阅读说明：本技术 一种生物活性物冻干固体面膜及其制备方法 (Bioactive substance freeze-dried solid mask and preparation method thereof ) 是由 杨桂花 赵进军 赵宇飞 于 2020-05-27 设计创作，主要内容包括：本发明涉及护肤品领域,具体涉及一种生物活性物冻干固体面膜及其制备方法,其中,一种生物活性物冻干固体面膜,由浸有面膜液的面膜布经低温干燥制得；所述面膜液包括A相、B相以及C相；所述A相包括水、甘油、丙二醇、植物提取物、透明质酸钠、传明酸、覆盆子酮葡糖苷、小核菌胶、赤藓醇、尿囊素、增稠剂以及对羟基苯乙酮；所述B相包括油橄榄果油、葡萄籽提取物、霍霍巴籽提取物、甜橙精油、维生素E、碳酸二辛酯以及氢化卵磷脂；所述C相为脂肪间充质干细胞裂解物。本发明的生物活性物固体面膜对皮肤具有美白、保湿、提高皮肤光泽度以及修复痘印、疤痕皮肤的效果。(The invention relates to the field of skin care products, in particular to a freeze-dried solid facial mask with a biological active substance and a preparation method thereof, wherein the freeze-dried solid facial mask with the biological active substance is prepared by drying facial mask cloth soaked with facial mask liquid at low temperature; the facial mask liquid comprises a phase A, a phase B and a phase C; the phase A comprises water, glycerol, propylene glycol, plant extract, sodium hyaluronate, tranexamic acid, raspberry ketone glucoside, sclerotium gum, erythritol, allantoin, thickener and p-hydroxyacetophenone; the phase B comprises olive fruit oil, grape seed extract, jojoba seed extract, sweet orange essential oil, vitamin E, dioctyl carbonate and hydrogenated lecithin; the phase C is adipose mesenchymal stem cell lysate. The bioactive substance solid facial mask disclosed by the invention has the effects of whitening, moisturizing, improving the skin glossiness and repairing acne marks and scar skin.)

1. A freeze-dried solid facial mask of biological active substances is characterized in that: is prepared by low-temperature drying of facial mask cloth soaked with facial mask liquid;

the facial mask liquid comprises a phase A, a phase B and a phase C;

the phase A comprises, by weight, 60-70 parts of water, 5-10 parts of glycerol, 3-5 parts of propylene glycol, 2-3 parts of plant extracts, 0.2-0.4 part of sodium hyaluronate, 0.06-0.08 part of tranexamic acid, 0.5-1 part of raspberry ketone glucoside, 0.3-0.5 part of sclerotinia sclerotiorum gum, 0.3-0.4 part of erythritol, 0.1-0.2 part of allantoin, 0.5-0.7 part of thickener and 0.2-0.3 part of p-hydroxyacetophenone;

the phase B comprises 0.2-0.4 part of olive oil, 0.2-0.4 part of grape seed extract, 0.2-0.4 part of jojoba seed extract, 0.1-0.2 part of sweet orange essential oil, 0.1-0.2 part of vitamin E, 0.4-0.6 part of dioctyl carbonate and 0.1-0.2 part of hydrogenated lecithin;

and the phase C is 2-3 parts of adipose mesenchymal stem cell lysate.

2. The freeze-dried solid mask pack of claim 1, wherein said solid mask pack comprises: the adipose-derived mesenchymal stem cell lysate is prepared by the following method:

A. solution preparation: adding a serum substitute to the basal medium to prepare a medium containing 4% of the serum substitute, and marking the medium as a solution C; adding 20mL of pancreatin with the concentration of 0.25% into 30mL of physiological saline, and uniformly mixing, wherein the label is solution D;

B. cell recovery: taking adipose tissue source seed mesenchymal stem cells, adding a basic culture solution, shaking, centrifuging to remove supernatant, collecting cell sediment, adding the solution C into the cell sediment, mixing to obtain cell suspension, inoculating the cell suspension into a culture bottle, and placing the culture bottle in a carbon dioxide incubator for culture;

