CN107125240A - A kind of skin spermatogonium protection liquid and preparation method thereof - Google Patents

Abstract

The invention provides a protective solution for skin spermatogonial stem cells and a preparation method thereof. The protective solution is mainly formed by dissolving aminoglycoside antibiotics in a DMEM medium aqueous solution, and each mL of the DMEM medium aqueous solution dissolves the aminoglycosides Antibiotic 80-120U, the concentration of the DMEM medium aqueous solution is 13-25mg/mL, the skin spermatogonial stem cell protection solution provided by the present invention fully simulates the environment in the human body, is more suitable for the survival of skin stem cells and spermatogonial stem cells, and the The protective solution is stable in nature and can significantly increase the survival rate of skin stem cells and spermatogonial stem cells; meanwhile, while preserving skin stem cells and spermatogonial stem cells, it can further expand the cultivation of skin stem cells and spermatogonial stem cells.

Description

A kind of skin spermatogonia protective liquid and its preparation method

technical field

The invention belongs to the field of skin spermatogonia preservation, and in particular relates to a skin spermatogonia protection solution and a preparation method thereof.

Background technique

Spermatogonia belong to the early developmental stage of male germ cells, which can continuously divide cells, increase the number of cells, and differentiate into spermatocytes. Primitive germ cells are differentiated to form spermatogonia, spermatogonia are replicated to form primary spermatocytes, primary spermatocytes undergo the first meiosis to form secondary spermatocytes, and then undergo the second meiosis to form sperm cells. Spermatogonial stem cells are the most studied at present.

As the largest organ of the human body, the skin has multiple functions such as sensation, temperature regulation, 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, and it is also an important component of the immune system part. The skin has a strong ability to repair and regenerate, which is directly related to the existence of skin stem cells. At present, epidermal stem cells are more researched.

At present, the research on skin cells or spermatogonia has gradually become a hotspot in the field of cell research, but based on the content disclosed in the prior art, the biggest problem in the process of their research is the preservation and maintenance of skin cells or spermatogonia. Transportation problem, the storage process includes many steps such as collection, transportation, separation, and cultivation. In actual operation, it needs to be stored for a long time. Once the skin cells or spermatogonia are collected, they are separated from the original in vivo environment. The rate is affected by many factors, such as time, temperature, osmotic pressure and so on. Therefore, the preservation or protection of the collected skin cells or spermatogonia is an essential step in the storage or research process. At present, skin cells or spermatogonia are mostly preserved by directly bottling or adding basal medium. The storage time of direct bottling is generally less than 12 hours, which brings considerable time pressure to the transportation, handover and detection of skin cells or spermatogonia, and also limits the scope of its storage business development; in addition, by adding basic culture To preserve skin cells or spermatogonial cells, because the basal medium cannot meet the requirements of clinical grade, which brings risks to their preservation and seriously affects the viability of epidermal stem cells or spermatogonial stem cells. In addition, the prior art has not disclosed a stem cell protection solution that can preserve skin cells or spermatogonia at the same time, and a placenta preservation solution of the prior art CN105532641 and its method of use are used to preserve 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 that can preserve both skin cells and spermatogonial cells.

Contents of the invention

In order to solve the above-mentioned technical problems, the present invention provides a protective solution for skin spermatogonia, which simulates the environment in the human body, is more suitable for the cultivation and preservation of skin cells or spermatogonia, and can significantly improve the quality of skin cells or spermatogonia. activity.

Concrete technical scheme of the present invention is as follows:

One aspect of the present invention provides a protective solution for skin spermatogonia. The protective solution is mainly formed by dissolving aminoglycoside antibiotics with DMEM medium aqueous solution, and 80% of the aminoglycoside antibiotics are dissolved in each mL of the DMEM medium aqueous solution. -120U, the concentration of the DMEM medium aqueous solution is 13-25mg/mL.

Among them, DMEM (Dulbecco's modified eagle medisum) medium is a medium containing various amino acids and glucose. The components and contents of the DMEM medium are as follows: anhydrous calcium chloride 265mg/L, ferric nitrate nonahydrate 0.1mg/L, potassium chloride 400mg/L, anhydrous magnesium sulfate 97.6mg/L 7, sodium chloride 6400mg /L, anhydrous sodium dihydrogen phosphate 109mg/L, succinic acid 75mg/L, sodium succinate 100mg/L, L-arginine hydrochloride 84mg/L, L-cystine hydrochloride 63mg/L, glycine 30mg /L, L-histidine hydrochloride 42mg/L, L-isoleucine 105mg/L, L-serine 42mg/L, L-threonine 95mg/L, L-tryptophan 16mg/L, L- Tyrosine 72mg/L, L-valine 94mg/L, D-calcium pantothenate 4mg/L, choline bitartrate 7.2mg/L, folic acid 4mg/L, inositol 7.2mg/L, nicotinamide 4mg/L, Riboflavin 0.4mg/L, thiamine hydrochloride 4mg/L, pyridoxine hydrochloride 4mg/L.

