueous solution of DMEM medium, and dissolving gentamicin 95 U per mL of the aqueous solution of DMEM medium, and the DMEM is cultured.
  • concentration of the base aqueous solution is 20 mg/mL
  • the components and weight of the activator dissolved per mL of the DMEM medium aqueous solution are as follows: 6.5 ⁇ g of deoxycholic acid, 15 ⁇ g of hydroxyethyl- ⁇ -cyclodextrin, and 1 ⁇ g of sodium caseinate. .
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin, a supplement and a stimulating agent in an aqueous solution of DMEM medium, and dissolving gentamicin 105 U per mL of the aqueous solution of DMEM medium.
  • concentration of the aqueous solution of DMEM medium is 15 mg/mL, and the components and weight of the supplement dissolved in the DMEM medium solution per mL are as follows: polyglutamic acid 16 mg, phenylalanine 8 mg, lysine 2 mg, and glutamine.
  • the amide is 0.6 mg, and the components and weight of the activator dissolved per mL of the aqueous solution of DMEM medium are as follows: 9 ⁇ g of deoxycholic acid, 20 ⁇ g of hydroxyethyl- ⁇ -cyclodextrin and 3 ⁇ g of sodium caseinate, wherein DMEM medium is aqueous solution.
  • the DMEM medium in the medium is a low glucose type DMEM medium.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin and a culture aid in an aqueous solution of DMEM medium, and dissolving gentamicin 100 U per mL of the DMEM medium solution, and the DMEM is cultured.
  • the concentration of the base aqueous solution is 18 mg/mL, per mL
  • the components and weights of the culture aid dissolved in the aqueous solution of DMEM medium are as follows: 5 ⁇ g of pullulan, 6 ⁇ g of glutathione, 20 ⁇ g of fatty acid lactyl ester, 30 ⁇ g of vitamin E, 1 ⁇ g of human serum albumin, 6 ⁇ g of ammonium nitrate, 3 ⁇ g of peptone and coconut. 15 ⁇ g of oleic acid monoethanolamide.
  • a skin spermatogonium cell protection solution which is prepared by dissolving gentamicin, a supplement and a culture aid in an aqueous solution of DMEM medium, and dissolving gentamicin 100 U per mL of the aqueous solution of DMEM medium.
  • concentration of the DMEM medium aqueous solution was 19 mg/mL, and the components and weights of the supplement dissolved in the DMEM medium aqueous solution per mL were as follows: polyglutamic acid 17 mg, phenylalanine 6 mg, lysine 1.5 mg, and valley.
  • DMEM medium aqueous solution 0.7 mg of aminoamide, and the components and weight of the culture aid dissolved per mL of the DMEM medium aqueous solution are as follows: 10 ⁇ g of pullulan, 10 ⁇ g of glutathione, 35 ⁇ g of fatty acid lactyl ester, 50 ⁇ g of vitamin E, and 3 ⁇ g of human serum albumin. 10 ⁇ g of ammonium nitrate, 5 ⁇ g of peptone and 20 ⁇ g of coconut oleic acid monoethanolamide, wherein the DMEM medium in the DMEM medium was a low-glucose DMEM medium.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin, a stimulating agent and a culture aid in an aqueous solution of DMEM medium, and dissolving gentamicin 100 U per mL of the aqueous solution of DMEM medium.
  • the concentration of the DMEM medium aqueous solution is 20 mg/mL, and the components and weight of the activator dissolved per mL of the DMEM medium aqueous solution are as follows: deoxycholic acid 7 ⁇ g, hydroxyethyl- ⁇ -cyclodextrin 17 ⁇ g, casein 2 ⁇ g of sodium, each component and weight of the culture aid dissolved in the DMEM medium solution per mL are as follows: 7.5 ⁇ g of pullulan, 8 ⁇ g of glutathione, 30 ⁇ g of fatty acid lactyl ester, 40 ⁇ g of vitamin E, 2 ⁇ g of human serum albumin, Ammonium nitrate 7.5 ⁇ g, peptone 4 ⁇ g, and coconut oleic acid monoethanolamide 17 ⁇ g.
  • a skin sperm cell protection solution for gentamicin and a time delay agent DMEM medium aqueous solution is dissolved, and gentamicin 110U is dissolved per mL of the DMEM medium aqueous solution, the concentration of the DMEM medium aqueous solution is 25 mg/mL, and each time the DMEM medium aqueous solution dissolves the delay agent
  • the ingredients and weights were as follows: 7.5 ⁇ g of lauroyl sarcosine, 50 ⁇ g of hydrogenated lecithin, and 1 ⁇ g of calcium monostearate.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin, a stimulating agent and a delaying agent in an aqueous solution of DMEM medium, and dissolving gentamicin 110 U per mL of the aqueous solution of DMEM medium.
  • concentration of the DMEM medium aqueous solution was 13 mg/mL, and the components and weight of the activator dissolved per mL of the DMEM medium aqueous solution were as follows: deoxycholic acid 8 ⁇ g, hydroxyethyl- ⁇ -cyclodextrin 18.5 ⁇ g, and cheese.
  • the components and weight of the delaying agent dissolved per mL of the aqueous solution of DMEM medium were as follows: 10 ⁇ g of lauroyl sarcosine, 35 ⁇ g of hydrogenated lecithin, and 3 ⁇ g of calcium monostearate.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin, a culture aid and a delay agent in an aqueous solution of DMEM medium, and dissolving gentamicin 110 U per mL of the aqueous solution of DMEM medium.
  • the concentration of the DMEM medium aqueous solution is 18 mg/mL
  • the components and weight of the culture aid dissolved in the DMEM medium aqueous solution per ml are as follows: 8 ⁇ g of pullulan, 7.5 ⁇ g of glutathione, 25 ⁇ g of fatty acid lactyl ester, Vitamin E37.5 ⁇ g, human serum albumin 2.5 ⁇ g, ammonium nitrate 9 ⁇ g, peptone 4.5 ⁇ g and coconut oleic acid monoethanolamide 17.5 ⁇ g
  • the components and weight of the delaying agent dissolved per mL of the DMEM medium aqueous solution are as follows: lauroyl 9 ⁇ g of sarcosine, 45 ⁇ g of hydrogenated lecithin, and 2 ⁇ g of calcium monostearate.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin, a stimulating agent, a culture aid and a delay agent in an aqueous solution of DMEM medium, and the DMEM is cultured per mL.