C. subculturing, adding solution D when cell confluency reaches 80% after cell recovery, digesting single cell, adding solution C, terminating digestion, centrifuging cell suspension, removing supernatant, collecting cell precipitate, adding basal medium into cell precipitate, re-suspending cell precipitate, adding solution C to obtain cell suspension, and adjusting cell density to 1 × 10⁶Adding into new culture flask, placing in carbon dioxide incubatorCulturing;

D. harvesting cell lysate: after subculture, when the confluence degree of the cells reaches 80%, adding the solution D to digest single cells by the cells; adding the solution C again to stop digestion; centrifuging the cell suspension, removing supernatant, and collecting cell precipitate; adding normal saline into the cell sediment, re-suspending the cell sediment, and adding the normal saline to obtain cell suspension; freezing the cell suspension at-80 deg.C for 30 min; repeating twice; centrifuging the cell lysate, and taking supernatant to obtain cell lysate; and (3) embedding the cell lysate with lipid, sterilizing, freezing and drying to obtain the adipose mesenchymal stem cell lysate.

3. The freeze-dried solid mask pack of claim 1, wherein said solid mask pack comprises: the plant extract is prepared by mixing centella extract, ribes nigrum fruit extract, opuntia ficus-indica stem extract, citron fruit extract and maple sugar extract according to the weight ratio of 4 (1-2) - (2-3) - (1-3) - (0.6-1.5).

4. The freeze-dried solid mask pack of claim 1, wherein said solid mask pack comprises: the thickening agent is carbomer.

5. A method for preparing a freeze-dried solid mask containing bioactive substances according to any one of claims 1 to 4, wherein the freeze-dried solid mask comprises the following steps: the method comprises the following steps:

s1, spreading a mask cloth, immersing the mask cloth in the mask liquid, and soaking for 10-20 min;

and S2, taking out the mask cloth soaked with the mask liquid, and carrying out vacuum freeze drying to obtain the bioactive substance freeze-dried solid mask.

6. The method for preparing the freeze-dried solid mask containing the bioactive substances according to claim 5, wherein the method comprises the following steps: the facial mask liquid of S1 is prepared by the following method: taking the phase A raw material according to the proportion, heating to 80-85 ℃, and stirring for 20-30min under the condition of heat preservation to obtain a phase A premix;

taking the phase B raw material, heating to 80-85 ℃, and stirring for 20-30min under the condition of heat preservation to obtain a phase B premix;

adding the B-phase premix into the A-phase premix, stirring at 80-85 deg.C for 10-15min, cooling to 40-45 deg.C, adding the C-phase raw material, stirring at 20-30min, and homogenizing to obtain the facial mask liquid.

7. The method for preparing the freeze-dried solid mask containing the bioactive substances according to claim 5, wherein the method comprises the following steps: the stirring speed is 300-500 r/min.

8. The method for preparing the freeze-dried solid mask containing the bioactive substances according to claim 5, wherein the method comprises the following steps: the homogenizing speed is 10000-.

9. The method for preparing the freeze-dried solid mask containing the bioactive substances according to claim 5, wherein the method comprises the following steps: vacuum freeze-drying of S2 refers to: taking out the mask cloth soaked with the mask liquid, placing the mask cloth in a vacuum freeze dryer, adjusting the temperature of the vacuum freeze dryer to-40 to-45 ℃, and pre-freezing for 10 to 16 hours; cooling the vacuum freeze dryer to-20 to-25 ℃ at the speed of 3 to 5 ℃, and preserving heat for 1 to 2 hours; then vacuumizing, and keeping the vacuum degree for 2-3 h under the condition of 20-25 Pa; heating to 20-25 ℃ at the temperature of 1-1.2 ℃, preserving heat for 2-3 h, and keeping for 3-4 h under the condition that the vacuum degree is 10-15 Pa.

Technical Field

The invention relates to the technical field of skin care products, in particular to a freeze-dried solid facial mask containing bioactive substances and a preparation method thereof.

