CN112353814A - Infant eczema-removing ointment containing mesenchymal stem cell lysate and preparation method and application thereof - Google Patents
Infant eczema-removing ointment containing mesenchymal stem cell lysate and preparation method and application thereof Download PDFInfo
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/06—Ointments; Bases therefor; Other semi-solid forms, e.g. creams, sticks, gels
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
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- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/06—Animal cells or tissues; Human cells or tissues
- C12N5/0602—Vertebrate cells
- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0662—Stem cells
- C12N5/0668—Mesenchymal stem cells from other natural sources
Abstract
The invention relates to the technical field of stem cell preparations, in particular to an infant eczema-removing ointment containing a mesenchymal stem cell lysate and a preparation method and application thereof, wherein the infant eczema-removing ointment containing the mesenchymal stem cell lysate contains the following substances in every 100ml of the ointment: mesenchymal stem cell lysate 4-10 × 108A lytic component of the individual cell; 10-20 mg of mesenchymal stem cell freeze-dried powder; 5-10 g of a stabilizer; 50-100 ml of electrolyte injection; 5-8 ml of a humectant; 5-10 ml of transdermal agent; 50-100 mg of propolis. The medicine of the invention has the advantages of definite curative effect,can promote the growth of epidermal cells for a long time, shorten the healing time of skin wounds, eliminate inflammation, have safety, no toxic or side effect, no relapse after healing, high healing rate, promote the healing of ulcer caused by chronic eczema and the like.
Description
Technical Field
The invention relates to the technical field of stem cell preparations, in particular to an infant eczema-removing ointment containing mesenchymal stem cell lysate and a preparation method and application thereof.
Background
Eczema is divided into three types, acute, subacute and chronic.
Acute eczema is characterized by its impairment and polymorphism, erythema, burning sensation and itching. Followed by the appearance of scattered or dense papules or small blisters on the erythema, which, after scratching or rubbing, are broken to form erosive, exudative surfaces; acute inflammation is relieved, skin damage is dry, scabbing and scaling appear after long-term treatment or treatment, and the acute stage is in a subacute stage.
Chronic eczema is developed by recurrent episodes of acute and subacute symptoms or is characterized by chronic inflammation at the beginning, usually confined to a same part for a long time, and is characterized by gradual thickening of the skin, deepening of skin wrinkles, infiltration, pigmentation and the like. The main symptom is severe pruritus.
At present, the oral antihistamine medicine is used for treating eczema. The dosage form of the external medicine is determined according to the clinical skin damage, if the red swelling is obvious, the exudation is performed by cold and wet compress with solution, and lotion, emulsion, mud paste, oil agent and the like can be used for erythema and pimple; oil solution is needed for blister and erosion; ointment for patients with scales and scabs; for lichen, mud ointment, emulsion, plastics, tincture and plaster are usually selected.
However, the medicines have side effects of different degrees, and have the problems of long treatment time, low cure rate and the like, thereby bringing pains to patients.
Disclosure of Invention
In view of the above technical problems, the present invention aims to provide a biological ointment for infant eczema removal, a preparation method and an application thereof, which have less side effects and better treatment effects.
In order to solve the technical problems, the specific technical scheme is as follows:
an ointment for removing eczema in infants containing mesenchymal stem cell lysate, which comprises the following components in every 100ml of ointment:
mesenchymal stem cell lysate 4-10 × 108A lytic component of the individual cell; 10-20 mg of mesenchymal stem cell freeze-dried powder; 5-10 g of a stabilizer; 50-100 ml of electrolyte injection; 5-8 ml of a humectant; 5-10 ml of transdermal agent; 50-100 mg of propolis.
Wherein the stabilizer is one or more of sodium hyaluronate, sodium alginate, chitosan and hydroxyethyl starch.
The humectant is one or more of propylene glycol or glycerol.
The electrolyte injection is compound electrolyte glucose MG3 injection.