As a further improvement, aminoglycoside antibiotics are selected from one or more of gentamicin, kanamycin, amikacin or tobramycin, preferably with a weight and number ratio of 5.5:1:1.2 A mixture of gentamicin, kanamycin and tobramycin.

The protective solution provided by the invention is closer to the environment in the human body, and is suitable for the preservation of skin cells or spermatogonia, and the aminoglycoside antibiotics added at the same time can also play a bactericidal effect.

As a further improvement, the DMEM medium in the DMEM medium aqueous solution is a low-sugar DMEM medium.

Preferably, the concentration of the aqueous DMEM medium solution is 16.9 mg.

As a further improvement, the protective solution also includes a supplement, and the ingredients and weight of the supplement dissolved in each mL of the DMEM medium aqueous solution are as follows: polyglutamic acid 15-20mg, phenylalanine 5-10mg, lysine 1-3mg and glutamine 0.5-1mg. Supplements that include a mixture of polyglutamic acid, phenylalanine, lysine, and glutamine provide more energy for the growth of skin cells or spermatogonia.

As a further improvement, the protection solution also includes a solubilizer, and the components and weight of the solubilizer dissolved per mL of the DMEM medium aqueous solution are as follows: 6.5-9 μg of deoxycholic acid, 15-20 μg of hydroxyethyl-β-cyclodextrin, Sodium caseinate 1-3 μg.

In the present invention, the activator composed of a mixture of deoxycholic acid, hydroxyethyl-β-cyclodextrin and sodium caseinate is added to the protective solution, which can penetrate deep into the inner structure of skin cells or spermatogonia, and can be combined with skin The activity rate peptides in the cells or spermatogonia form clathrates, which can lower the freezing point, reduce the formation of ice crystals, reduce the degree of cell shrinkage, and then play a role in increasing the activity rate of skin cells or spermatogonia.

As a further improvement, the protective solution also includes a culture aid, and the ingredients and weight of the culture aid dissolved in each mL of the DMEM medium aqueous solution are as follows: 5-10 μg of pullulan, 6-10 μg of glutathione, fatty acid lactoyl ester 20-35μg, vitamin E30-50μg, human serum albumin 1-3μg, ammonium nitrate 6-10μg, peptone 3-5μg and coconut oleic acid monoethanolamide 15-20μg.

By adding a culture aid consisting of the above substances to the protective solution, not only can the viability of epidermal stem cells or spermatogonial stem cells be maintained, but also the number of skin cells or spermatogonial cells can be expanded together with the DMEM medium aqueous solution .

As a further improvement, the protective solution also includes a time-delay agent, and the components and weight of the time-delay agent dissolved in each mL of the DMEM medium aqueous solution are as follows: 7.5-10 μg of lauroyl sarcosine, 35-50 μg of hydrogenated lecithin, 35-50 μg of monostearin Calcium acid 1-3μg.

The present invention can significantly increase the preservation time of skin cells or spermatogonia by adding a time-delay agent composed of a mixture of lauroyl sarcosine, hydrogenated lecithin and calcium monostearate in the protection solution, and can improve the preservation time of each component. solubility.

As a further improvement, the protection solution also includes a stabilizer, and the components and weight of the stabilizer dissolved in each mL of the DMEM medium aqueous solution are as follows: 15-20 μg of sodium ascorbate, 5-10 μg of sodium starch phosphate and 3-6 μg of potassium citrate.

The present invention mainly plays a role in maintaining the osmotic balance in skin cells or spermatogonia by adding a stabilizer composed of a mixture of sodium ascorbate, sodium starch phosphate and potassium citrate in the protective solution, and improving the osmotic balance of skin cells or spermatogonia. At the same time, it can increase the viscosity of the protective solution and improve the stability of the protective solution.

Another aspect of the present invention provides a method for preparing a skin spermatogonia protective solution, the method comprising the steps of:

1) Take 1/3 volume of DMEM medium aqueous solution, add 1/4 unit of aminoglycoside antibiotics at 10°C, mix well, and let stand for 5 minutes;

2) Add 1/3 volume of DMEM medium aqueous solution, mix well, add 1/3 unit of aminoglycoside antibiotics at 10°C, mix well, and let stand for 7 minutes;

3) Add the remaining 1/3 volume of DMEM medium aqueous solution, mix well, add the remaining 5/12 units of aminoglycoside antibiotics at 10°C, mix well, and obtain the skin spermatogonial cell protection solution.

The clarification of the protective solution prepared by the invention can improve the viability of epidermal stem cells or spermatogonial stem cells.

In the protective solution provided by the present invention, if there is no special preparation method specified, all the components are directly dissolved in the DMEM medium aqueous solution.