Background

The facial mask is a category of skin care products, is a carrier of the beauty care products, and is applied on the face for beauty functions such as moisturizing, whitening, anti-aging, grease balancing and the like. The facial mask mainly has the forms of a paste type, a tearing type, a jelly type and a wet tissue type; the mud paste type facial mask is commonly an alga facial mask, a mud facial mask and the like, the tearing type facial mask is commonly a special nasal paste for blackhead, the jelly glue type facial mask is mainly a sleep facial mask, and the wet tissue type facial mask is generally a facial mask paper which is packaged by a single piece and is soaked with a beautifying liquid. In recent years, with the diversified needs of people for skin care, a novel facial mask different from the conventional facial mask appears, wherein the freeze-dried facial mask is a novel facial mask.

Disclosure of Invention

Aiming at the defects in the prior art, the first object of the invention is to provide a freeze-dried solid facial mask containing bioactive substances, which has the advantages of whitening, moisturizing, improving the skin glossiness and repairing acne scar skin.

The second purpose of the invention is to provide a preparation method of a freeze-dried solid mask containing biological active substances, which has the advantage of keeping the activity of biological factors by blocking the mask liquid in a mask cloth through a freeze-drying technology.

In order to achieve the first object, the invention provides the following technical scheme: a bioactive lyophilized solid facial mask is prepared by soaking facial mask cloth in facial mask solution, and drying at low temperature;

the facial mask liquid comprises a phase A, a phase B and a phase C;

the phase A comprises, by weight, 60-70 parts of water, 5-10 parts of glycerol, 3-5 parts of propylene glycol, 2-3 parts of plant extracts, 0.2-0.4 part of sodium hyaluronate, 0.06-0.08 part of tranexamic acid, 0.5-1 part of raspberry ketone glucoside, 0.3-0.5 part of sclerotinia sclerotiorum gum, 0.3-0.4 part of erythritol, 0.1-0.2 part of allantoin, 0.5-0.7 part of thickener and 0.2-0.3 part of p-hydroxyacetophenone;

the phase B comprises 0.2-0.4 part of olive oil, 0.2-0.4 part of grape seed extract, 0.2-0.4 part of jojoba seed extract, 0.1-0.2 part of sweet orange essential oil, 0.1-0.2 part of vitamin E, 0.4-0.6 part of dioctyl carbonate and 0.1-0.2 part of hydrogenated lecithin; and the phase C is 2-3 parts of adipose mesenchymal stem cell lysate.

By adopting the technical scheme, the fat mesenchymal stem cell lysate is used as a biological factor, the raspberry ketone glucoside, the tranexamic acid, the plant extract, the moisturizing component glycerol, the propylene glycol, the sodium hyaluronate, the sclerotium rolfsii gum, the erythritol and the allantoin are matched to play a role in moisturizing, moisturizing and whitening the skin, the olive oil, the grape seed extract, the jojoba seed extract, the sweet orange essential oil and the vitamin E can further lock water for the skin, and the skin can be moisturized, moisturized and whitened by matching the raw materials, has a good repairing function on scars and acne printing skin, can promote tissue growth, improve cell metabolism, soften cuticle protein and reduce wrinkle growth, and can not only repair and maintain the skin intensively when being used at night, the added sweet orange essential oil can relieve pressure and has the functions of relieving and soothing nerves; after the skin is repaired at night, the water-oil balance of the skin can be adjusted, and the phenomena of powder blocking and dressing non-sticking are not easy to occur in the next day of makeup.

The adipose-derived mesenchymal stem cell lysate is prepared by the adipose-derived mesenchymal stem cells after cell recovery, subculture and cell lysis, wherein the adipose-derived mesenchymal stem cell lysate contains biological factors which have good repairing effect on damaged cells, scars and acne marks after being exposed to the sun, and can promote the growth of epidermal cells and accelerate the metabolic death of aged cells, thereby avoiding the acne marks and scars caused by the repair of dermal cells. The whitening component can inhibit the formation of skin melanin, promote skin metabolism, accelerate melanin removal, and reduce black pox mark and pit pox mark.

The sodium hyaluronate has good effects of moisturizing and promoting tissue repair and regeneration, has strong lubricating feeling and film forming property, and can protect skin; the raspberry ketone glucoside is also called raspberry glucoside, and has good melanin inhibiting effect by compounding with tranexamic acid and vitamin E, and has whitening, skin brightening and anti-aging effects; the olive fruit oil, the grape seed extract, the jojoba seed extract, the sweet orange essential oil, the vitamin E, the dioctyl carbonate and the hydrogenated lecithin are used as oil phases, have good ductility and permeability, can penetrate into skin cells on one hand, adjust the water-oil balance of the skin, improve the moistening performance of the skin, and form a water-locking protective film on the surface layer of the skin after water replenishing and moisture retaining on the other hand, so that the soft and elastic feeling of the skin is improved.

