Cell exosome for promoting hair growth and preparation method thereof
The exosomes prepared by hypoxia culture and icariin liposome treatment solved the problem of poor hair growth effect of stem cell exosomes and achieved significant hair growth effect.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-11-17
- Publication Date
- 2026-04-03
AI Technical Summary
The existing stem cell exosomes have a slow onset of action and poor hair growth effect, which has become a factor restricting their application in the treatment of hair loss.
Umbilical cord mesenchymal stem cells were cultured in a low-oxygen medium with the addition of icariin liposomes. Exosomes were prepared by gradient centrifugation to promote the secretion of active cytokines such as VGEF, activate hair follicle development, and exert anti-inflammatory effects.
It significantly increased the content of active cytokines such as VGEF in exosomes, promoted hair follicle development and relieved scalp inflammation, improved the hair follicle microenvironment, and achieved a more effective hair growth effect.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of biotechnology, and in particular to a cell exosome that promotes hair growth and a method for preparing the same. Background Technology
[0002] Hair loss, especially androgenetic alopecia, is a common health problem primarily caused by hair follicle atrophy. Current traditional treatments for hair loss mainly include surgery and medication. Surgery, which involves hair follicle transplantation, carries some potential risks of trauma and cannot stop hair loss in untransplanted areas. Medications for hair loss, such as minoxidil, can be irritating and may cause side effects. Given the limitations of these traditional therapies, it is necessary to develop new treatments that can improve hair loss.
[0003] Stem cell exosomes are nanoscale vesicles secreted by stem cells, containing bioactive substances such as proteins, nucleic acids, and lipids. Compared with traditional drugs, stem cell exosomes have the advantages of being less irritating, having no toxic side effects, and being highly safe. Studies have shown that stem cell exosomes have the potential to repair and regenerate hair follicles, and can promote hair regeneration. However, the application of stem cell exosome products in the treatment of hair loss still suffers from slow onset of action and poor hair regrowth effects, which has become a major factor restricting the application of stem cell exosomes in the field of hair regrowth. Summary of the Invention
[0004] Based on the technical problems existing in the background art, the present invention proposes a cell exosome that promotes hair growth and its preparation method.
[0005] The present invention proposes a method for preparing exosomes that promote hair growth, comprising the following steps:
[0006] S1. Umbilical cord mesenchymal stem cells are seeded into a culture dish containing the first culture medium and cultured under hypoxic conditions. The culture conditions are: 1%~2% O2, 5% CO2, temperature of 37℃, and culture time of 24~48h. The first culture medium is high glucose DMEM medium.
[0007] S2. Replace the culture medium in the culture dish with the second culture medium and culture under normoxic conditions: 5% CO2, temperature 37℃, culture time 36~48h. Then, perform gradient centrifugation at 2~4℃ to obtain the exosomes. The second culture medium includes a basal medium and additives. The basal medium is serum-free DMEM / F12 medium. The additives include vitamin C, L-glutathione, transferrin, and icariin liposome suspension. The amount of vitamin C is 20~50mg / L, the amount of L-glutathione is 1~5mg / L, the amount of transferrin is 5~10μg / mL, and the amount of icariin liposome suspension is 2~5mL / L. The solid content of the icariin liposome suspension is 1~2%.
[0008] In this invention, the concentrations of O2 and CO2 refer to the volume percentage content. For example, 1%~2% O2 means that the volume percentage content of O2 is 1%~2%.
[0009] Preferably, in S1, the seeding density of the umbilical cord mesenchymal stem cells is 1~3×10⁻⁶. 4 pcs / cm 2 .
[0010] Preferably, the umbilical cord mesenchymal stem cells are P3-P5 generation umbilical cord mesenchymal stem cells.