The beneficial effects of the present invention are: the protective solution for skin spermatogonia provided by the present invention fully simulates the environment in the human body, is more suitable for the survival of skin cells or spermatogonia, and the protective solution is stable in nature, and can significantly improve the concentration of epidermal stem cells or spermatogonia. The activity 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.

detailed description

Example 1

A kind of skin spermatogonia protection liquid, this protection liquid is that gentamicin is dissolved with DMEM medium aqueous solution, every mL described DMEM medium aqueous solution dissolves described gentamycin 80U, and described DMEM medium aqueous solution The concentration is 13mg/mL.

Example 2

A kind of skin spermatogonia protection liquid, this protection liquid is that kanamycin is dissolved with DMEM medium aqueous solution, every mL described DMEM medium aqueous solution dissolves 120U of described gentamycin, and described DMEM medium aqueous solution The concentration is 25mg/mL, and the preparation method of this protective solution comprises the steps:

1) Take 1/3 volume of DMEM medium aqueous solution, add 1/4 unit of kanamycin at 10°C, mix well, and let stand for 5 minutes;

2) Add 1/3 volume of DMEM medium aqueous solution, mix well, add 1/3 unit of kanamycin at 10°C, mix well, and let stand for 7 minutes;

3) Add the remaining 1/3 volume of DMEM medium aqueous solution, mix well, add the remaining 5/12 units of kanamycin at 10°C, mix well, and obtain the skin spermatogonial cell protection solution.

Example 3

A skin spermatogonial cell protection solution, which is formed by dissolving gentamicin, kanamycin and tobramycin with DMEM medium aqueous solution, and dissolving gentamicin in every mL of the DMEM medium aqueous solution 82.5U, kanamycin 15U, tobramycin 18U, the concentration of the DMEM medium aqueous solution is 16.9mg/mL.

Example 4

A kind of skin spermatogonia protection solution, this protection solution is that gentamicin is dissolved with DMEM medium aqueous solution, every mL of described DMEM medium aqueous solution dissolves gentamicin 90U, the concentration of described DMEM medium aqueous solution 16.9mg/mL, the DMEM medium in the DMEM medium aqueous solution is a low-sugar DMEM medium.

Example 5

A kind of skin spermatogonia protection solution, this protection solution is that tobramycin and supplement are dissolved with DMEM medium aqueous solution, and per mL of described DMEM medium aqueous solution dissolves tobramycin 90U, and described DMEM medium The concentration of the aqueous solution is 21 mg/mL, and the components and weights of supplements dissolved in each mL of the DMEM medium aqueous solution are as follows: polyglutamic acid 15 mg, phenylalanine 5 mg, lysine 1 mg and glutamine 0.5 mg.

Example 6

A kind of skin spermatogonia protection liquid, this protection liquid is that gentamicin and supplement are dissolved with DMEM medium aqueous solution, every mL described DMEM medium aqueous solution dissolves 100U of gentamycin, described DMEM medium The concentration of the aqueous solution is 20mg/mL, and the DMEM medium in the DMEM medium aqueous solution is a low-sugar DMEM medium, and the ingredients and weight of the supplement dissolved in the DMEM medium aqueous solution per mL are as follows: polyglutamic acid 20mg, benzene Alanine 10mg, Lysine 3mg and Glutamine 1mg.

Example 7

A kind of skin spermatogonia protection liquid, this protection liquid is that gentamicin and activator are dissolved with DMEM culture medium aqueous solution, every mL described DMEM medium aqueous solution dissolves gentamicin 95U, and described DMEM culture The concentration of the base aqueous solution is 20 mg/mL, and the components and weights of the activator dissolved in each mL of the DMEM medium aqueous solution are as follows: 6.5 μg of deoxycholic acid, 15 μg of hydroxyethyl-β-cyclodextrin and 1 μg of sodium caseinate .

Example 8

A kind of skin spermatogonia protection liquid, this protection liquid is that gentamicin, replenisher and activator are dissolved with DMEM medium aqueous solution, and every mL of described DMEM medium aqueous solution dissolves gentamicin 105U, so The concentration of the aqueous DMEM medium solution is 15 mg/mL, and the components and weight of supplements dissolved in the aqueous DMEM medium solution per mL are as follows: 16 mg polyglutamic acid, 8 mg phenylalanine, 2 mg lysine and glutamic acid Amide 0.6 mg, the ingredients and weight of the activator dissolved in the aqueous DMEM medium solution per mL are as follows: 9 μg of deoxycholic acid, 20 μg of hydroxyethyl-β-cyclodextrin and 3 μg of sodium caseinate, wherein the aqueous solution of DMEM medium The DMEM medium in is a low-sugar DMEM medium.

Example 9

A kind of skin spermatogonia protection solution, this protection solution is to dissolve gentamicin and culture aid with DMEM medium aqueous solution, every mL of described DMEM medium aqueous solution dissolves 100U of gentamycin, and described DMEM culture The concentration of the base aqueous solution is 18 mg/mL, and the ingredients and weights of the culture aids dissolved in the DMEM medium aqueous solution per mL are as follows: 5 μg of pullulan, 6 μg of glutathione, 20 μg of fatty acid lactoyl ester, 30 μg of vitamin E, human serum Albumin 1 μg, ammonium nitrate 6 μg, peptone 3 μg and coconut oleic acid monoethanolamide 15 μg.