  • the aqueous solution of the base solution dissolves gentamicin 110 U, the concentration of the aqueous solution of the DMEM medium is 16.9 mg/mL, and the components and weight of the activator dissolved per mL of the aqueous solution of the DMEM medium are as follows: deoxycholic acid 7.2 ⁇ g, hydroxy 18.5 ⁇ g of ethyl- ⁇ -cyclodextrin and 2.2 ⁇ g of sodium caseinate, the components and weight of the culture aid dissolved per mL of the aqueous solution of DMEM medium were as follows: 9 ⁇ g of pullulan, 6.5 ⁇ g of glutathione, and fatty acid 22.5 ⁇ g of lactyl ester, 35 ⁇ g of vitamin E, 2.6 ⁇ g of human serum albumin, 6.5 ⁇ g of ammonium nitrate, 4.5 ⁇ g of peptone, and 16.5 ⁇ g of coconut oleic acid monoethanolamide, the components of the delaying agent
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin, a supplement, a stimulating agent and a delaying agent in an aqueous solution of DMEM medium, and dissolving G. glabrata per mL of the aqueous solution of DMEM medium.
  • the concentration of the DMEM medium aqueous solution is 23 mg/mL
  • the supplement component and weight dissolved per mL of the DMEM medium aqueous solution are as follows: polyglutamic acid 17 mg, phenylalanine 6 mg, lysine 1.5 Mg and glutamine 0.7 mg
  • each component and weight of the activator dissolved per mL of the DMEM medium aqueous solution are as follows: deoxycholic acid 7.2 ⁇ g, hydroxyethyl- ⁇ -cyclodextrin 18.5 ⁇ g and sodium caseinate 2.2 ⁇ g
  • each component and weight of the culture aid dissolved in the DMEM medium solution per mL are as follows: 9 ⁇ g of pullulan, 6.5 ⁇ g of glutathione, 22.5 ⁇ g of fatty acid lactyl ester, 35 ⁇ g of vitamin E, and 2.6 ⁇ g of human serum albumin.
  • DMEM medium 6.5 ⁇ g of ammonium nitrate, 4.5 ⁇ g of peptone, and 16.5 ⁇ g of coconut oleic acid monoethanolamide, the components and weight of the retarder dissolved per mL of the aqueous solution of DMEM medium are as follows: 9 ⁇ g of lauroyl sarcosine, 45 ⁇ g of hydrogenated lecithin, and single 1.5 ⁇ g calcium stearate, of which DMEM medium Low glucose DMEM media is DMEM medium type.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin and a stabilizer in an aqueous solution of DMEM medium, and dissolving the solution of the DMEM medium per mL.
  • concentration of the DMEM medium aqueous solution is 17.5 mg/mL
  • the components and weights of the stabilizer dissolved per mL of the DMEM medium aqueous solution are as follows: 15 ⁇ g of sodium ascorbate, 10 ⁇ g of sodium starch phosphate, and potassium citrate 6 ⁇ g.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin, a stimulating agent and a stabilizer in an aqueous solution of DMEM medium, and dissolving gentamicin 100 U per mL of the aqueous solution of DMEM medium.
  • concentration of the aqueous solution of DMEM medium is 15 mg/mL, and the components and weight of the activator dissolved per mL of the aqueous solution of DMEM medium are as follows: 7 ⁇ g of deoxycholic acid, 17 ⁇ g of hydroxyethyl- ⁇ -cyclodextrin and casein acid.
  • each component and weight of the stabilizer dissolved per mL of the DMEM medium aqueous solution were as follows: 20 ⁇ g of sodium ascorbate, 5 ⁇ g of sodium starch phosphate, and 3 ⁇ g of potassium citrate.
  • a skin spermatogonium cell protection solution which is prepared by dissolving gentamicin, a culture aid and a stabilizer in an aqueous solution of DMEM medium, and dissolving gentamicin 100 U per mL of the aqueous solution of DMEM medium.
  • concentration of the DMEM medium aqueous solution is 22 mg/mL
  • the components and weight of the culture aid dissolved in the DMEM medium aqueous solution per ml are as follows: 6 ⁇ g of pullulan, 7 ⁇ g of glutathione, 30 ⁇ g of fatty acid lactyl ester, and 40 ⁇ g of vitamin E.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin, a supplement, a culture aid, a stimulating agent and a stabilizer in an aqueous solution of DMEM medium, and is dissolved per mL of the DMEM medium solution.
  • Gentamicin 100 U, the aqueous solution of DMEM medium
  • the concentration of the supplement was 16.9 mg/mL
  • the components and weight of the supplement dissolved in the DMEM medium solution per mL were as follows: polyglutamic acid 17 mg, phenylalanine 6 mg, lysine 1.5 mg, and glutamine 0.7 mg.
  • the components and weight of the activator dissolved per mL of the aqueous solution of DMEM medium are as follows: 7 ⁇ g of deoxycholic acid, 17 ⁇ g of hydroxyethyl- ⁇ -cyclodextrin and 2 ⁇ g of sodium caseinate, dissolved in the aqueous solution of DMEM medium per mL.
  • the ingredients and weight of the culture aid are as follows: 6 ⁇ g of pullulan, 7 ⁇ g of glutathione, 30 ⁇ g of fatty acid lactyl ester, 40 ⁇ g of vitamin E, 2 ⁇ g of human serum albumin, 6 ⁇ g of ammonium nitrate, 5 ⁇ g of peptone, and 17 ⁇ g of coconut oleic acid monoethanolamide.
  • the components and weight of the delaying agent dissolved per mL of the DMEM medium aqueous solution are as follows: 9 ⁇ g of lauroyl sarcosine, 40 ⁇ g of hydrogenated lecithin, and 2 ⁇ g of calcium monostearate, and the solution is stable per mL of the aqueous solution of DMEM medium.
  • the components and weights of the agent were as follows: 18 ⁇ g of sodium ascorbate, 8 ⁇ g of sodium starch phosphate, and 4 ⁇ g of potassium citrate, wherein the DMEM medium in the aqueous solution of DMEM was a low-glucose DMEM medium.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving penicillin in an aqueous solution of DMEM medium, and dissolving the penicillin 80 U per mL of the DMEM medium aqueous solution, and the concentration of the DMEM medium aqueous solution is 13 mg/mL. .
  • a skin spermatogonium cell protection solution which is prepared by dissolving gentamicin and a supplement in an aqueous solution of DMEM medium, and dissolving the gentamicin 115 U per mL of the DMEM medium solution, the DMEM
  • the concentration of the aqueous medium solution was 21 mg/mL, and the components and weights of the supplement dissolved per mL of the DMEM medium aqueous solution were as follows: polyglutamic acid 20 mg, phenylalanine 6 mg, and lysine 1.5 mg.
  • a skin sperm cell protection solution for gentamicin and a stimulating agent The DMEM medium aqueous solution is dissolved, and the gentamicin 95U is dissolved per mL of the DMEM medium aqueous solution, and the concentration of the DMEM medium aqueous solution is 20 mg/mL, and each of the DMEM medium aqueous solution dissolved in each ml of the activator is dissolved.