The transdermal agent is menthol.
The preparation method of the infant de-eczema ointment containing the mesenchymal stem cell lysate comprises the following steps:
(1) carrying out starvation culture after culturing the human mesenchymal stem cells; collecting cell culture supernatant, adding water-soluble chitosan into the supernatant after filtration by a filter membrane, mixing uniformly to obtain mesenchymal stem cell lysate, and storing in a refrigerator at 4 ℃ for later use;
(2) the preparation method of the mesenchymal stem cell freeze-dried powder comprises the following steps of sequentially filtering a mesenchymal stem cell supernatant through 0.45 mu m and 0.22 mu m filter membranes, concentrating and freeze-drying;
(3) dissolving a stabilizer, a humectant and a transdermal agent in an electrolyte solution to prepare a uniform solution;
(4) and (4) uniformly mixing the mesenchymal stem cell lysate, the mesenchymal stem cell freeze-dried powder and the solution in the step (3) according to the proportion, and putting the mixture into a refrigerator at the temperature of-80 ℃ for freezing storage.
Wherein, the step (1) comprises the following steps:
(a) culturing human mesenchymal stem cells: selecting human mesenchymal stem cells from a cell bank from P2-P6, and inoculating the cells in three batches after recovery, wherein the inoculation densities of the three batches are respectively 50-60%, 30-40% and 20-30% of the maximum fusion degree;
the complete culture medium for inoculating and culturing the cells is phenol red-free DMEM/DF12, and 10% fetal calf serum in volume ratio concentration is added;
after the inoculation of the cells is finished, putting the cells into an incubator for culture, wherein the culture conditions are that the temperature is 37 ℃ and the volume concentration of carbon dioxide is 5%;
(b) when the fusion degree of the first batch of cell culture reaches 70-80%, cleaning the cells for 2 times by using normal saline, and then carrying out starvation culture by using compound electrolyte injection; after culturing for 18-24 hours, collecting cell culture supernatant when the cell fusion degree of the first batch reaches 90-95%, centrifuging for 6-10 min under the condition of 3000-4000 g to remove precipitates, and collecting centrifuged supernatant;
(c) when the fusion degree of the second batch of cell culture reaches 70-80%, washing the cells for 2 times by using physiological saline, and performing starvation culture by using the supernatant obtained by the first batch of cell culture in the step (b); after culturing for 18-24 hours, collecting cell culture supernatant when the cell fusion degree of the second batch reaches 90% -95%, centrifuging for 6-10 min under the condition of 3000-4000 g to remove precipitates, and collecting centrifuged supernatant;
(d) when the fusion degree of the third batch of cell culture reaches 70-80%, washing the cells for 2 times by using normal saline, and performing starvation culture by using the supernatant obtained by the second batch of cell culture in the step (c); after culturing for 18-24 hours, collecting cell culture supernatant when the cell fusion degree of the second batch reaches 90% -95%, centrifuging for 6-10 min under the condition of 3000-4000 g to remove precipitates, and collecting centrifuged supernatant;
(e) filtering the supernatant obtained in the step (d) by using a 0.22um filter membrane, adding water-soluble chitosan into the filtered supernatant, and uniformly mixing; the mass concentration of the chitosan in the water-soluble chitosan is 0.05-0.08%.
The mesenchymal stem cell is human umbilical cord mesenchymal stem cell, adipose mesenchymal stem cell or skin mesenchymal stem cell.
The ointment containing mesenchymal stem cell lysate for removing eczema in infants is used for treating infantile eczema.
The invention has the beneficial effects that:
the medicine has definite curative effect, can promote the growth of epidermal cells for a long time, can shorten the healing time of skin wounds, has the effects of eliminating inflammation, is safe, has no toxic or side effect, does not relapse after healing, has high healing rate, promotes the healing of ulcer caused by eczema which is not healed for a long time, and the like.