Example 10

A kind of skin spermatogonia protection liquid, this protection liquid is that gentamycin, replenisher and culture aid are dissolved with DMEM medium aqueous solution and form, every mL described DMEM medium aqueous solution dissolves gentamycin 100U, so The concentration of the aqueous DMEM medium solution is 19 mg/mL, and the ingredients and weight of the supplements dissolved in the aqueous DMEM medium solution per mL are as follows: polyglutamic acid 17mg, phenylalanine 6mg, lysine 1.5mg and glutamic acid Aminoamide 0.7 mg, the components and weights of the culture aids dissolved in the DMEM medium aqueous solution per mL are as follows: 10 μg of pullulan, 10 μg of glutathione, 35 μg of fatty acid lactoyl ester, 50 μg of vitamin E, 3 μg of human serum albumin, 10 μg of ammonium nitrate, 5 μg of peptone and 20 μg of coconut oleic acid monoethanolamide, wherein the DMEM medium in the DMEM medium aqueous solution is a low-sugar DMEM medium.

Example 11

A kind of skin spermatogonia protective liquid, this protective liquid is that gentamycin, activator and culture aid are dissolved with DMEM medium aqueous solution, and per mL described DMEM medium aqueous solution dissolves 100U of gentamycin, The concentration of the aqueous DMEM medium solution is 20 mg/mL, and the components and weights of the activators dissolved in the aqueous DMEM medium solution per mL are as follows: 7 μg of deoxycholic acid, 17 μg of hydroxyethyl-β-cyclodextrin, casein The components and weights of the culture aids dissolved in each mL of the DMEM medium aqueous solution are as follows: 7.5 μg of pullulan, 8 μg of glutathione, 30 μg of fatty acid lactoyl ester, 40 μg of vitamin E, 2 μg of human serum albumin, Ammonium nitrate 7.5 μg, peptone 4 μg and coconut oleic acid monoethanolamide 17 μg.

Example 12

A kind of skin spermatogonia protection solution, this protection solution is to dissolve gentamicin and time-delay agent with DMEM medium aqueous solution, every mL of described DMEM medium aqueous solution dissolves 110U of gentamycin, and described DMEM culture The concentration of the base aqueous solution is 25 mg/mL, and the components and weights of the delay agent dissolved in each mL of the DMEM medium aqueous solution are as follows: 7.5 μg of lauroyl sarcosine, 50 μg of hydrogenated lecithin and 1 μg of calcium monostearate.

Example 13

A kind of skin spermatogonia protection liquid, this protection liquid is that gentamicin, activator and time-delay agent are dissolved with DMEM medium aqueous solution, and every mL described DMEM medium aqueous solution dissolves 110U of gentamicin, The concentration of the aqueous DMEM medium solution is 13 mg/mL, and the components and weights of the activators dissolved in the aqueous DMEM medium solution per mL are as follows: 8 μg of deoxycholic acid, 18.5 μg of hydroxyethyl-β-cyclodextrin and phenolic acid 2.5 μg of sodium proteinate, and the ingredients and weight of the time-delay agent dissolved in the DMEM medium aqueous solution per mL are as follows: 10 μg of lauroyl sarcosine, 35 μg of hydrogenated lecithin and 3 μg of calcium monostearate.

Example 14

A kind of skin spermatogonia protection liquid, this protection liquid is to form by dissolving gentamicin, culture aid and time delay agent with DMEM medium aqueous solution, every mL of described DMEM medium aqueous solution dissolves 110U of gentamicin, The concentration of the aqueous DMEM medium solution is 18 mg/mL, and the components and weights of the culture aids dissolved in the aqueous DMEM medium solution per mL are as follows: 8 μg of pullulan, 7.5 μg of glutathione, 25 μg of fatty acid lactoyl ester, 37.5 μg of vitamin E, 2.5 μg of human serum albumin, 9 μg of ammonium nitrate, 4.5 μg of peptone and 17.5 μg of coconut oleic acid monoethanolamide, the ingredients and weight of the delay agent dissolved in the DMEM medium aqueous solution per mL are as follows: Sarcosine 9μg, hydrogenated lecithin 45μg and calcium monostearate 2μg.