  • the ingredients and weights were as follows: 6.5 ⁇ g of deoxycholic acid, 15 ⁇ g of hydroxyethyl- ⁇ -cyclodextrin, and 1 ⁇ g of trisodium citrate.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin and a stimulating agent in an aqueous solution of DMEM medium, and dissolving gentamicin 95 U per mL of the aqueous solution of DMEM medium, and the DMEM is cultured.
  • concentration of the base aqueous solution was 20 mg/mL
  • the components and weight of the activator dissolved per mL of the DMEM medium aqueous solution were as follows: 6.5 ⁇ g of deoxycholic acid and 1 ⁇ g of sodium caseinate.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin and a delay agent in an aqueous solution of DMEM medium, and dissolving gentamicin 110 U per mL of the aqueous solution of DMEM medium, and the DMEM is cultured.
  • concentration of the base aqueous solution was 25 mg/mL
  • the components and weights of the delayed agent dissolved per mL of the DMEM medium aqueous solution were as follows: 7.5 ⁇ g of lauroyl lysine, 50 ⁇ g of lecithin, and 1 ⁇ g of calcium monostearate.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin and a delay agent in an aqueous solution of DMEM medium, and dissolving gentamicin 110 U per mL of the aqueous solution of DMEM medium, and the DMEM is cultured.
  • concentration of the base aqueous solution was 25 mg/mL
  • the components and weights of the delayed agent dissolved per mL of the DMEM medium aqueous solution were as follows: 7.5 ⁇ g of lauroyl sarcosine and 50 ⁇ g of hydrogenated lecithin.
  • a skin sperm cell protection solution for gentamicin and culture aids DMEM medium aqueous solution is dissolved, and gentamicin 100 U is dissolved per mL of the DMEM medium aqueous solution, and the concentration of the DMEM medium aqueous solution is 18 mg/mL, and each of the DMEM medium aqueous solution dissolved in each of the culture aids
  • the components and weights were as follows: 5 ⁇ g of pullulan, 6 ⁇ g of glutathione, 20 ⁇ g of fatty acid lactyl ester, 30 ⁇ g of vitamin C, 1 ⁇ g of human serum albumin, 6 ⁇ g of ammonium nitrate, 3 ⁇ g of sodium pyruvate, and 15 ⁇ g of coconut oleic acid monoethanolamide.
  • the invention relates to a skin spermatogonium cell protection solution, which is prepared by dissolving gentamicin and a culture aid in an aqueous solution of DMEM medium, and dissolving gentamicin 100 U per mL of the DMEM medium solution, and the DMEM is cultured.
  • concentration of the base aqueous solution was 18 mg/mL
  • the components and weights of the culture aid dissolved per mL of the DMEM medium aqueous solution were as follows: glutathione 6 ⁇ g, fatty acid lactyl ester 20 ⁇ g, vitamin E 30 ⁇ g, ammonium nitrate 6 ⁇ g, peptone 3 ⁇ g and Coconut oleic acid monoethanolamide 15 ⁇ g.
  • a skin spermatogonium cell protection solution which is prepared by dissolving gentamicin and a stabilizer in an aqueous solution of DMEM medium, and dissolving gentamicin 100 U per mL of the DMEM medium solution, the DMEM medium
  • the concentration of the aqueous solution was 17.5 mg/mL
  • the components and weights of the stabilizer dissolved per mL of the aqueous DMEM medium solution were as follows: 15 ⁇ g of ascorbic acid, 10 ⁇ g of sodium starch phosphate, and 6 ⁇ g of citric acid.
  • a skin spermatogonium cell protection solution which is prepared by dissolving gentamicin and a stabilizer in an aqueous solution of DMEM medium, and dissolving gentamicin 100 U per mL of the DMEM medium solution, the DMEM medium
  • concentration of the aqueous solution was 17.5 mg/mL
  • the components and weights of the stabilizer dissolved per mL of the DMEM medium aqueous solution were as follows: 15 ⁇ g of sodium ascorbate and 6 ⁇ g of potassium citrate.
  • the spermatogonial cells were control 1-6 groups.
  • the skin cells and spermatogonia of each experimental group and control group entered the test procedure at 24h, 48h, 96h, 240h and 480h, respectively, and the density of skin cells and spermatogonia was adjusted to 1 ⁇ 10 6 cells/mL.
  • Mix well with cell suspension: 0.4% trypan blue 3:1 (v:v)
  • the viability of spermatogonial stem cells is shown in Table 1.
  • the cell viability of the skin cells and spermatogonia stored in the protective solution of the experimental group 1-2 was higher than that of the skin cells and spermatogonia stored separately using the protective solution used in the control group 1.
  • the cell viability of the skin cells and spermatogonia preserved by the protective solution used in the experimental group 3-5 was higher than that of the skin cells and spermatogonia stored separately using the protective solution used in the control group 2-4;
  • the skin cells and spermatogonia cells stored in the protective liquid used in the experimental group 6-7 were longer than the control group 5-6 and the experimental group 1-4, and reached 480 hours, and the survival rate still reached 50% or more. .
  • a surfactant composed of a mixture of sodium After that, it can significantly increase the activity of the protective liquid to preserve skin cells or spermatogonia, when polyglutamic acid, phenylalanine, lysine and glutamine and deoxycholic acid, hydroxyethyl- ⁇ -cyclodextrin
  • the survival rate of skin cells or spermatogonia is reduced after the lack of one of the components of sodium and sodium caseinate is replaced by other components; and lauroyl sarcosine, hydrogenated lecithin and single are added to the protective solution.
  • the mixture of calcium stearate can also increase the preservation time of the protective solution on skin cells or spermatogonia, and can save the storage time to 480h, when the three components of lauroyl sarcosine, hydrogenated lecithin and calcium monostearate
  • the skin cells or spermatogonia can only be preserved for 96 hours after they are either replaced or replaced by other components.
  • CN1935984 discloses an in vitro culture method for avian spermatogonia.
  • the cultured spermatogonia is a control group 1-3.
  • the skin cells and spermatogonia of each experimental group and control group were sampled for 2 days, and the cells were counted (Trypan blue staining method), and the total number of cells and amplification of the epidermal stem cells and spermatogonial stem cells in the protective solution were respectively performed. The multiples and cell viability were measured, and the results are shown in Table 2.
  • the protective solution provided by the embodiment 9 and the embodiment 11 of the present invention can significantly increase the expansion factor of the stem cells, and the amplification factor is significantly higher than that of the embodiment 7 and the control 1-3 group, and the protective solution
  • the above-mentioned ingredients added do not affect the viability of the stem cells, and it is concluded that the addition of a mixture of pullulan and glutathione in the protective solution provided by the present invention can significantly increase the expansion factor of the stem cells, but when When the composition changes, or a certain component is omitted, the expansion ratio of the protective solution to the stem cells is remarkably lowered.