Detailed Description
In order to make those skilled in the art better understand the technical solutions of the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1
An ointment for removing eczema in infants containing mesenchymal stem cell lysate, which comprises the following components in every 100ml of ointment: mesenchymal stem cell lysate of about 5X 108A lytic component of the individual cell; 15mg of mesenchymal stem cell freeze-dried powder; 7g of a stabilizer; 80ml of electrolyte injection; 8ml of humectant; 5ml of transdermal agent; propolis 100 mg.
Wherein the stabilizer is sodium hyaluronate. The humectant is propylene glycol. The electrolyte injection is compound electrolyte glucose MG3 injection. The transdermal agent is menthol.
The preparation method of the infant de-eczema ointment containing the mesenchymal stem cell lysate comprises the following steps:
(1) carrying out starvation culture after culturing the human adipose mesenchymal stem cells; collecting cell culture supernatant, adding water-soluble chitosan into the supernatant after filtration by a filter membrane, mixing uniformly to obtain mesenchymal stem cell lysate, and storing in a refrigerator at 4 ℃ for later use;
(2) the preparation method of the mesenchymal stem cell freeze-dried powder comprises sequentially filtering the supernatant of the adipose mesenchymal stem cell with 0.45 μm and 0.22 μm filter membranes, concentrating and freeze-drying;
(3) dissolving a stabilizer, a humectant and a transdermal agent in an electrolyte solution to prepare a uniform solution;
(4) and (4) uniformly mixing the mesenchymal stem cell lysate, the mesenchymal stem cell freeze-dried powder and the solution in the step (3) according to the proportion, and putting the mixture into a refrigerator at the temperature of-80 ℃ for freezing storage.
Wherein, the step (1) comprises the following steps:
(a) culturing human mesenchymal stem cells: selecting human adipose-derived mesenchymal stem cells of P2-P6 from a cell bank, and inoculating the cells in three batches after recovery, wherein the inoculation densities of the three batches are respectively 50-60%, 30-40% and 20-30% of the maximum fusion degree;
the complete culture medium for inoculating and culturing the cells is phenol red-free DMEM/DF12, and 10% fetal calf serum in volume ratio concentration is added;
after the inoculation of the cells is finished, putting the cells into an incubator for culture, wherein the culture conditions are that the temperature is 37 ℃ and the volume concentration of carbon dioxide is 5%;
(b) when the fusion degree of the first batch of cell culture reaches 70-80%, cleaning the cells for 2 times by using normal saline, and then carrying out starvation culture by using compound electrolyte injection; after 24 hours of culture, when the cell fusion degree of the first batch reaches 90-95%, collecting cell culture supernatant, centrifuging for 6min under the condition of 4000g to remove precipitates, and collecting centrifuged supernatant;
(c) when the fusion degree of the second batch of cell culture reaches 70-80%, washing the cells for 2 times by using physiological saline, and performing starvation culture by using the supernatant obtained by the first batch of cell culture in the step (b); after culturing for 24 hours, collecting cell culture supernatant when the cell fusion degree of the second batch reaches 90-95%, centrifuging for 6min under the condition of 4000g to remove precipitates, and collecting centrifuged supernatant;
(d) when the fusion degree of the third batch of cell culture reaches 70-80%, washing the cells for 2 times by using normal saline, and performing starvation culture by using the supernatant obtained by the second batch of cell culture in the step (c); after culturing for 24 hours, collecting cell culture supernatant when the cell fusion degree of the second batch reaches 90-95%, centrifuging for 6min under the condition of 4000g to remove precipitates, and collecting centrifuged supernatant;
(e) filtering the supernatant obtained in the step (d) by using a 0.22um filter membrane, adding water-soluble chitosan into the filtered supernatant, and uniformly mixing; the mass concentration of the chitosan in the water-soluble chitosan is 0.08%.