Example 15

A skin spermatogonia protective solution, which is formed by dissolving gentamicin, activator, culture aid and delay agent with DMEM medium aqueous solution, and the DMEM aqueous medium solution of each mL dissolves gentamicin Mycin 110U, the concentration of the DMEM medium aqueous solution is 16.9mg/mL, the components and weight of the activator dissolved in each mL of the DMEM medium aqueous solution are as follows: 7.2 μg of deoxycholic acid, hydroxyethyl-β-cyclo Dextrin 18.5 μg and sodium caseinate 2.2 μg, the components and weights of the culture aids dissolved in each mL of the DMEM medium aqueous solution are as follows: pullulan 9 μg, glutathione 6.5 μg, fatty acid lactoyl ester 22.5 μg, 35 μg of vitamin E, 2.6 μg of human serum albumin, 6.5 μg of ammonium nitrate, 4.5 μg of peptone and 16.5 μg of coconut oleic acid monoethanolamide, the ingredients and weight of the delay agent dissolved in the DMEM medium aqueous solution per mL are as follows: Amino acid 9μg, hydrogenated lecithin 45μg and calcium monostearate 1.5μg.

Example 16

A kind of skin spermatogonia protection liquid, this protection liquid is to dissolve gentamicin, supplement, activator and time-delay agent with DMEM medium aqueous solution, every mL of described DMEM medium aqueous solution dissolves gentamicin Supplement 110U, the concentration of described DMEM medium aqueous solution is 23mg/mL, and the replenisher composition and the weight that every mL described DMEM medium aqueous solution dissolves are as follows: Polyglutamic acid 17mg, phenylalanine 6mg, lysine 1.5 mg and glutamine 0.7 mg, the components and weight of the activator dissolved in the DMEM medium aqueous solution per mL are as follows: 7.2 μg of deoxycholic acid, 18.5 μg of hydroxyethyl-β-cyclodextrin and 2.2 μg of sodium caseinate μg, the components and weights of the culture aids dissolved in the DMEM medium aqueous solution per mL are as follows: 9 μg of pullulan, 6.5 μg of glutathione, 22.5 μg of fatty acid lactoyl ester, 35 μg of vitamin E, 2.6 μg of human serum albumin, 6.5 μg of ammonium nitrate, 4.5 μg of peptone and 16.5 μg of coconut oleic acid monoethanolamide, the components and weight of the delay agent dissolved in the DMEM medium aqueous solution per mL are as follows: 9 μg of lauroyl sarcosine, 45 μg of hydrogenated lecithin and mono Calcium stearate 1.5 μg, wherein the DMEM medium in the DMEM medium aqueous solution is a low-sugar DMEM medium.

Example 17

A kind of skin spermatogonia protection solution, this protection solution is that gentamicin and stabilizer are dissolved with DMEM medium aqueous solution, every mL of described DMEM medium aqueous solution dissolves gentamycin 100U, described DMEM medium The concentration of the aqueous solution is 17.5 mg/mL, and the components and weights of the stabilizers dissolved in each mL of the DMEM medium aqueous solution are as follows: 15 μg of sodium ascorbate, 10 μg of sodium starch phosphate and 6 μg of potassium citrate.

Example 18

A kind of skin spermatogonia protection solution, this protection solution is that gentamicin, activator and stabilizer are dissolved with DMEM medium aqueous solution, and the DMEM medium aqueous solution of every mL dissolves gentamycin 100U, so The concentration of the aqueous DMEM medium solution is 15 mg/mL, and the components and weight of the activator dissolved in the aqueous DMEM medium solution per mL are as follows: 7 μg of deoxycholic acid, 17 μg of hydroxyethyl-β-cyclodextrin and caseinic acid Sodium 2 μg, the components and weights of stabilizers dissolved per mL of the DMEM medium aqueous solution are as follows: 20 μg sodium ascorbate, 5 μg sodium starch phosphate and 3 μg potassium citrate.

Example 19

A kind of skin spermatogonia protection liquid, this protection liquid is that gentamicin, culture aid and stabilizing agent are dissolved with DMEM medium aqueous solution, and the DMEM medium aqueous solution of every mL dissolves gentamycin 100U, so The concentration of the aqueous DMEM medium solution is 22 mg/mL, and the components and weights of the culture aids dissolved in the aqueous DMEM medium solution per mL are as follows: 6 μg of pullulan, 7 μg of glutathione, 30 μg of fatty acid lactoyl ester, and 40 μg of vitamin E , 2 μg of human serum albumin, 6 μg of ammonium nitrate, 5 μg of peptone and 17 μg of coconut oleic acid monoethanolamide. and potassium citrate 5 μg.

Example 20

A kind of skin spermatogonia protection liquid, this protection liquid is that gentamicin, supplement, culture aid, activator and stabilizer are dissolved with DMEM medium aqueous solution, and every mL of described DMEM medium aqueous solution dissolves Gentamicin 100U, the concentration of the DMEM medium aqueous solution is 16.9mg/mL, the ingredients and weight of the supplement dissolved in each mL of the DMEM medium aqueous solution are as follows: polyglutamic acid 17mg, phenylalanine 6mg , 1.5 mg of lysine and 0.7 mg of glutamine, each component and weight of the activator dissolved in the DMEM medium aqueous solution per mL are as follows: 7 μg of deoxycholic acid, 17 μg of hydroxyethyl-β-cyclodextrin and casein The components and weights of the culture aids dissolved in each mL of the DMEM medium aqueous solution are as follows: 6 μg of pullulan, 7 μg of glutathione, 30 μg of fatty acid lactoyl ester, 40 μg of vitamin E, 2 μg of human serum albumin, nitric acid Ammonium 6 μg, peptone 5 μg and coconut oleic acid monoethanolamide 17 μg, the ingredients and weight of the delay agent dissolved in the DMEM medium aqueous solution per mL are as follows: 9 μg of lauroyl sarcosine, 40 μg of hydrogenated lecithin, calcium monostearate 2 μg, the components and weight of the stabilizer dissolved in the DMEM medium aqueous solution per mL are as follows: 18 μg sodium ascorbate, 8 μg sodium starch phosphate and 4 μg potassium citrate, wherein the DMEM medium in the DMEM medium aqueous solution is a low-sugar DMEM culture base.