[0011] In this invention, P3-P5 generation umbilical cord mesenchymal stem cells can be prepared using conventional methods. Preferably, the method for preparing the P3-P5 generation umbilical cord mesenchymal stem cells includes: cutting umbilical cord tissue into 1-3 mm pieces. 2 Small pieces of the umbilical cord mesenchymal stem cells were placed in a culture dish containing 10% fetal bovine serum in DMEM / F12 medium and cultured adherently at 37°C and 5% CO2 for 10-14 days. Then, the cells were digested with trypsin and centrifuged to obtain P0 generation umbilical cord mesenchymal stem cells. The P0 generation umbilical cord mesenchymal stem cells were passaged under the conditions of 37°C and 5% CO2 in DMEM / F12 medium containing 10% fetal bovine serum to obtain P3-P5 generation umbilical cord mesenchymal stem cells.
[0012] Preferably, the preparation method of the icariin liposome suspension includes: dissolving icariin, soybean lecithin and cholesterol in methanol, removing the solvent by rotary evaporation at 45-55°C, and then vacuum drying to obtain a lipid complex; adding phosphate buffer to the lipid complex, heating and stirring at 45-55°C for 10-20 min, and then sonicating at 150-250 W for 5-10 min to obtain the final product.
[0013] Preferably, the pH of the phosphate (PBS) buffer is 6.6-7.4 and the concentration is 10 mM.
[0014] Preferably, the mass ratio of icariin, soybean lecithin, and cholesterol is 1:5~10:1:5~10.
[0015] Preferably, the ratio of icariin to methanol is 1g: 50~100mL.
[0016] Preferably, in step S3, the specific steps of gradient centrifugation include: first centrifuging at 250g~350g for 5~15min and collecting the supernatant; then centrifuging at 1500g~2500g for 15~25min and collecting the supernatant; then centrifuging at 8000g~12000g for 20~30min and collecting the supernatant; and finally centrifuging at 100000g~120000g for 80~120min and collecting the precipitate, thus obtaining the exosomes.
[0017] The present invention also proposes a cell exosome that promotes hair growth, which is prepared by the aforementioned preparation method.
[0018] The beneficial effects of this invention are as follows:
[0019] This invention first culturees umbilical cord mesenchymal stem cells under hypoxia, effectively promoting stem cell proliferation and activation, increasing exosome secretion and the content of active cytokines such as VGEF that promote hair follicle development. Then, the culture is adjusted to normoxic conditions, and icariin liposomes are added to the culture medium for further culture. This further stimulates stem cells to produce cytokines such as VGEF that promote hair follicle development and anti-inflammatory cytokines, thereby promoting hair follicle development, providing necessary nutrients for hair follicle growth, relieving scalp inflammation, and improving the hair follicle microenvironment, thus effectively improving hair regrowth. Detailed Implementation
[0020] The technical solution of the present invention will now be described in detail through specific embodiments.
[0021] Unless otherwise specified, the test materials used in the following examples are all commercially available reagents.
[0022] Example 1
[0023] Preparation of icariin liposome suspension:
[0024] Icariin, soybean lecithin, and cholesterol were dissolved in methanol at a mass ratio of 1:5:5. The solvent was removed by rotary evaporation at 50°C, followed by vacuum drying to obtain a lipid complex with an icariin to methanol ratio of 1 g:50 mL. PBS buffer (pH 7.2, 10 mM) was added to the lipid complex, and the mixture was heated and stirred at 50°C for 15 min, followed by sonication at 200 W for 8 min to obtain an icariin liposome suspension with a solid content of 1.5%.
[0025] Preparation of cell exosomes:
[0026] S1. Cut the human umbilical cord tissue into 1-3mm pieces. 2 Small pieces of the stem cells were placed in a culture dish containing DMEM / F12 medium with 10% fetal bovine serum and cultured adherently at 37°C and 5% CO2 for 12 days. Then, the cells were digested with 0.25% trypsin and centrifuged to obtain P0 generation umbilical cord mesenchymal stem cells. The P0 generation umbilical cord mesenchymal stem cells were passaged at 37°C and 5% CO2 in DMEM / F12 medium containing 10% fetal bovine serum to obtain P3 generation umbilical cord mesenchymal stem cells.