  • the skin cells and spermatogonia which were separately stored in the protective solution of Example 17 and Example 20 were the experimental group 1-2, and the skin cells and spermatogonia cells respectively stored in the protective solution of Comparative Example 9-10 were used as the control 1-2. group.
  • the skin cells and spermatogonia of each experimental group and control group entered the test procedure at 24h, 48h, 96h, 240h and 480h, respectively.
  • the density of skin cells and spermatogonia was adjusted to 5 ⁇ 10 5 cells/mL, and 8 mL was inoculated.

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Abstract

一种皮肤精原干细胞保护液及其制备方法，该保护液主要是将氨基糖苷类抗生素用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解所述氨基糖糖苷类抗生素80-120U，该DMEM培养基水溶液的浓度为13-25mg/mL，上述皮肤精原干细胞保护液充分模拟了人体体内环境，更适合皮肤干细胞和精原干细胞的生存，并且该保护液性质稳定，能够显著提高皮肤干细胞和精原干细胞的存活率；同时在保存皮肤干细胞和精原干细胞的同时，还可以进一步扩大皮肤干细胞和精原干细胞的培养。

Description

一种皮肤精原细胞保护液及其制备方法 技术领域
本发明属于皮肤精原细胞保存领域，特别涉及一种皮肤精原细胞保护液及其制备方法。
背景技术
精原细胞属于雄性生殖细胞的早期发育阶段，能不断地进行细胞分裂，增加细胞数量，并分化为精母细胞。原始的胚细胞经分化形成精原细胞，精原细胞经复制形成初级精母细胞，初级经母细胞经过第一次减数分裂后形成次级精母细胞，再经过第二次减数分裂形成精细胞。目前研究较多的为精原干细胞。
皮肤作为人体最大的器官，具有感觉、调节体温、分泌及排泄等多种功能，并且其还可以作为人体与外界环境的屏障起到维持体内环境稳定性的作用，同时其也是免疫系统的重要组成部分。皮肤具有极强的修复和再生能力，这与皮肤干细胞的存在具有直接的关系，目前研究较多的有表皮干细胞。
目前，对于皮肤细胞或精原细胞的研究逐渐成为细胞领域研究的一个热点，但是基于现有技术公开的内容可知，对它们的研究过程中存在的最大问题就是皮肤细胞或精原细胞的保存和运输问题，其在存储过程中包括采集、运输、分离、培养等诸多步骤，实际操作中需要存储较长的时间，一旦皮肤细胞或精原细胞采集后，脱离了原有的体内环境，其活率就受到诸多因素的影响，诸如时间、温度、渗透压等。因此对于采集后的皮肤细胞或精原细胞的保存或保护是其存储或研究过程中必不可少的步骤。目前皮肤细胞或精原细胞的保存多采用直接装瓶或者补加基础培养基的方法来保存。直接装瓶的保存时间一般少于12小时，给皮肤 细胞或精原细胞的运输、交接和检测带来了不小的时间压力，也限制了其保存业务发展的范围；另外，通过加入基础培养基来保存皮肤细胞或精原细胞，因为基础培养基不能达到临床级别的要求，为它们的保存带来了风险，严重影响了表皮干细胞或精原干细胞的活率。此外，现有技术还没有公开一种能够同时保存皮肤细胞或精原细胞的干细胞保护液，并且现有技术CN105532641的一种胎盘保存液及其使用方法在保存皮肤细胞或精原细胞时，保存12h后，表皮干细胞或精原干细胞的活率就已消失；因此目前急需提供一种既能够保存皮肤细胞有能保存精原细胞的保护液。
发明内容
为了解决上述技术问题，本发明提供一种皮肤精原细胞保护液，该保护液模拟了人体体内环境，更适合皮肤细胞或精原细胞的培养和保存，并且能够显著提高皮肤细胞或精原细胞的活性。
本发明具体技术方案如下：
本发明一方面提供一种皮肤精原细胞保护液，该保护液主要是将氨基糖苷类抗生素用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解所述氨基糖糖苷类抗生素80-120U，所述DMEM培养基水溶液的浓度为13-25mg/mL。
其中，DMEM(Dulbecco’s modified eagle medisum)培养基为一种含各种氨基酸和葡萄糖的培养基。该DMEM培养基内各成分及含量如下：无水氯化钙265mg/L、九水硝酸铁0.1mg/L、氯化钾400mg/L、无水硫酸镁97.6mg/L 7、氯化钠6400mg/L、无水磷酸二氢钠109mg/L、丁二酸75mg/L、丁二酸钠100mg/L、L-盐酸精氨酸84mg/L、L-盐酸胱氨酸63mg/L、甘氨酸30mg/L、L-盐酸组氨酸42mg/L、L-异亮氨酸105mg/L、L-丝氨酸42mg/L、L-苏氨酸95mg/L、L-色氨酸16mg/L、L-酪氨酸72mg/L、L-缬氨酸94mg/L、D-泛酸钙4mg/L、酒石酸胆碱 7.2mg/L、叶酸4mg/L、肌醇7.2mg/L、烟酰胺4mg/L、核黄素0.4mg/L、盐酸硫胺4mg/L、盐酸吡哆辛4mg/L。
进一步的改进，氨基糖苷类抗生素选自庆大霉素、卡那霉素、阿米卡星或妥布霉素中的一种或多种，优选为重量份数比为5.5∶1∶1.2的庆大霉素、卡那霉素和妥布霉素的混合物。
本发明提供的保护液更接近人体体内环境，适合皮肤细胞或精原细胞的保存，同时加入的氨基糖苷类抗生素还可起到杀菌的效果。
进一步的改进，DMEM培养基水溶液中的DMEM培养基为低糖型DMEM培养基。
优选地，DMEM培养基水溶液的浓度为16.9mg。
进一步的改进，该保护液还包括补充剂，每mL所述DMEM培养基水溶液溶解的补充剂各成分及重量如下：多聚谷氨酸15-20mg、苯丙氨酸5-10mg、赖氨酸1-3mg和谷氨酰胺0.5-1mg。加入多聚谷氨酸、苯丙氨酸、赖氨酸和谷氨酰胺的混合物组成的补充剂可为皮肤细胞或精原细胞的生长提供更多的能量。
进一步的改进，保护液还包括增活剂，每mL所述DMEM培养基水溶液溶解的增溶剂各成分及重量如下：脱氧胆酸6.5-9μg、羟乙基-β-环糊精15-20μg、酪蛋白酸钠1-3μg。