Further, the adipose mesenchymal stem cell lysate is prepared by the following method:

A. solution preparation: adding a serum substitute to the basal medium to prepare a medium containing 4% of the serum substitute, and marking the medium as a solution C; adding 20mL of pancreatin with the concentration of 0.25% into 30mL of physiological saline, and uniformly mixing, wherein the label is solution D;

B. cell recovery: taking adipose tissue source seed mesenchymal stem cells, adding a basic culture solution, shaking, centrifuging to remove supernatant, collecting cell sediment, adding the solution C into the cell sediment, mixing to obtain cell suspension, inoculating the cell suspension into a culture bottle, and placing the culture bottle in a carbon dioxide incubator for culture;

C. subculturing, adding solution D when cell confluency reaches 80% after cell recovery, digesting single cell, adding solution C, terminating digestion, centrifuging cell suspension, removing supernatant, collecting cell precipitate, adding basal medium into cell precipitate, re-suspending cell precipitate, adding solution C to obtain cell suspension, and adjusting cell density to 1 × 10⁶The amount of the active carbon is one/mL,adding the mixture into a new culture bottle, and placing the new culture bottle in a carbon dioxide incubator for culture;

D. harvesting cell lysate: after subculture, when the confluence degree of the cells reaches 80%, adding the solution D to digest single cells by the cells; adding the solution C again to stop digestion; centrifuging the cell suspension, removing supernatant, and collecting cell precipitate; adding normal saline into the cell sediment, re-suspending the cell sediment, and adding the normal saline to obtain cell suspension; freezing the cell suspension at-80 deg.C for 30 min; repeating twice; centrifuging the cell lysate, and taking supernatant to obtain cell lysate; and (3) embedding the cell lysate with lipid, sterilizing, freezing and drying to obtain the adipose mesenchymal stem cell lysate.

By adopting the technical scheme, the mesenchymal stem cells are a group of heterogeneous cell groups derived from the matrix, the adipose mesenchymal stem cells have wide sources, and contain a large amount of active ingredients, such as stem cell growth factors, fibroblast growth factors, endothelial cell growth factors, epidermal cell growth factors, collagen, hyaluronic acid and the like, so that the growth of epidermal keratinocytes can be promoted, damaged cells can be repaired, and the metabolism of the cells can be improved; the adipose tissue-derived seed mesenchymal stem cells are adopted to obtain cell lysate after cell recovery, subculture and cell lysis, and the cell lysate is subjected to lipid embedding, sterilization and freeze drying, so that the active retention period of growth factors in the cell lysate can be improved, the water in the cell lysate is sublimated in a vacuum sterile environment, the damage to biological tissues and cell structures and characteristics is small, and the biological tissues and cell structures can rapidly enter a dormant state, so that the biological activity of the cell lysate can be effectively prolonged, and the using effect of the freeze-dried solid mask can be improved.

Further, the plant extract is prepared by mixing centella extract, ribes nigrum fruit extract, opuntia ficus-indica stem extract, citron fruit extract and maple extract according to the weight ratio of 4 (1-2): (2-3): 1-3): 0.6-1.5.