[0027] The above-mentioned P3 generation umbilical cord mesenchymal stem cells were used at a dose of 2.5 × 10⁻⁶. 4 pcs / cm 2 The inoculation density was inoculated into culture dishes containing the first culture medium and cultured under hypoxic conditions: 1% O2, 5% CO2, temperature 37℃, and culture time 36h; the first culture medium was high-glucose DMEM medium (Thermo Fisher 12430054).
[0028] S2. Replace the culture medium in the petri dish with the second culture medium and culture under normoxic conditions: 5% CO2, 21% O2, temperature 37℃, and culture time 36h. After culture, perform gradient centrifugation at 4℃. The gradient centrifugation steps are as follows: first, centrifuge at 300g for 10min and collect the supernatant; then, centrifuge at 2000g for 20min and collect the supernatant; then, centrifuge at 10000g for 25min and collect the supernatant; finally, centrifuge at 100000g for 90min and collect the precipitate, which is the exosome. The second culture medium consists of basal medium and additives. The basal medium is serum-free DMEM / F12 medium (Thermo Fisher Scientific). (12634010), the additives are vitamin C, L-glutathione, transferrin and the above-mentioned icariin liposome suspension, the amount of vitamin C is 40 mg / L, the amount of L-glutathione is 2 mg / L, the amount of transferrin is 8 μg / mL, and the amount of icariin liposome suspension is 3 mL / L.
[0029] Example 2
[0030] Preparation of icariin liposome suspension:
[0031] Icariin, soybean lecithin, and cholesterol were dissolved in methanol at a mass ratio of 1:5:5. The solvent was removed by rotary evaporation at 45°C, followed by vacuum drying to obtain a lipid complex with an icariin to methanol ratio of 1 g:50 mL. PBS buffer (pH 7.2, 10 mM) was added to the lipid complex. The mixture was first heated and stirred at 45°C for 10 min, and then sonicated at 150 W for 5 min to obtain an icariin liposome suspension with a solid content of 1%.
[0032] Preparation of cell exosomes:
[0033] S1. Cut the human umbilical cord tissue into 1-3mm pieces. 2 Small pieces of the umbilical cord mesenchymal stem cells were placed in a culture dish containing DMEM / F12 medium with 10% fetal bovine serum and cultured adherently at 37°C and 5% CO2 for 10 days. Then, the cells were digested with 0.25% trypsin and centrifuged to obtain P0 generation umbilical cord mesenchymal stem cells. The P0 generation umbilical cord mesenchymal stem cells were passaged at 37°C and 5% CO2 in DMEM / F12 medium containing 10% fetal bovine serum to obtain P3-P5 generation umbilical cord mesenchymal stem cells.
[0034] The above-mentioned P3~P5 generation umbilical cord mesenchymal stem cells were used at a rate of 1×10⁻⁶. 4 pcs / cm 2 The inoculation density was inoculated into culture dishes containing the first culture medium and cultured under hypoxic conditions: 1% O2, 5% CO2, temperature 37℃, and culture time 24h; the first culture medium was high-glucose DMEM medium (Thermo Fisher 12430054).
[0035] S2. Replace the culture medium in the petri dish with the second culture medium and culture under normoxic conditions: 5% CO2, 21% O2, temperature 37℃, and culture time 48h. After culture, perform gradient centrifugation at 4℃. The gradient centrifugation steps are as follows: first, centrifuge at 250g for 5min and collect the supernatant; then, centrifuge at 1500g for 15min and collect the supernatant; then, centrifuge at 8000g for 20min and collect the supernatant; finally, centrifuge at 100000g for 80min and collect the precipitate, which is the exosome. The second culture medium consists of basal medium and additives. The basal medium is serum-free DMEM / F12 medium (ThermoFisher). (12634010), the additives are vitamin C, L-glutathione, transferrin and the above-mentioned icariin liposome suspension, the amount of vitamin C is 20 mg / L, the amount of L-glutathione is 1 mg / L, the amount of transferrin is 5 μg / mL, and the amount of icariin liposome suspension is 2 mL / L.