本发明通过在保护液内加入脱氧胆酸、羟乙基-β-环糊精和酪蛋白酸钠的混合物组成的增活剂，能够深入到皮肤细胞或精原细胞内部结构中，能够与皮肤细胞或精原细胞内的活率肽形成包合物，具有降低冰点，减少冰晶形成、减轻细胞缩水程度、进而起到提高皮肤细胞或精原细胞活率的作用。
进一步的改进，保护液还包括培养助剂，每mL所述DMEM培养基 水溶液溶解的培养助剂各成分及重量如下：普鲁兰5-10μg、谷胱甘肽6-10μg、脂肪酸乳酰酯20-35μg、维生素E30-50μg、人血清白蛋白1-3μg、硝酸铵6-10μg、蛋白胨3-5μg和椰子油酸单乙醇酰胺15-20μg。
通过在保护液中加入由以上物质组成的培养助剂，不但能够保持表皮干细胞或精原干细胞的活率，同时能够与DMEM培养基水溶液一起，起到扩增皮肤细胞或精原细胞数目的目的。
进一步的改进，保护液还包括延时剂，每mL所述DMEM培养基水溶液溶解的延时剂各成分及重量如下：月桂酰肌氨酸7.5-10μg、氢化卵磷脂35-50μg、单硬脂酸钙1-3μg。
本发明通过在保护液内加入由月桂酰肌氨酸、氢化卵磷脂和单硬脂酸钙的混合物组成的延时剂，能够显著提高皮肤细胞或精原细胞的保存时间，并且能够提高各成分的溶解度。
进一步的改进，保护液还包括稳定剂，每mL所述DMEM培养基水溶液溶解的稳定剂各成分及重量如下：抗坏血酸钠15-20μg、磷酸淀粉钠5-10μg和枸橼酸钾3-6μg。
本发明通过在保护液内加入由抗坏血酸钠、磷酸淀粉钠和枸橼酸钾的混合物组成的稳定剂，主要起到维持皮肤细胞或精原细胞内的渗透平衡，提高皮肤细胞或精原细胞的贴壁率，同时能增加保护液的粘度，提高该保护液的稳定性。
本发明另一方面提供了一种皮肤精原细胞保护液的制备方法，该方法包括如下步骤：
1)取1/3体积的DMEM培养基水溶液，于10℃加入1/4单位的氨基糖苷类抗生素，混合均匀，静置5min；
2)再补加1/3体积的DMEM培养基水溶液，混匀，于10℃加入1/3 单位的氨基糖苷类抗生素，混合均匀，静置7min；
3)再补加剩余1/3体积的DMEM培养基水溶液，混匀，于10℃加入剩余5/12单位的氨基糖苷类抗生素，混合均匀，即得皮肤精原细胞保护液。
通过本发明制备的保护液澄清，能够提高表皮干细胞或精原干细胞的活率。
本发明所提供的保护液中，没有特殊指明制备方法的，所有的组分就是直接溶解到DMEM培养基水溶液中。
本发明的有益效果是：本发明提供的皮肤精原细胞保护液充分模拟了人体体内环境，更适合皮肤细胞或精原细胞的生存，并且该保护液性质稳定，能够显著提高表皮干细胞或精原干细胞的活率；同时在保存皮肤细胞或精原细胞的同时，还可以进一步扩增皮肤细胞或精原细胞的数目。
具体实施方式
实施例1
一种皮肤精原细胞保护液，该保护液是将庆大霉素用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解所述庆大霉素80U，所述DMEM培养基水溶液的浓度为13mg/mL。
实施例2
一种皮肤精原细胞保护液，该保护液是将卡那霉素用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解所述庆大霉素120U，所述DMEM培养基水溶液的浓度为25mg/mL，该保护液的制备方法包括如下步骤：
1)取1/3体积的DMEM培养基水溶液，于10℃加入1/4单位的卡那 霉素，混合均匀，静置5min；
2)再补加1/3体积的DMEM培养基水溶液，混匀，于10℃加入1/3单位的卡那霉素，混合均匀，静置7min；
3)再补加剩余1/3体积的DMEM培养基水溶液，混匀，于10℃加入剩余5/12单位的卡那霉素，混合均匀，即得皮肤精原细胞保护液。
实施例3
一种皮肤精原细胞保护液，该保护液是将庆大霉素、卡那霉素和妥布霉素用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素82.5U、卡那霉素15U、妥布霉素18U，所述DMEM培养基水溶液的浓度为16.9mg/mL。
实施例4
一种皮肤精原细胞保护液，该保护液是将庆大霉素用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素90U，所述DMEM培养基水溶液的浓度为16.9mg/mL，DMEM培养基水溶液中的DMEM培养基为低糖型DMEM培养基。
实施例5
一种皮肤精原细胞保护液，该保护液是将妥布霉素和补充剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解妥布霉素90U，所述DMEM培养基水溶液的浓度为21mg/mL，每mL所述DMEM培养基水溶液溶解的补充剂各成分及重量如下：多聚谷氨酸15mg、苯丙氨酸5mg、赖氨酸1mg和谷氨酰胺0.5mg。
实施例6
一种皮肤精原细胞保护液，该保护液是将庆大霉素和补充剂用 DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素100U，所述DMEM培养基水溶液的浓度为20mg/mL，DMEM培养基水溶液中的DMEM培养基为低糖型DMEM培养基，每mL所述DMEM培养基水溶液溶解的补充剂各成分及重量如下：多聚谷氨酸20mg、苯丙氨酸10mg、赖氨酸3mg和谷氨酰胺1mg。
实施例7
一种皮肤精原细胞保护液，该保护液是将庆大霉素和增活剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素95U，所述DMEM培养基水溶液的浓度为20mg/mL，每mL所述DMEM培养基水溶液溶解的增活剂各成分及重量如下：脱氧胆酸6.5μg、羟乙基-β-环糊精15μg和酪蛋白酸钠1μg。
实施例8
一种皮肤精原细胞保护液，该保护液是将庆大霉素、补充剂和增活剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素105U，所述DMEM培养基水溶液的浓度为15mg/mL，每mL所述DMEM培养基水溶液溶解的补充剂各成分及重量如下：多聚谷氨酸16mg、苯丙氨酸8mg、赖氨酸2mg和谷氨酰胺0.6mg，每mL所述DMEM培养基水溶液溶解的增活剂各成分及重量如下：脱氧胆酸9μg、羟乙基-β-环糊精20μg和酪蛋白酸钠3μg，其中DMEM培养基水溶液中的DMEM培养基为低糖型DMEM培养基。
实施例9
一种皮肤精原细胞保护液，该保护液是将庆大霉素和培养助剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素100U，所述DMEM培养基水溶液的浓度为18mg/mL，每mL所 述DMEM培养基水溶液溶解的培养助剂各成分及重量如下：普鲁兰5μg、谷胱甘肽6μg、脂肪酸乳酰酯20μg、维生素E30μg、人血清白蛋白1μg、硝酸铵6μg、蛋白胨3μg和椰子油酸单乙醇酰胺15μg。