By adopting the technical scheme, the centella asiatica extract has the effects of whitening, moisturizing, diminishing inflammation, resisting acne, promoting skin repair, tightening the connecting part of the epidermis and the dermis, softening the skin, relieving the skin relaxation phenomenon and improving the skin elasticity; the RIBES NIGRUM FRUIT EXTRACT is called RIBES NIGRUM (BLACK CURRANT) FRUIT EXTRACT, and has effects of whitening skin, resolving macula, and astringing skin; the OPUNTIA FICUS-indica stem EXTRACT, which is named as OPUNTIA FICUS-INDICA STEM EXTRACT in English, can inhibit secretion of male hormone, has a prevention and treatment effect on acne caused by high male hormone, can promote generation of skin growth factors, and has a good anti-aging effect; acer SACCHARUM EXTRACT (Acer SACCHARUM) (sugarmeable) EXTRACT in English name, and the Acer SACCHARUM EXTRACT has good antioxidant and anti-inflammatory effects; the Citrus Medica Fruit Extract has good antibacterial and anti-inflammatory effects, and is named as Citrus Medica Limonum (Lemon) Fruit Extract. The plant extract compounded by the centella extract, the ribes nigrum fruit extract, the opuntia ficus-indica stem extract, the citron fruit extract and the maple extract has good effects of relieving, diminishing inflammation and astringing the skin, can relieve vasodilatation caused by skin inflammation, and is beneficial to fading red pox marks and crater pox marks; the components of the plant extract are safe and natural, and the plant extract is matched with adipose mesenchymal stem cells, tranexamic acid, raspberry ketone glucoside and sclerotium rolfsii gum, so that the skin damage such as acne marks and scars can be repaired, and the skin glossiness can be improved.

Further, the thickener is carbomer.

By adopting the technical scheme, carbomer serving as a hydrophilic thickening agent has good compatibility with other components, and is beneficial to improving the stability of a system.

In order to achieve the second object, the invention provides the following technical scheme:

a preparation method of a freeze-dried solid facial mask containing bioactive substances comprises the following steps:

s1, spreading a mask cloth, immersing the mask cloth in the mask liquid, and soaking for 10-20 min;

and S2, taking out the mask cloth soaked with the mask liquid, and carrying out vacuum freeze drying to obtain the bioactive substance freeze-dried solid mask.

Further, the facial mask solution of S1 is prepared by the following method: taking the phase A raw material according to the proportion, heating to 80-85 ℃, and stirring for 20-30min under the condition of heat preservation to obtain a phase A premix;

taking the phase B raw material, heating to 80-85 ℃, and stirring for 20-30min under the condition of heat preservation to obtain a phase B premix;

adding the B-phase premix into the A-phase premix, stirring at 80-85 deg.C for 10-15min, cooling to 40-45 deg.C, adding the C-phase raw material, stirring at 20-30min, and homogenizing to obtain the facial mask liquid.

By adopting the technical scheme, the A-phase raw material and the B-phase raw material are respectively mixed and then are mixed, so that the uniformity of raw material mixing can be improved, and the stability of a system can be improved.

Further, the stirring speed is 300-.

Furthermore, the homogenizing speed is 10000-.

By adopting the technical scheme, the raw materials are homogenized, so that the dispersion in the system is uniform without micronization, and the stability of the system is improved.

Further, vacuum freeze-drying of S2 means: taking out the mask cloth soaked with the mask liquid, placing the mask cloth in a vacuum freeze dryer, adjusting the temperature of the vacuum freeze dryer to-40 to-45 ℃, and pre-freezing for 10 to 16 hours; cooling the vacuum freeze dryer to-20 to-25 ℃ at the speed of 3 to 5 ℃, and preserving heat for 1 to 2 hours; then vacuumizing, and keeping the vacuum degree for 2-3 h under the condition of 20-25 Pa; heating to 20-25 ℃ at the temperature of 1-1.2 ℃, preserving heat for 2-3 h, and keeping for 3-4 h under the condition that the vacuum degree is 10-15 Pa.

By adopting the technical scheme, the water in the cell lysate is sublimated in a vacuum sterile environment, the damage to biological tissues and cell structures and characteristics is small, and the biological tissues and the cell structures and the characteristics are enabled to rapidly enter a dormant state, so that the biological activity of the freeze-dried solid mask is effectively prolonged, and the using effect of the freeze-dried solid mask is improved.