Comparative example 1

A skin spermatogonia protective solution, which is formed by dissolving penicillin with an aqueous DMEM medium solution, 80 U of penicillin is dissolved in each mL of the aqueous DMEM medium solution, and the concentration of the aqueous DMEM medium solution is 13 mg/mL .

Comparative example 2

A kind of skin spermatogonia protection liquid, this protection liquid is that gentamicin and supplement are dissolved with DMEM medium aqueous solution, every mL described DMEM medium aqueous solution dissolves described gentamycin 115U, and described DMEM The concentration of the aqueous medium solution is 21 mg/mL, and the components and weights of supplements dissolved in each mL of the DMEM aqueous medium solution are as follows: 20 mg polyglutamic acid, 6 mg phenylalanine, and 1.5 mg lysine.

Comparative example 3

A kind of skin spermatogonia protection liquid, this protection liquid is that gentamicin and activator are dissolved with DMEM culture medium aqueous solution, every mL described DMEM medium aqueous solution dissolves gentamicin 95U, and described DMEM culture The concentration of the base aqueous solution is 20 mg/mL, and the components and weights of the activator dissolved in each mL of the DMEM medium aqueous solution are as follows: 6.5 μg of deoxycholic acid, 15 μg of hydroxyethyl-β-cyclodextrin and 1 μg of trisodium citrate .

Comparative example 4

A kind of skin spermatogonia protection liquid, this protection liquid is that gentamicin and activator are dissolved with DMEM culture medium aqueous solution, every mL described DMEM medium aqueous solution dissolves gentamicin 95U, and described DMEM culture The concentration of the base aqueous solution is 20 mg/mL, and the components and weights of the activator dissolved in each mL of the DMEM medium aqueous solution are as follows: 6.5 μg of deoxycholic acid and 1 μg of sodium caseinate.

Comparative example 5

A kind of skin spermatogonia protection solution, this protection solution is to dissolve gentamicin and time-delay agent with DMEM medium aqueous solution, every mL of described DMEM medium aqueous solution dissolves 110U of gentamycin, and described DMEM culture The concentration of the base aqueous solution is 25 mg/mL, and the ingredients and weight of the delay agent dissolved in the DMEM medium aqueous solution per mL are as follows: 7.5 μg of lauroyl lysine, 50 μg of lecithin and 1 μg of calcium monostearate.

Comparative example 6

A kind of skin spermatogonia protection solution, this protection solution is to dissolve gentamicin and time-delay agent with DMEM medium aqueous solution, every mL of described DMEM medium aqueous solution dissolves 110U of gentamycin, and described DMEM culture The concentration of the base aqueous solution is 25 mg/mL, and the components and weights of the delay agent dissolved in each mL of the DMEM medium aqueous solution are as follows: 7.5 μg of lauroyl sarcosine and 50 μg of hydrogenated lecithin.

Comparative example 7

A kind of skin spermatogonia protection solution, this protection solution is to dissolve gentamicin and culture aid with DMEM medium aqueous solution, every mL of described DMEM medium aqueous solution dissolves 100U of gentamycin, and described DMEM culture The concentration of the aqueous base solution is 18 mg/mL, and the ingredients and weights of the culture aids dissolved in each mL of the DMEM medium aqueous solution are as follows: 5 μg of pullulan, 6 μg of glutathione, 20 μg of fatty acid lactoyl esters, 30 μg of vitamin C, and human serum Albumin 1 μg, ammonium nitrate 6 μg, sodium pyruvate 3 μg and coconut oleic acid monoethanolamide 15 μg.

Comparative example 8

A kind of skin spermatogonia protection solution, this protection solution is to dissolve gentamicin and culture aid with DMEM medium aqueous solution, every mL of described DMEM medium aqueous solution dissolves 100U of gentamycin, and described DMEM culture The concentration of the base aqueous solution is 18 mg/mL, and the ingredients and weights of the culture aids dissolved in each mL of the DMEM medium aqueous solution are as follows: 6 μg of glutathione, 20 μg of fatty acid lactoyl ester, 30 μg of vitamin E, 6 μg of ammonium nitrate, 3 μg of peptone and Coconut Oleic Acid Monoethanolamide 15 μg.