实施例10
一种皮肤精原细胞保护液，该保护液是将庆大霉素、补充剂和培养助剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素100U，所述DMEM培养基水溶液的浓度为19mg/mL，每mL所述DMEM培养基水溶液溶解的补充剂各成分及重量如下：多聚谷氨酸17mg、苯丙氨酸6mg、赖氨酸1.5mg和谷氨酰胺0.7mg，每mL所述DMEM培养基水溶液溶解的培养助剂各成分及重量如下：普鲁兰10μg、谷胱甘肽10μg、脂肪酸乳酰酯35μg、维生素E50μg、人血清白蛋白3μg、硝酸铵10μg、蛋白胨5μg和椰子油酸单乙醇酰胺20μg，其中DMEM培养基水溶液中的DMEM培养基为低糖型DMEM培养基。
实施例11
一种皮肤精原细胞保护液，该保护液是将庆大霉素、增活剂和培养助剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素100U，所述DMEM培养基水溶液的浓度为20mg/mL，每mL所述DMEM培养基水溶液溶解的增活剂各成分及重量如下：脱氧胆酸7μg、羟乙基-β-环糊精17μg、酪蛋白酸钠2μg，每mL所述DMEM培养基水溶液溶解的培养助剂各成分及重量如下：普鲁兰7.5μg、谷胱甘肽8μg、脂肪酸乳酰酯30μg、维生素E40μg、人血清白蛋白2μg、硝酸铵7.5μg、蛋白胨4μg和椰子油酸单乙醇酰胺17μg。
实施例12
一种皮肤精原细胞保护液，该保护液是将庆大霉素和延时剂用 DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素110U，所述DMEM培养基水溶液的浓度为25mg/mL，每mL所述DMEM培养基水溶液溶解的延时剂各成分及重量如下：月桂酰肌氨酸7.5μg、氢化卵磷脂50μg和单硬脂酸钙1μg。
实施例13
一种皮肤精原细胞保护液，该保护液是将庆大霉素、增活剂和延时剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素110U，所述DMEM培养基水溶液的浓度为13mg/mL，每mL所述DMEM培养基水溶液溶解的增活剂各成分及重量如下：脱氧胆酸8μg、羟乙基-β-环糊精18.5μg和酪蛋白酸钠2.5μg，每mL所述DMEM培养基水溶液溶解的延时剂各成分及重量如下：月桂酰肌氨酸10μg、氢化卵磷脂35μg和单硬脂酸钙3μg。
实施例14
一种皮肤精原细胞保护液，该保护液是将庆大霉素、培养助剂和延时剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素110U，所述DMEM培养基水溶液的浓度为18mg/mL，每mL所述DMEM培养基水溶液溶解的培养助剂各成分及重量如下：普鲁兰8μg、谷胱甘肽7.5μg、脂肪酸乳酰酯25μg、维生素E37.5μg、人血清白蛋白2.5μg、硝酸铵9μg、蛋白胨4.5μg和椰子油酸单乙醇酰胺17.5μg，每mL所述DMEM培养基水溶液溶解的延时剂各成分及重量如下：月桂酰肌氨酸9μg、氢化卵磷脂45μg和单硬脂酸钙2μg。
实施例15
一种皮肤精原细胞保护液，该保护液是将庆大霉素、增活剂、培养助剂和延时剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培 养基水溶液溶解庆大霉素110U，所述DMEM培养基水溶液的浓度为16.9mg/mL，每mL所述DMEM培养基水溶液溶解的增活剂各成分及重量如下：脱氧胆酸7.2μg、羟乙基-β-环糊精18.5μg和酪蛋白酸钠2.2μg，每mL所述DMEM培养基水溶液溶解的培养助剂各成分及重量如下：普鲁兰9μg、谷胱甘肽6.5μg、脂肪酸乳酰酯22.5μg、维生素E35μg、人血清白蛋白2.6μg、硝酸铵6.5μg、蛋白胨4.5μg和椰子油酸单乙醇酰胺16.5μg，每mL所述DMEM培养基水溶液溶解的延时剂各成分及重量如下：月桂酰肌氨酸9μg、氢化卵磷脂45μg和单硬脂酸钙1.5μg。
实施例16
一种皮肤精原细胞保护液，该保护液是将庆大霉素、补充剂、增活剂和延时剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素110U，所述DMEM培养基水溶液的浓度为23mg/mL，每mL所述DMEM培养基水溶液溶解的补充剂成分及重量如下：多聚谷氨酸17mg、苯丙氨酸6mg、赖氨酸1.5mg和谷氨酰胺0.7mg，每mL所述DMEM培养基水溶液溶解的增活剂各成分及重量如下：脱氧胆酸7.2μg、羟乙基-β-环糊精18.5μg和酪蛋白酸钠2.2μg，每mL所述DMEM培养基水溶液溶解的培养助剂各成分及重量如下：普鲁兰9μg、谷胱甘肽6.5μg、脂肪酸乳酰酯22.5μg、维生素E35μg、人血清白蛋白2.6μg、硝酸铵6.5μg、蛋白胨4.5μg和椰子油酸单乙醇酰胺16.5μg，每mL所述DMEM培养基水溶液溶解的延时剂各成分及重量如下：月桂酰肌氨酸9μg、氢化卵磷脂45μg和单硬脂酸钙1.5μg，其中DMEM培养基水溶液中的DMEM培养基为低糖型DMEM培养基。
实施例17
一种皮肤精原细胞保护液，该保护液是将庆大霉素和稳定剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆 大霉素100U，所述DMEM培养基水溶液的浓度为17.5mg/mL，每mL所述DMEM培养基水溶液溶解的稳定剂各成分及重量如下：抗坏血酸钠15μg、磷酸淀粉钠10μg和枸橼酸钾6μg。
实施例18
一种皮肤精原细胞保护液，该保护液是将庆大霉素、增活剂和稳定剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素100U，所述DMEM培养基水溶液的浓度为15mg/mL，每mL所述DMEM培养基水溶液溶解的增活剂各成分及重量如下：脱氧胆酸7μg、羟乙基-β-环糊精17μg和酪蛋白酸钠2μg，每mL所述DMEM培养基水溶液溶解的稳定剂各成分及重量如下：抗坏血酸钠20μg、磷酸淀粉钠5μg和枸橼酸钾3μg。
实施例19