In summary, compared with the prior art, the invention has the following beneficial effects:

the invention takes fat mesenchymal stem cell lysate as a biological factor, can play the roles of moisturizing, moisturizing and whitening the skin through the matching of whitening components such as raspberry ketone glucoside, tranexamic acid, plant extract, moisturizing components such as glycerin, propylene glycol, sodium hyaluronate, sclerotium rolfsii gum, erythritol and allantoin, can further lock water for the skin through skin moistening grease such as olive oil, grape seed extract, jojoba seed extract, sweet orange essential oil and vitamin E, has the functions of moisturizing, moisturizing and whitening the skin, has good repairing effect on scars and acne printing skin through the matching of the raw materials, can promote tissue growth, improve cell metabolism, soften cuticle protein and reduce wrinkle growth, can not only carry out intensive repair and maintenance on the skin when being used at night, but also can relieve pressure through the added sweet orange essential oil, has effects in relieving anxiety; after the skin is repaired at night, the water-oil balance of the skin can be adjusted, and the phenomena of powder blocking and dressing non-sticking are not easy to occur in the next day of makeup.

Detailed Description

The present invention will be described in further detail below.

Preparation example of adipose-derived mesenchymal Stem cell lysate

Preparation of adipose mesenchymal stem cell lysate example 1: A. solution preparation: adding serum substitute into DMEM/F12 basal medium to prepare a medium containing 4% serum substitute, and marking the medium as solution C; 20mL of 0.25% pancreatin was added to 30mL of physiological saline and mixed, and the mixture was labeled as solution D.

B. Cell recovery:

b1, adding 10mL of DMEM/F12 basal medium into a 15mL centrifuge tube, preheating to 37 ℃, and standing for later use;

b2, taking out the cryopreservation tube containing the human adipose tissue-derived seed mesenchymal stem cells from the liquid nitrogen tank, quickly putting the tube into a 37 ℃ water bath kettle, vertically placing the tube cover of the cryopreservation tube upwards, enabling the tube cover to be higher than the water surface, and quickly shaking the cryopreservation tube to quickly melt the cryopreservation liquid in the cryopreservation tube;

b3, after the frozen stock solution (the seed mesenchymal stem cells) of b2 is melted, unscrewing a tube cover, and sucking the seed mesenchymal stem cells into a b1 centrifuge tube by using a 5mL pipette;

b4, adding 1mL of basic culture medium into the frozen tube emptied from b3 by using a 5mL pipette, slightly shaking to wash the tube wall, pouring the washing liquid into the same centrifugal tube b1, screwing a tube cover, and reversing and uniformly mixing;

b5, placing the centrifugal tube with the seed mesenchymal stem cells in b4 in a centrifuge, and centrifuging for 5min at 300 g;

b6, sucking and removing supernatant by using a pipette, screwing a tube cover, and gently shaking and scattering cell sediment;

b7, unscrewing a tube cover, adding a basic culture medium into the centrifuge tube b6, fixing the volume to 10mL, screwing the tube cover, and reversing and uniformly mixing;

b8, putting the centrifuge tube in b7 into a centrifuge, and centrifuging for 5min at 300 g;

b9, removing the supernatant of the b8 centrifuge tube by a pipette, screwing a tube cover, and gently shaking to disperse cell sediments;

b10, unscrewing a tube cover, adding 5mL of solution C into the b9 centrifuge tube, screwing the tube cover, and reversing and uniformly mixing to obtain a cell suspension; 0.5mL of cell suspension was aspirated with a 5mL pipette for cell counting;

b11, sucking the solution C by a 25mL pipette, and adding the solution C into a T-175 culture bottle according to the amount of 28 mL/bottle;

b12, sucking b10 cell suspension by a 5mL pipette, and inoculating the cell suspension into a b 11T-175 culture bottle, wherein the inoculation amount of the cells is 1.2 × 10⁶T-175, putting a T-175 culture bottle in a carbon dioxide incubator for culturing for 72 hours, wherein the temperature is 37 ℃, and the volume ratio concentration of carbon dioxide is 5%;