[0036] Example 3
[0037] Preparation of icariin liposome suspension:
[0038] Icariin, soybean lecithin, and cholesterol were dissolved in methanol at a mass ratio of 1:10:10. The solvent was removed by rotary evaporation at 55°C, followed by vacuum drying to obtain a lipid complex with an icariin to methanol ratio of 1 g:100 mL. PBS buffer (pH 7.2, 10 mM) was added to the lipid complex, and the mixture was heated and stirred at 55°C for 20 min, followed by sonication at 250 W for 10 min to obtain an icariin liposome suspension with a solid content of 2%.
[0039] Preparation of cell exosomes:
[0040] S1. Cut the human umbilical cord tissue into 1-3mm pieces. 2 Small pieces of the umbilical cord mesenchymal stem cells were placed in a culture dish containing DMEM / F12 medium with 10% fetal bovine serum and cultured adherently at 37°C and 5% CO2 for 14 days. Then, the cells were digested with 0.25% trypsin and centrifuged to obtain P0 generation umbilical cord mesenchymal stem cells. The P0 generation umbilical cord mesenchymal stem cells were passaged at 37°C and 5% CO2 in DMEM / F12 medium containing 10% fetal bovine serum to obtain P3-P5 generation umbilical cord mesenchymal stem cells.
[0041] The above-mentioned P3~P5 generation umbilical cord mesenchymal stem cells were used at a rate of 3×10⁻⁶. 4 pcs / cm 2The inoculation density was inoculated into culture dishes containing the first culture medium and cultured under hypoxic conditions: 2% O2, 5% CO2, temperature 37℃, and culture time 48h; the first culture medium was high-glucose DMEM medium (Thermo Fisher 12430054).
[0042] S2. Replace the culture medium in the petri dish with the second culture medium and culture under normoxic conditions: 5% CO2, 21% O2, temperature 37℃, and culture time 48h. After culture, perform gradient centrifugation at 4℃. The gradient centrifugation steps are as follows: first, centrifuge at 350g for 15min and collect the supernatant; then, centrifuge at 2500g for 25min and collect the supernatant; then, centrifuge at 12000g for 30min and collect the supernatant; finally, centrifuge at 120000g for 120min and collect the precipitate, which is the exosome. The second culture medium consists of basal medium and additives. The basal medium is serum-free DMEM / F12 medium (Thermo Fisher Scientific). (12634010), the additives are vitamin C, L-glutathione, transferrin and the above-mentioned icariin liposome suspension, the amount of vitamin C is 50 mg / L, the amount of L-glutathione is 5 mg / L, the amount of transferrin is 10 μg / mL, and the amount of icariin liposome suspension is 5 mL / L.
[0043] Comparative Example 1
[0044] The only difference between Comparative Example 1 and Example 1 is the second culture medium. The second culture medium in Comparative Example 1 consists of a basal medium and additives. The basal medium is serum-free DMEM / F12 medium (Thermo Fisher 12634010), and the additives are vitamin C, L-glutathione, and transferrin. The amount of vitamin C is 40 mg / L, the amount of L-glutathione is 2 mg / L, and the amount of transferrin is 8 μg / mL.
[0045] Comparative Example 2
[0046] The only difference between Comparative Example 2 and Example 1 is the second culture medium. The second culture medium in Comparative Example 2 consists of a basal medium and additives. The basal medium is serum-free DMEM / F12 medium (Thermo Fisher 12634010), and the additives are vitamin C, L-glutathione, transferrin, and icariin. The dosage of vitamin C is 40 mg / L, the dosage of L-glutathione is 2 mg / L, the dosage of transferrin is 8 μg / mL, and the dosage of icariin is 3 μg / mL.