Comparative example 9

A kind of skin spermatogonia protection solution, this protection solution is that gentamicin and stabilizer are dissolved with DMEM medium aqueous solution, every mL of described DMEM medium aqueous solution dissolves gentamycin 100U, described DMEM medium The concentration of the aqueous solution is 17.5 mg/mL, and the components and weights of the stabilizers dissolved in each mL of the DMEM medium aqueous solution are as follows: 15 μg of ascorbic acid, 10 μg of sodium starch phosphate and 6 μg of citric acid.

Comparative example 10

A kind of skin spermatogonia protection solution, this protection solution is that gentamicin and stabilizer are dissolved with DMEM medium aqueous solution, every mL of described DMEM medium aqueous solution dissolves gentamycin 100U, described DMEM medium The concentration of the aqueous solution is 17.5 mg/mL, and the components and weights of the stabilizers dissolved in each mL of the DMEM medium aqueous solution are as follows: 15 μg of sodium ascorbate and 6 μg of potassium citrate.

Test example 1 Viability detection

The skin cells and spermatogonia preserved respectively with the protective solutions of Examples 1, 3, 5, 7-8 and 12-13 were used as experimental 1-7 groups, and the skin cells and spermatogonia preserved respectively with the protective solutions of Comparative Examples 1-6 Spermatogonia were the control group 1-6. The skin cells and spermatogonia of each experimental group and control group entered the test program at 24h, 48h, 96h, 240h and 480h respectively, and the density of skin cells and spermatogonia was adjusted to 1×10 ⁶ cells/mL. According to the cell suspension: 0.4% trypan blue = 3:1 (v:v), mix thoroughly, take 20 μL of the cell suspension and add it to the cell counting plate, and use the Countstar cell counter to detect the epidermal stem cells in the protection solution of each experimental group and control group and the activity rate of spermatogonial stem cells, the results are shown in Table 1.

Table 1 The results of the cell viability of the experimental group and the control group

Note: "--" indicates no survival rate.

It can be seen from Table 1 that the cell viability of the skin cells and spermatogonia preserved in the protective solution of the experimental group 1-2 is higher than the cell viability of the skin cells and spermatogonia preserved in 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 experiment 3-5 groups is higher than the cell viability of the skin cells and spermatogonia preserved by the protective solution used by the control group 2-4; In addition, the skin cells and spermatogonia preserved by the protective solution used in the experiment 6-7 group are longer than those of the control group 5-6 and the experiment 1-4 group, up to 480 hours, and the activity rate still reaches more than 50%. .

It can be concluded that, after the aminoglycoside antibiotics in the protective solution provided by the present invention are replaced with antibiotics such as penicillin, the viability of skin cells or spermatogonia is significantly reduced. When a supplement consisting of polyglutamic acid, phenylalanine, lysine, and glutamine is added to the protective solution or deoxycholic acid, hydroxyethyl-β-cyclodextrin, and casein are added to the protective solution After the activator composed of sodium acid mixture, it can significantly improve the activity rate of skin cells or spermatogonia preserved in the protective solution. When polyglutamic acid, phenylalanine, lysine and glutamine and deoxycholic acid, After one of the components of hydroxyethyl-β-cyclodextrin and sodium caseinate is missing or replaced by other components, the vitality of skin cells or spermatogonia is reduced; and adding lauroyl muscle to the protective solution The mixture of amino acid, hydrogenated lecithin and calcium monostearate can also improve the preservation time of the protective solution on skin cells or spermatogonia, which can increase the preservation time to 480h. When lauroyl sarcosine, hydrogenated lecithin and monostearin After one of the three components of calcium stearate is missing or replaced by other components, the preservation time of skin cells or spermatogonia can only be maintained within 96 hours.

Test Example 2 Stem Cell Expansion and Culture Detection

The skin cells and spermatogonia preserved respectively with the protective solution of Example 7, Example 9 and Example 11 were used as experimental 1-3 groups, and the skin cells and spermatogonia preserved respectively with the protective solution of Comparative Example 7-8 and CN1935984 discloses a method for culturing poultry spermatogonia in vitro. The cultivated spermatogonia are the control groups 1-3. The skin cells and spermatogonia of each experimental group and control group were stored for 2 days and then sampled for cell counting (trypan blue staining method). See Table 2 for the determination of multiples and cell viability.

Table 2 Expansion culture results of experimental group and control group

As can be seen from the table, the protection solution provided by Example 9 and Example 11 of the present invention can significantly increase the expansion multiple of stem cells, which is significantly higher than that of Example 7 and the control group 1-3, and the protection solution The above-mentioned ingredients added in the method do not affect the viability of stem cells, thus, after adding pullulan, glutathione and other mixtures in the protection solution provided by the present invention, the amplification factor of stem cells can be significantly improved, but when the above When the components are changed, or a certain component is omitted, the amplification factor of the protection solution for stem cells is significantly reduced.