C. subculturing:

c1, after the cells are recovered, when the confluence degree of the cells reaches 80%, taking the T-175 culture bottle of b12 out of the carbon dioxide incubator;

c2, transferring the T-175 culture bottle to a biological safety cabinet, stacking stably, unscrewing the bottle cap, and transferring the supernatant in the culture bottle to a sterile serum bottle by using a 25mL pipette;

c3, preparation of a sample: sucking 4mL of culture supernatant from a sterile serum bottle by using a 5mL pipette, adding the culture supernatant into a 5mL centrifuge tube, labeling to prepare a sample to be detected, and conveying the sample to a quality detection part for detection;

c4, sucking the physiological saline by using a 10mL pipette, adding the physiological saline into a T-175 culture bottle with the volume of 10 mL/bottle, shaking the bottle body, rinsing the bottom of the bottle, and sucking away the lotion;

c5, sucking the solution D by using a 25mL pipette, adding the solution D along the inner wall of the non-cell surface of the T-175 culture bottle according to the amount of 5 mL/bottle, quickly shaking the bottle body, and flatly placing the bottle body after fully infiltrating the cell surface;

c6, observing cell rounding under a microscope, erecting a bottle body, sucking the solution C by using a 25mL pipette, adding the solution C along the inner wall of the cell surface of the T-175 culture bottle according to the amount of 5 mL/bottle, shaking the bottle body, uniformly infiltrating the cell surface, and stopping digestion;

c7, sucking out the cell suspension in the T-175 culture bottle by using a 25mL pipette and collecting the cell suspension in a 50mL centrifuge tube;

c8, screwing a centrifugal tube cover of c7, putting the centrifugal tube cover into a centrifugal machine, and centrifuging for 5min at 300 g;

c9, taking out the centrifuge tube, pouring out the supernatant, and shaking up the cell sediment;

c10, pouring a basic culture medium into the c9 centrifuge tube, fixing the volume to 50ml, screwing down the tube cover, and reversing and uniformly mixing;

c11, putting the c10 centrifuge tube into a centrifuge, and centrifuging for 5min at 300 g;

c12, taking out the c11 centrifuge tube, pouring out the supernatant, and shaking up the cell sediment;

c13, union tube: taking a c12 centrifuge tube, adding 20mL of basal medium by using a 25mL pipette, shaking the tube body, transferring the cell sediment to another centrifuge tube after the cell sediment is suspended, and repeating the transfer until all cells are merged into 150 mL centrifuge tube;

c14, washing the tube: adding a basic culture medium into the centrifugal tube emptied by the c13, slightly shaking to wash the tube wall, and collecting the washing liquid into the c13 centrifugal tube;

c15, adding 5mL of solution C into a C13 centrifuge tube, screwing down a tube cover, and reversing and uniformly mixing;

c16, unscrewing the tube cover, taking 0.5mL of cell suspension from the c15 centrifuge tube by using a 1mL pipette, and counting cells;

c17, adding the solution C into a C14 centrifuge tube, and adjusting the cell density to 1 × 10⁶Per mL;

c18, sucking the solution C by using a 25mL pipette, adding the solution C into a new T-175 culture bottle according to the amount of 28 mL/bottle, and stacking the solution C stably for later use;

c19, pipette the cell suspension from the c17 centrifuge tube with a 5mL pipette, and inoculate 1.5 mL/vial into a C18T-175 flask (1.5 × 10)⁶Person/bottle);

c20, screwing a bottle cover, shaking uniformly, and putting the T-175 culture bottle into a carbon dioxide incubator for culturing for 72 hours.

D. Harvesting cell lysate:

d1, carrying out subculture on the cells for 72 hours, and taking out the T-175 culture bottle from the carbon dioxide incubator when the cell confluency reaches 80%;

d2, transferring the T-175 culture bottle to a biological safety cabinet, stacking stably, unscrewing the bottle cap, and transferring the supernatant in the culture bottle to a sterile serum bottle by using a 25mL pipette;

d3, sucking the physiological saline by using a 10mL pipette, adding the physiological saline into a T-175 culture bottle with the volume of 10 mL/bottle, shaking the bottle body, rinsing the bottom of the bottle, and sucking away the lotion;

d4, sucking the solution D by using a 25mL pipette, adding the solution D along the inner wall of the non-cell surface of the T-175 culture bottle according to the amount of 5 mL/bottle, quickly shaking the bottle body, and flatly placing the bottle body after fully infiltrating the cell surface;

d5, observing cell rounding under a microscope, erecting a bottle body, sucking the solution C by using a 25mL pipette, adding the solution C along the inner wall of the cell surface of the T-175 culture bottle according to the amount of 5 mL/bottle, shaking the bottle body, uniformly infiltrating the cell surface, and stopping digestion;