[0047] Comparative Example 3
[0048] Preparation of icariin liposome suspension:
[0049] Icariin, soybean lecithin, and cholesterol were dissolved in methanol at a mass ratio of 1:5:5. The solvent was removed by rotary evaporation at 50°C, followed by vacuum drying to obtain a lipid complex with an icariin to methanol ratio of 1 g:50 mL. PBS buffer (pH 7.2, 10 mM) was added to the lipid complex, and the mixture was heated and stirred at 50°C for 15 min, followed by sonication at 200 W for 8 min to obtain an icariin liposome suspension with a solid content of 1.5%.
[0050] Preparation of cell exosomes:
[0051] S1. Cut the human umbilical cord tissue into 1-3mm pieces. 2 Small pieces of the stem cells were placed in a culture dish containing DMEM / F12 medium with 10% fetal bovine serum and cultured adherently at 37°C and 5% CO2 for 12 days. Then, the cells were digested with 0.25% trypsin and centrifuged to obtain P0 generation umbilical cord mesenchymal stem cells. The P0 generation umbilical cord mesenchymal stem cells were passaged at 37°C and 5% CO2 in DMEM / F12 medium containing 10% fetal bovine serum to obtain P3 generation umbilical cord mesenchymal stem cells.
[0052] The above-mentioned P3 generation umbilical cord mesenchymal stem cells were used at a dose of 2.5 × 10⁻⁶. 4 pcs / cm 2 The inoculation density was inoculated into culture dishes containing stem cell culture medium and cultured under normoxic conditions: 5% CO2, 21% O2, temperature 37℃, and culture time 72h. After culture, the cells were centrifuged at 4℃ using a gradient centrifugation process: first centrifuged at 300g for 10min and collected the supernatant, then centrifuged at 2000g for 20min and collected the supernatant, then centrifuged at 10000g for 25min and collected the supernatant, and finally centrifuged at 100000g for 90min and collected the precipitate, which yielded exosomes. The second culture medium consisted of basal medium and additives. The basal medium was serum-free DMEM / F12 medium (Thermo Fisher 12634010), and the additives were vitamin C, L-glutathione, transferrin, and the above-mentioned icariin liposome suspension. The amount of vitamin C used was 40mg / L, and the amount of L-glutathione used was...
[0053] Test case
[0054] The exosomes collected in Examples 1-3 and Comparative Examples 1-3 were resuspended in 10 mL of PBS buffer (pH 7.2, 10 mM) to obtain exosome suspensions. The concentration of VGEF (vascular endothelial growth factor) in the exosome suspensions was then detected by ELISA. Three parallel samples were tested in each group, and the average was taken. The results are shown in Table 1.
[0055] Table 1
[0056]
[0057] C57BL / 6J mice were randomly divided into a control group, a positive control group, Example 1-3 groups, and Comparative Example 1-3 groups, with 10 mice in each group. A 1:1 mixture of rosin and paraffin was heated and applied evenly to the back area of the mice. After the mixture cooled and dried, the mice underwent hair removal. The back skin was pink and undamaged, confirming that the hair follicles were in the resting phase, thus establishing a mouse hair removal model. For Example 1-3 groups and Comparative Example 1-3 groups, 0.2 mL of the above exosome suspension was applied daily to the hair removal area (2 cm × 2 cm). The positive control group received an equal volume of 1% minoxidil, and the blank control group received an equal volume of deionized water. This treatment was continued for 14 days. Newly grown hair was then scraped from the hair removal area and weighed, with the average weight recorded. The results are shown in Table 2.
[0058] Table 2
[0059]
[0060] As shown in Table 1, the VGEF content in the exosomes obtained in Examples 1-3 was significantly increased. This indicates that by first culturing under hypoxia and then culturing under normoxic conditions with the addition of icariin liposomes, the paracrine mechanism of stem cells can be activated more effectively, thereby greatly increasing the secretion of active cytokines such as VGEF.
[0061] As shown in Table 2, the exosomes obtained using Examples 1-3 can achieve better hair growth effects compared with the commonly used hair growth drug minoxidil group. This may be related to the increased content of active cytokines such as VGEF in the exosomes obtained in this invention.