一种皮肤精原细胞保护液，该保护液是将庆大霉素、培养助剂和稳定剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素100U，所述DMEM培养基水溶液的浓度为22mg/mL，每mL所述DMEM培养基水溶液溶解的培养助剂各成分及重量如下：普鲁兰6μg、谷胱甘肽7μg、脂肪酸乳酰酯30μg、维生素E40μg、人血清白蛋白2μg、硝酸铵6μg、蛋白胨5μg和椰子油酸单乙醇酰胺17μg，每mL所述DMEM培养基水溶液溶解的稳定剂各成分及重量如下：抗坏血酸钠17.5μg、磷酸淀粉钠7.5μg和枸橼酸钾5μg。
实施例20
一种皮肤精原细胞保护液，该保护液是将庆大霉素、补充剂、培养助剂、增活剂和稳定剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素100U，所述DMEM培养基水溶液的 浓度为16.9mg/mL，每mL所述DMEM培养基水溶液溶解的补充剂各成分及重量如下：多聚谷氨酸17mg、苯丙氨酸6mg、赖氨酸1.5mg和谷氨酰胺0.7mg，每mL所述DMEM培养基水溶液溶解的增活剂各成分及重量如下：脱氧胆酸7μg、羟乙基-β-环糊精17μg和酪蛋白酸钠2μg，每mL所述DMEM培养基水溶液溶解的培养助剂各成分及重量如下：普鲁兰6μg、谷胱甘肽7μg、脂肪酸乳酰酯30μg、维生素E40μg、人血清白蛋白2μg、硝酸铵6μg、蛋白胨5μg和椰子油酸单乙醇酰胺17μg，每mL所述DMEM培养基水溶液溶解的延时剂各成分及重量如下：月桂酰肌氨酸9μg、氢化卵磷脂40μg、单硬脂酸钙2μg，每mL所述DMEM培养基水溶液溶解的稳定剂各成分及重量如下：抗坏血酸钠18μg、磷酸淀粉钠8μg和枸橼酸钾4μg，其中DMEM培养基水溶液中的DMEM培养基为低糖型DMEM培养基。
对照例1
一种皮肤精原细胞保护液，该保护液是将青霉素用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解所述青霉素80U，所述DMEM培养基水溶液的浓度为13mg/mL。
对照例2
一种皮肤精原细胞保护液，该保护液是将庆大霉素和补充剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解所述庆大霉素115U，所述DMEM培养基水溶液的浓度为21mg/mL，每mL所述DMEM培养基水溶液溶解的补充剂各成分及重量如下：多聚谷氨酸20mg、苯丙氨酸6mg、赖氨酸1.5mg。
对照例3
一种皮肤精原细胞保护液，该保护液是将庆大霉素和增活剂用 DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素95U，所述DMEM培养基水溶液的浓度为20mg/mL，每mL所述DMEM培养基水溶液溶解的增活剂各成分及重量如下：脱氧胆酸6.5μg、羟乙基-β-环糊精15μg和柠檬酸三钠1μg。
对照例4
一种皮肤精原细胞保护液，该保护液是将庆大霉素和增活剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素95U，所述DMEM培养基水溶液的浓度为20mg/mL，每mL所述DMEM培养基水溶液溶解的增活剂各成分及重量如下：脱氧胆酸6.5μg和酪蛋白酸钠1μg。
对照例5
一种皮肤精原细胞保护液，该保护液是将庆大霉素和延时剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素110U，所述DMEM培养基水溶液的浓度为25mg/mL，每mL所述DMEM培养基水溶液溶解的延时剂各成分及重量如下：月桂酰赖氨酸7.5μg、卵磷脂50μg和单硬脂酸钙1μg。
对照例6
一种皮肤精原细胞保护液，该保护液是将庆大霉素和延时剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素110U，所述DMEM培养基水溶液的浓度为25mg/mL，每mL所述DMEM培养基水溶液溶解的延时剂各成分及重量如下：月桂酰肌氨酸7.5μg和氢化卵磷脂50μg。
对照例7
一种皮肤精原细胞保护液，该保护液是将庆大霉素和培养助剂用 DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素100U，所述DMEM培养基水溶液的浓度为18mg/mL，每mL所述DMEM培养基水溶液溶解的培养助剂各成分及重量如下：普鲁兰5μg、谷胱甘肽6μg、脂肪酸乳酰酯20μg、维生素C30μg、人血清白蛋白1μg、硝酸铵6μg、丙酮酸钠3μg和椰子油酸单乙醇酰胺15μg。
对照例8
一种皮肤精原细胞保护液，该保护液是将庆大霉素和培养助剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素100U，所述DMEM培养基水溶液的浓度为18mg/mL，每mL所述DMEM培养基水溶液溶解的培养助剂各成分及重量如下：谷胱甘肽6μg、脂肪酸乳酰酯20μg、维生素E30μg、硝酸铵6μg、蛋白胨3μg和椰子油酸单乙醇酰胺15μg。
对照例9
一种皮肤精原细胞保护液，该保护液是将庆大霉素和稳定剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素100U，所述DMEM培养基水溶液的浓度为17.5mg/mL，每mL所述DMEM培养基水溶液溶解的稳定剂各成分及重量如下：抗坏血酸15μg、磷酸淀粉钠10μg和枸橼酸6μg。
对照例10
一种皮肤精原细胞保护液，该保护液是将庆大霉素和稳定剂用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解庆大霉素100U，所述DMEM培养基水溶液的浓度为17.5mg/mL，每mL所述DMEM培养基水溶液溶解的稳定剂各成分及重量如下：抗坏血酸钠15μg和枸橼酸钾6μg。
试验例1活率检测
以实施例1、3、5、7-8和12-13的保护液分别保存的皮肤细胞和精原细胞为实验1-7组，以对照例1-6的保护液分别保存的皮肤细胞和精原细胞为对照1-6组。各实验组和对照组的皮肤细胞和精原细胞分别在24h、48h、96h、240h和480h进入试验程序，调整皮肤细胞和精原细胞的密度均为1×106cells/mL。按细胞悬液∶0.4％台盼蓝＝3∶1(v∶v)充分混匀，取20μL细胞悬液加入细胞计数板中，用Countstar细胞计数器检测各实验组和对照组保护液内表皮干细胞和精原干细胞的活率，结果如表1所示。
表1实验组和对照组细胞活率结果
Figure PCTCN2016094676-appb-000001
Figure PCTCN2016094676-appb-000002
注：“--”表示没有活率。