Example 2
An ointment for removing eczema in infants containing mesenchymal stem cell lysate, which comprises the following components in every 100ml of ointment:
mesenchymal stem cell lysate of about 10X 108A lytic component of the individual cell; 10mg of mesenchymal stem cell freeze-dried powder; 5-10 g of a stabilizer; 50ml of electrolyte injection; humectant 5m 1; 8ml of transdermal agent; propolis 60 mg.
Wherein the stabilizing agent is sodium alginate. The humectant is one or more of glycerol. The electrolyte injection is compound electrolyte glucose MG3 injection. The transdermal agent is menthol.
The preparation method of the infant de-eczema ointment containing the mesenchymal stem cell lysate comprises the following steps:
(1) carrying out starvation culture after culturing the human umbilical cord mesenchymal stem cells; collecting cell culture supernatant, adding water-soluble chitosan into the supernatant after filtration by a filter membrane, mixing uniformly to obtain mesenchymal stem cell lysate, and storing in a refrigerator at 4 ℃ for later use;
(2) the preparation method of the umbilical cord mesenchymal stem cell freeze-dried powder comprises the following steps of sequentially filtering umbilical cord mesenchymal stem cell supernatant through 0.45 mu m and 0.22 mu m filter membranes, concentrating and freeze-drying;
(3) dissolving a stabilizer, a humectant and a transdermal agent in an electrolyte solution to prepare a uniform solution;
(4) and (4) uniformly mixing the umbilical cord mesenchymal stem cell lysate, the umbilical cord mesenchymal stem cell freeze-dried powder and the solution in the step (3) according to the proportion, and freezing and storing in a refrigerator at the temperature of-80 ℃.
Wherein, the step (1) comprises the following steps:
(a) culturing human umbilical cord mesenchymal stem cells: selecting human umbilical cord mesenchymal stem cells of P2-P6 from a cell bank, and inoculating the cells in three batches after recovery, wherein the inoculation densities of the three batches are respectively 50-60%, 30-40% and 20-30% of the maximum fusion degree;
the complete culture medium for inoculating and culturing the cells is phenol red-free DMEM/DF12, and 10% fetal calf serum in volume ratio concentration is added;
after the inoculation of the cells is finished, putting the cells into an incubator for culture, wherein the culture conditions are that the temperature is 37 ℃ and the volume concentration of carbon dioxide is 5%;
(b) when the fusion degree of the first batch of cell culture reaches 70-80%, cleaning the cells for 2 times by using normal saline, and then carrying out starvation culture by using compound electrolyte injection; after culturing for 18 hours, collecting cell culture supernatant when the cell fusion degree of the first batch reaches 90-95%, centrifuging for 10min under the condition of 3000g to remove precipitates, and collecting centrifuged supernatant;
(c) when the fusion degree of the second batch of cell culture reaches 70-80%, washing the cells for 2 times by using physiological saline, and performing starvation culture by using the supernatant obtained by the first batch of cell culture in the step (b); after culturing for 18 hours, collecting cell culture supernatant when the cell fusion degree of the second batch reaches 90-95%, centrifuging for 10min under the condition of 3000g to remove precipitates, and collecting centrifuged supernatant;
(d) when the fusion degree of the third batch of cell culture reaches 70-80%, washing the cells for 2 times by using normal saline, and performing starvation culture by using the supernatant obtained by the second batch of cell culture in the step (c); after culturing for 18 hours, collecting cell culture supernatant when the cell fusion degree of the second batch reaches 90-95%, centrifuging for 10min under the condition of 3000g to remove precipitates, and collecting centrifuged supernatant;
(e) filtering the supernatant obtained in the step (d) by using a 0.22um filter membrane, adding water-soluble chitosan into the filtered supernatant, and uniformly mixing; the mass concentration of the chitosan in the water-soluble chitosan is 0.05%.