[0062] Based on Tables 1 and 2, compared with Comparative Example 1 (without icariin), Comparative Example 2 (with icariin), and Comparative Example 3 (without hypoxia culture), the VGEF content and hair growth were significantly increased in Examples 1-3. This is because the initial hypoxia activation followed by icariin activation has a synergistic effect, which can more effectively promote the secretion of exosomes and active cytokines. At the same time, liposomes of icariin are beneficial for the absorption and utilization of stem cells, and can more effectively activate the paracrine pathway of stem cells.
[0063] The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.
Claims
1. A method for preparing exosomes that promote hair growth, characterized in that, Includes the following steps: S1. Umbilical cord mesenchymal stem cells are seeded into a culture dish containing the first culture medium and cultured under hypoxic conditions. The culture conditions are: 1%~2% O2, 5% CO2, temperature of 37℃, and culture time of 24~48h. The first culture medium is high glucose DMEM medium. S2. Replace the culture medium in the culture dish with the second culture medium and culture under normoxic conditions: 5% CO2, temperature 37℃, culture time 36~48h. Then, perform gradient centrifugation at 2~4℃ to obtain the exosomes. The second culture medium includes a basal medium and additives. The basal medium is serum-free DMEM / F12 medium. The additives include vitamin C, L-glutathione, transferrin, and icariin liposome suspension. The amount of vitamin C is 20~50mg / L, the amount of L-glutathione is 1~5mg / L, the amount of transferrin is 5~10μg / mL, and the amount of icariin liposome suspension is 2~5mL / L. The solid content of the icariin liposome suspension is 1~2%.
2. The method for preparing exosomes that promote hair growth according to claim 1, characterized in that, In S1, the seeding density of the umbilical cord mesenchymal stem cells is 1~3×10⁻⁶. 4 pcs / cm 2 .
3. The method for preparing exosomes that promote hair growth according to claim 1, characterized in that, The umbilical cord mesenchymal stem cells are P3-P5 generation umbilical cord mesenchymal stem cells.
4. The method for preparing exosomes that promote hair growth according to claim 3, characterized in that, The method for preparing P3-P5 generation umbilical cord mesenchymal stem cells includes: cutting umbilical cord tissue into 1-3 mm pieces. 2 Small pieces of the umbilical cord mesenchymal stem cells were placed in a culture dish containing 10% fetal bovine serum in DMEM / F12 medium and cultured adherently at 37°C and 5% CO2 for 10-14 days. Then, the cells were digested with trypsin and centrifuged to obtain P0 generation umbilical cord mesenchymal stem cells. The P0 generation umbilical cord mesenchymal stem cells were passaged under the conditions of 37°C and 5% CO2 in DMEM / F12 medium containing 10% fetal bovine serum to obtain P3-P5 generation umbilical cord mesenchymal stem cells.
5. The method for preparing exosomes that promote hair growth according to claim 1, characterized in that, The preparation method of the icariin liposome suspension includes: dissolving icariin, soybean lecithin and cholesterol in methanol, removing the solvent by rotary evaporation at 45-55℃, and then vacuum drying to obtain a lipid complex; adding phosphate buffer to the lipid complex, heating and stirring at 45-55℃ for 10-20 min, and then sonicating at 150-250W for 5-10 min to obtain the final product.
6. The method for preparing exosomes that promote hair growth according to claim 5, characterized in that, The mass ratio of icariin, soybean lecithin, and cholesterol is 1:5~10:1:5~10.
7. The method for preparing exosomes that promote hair growth according to claim 1, characterized in that, In S3, the specific steps of gradient centrifugation include: first centrifuging at 250g~350g for 5~15min and collecting the supernatant; then centrifuging at 1500g~2500g for 15~25min and collecting the supernatant; then centrifuging at 8000g~12000g for 20~30min and collecting the supernatant; and finally centrifuging at 100000g~120000g for 80~120min and collecting the precipitate, thus obtaining the exosomes.
8. An exosome that promotes hair growth, characterized in that, It is prepared by the preparation method according to any one of claims 1 to 7.