Test example 3 detection of adhesion rate

The skin cells and spermatogonia preserved in the protective solution of Example 17 and Example 20 were used as experimental groups 1-2, and the skin cells and spermatogonia preserved in the protective solution of Comparative Example 9-10 were used as control 1-2 Group. The skin cells and spermatogonia of each experimental group and control group entered the test program at 24h, 48h, 96h, 240h and ^480h respectively. In a plate with a diameter of 9cm, after 48 hours of natural adherent growth, remove unattached cells, and then use 0.25m/v% trypsin to perform enzymatic hydrolysis, and calculate the number of adherent epidermal stem cells and spermatogonial stem cells , to obtain the attachment rate. The results are shown in Table 3.

Table 3 Results of cell adhesion rate in experimental group and control group

It can be seen from Table 3 that after 96 hours of storage in Experiment 1-2, the cell adhesion rate remained above 80%, and the adhesion effect was significantly better than that of the control 1-2 group, and the cell adhesion rate of Experiment 2 group was 480 hours after storage. Also remain at more than 50%, draw thus, the mixture of sodium ascorbate, sodium starch phosphate and potassium citrate can improve the adherence rate of skin cells or spermatogonia, when one of them changes or omits after composition, skin cells or The attachment rate of spermatogonia decreased significantly.

The above-mentioned embodiments only express several implementation modes of the present invention, and the descriptions thereof are relatively specific and detailed, but should not be construed as limiting the patent scope of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent for the present invention should be based on the appended claims.

Claims (10)

1. a kind of skin spermatogonia protective liquid, it is characterized in that, described protective liquid is mainly that aminoglycoside antibiotics is dissolved with DMEM medium aqueous solution, and every mL described DMEM aqueous medium solution dissolves described aminoglycosides Antibiotic 80-120U, the concentration of the DMEM medium aqueous solution is 13-25mg/mL. 2. skin spermatogonia protective solution as claimed in claim 1, is characterized in that, described aminoglycoside antibiotic is selected from the one in gentamicin, kanamycin, amikacin or tobramycin one or more species. 3. skin spermatogonia protective solution as claimed in claim 1, is characterized in that, the DMEM medium in described DMEM medium aqueous solution is low-sugar type DMEM medium. 4. skin spermatogonia protection solution as claimed in claim 1, is characterized in that, the concentration of described DMEM medium aqueous solution is 16.9mg/mL. 5. skin spermatogonia protection solution as claimed in claim 3, is characterized in that, described protection solution also comprises replenisher, and each component and the weight of the supplement that every mL described DMEM medium aqueous solution dissolves are as follows: Amino acid 15-20mg, phenylalanine 5-10mg, lysine 1-3mg and glutamine 0.5-1mg. 6. skin spermatogonia protection solution as claimed in claim 1, is characterized in that, described protection solution also comprises activator, each component and the weight of the activator that every mL described DMEM medium aqueous solution dissolves are as follows: deoxygenation Cholic acid 6.5-9 μg, hydroxyethyl-β-cyclodextrin 15-20 μg, sodium caseinate 1-3 μg. 7. skin spermatogonia protection solution as claimed in claim 1, is characterized in that, described protection solution also comprises culture aid, and each composition and the weight of the culture aid of per mL described DMEM culture medium aqueous solution dissolving are as follows: Lulan 5-10μg, glutathione 6-10μg, fatty acid lactoyl ester 20-35μg, vitamin E30-50μg, human serum albumin 1-3μg, ammonium nitrate 6-10μg, peptone 3-5μg and coconut oleic acid mono Ethanolamide 15-20 μg. 8. skin spermatogonia protection solution as claimed in claim 1, is characterized in that, described protection solution also comprises time-delay agent, and each composition and weight of the time-delay agent that per mL described DMEM culture medium aqueous solution dissolves are as follows: Acyl sarcosine 7.5-10μg, hydrogenated lecithin 35-50μg, calcium monostearate 1-3μg. 9. skin spermatogonia protection liquid as claimed in claim 1, is characterized in that, described protection liquid also comprises stabilizer, each component and the weight of the stabilizer that every mL described DMEM medium aqueous solution dissolves are as follows: sodium ascorbate 15 -20 μg, sodium starch phosphate 5-10 μg and potassium citrate 3-6 μg. 10. A preparation method of the skin spermatogonia protective solution according to claim 1, characterized in that, said method comprises the steps of: 1) Take 1/3 volume of DMEM medium aqueous solution, add 1/4 unit of aminoglycoside antibiotics at 10°C, mix well, and let stand for 5 minutes; 2) Add 1/3 volume of DMEM medium aqueous solution, mix well, add 1/3 unit of aminoglycoside antibiotics at 10°C, mix well, and let stand for 7 minutes; 3) Add the remaining 1/3 volume of DMEM medium aqueous solution, mix well, add the remaining 5/12 units of aminoglycoside antibiotics at 10°C, mix well, and obtain the skin spermatogonial cell protection solution.