d6, pipette the cell suspension from the T-175 flask with a 25mL pipette and collect into a 50mL centrifuge tube;

d7, screwing a centrifugal tube cover of d6, putting the centrifugal tube cover into a centrifugal machine, and centrifuging for 5min at 300 g;

d8, taking out the centrifuge tube, pouring out the supernatant, and shaking up the cell sediment;

d9, pouring normal saline into the d8 centrifuge tube, fixing the volume to 50ml, screwing down the tube cover, and reversing and uniformly mixing;

d10, putting the d9 centrifuge tube into a centrifuge, and centrifuging for 5min at 300 g;

d11, taking out the centrifuge tube, pouring out the supernatant, and shaking up the cell sediment;

d12, union tube: taking a d11 centrifuge tube, adding 20mL of physiological saline by using a 25mL pipette, shaking the tube body, transferring the resuspended cells to another centrifuge tube after precipitation, and repeating the transfer until all the cells are merged into 150 mL centrifuge tube;

d13, washing the tube: adding a basic culture medium into the centrifugal tube emptied by the d12, slightly shaking to wash the tube wall, and collecting the washing liquid into the d12 centrifugal tube;

d14, adding physiological saline into a d12 centrifuge tube, fixing the volume to 50mL, screwing down a tube cover, and reversing and uniformly mixing;

d15, putting a 50mL centrifuge tube of d14 into a refrigerator with the temperature of 80 ℃ below zero, freezing for 30min, taking out after completely freezing, and melting at room temperature;

d16, repeating the operation of d15 twice;

d17, putting the d16 centrifuge tube into a centrifuge, centrifuging for 5min at 300g, and taking supernatant as cell lysate;

d18, adding 2kg of cell lysate, 0.8kg of beta-cyclodextrin and 0.2kg of trehalose into 0.2mol/L disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution with the concentration of 10L, pH being 7.2, and stirring at the speed of 500r/min for 2 hours to obtain a pretreatment solution;

adding 0.57kg of lecithin, 0.4kg of vitamin E and 0.03kg of mannitol into 1L of absolute ethyl alcohol, and stirring at the speed of 500r/min for 30min to obtain an embedding solution;

dropwise adding the embedding solution into the pretreatment solution for 1h, stirring for 1h after dropwise adding is finished, performing rotary evaporation for 10min at the temperature of 30 ℃ and the vacuum degree of 0.1MPa to remove anhydrous and disodium hydrogen phosphate-sodium dihydrogen phosphate buffer solution, and performing filtration sterilization by using a sterile filter of 0.22 mu m to obtain an embedded cell lysate;

d19, placing the embedded cell lysate in a vacuum freeze dryer, keeping the temperature at 4 ℃ for 30min, then cooling the vacuum freeze dryer to-10 ℃ at the speed of 5 ℃, and keeping the temperature for 1 h; cooling the vacuum freeze dryer to-20-degree centigrade at the speed of 5 degree centigrade, and keeping the temperature for 1 h; cooling the vacuum freeze dryer to-35 ℃ at the speed of 5 ℃, and preserving heat for 2 hours; then vacuumizing until the vacuum degree is 20Pa, heating to-25 ℃ at the temperature of 5 ℃, and preserving heat for 5 hours; heating to-5 deg.C at 5 deg.C, and maintaining for 1 h; heating to 255 ℃ at the temperature of 1 ℃, preserving heat for 2h, and keeping for 3h under the condition that the vacuum degree is 10Pa to obtain the adipose-derived mesenchymal stem cell lysate.

Preparation of adipose mesenchymal stem cell lysate 2: the difference between the preparation example and the preparation example 1 of the adipose tissue-derived mesenchymal stem cell lysate is that the step d18 is not included, and the adipose tissue-derived mesenchymal stem cell lysate is obtained by directly performing vacuum freeze-drying on the cell lysate after the cell lysate is obtained at the step d 17.

Preparation of adipose mesenchymal stem cell lysate 3: the difference between this preparation example and preparation example 1 of adipose-derived mesenchymal stem cell lysate is that in step d18, beta-cyclodextrin and trehalose are not added to the cell lysate.