由表1可知，实验1-2组的保护液分别保存的皮肤细胞和精原细胞的细胞活率要比使用对照1组使用的保护液分别保存的皮肤细胞和精原细胞的细胞活率高；实验3-5组使用的保护液分别保存的皮肤细胞和精原细胞的细胞活率要比使用对照2-4组使用的保护液分别保存的皮肤细胞和精原细胞的细胞活率高；并且实验6-7组使用的保护液分别保存的皮肤细胞和精原细胞的时间要比对照5-6组和实验1-4组的时间长，可达到480h，其活率仍达到50％以上。
由此得出，本发明提供的保护液中的氨基糖苷类抗生素换成了青霉素等抗生素后，皮肤细胞或精原细胞的活率显著降低。当保护液中加入由多聚谷氨酸、苯丙氨酸、赖氨酸和谷氨酰胺组成的补充剂或在保护液中加入脱氧胆酸、羟乙基-β-环糊精和酪蛋白酸钠混合物组成的增活剂 后，能够显著提高保护液保存皮肤细胞或精原细胞的活率，当多聚谷氨酸、苯丙氨酸、赖氨酸和谷氨酰胺及脱氧胆酸、羟乙基-β-环糊精和酪蛋白酸钠的成分中缺少其一或者被别的成分替代后，皮肤细胞或精原细胞的活率有所降低；并且在保护液中加入月桂酰肌氨酸、氢化卵磷脂和单硬脂酸钙的混合物还可提高保护液对皮肤细胞或精原细胞的保存时间，可将保存时间提高到480h，当月桂酰肌氨酸、氢化卵磷脂和单硬脂酸钙的三种成分缺少其一或被别的成分替代后，皮肤细胞或精原细胞的保存时间也只能维持在96h内。
试验例2干细胞扩增培养检测
以实施例7、实施例9和实施例11的保护液分别保存的皮肤细胞和精原细胞为实验1-3组，以对照例7-8的保护液分别保存的皮肤细胞和精原细胞和CN1935984公开的一种禽类精原细胞的体外培养方法培养的精原细胞为对照1-3组。各实验组和对照组的皮肤细胞和精原细胞各保存2天后取样，进行细胞计数(台盼蓝染色法)，对保护液内表皮干细胞和精原干细胞的活率分别进行细胞总数、扩增倍数、细胞活率的测定，结果见表2。
表2实验组和对照组的扩增培养结果
Figure PCTCN2016094676-appb-000003
Figure PCTCN2016094676-appb-000004
从表中可以看出，本发明实施例9和实施例11提供的保护液可显著提高干细胞的扩增倍数，其扩增倍数显著高于实施例7、对照1-3组，并且该保护液中加入的上述成分不影响干细胞的活率，由此得出，在本发明提供的保护液内加入普鲁兰、谷胱甘肽等混合物后，可以显著提高干细胞的扩增倍数，但当以上成分发生变化，或者省略某一成分后，该保护液对干细胞的扩增倍数显著降低。
试验例3贴壁率检测
以实施例17和实施例20的保护液分别保存的皮肤细胞和精原细胞为实验1-2组，以对照例9-10的保护液分别保存的皮肤细胞和精原细胞为对照1-2组。各实验组和对照组的皮肤细胞和精原细胞分别在24h、48h、96h、240h和480h进入试验程序，调整皮肤细胞和精原细胞的密度均为5×105cells/mL，接种8mL至直径为9cm的平皿中，待其自然贴壁生长48小时后，去除未贴壁的细胞后，用0.25m/v％胰蛋白酶进行酶解，计算出贴壁的表皮干细胞和精原干细胞的数量，得出贴壁率。结果如表3所示。
表3实验组和对照组细胞贴壁率结果
Figure PCTCN2016094676-appb-000005
Figure PCTCN2016094676-appb-000006
由表3可知，实验1-2组在保存96h后，细胞贴壁率仍保持在80％以上，贴壁效果明显好于对照1-2组，并且实验2组在保存480h后细胞贴壁率还保持在50％以上，由此得出，抗坏血酸钠、磷酸淀粉钠和枸橼酸钾的混合物可提高皮肤细胞或精原细胞的贴壁率，当其中一个成分变化或省略后，皮肤细胞或精原细胞的贴壁率显著降低。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对本发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。

Claims (10)

  1. 一种皮肤精原细胞保护液，其特征在于，所述保护液主要是将氨基糖苷类抗生素用DMEM培养基水溶液溶解而成，每mL所述DMEM培养基水溶液溶解所述氨基糖糖苷类抗生素80-120U，所述DMEM培养基水溶液的浓度为13-25mg/mL。
  2. 如权利要求1所述的皮肤精原细胞保护液，其特征在于，所述氨基糖苷类抗生素选自庆大霉素、卡那霉素、阿米卡星或妥布霉素中的一种或多种。
  3. 如权利要求1所述的皮肤精原细胞保护液，其特征在于，所述DMEM培养基水溶液中的DMEM培养基为低糖型DMEM培养基。
  4. 如权利要求1所述皮肤精原细胞保护液，其特征在于，所述DMEM培养基水溶液的浓度为16.9mg/mL。
  5. 如权利要求3所述的皮肤精原细胞保护液，其特征在于，所述保护液还包括补充剂，每mL所述DMEM培养基水溶液溶解的补充剂各成分及重量如下：多聚谷氨酸15-20mg、苯丙氨酸5-10mg、赖氨酸1-3mg和谷氨酰胺0.5-1mg。
  6. 如权利要求1所述的皮肤精原细胞保护液，其特征在于，所述保护液还包括增活剂，每mL所述DMEM培养基水溶液溶解的增活剂各成分及重量如下：脱氧胆酸6.5-9μg、羟乙基-β-环糊精15-20μg、酪蛋白酸钠1-3μg。
  7. 如权利要求1所述的皮肤精原细胞保护液，其特征在于，所述保护液还包括培养助剂，每mL所述DMEM培养基水溶液溶解的培养助剂各成 分及重量如下：普鲁兰5-10μg、谷胱甘肽6-10μg、脂肪酸乳酰酯20-35μg、维生素E30-50μg、人血清白蛋白1-3μg、硝酸铵6-10μg、蛋白胨3-5μg和椰子油酸单乙醇酰胺15-20μg。
  8. 如权利要求1所述的皮肤精原细胞保护液，其特征在于，所述保护液还包括延时剂，每mL所述DMEM培养基水溶液溶解的延时剂各成分及重量如下：月桂酰肌氨酸7.5-10μg、氢化卵磷脂35-50μg、单硬脂酸钙1-3μg。
  9. 如权利要求1所述的皮肤精原细胞保护液，其特征在于，所述保护液还包括稳定剂，每mL所述DMEM培养基水溶液溶解的稳定剂各成分及重量如下：抗坏血酸钠15-20μg、磷酸淀粉钠5-10μg和枸橼酸钾3-6μg。
  10. 一种权利要求1所述的皮肤精原细胞保护液的制备方法，其特征在于，所述方法包括如下步骤：
    1)取1/3体积的DMEM培养基水溶液，于10℃加入1/4单位的氨基糖苷类抗生素，混合均匀，静置5min；
    2)再补加1/3体积的DMEM培养基水溶液，混匀，于10℃加入1/3单位的氨基糖苷类抗生素，混合均匀，静置7min；
    3)再补加剩余1/3体积的DMEM培养基水溶液，混匀，于10℃加入剩余5/12单位的氨基糖苷类抗生素，混合均匀，即得皮肤精原细胞保护液。
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