Example 3
An ointment for removing eczema in infants containing mesenchymal stem cell lysate, which comprises the following components in every 100ml of ointment:
mesenchymal stem cell lysate 4-10 × 108A lytic component of the individual cell; 10-20 mg of mesenchymal stem cell freeze-dried powder; 5-10 g of a stabilizer; 50-100 ml of electrolyte injection; 5-8 m of a humectant 1; 5-10 ml of transdermal agent; 50-100 mg of propolis.
Wherein the stabilizer is chitosan. The humectant is glycerol. The electrolyte injection is compound electrolyte glucose MG3 injection. The transdermal agent is menthol.
The preparation method of the infant de-eczema ointment containing the mesenchymal stem cell lysate comprises the following steps:
(1) starvation culture is carried out after human skin mesenchymal stem cells are cultured; collecting cell culture supernatant, adding water-soluble chitosan into the supernatant after filtration by a filter membrane, mixing uniformly to obtain mesenchymal stem cell lysate, and storing in a refrigerator at 4 ℃ for later use;
(2) the preparation method of the skin mesenchymal stem cell freeze-dried powder comprises sequentially filtering the supernatant of the skin mesenchymal stem cell with 0.45 μm and 0.22 μm filter membranes, concentrating, and freeze-drying;
(3) dissolving a stabilizer, a humectant and a transdermal agent in an electrolyte solution to prepare a uniform solution;
(4) and (3) uniformly mixing the skin mesenchymal stem cell lysate, the skin mesenchymal stem cell freeze-dried powder and the solution in the step (3) according to the proportion, and freezing and storing in a refrigerator at the temperature of-80 ℃.
Wherein, the step (1) comprises the following steps:
(a) human skin mesenchymal stem cell culture: selecting human skin mesenchymal stem cells of P2-P6 from a cell bank, inoculating the cells in three batches after recovery, wherein the inoculation densities of the three batches are respectively 50% -60%, 35% -45% and 20% -30% of the maximum fusion degree;
the complete culture medium for inoculating and culturing the cells is phenol red-free DMEM/DF12, and 10% fetal calf serum in volume ratio concentration is added;
after the inoculation of the cells is finished, putting the cells into an incubator for culture, wherein the culture conditions are that the temperature is 37 ℃ and the volume concentration of carbon dioxide is 5%;
(b) when the fusion degree of the first batch of cell culture reaches 70-80%, cleaning the cells for 2 times by using normal saline, and then carrying out starvation culture by using compound electrolyte injection; after 24 hours of culture, when the cell fusion degree of the first batch reaches 90-95%, collecting cell culture supernatant, centrifuging for 6min under the condition of 4000g to remove precipitates, and collecting centrifuged supernatant;
(c) when the fusion degree of the second batch of cell culture reaches 70-80%, washing the cells for 2 times by using physiological saline, and performing starvation culture by using the supernatant obtained by the first batch of cell culture in the step (b); after culturing for 12-24 hours, collecting cell culture supernatant when the cell fusion degree of the second batch reaches 90% -95%, centrifuging for 6min under the condition of 4000g to remove precipitates, and collecting centrifuged supernatant;
(d) when the fusion degree of the third batch of cell culture reaches 70-80%, washing the cells for 2 times by using physiological saline, and performing starvation culture by using the supernatant obtained by the second batch of cell culture in the step (c); after culturing for 24 hours, collecting cell culture supernatant when the cell fusion degree of the second batch reaches 90-95%, centrifuging for 6min under the condition of 4000g to remove precipitates, and collecting centrifuged supernatant;
(e) filtering the supernatant obtained in the step (d) by using a 0.22um filter membrane, adding water-soluble chitosan into the filtered supernatant, and uniformly mixing; the mass concentration of the chitosan in the water-soluble chitosan is 0.06%.
In order to verify the effect, clinical trials were undertaken:
the ointments prepared in examples 1 to 3 were evaluated for efficacy, and specifically tested as follows:
the test method for the eczema-removing efficacy is as follows: four groups of male and female, wherein 10 volunteers are selected in each group, and the patients with eczema all have the age of 2-48 months; three groups of the above were each prepared using the ointments of examples 1 to 3, and the comparative example was prepared using commercially available ointments.
The treatment method comprises the following steps: continuously trying for 2 weeks, and taking pictures and archiving in the morning and evening; comparing the photographs at day 0 and week 2, the evaluation of complete elimination of eczema symptoms was significant, and the evaluation of remission of eczema symptoms was effective; no significant change in eczema symptoms was assessed as ineffective.
The specific test results are shown in table 1.
TABLE 1 efficacy test record chart for eliminating eczema
| Show effect | Is effective | Invalidation | |
| Example 1 | 5 | 5 | 0 |
| Example 2 | 5 | 5 | 0 |
| Example 3 | 6 | 4 | 0 |
| Comparative example | 3 | 6 | 1 |
As can be seen from the comparison of the examples and the comparative examples, the ointment prepared in the examples of the present invention has a good eczema-removing effect.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (8)
1. An ointment for removing eczema in infants containing mesenchymal stem cell lysate, which is characterized by comprising the following components in each 100ml of ointment:
mesenchymal stem cell lysate 4-10 × 108A lytic component of the individual cell; 10-20 mg of mesenchymal stem cell freeze-dried powder; 5-10 g of a stabilizer; 50-100 ml of electrolyte injection; 5-8 ml of a humectant; 5-10 ml of transdermal agent; 50-100 mg of propolis.
2. The ointment of claim 1, wherein the stabilizer is one or more of sodium hyaluronate, sodium alginate, chitosan and hydroxyethyl starch.
3. The ointment of claim 1, wherein the humectant is one or more of propylene glycol or glycerin.
4. The ointment of claim 1, wherein the electrolyte injection is a compound electrolyte glucose MG3 injection.
5. The ointment of claim 1, wherein the transdermal agent is menthol.
6. The method for preparing infant de-eczematous ointment containing mesenchymal stem cell lysate according to claim 1, comprising the steps of:
(1) carrying out starvation culture after culturing the human mesenchymal stem cells; collecting cell culture supernatant, adding water-soluble chitosan into the supernatant after filtration by a filter membrane, mixing uniformly to obtain mesenchymal stem cell lysate, and storing in a refrigerator at 4 ℃ for later use;
(2) the preparation method of the mesenchymal stem cell freeze-dried powder comprises the following steps of sequentially filtering a mesenchymal stem cell supernatant through 0.45 mu m and 0.22 mu m filter membranes, concentrating and freeze-drying;
(3) dissolving a stabilizer, a humectant and a transdermal agent in an electrolyte solution to prepare a uniform solution;
(4) and (4) uniformly mixing the mesenchymal stem cell lysate, the mesenchymal stem cell freeze-dried powder and the solution in the step (3) according to the proportion, and putting the mixture into a refrigerator at the temperature of-80 ℃ for freezing storage.
7. The method for preparing infant de-eczema ointment containing mesenchymal stem cell lysate according to claim 6, wherein the step (1) comprises the following steps:
(a) culturing human mesenchymal stem cells: selecting human mesenchymal stem cells from a cell bank from P2-P6, and inoculating the cells in three batches after recovery, wherein the inoculation densities of the three batches are respectively 50-60%, 30-40% and 20-30% of the maximum fusion degree;
the complete culture medium for inoculating and culturing the cells is phenol red-free DMEM/DF12, and 10% fetal calf serum in volume ratio concentration is added;
after the inoculation of the cells is finished, putting the cells into an incubator for culture, wherein the culture conditions are that the temperature is 37 ℃ and the volume concentration of carbon dioxide is 5%;
(b) when the fusion degree of the first batch of cell culture reaches 70-80%, cleaning the cells for 2 times by using normal saline, and then carrying out starvation culture by using compound electrolyte injection; after culturing for 18-24 hours, collecting cell culture supernatant when the cell fusion degree of the first batch reaches 90-95%, centrifuging for 6-10 min under the condition of 3000-4000 g to remove precipitates, and collecting centrifuged supernatant;
(c) when the fusion degree of the second batch of cell culture reaches 70-80%, washing the cells for 2 times by using physiological saline, and performing starvation culture by using the supernatant obtained by the first batch of cell culture in the step (b); after culturing for 18-24 hours, collecting cell culture supernatant when the cell fusion degree of the second batch reaches 90% -95%, centrifuging for 6-10 min under the condition of 3000-4000 g to remove precipitates, and collecting centrifuged supernatant;
(d) when the fusion degree of the third batch of cell culture reaches 70-80%, washing the cells for 2 times by using normal saline, and performing starvation culture by using the supernatant obtained by the second batch of cell culture in the step (c); after culturing for 18-24 hours, collecting cell culture supernatant when the cell fusion degree of the second batch reaches 90% -95%, centrifuging for 6-10 min under the condition of 3000-4000 g to remove precipitates, and collecting centrifuged supernatant;
(e) filtering the supernatant obtained in the step (d) by using a 0.22um filter membrane, adding water-soluble chitosan into the filtered supernatant, and uniformly mixing; the mass concentration of the chitosan in the water-soluble chitosan is 0.05-0.08%.
8. Use of an ointment containing mesenchymal stem cell lysate for eliminating eczema in infants according to claim 1, for treating infant eczema.
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| CN106039293A (en) * | 2016-07-28 | 2016-10-26 | 广州赛莱拉干细胞科技股份有限公司 | Composition for repairing skin ulcers and preparation method thereof |
| CN106176813A (en) * | 2016-07-28 | 2016-12-07 | 广州赛莱拉干细胞科技股份有限公司 | A kind of compositions repairing skin ulcer and preparation method thereof |
| CN106474155A (en) * | 2016-10-19 | 2017-03-08 | 天津普瑞赛尔生物科技有限公司 | External-use gel preparation containing human umbilical cord mesenchymal stem cells extract and its production and use |
| CN107158035A (en) * | 2017-05-17 | 2017-09-15 | 天津普瑞赛尔生物科技有限公司 | Treatment bedsore external-use gel containing high concentration human mesenchymal stem cell excretion body extract and preparation method thereof |
| CN107174590A (en) * | 2017-05-17 | 2017-09-19 | 天津普瑞赛尔生物科技有限公司 | For small-sized pet containing reparation spray of people's mesenchymal stem cells extract and preparation method thereof between high concentration |
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2019
- 2019-07-26 CN CN201910727743.1A patent/CN112353814A/en active Pending
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104762260A (en) * | 2015-04-23 | 2015-07-08 | 广州赛莱拉干细胞科技股份有限公司 | Preparation method and application of adipose-derived mesenchymal stem cells and preparation thereof |
| CN106039293A (en) * | 2016-07-28 | 2016-10-26 | 广州赛莱拉干细胞科技股份有限公司 | Composition for repairing skin ulcers and preparation method thereof |
| CN106176813A (en) * | 2016-07-28 | 2016-12-07 | 广州赛莱拉干细胞科技股份有限公司 | A kind of compositions repairing skin ulcer and preparation method thereof |
| CN106474155A (en) * | 2016-10-19 | 2017-03-08 | 天津普瑞赛尔生物科技有限公司 | External-use gel preparation containing human umbilical cord mesenchymal stem cells extract and its production and use |
| CN107158035A (en) * | 2017-05-17 | 2017-09-15 | 天津普瑞赛尔生物科技有限公司 | Treatment bedsore external-use gel containing high concentration human mesenchymal stem cell excretion body extract and preparation method thereof |
| CN107174590A (en) * | 2017-05-17 | 2017-09-19 | 天津普瑞赛尔生物科技有限公司 | For small-sized pet containing reparation spray of people's mesenchymal stem cells extract and preparation method thereof between high concentration |
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