CN115369073A - Preparation method of epidermal stem cell exosome - Google Patents
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Abstract
The invention provides a preparation method of an epidermal stem cell exosome, belonging to the field of biotechnology and medicine, based on the characteristics of Wnts signal paths, wnts protein is utilized to culture high-activity high-dry basal stem cells, and then the cell exosome is obtained. The obtained exosome has Wnts protein for promoting epidermal stem cell proliferation, and has strong capacity of promoting epidermal stem cell regeneration and growth activity; and the enzyme system is complete, so that the problem of fibrotic scars or damaged skin appendages caused by injury repair can be repaired.
Description
Technical Field
The invention belongs to the field of biotechnology and medicine, and particularly relates to a preparation method of high-activity high-dry epidermal stem cell exosome.
Background
Skin aging and diseases caused by mutation or damage have wide influence on human health, and how to repair damage to keep skin healthy and delay aging is one of the difficulties in medical research. With the development of the stem cell field, the development of dermatologic treatment or drug screening through functional seed cells is an emerging development direction. Stem cell-derived cell-specific cell exosomes may modulate biological function and enhance tissue regeneration by mediating cell-to-cell communication by cell-specific mRNA or proteins. However, most of the stem cells of the obtained exosomes are derived from pluripotent stem cells or mesenchymal stem cells, lack of skin specificity and related functionality, and do not meet the requirements of clinical treatment and drug screening experiments on skin problems.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention provides a preparation method of an epidermal stem cell exosome, which is a method for directionally differentiating functional epidermal stem cells into extracellular vesicles (exosomes), and based on the characteristics of a Wnt signal path, high-activity and high-dryness basal stem cells are cultured by using Wnts protein, so that the extracellular exosomes are obtained.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a preparation method of epidermal stem cell exosomes comprises the following steps:
removing subcutaneous tissues and fat from skin tissue samples, digesting by Dispase II (also called neutral protease) to obtain epidermal layers, and digesting by Trypsin/EDTA (ethylene diamine tetraacetic acid) prepared from Trypsin and EDTA to obtain epidermal stem cells;
preparing a culture medium, wherein the culture medium is Advanced TM DMEM/F12 culture medium or RPMI-1640 cell culture medium, wherein the components of the culture medium contain recombinant human Wnts protein;
pre-coating the well plate by using Matrigel, and adding a culture medium into each well of the well plate after waiting for a period of time; inoculating the epidermal stem cells into a pore plate containing a culture medium for culturing;
separating the extracellular vesicles of the epidermal stem cells from the supernatant of the cultured epidermal stem cells, putting the extracellular vesicles into a fetal bovine serum culture medium for growth, taking out the extracellular vesicles after 70-80% of the extracellular vesicles are fused, and adding the extracellular vesicles into another fetal bovine serum culture medium without bovine extracellular vesicles for culture for a period of time;
centrifuging the culture medium for the first time to remove dead cells; centrifuging the supernatant for the second time to eliminate contaminated cell debris; then carrying out third centrifugal precipitation to obtain extracellular vesicles, and removing polluted protein through washing and precipitation; and centrifuging for the fourth time to remove the supernatant and separating the extracellular secretion.
Further, the conditions for digestion with Dispase II were: the tissue was digested with Dispase II at 37 ℃ for several hours.
Further, the conditions for digestion with Trypsin/EDTA were: the epidermal layer is digested with preheated Trypsin/EDTA at 37 deg.C for 8-15 min, centrifuged at 1000 Xg-1500 Xg for 2.5-3.5 min, and washed several times with PBS at 4 deg.C.
Further, advanced TM Components of the DMEM/F12 medium include non-essential amino acids NEAA and B-27 in addition to the recombinant human Wnts protein TM Additive, advanced cell culture additive GlutaMAX TM The recombinant human epidermal growth factor protein HEPES comprises a non-ionic amphoteric buffer solution HEPES, N-acetylcysteine N-Ace, recombinant human epidermal growth factor protein hEGF, a pathway inhibitor A83-01, a eukaryotic adenylate cyclase activator Forskolin and a penicillin streptomycin double-resistant solution.
Further, advanced TM Preferred components of the DMEM/F12 medium comprise 1% of the non-essential amino acids NEAA, 2% B-27 TM Additive, 1% advanced cell culture additive GlutaMAX TM The recombinant human epidermal growth factor protein HEPES comprises 1% of non-ionic amphoteric buffer solution HEPES, 1mM N-acetylcysteine N-Ace, 50ng/mL of recombinant human epidermal growth factor protein hEGF, 2 mu M of pathway inhibitor A83-01, 10 mu M of eukaryotic adenylate cyclase activator Forskolin, 100U/L of penicillin streptomycin double-resistant solution and 50ng/mL of recombinant human Wnts protein.
Further, the well plates were pre-coated with Matrigel, and were used after waiting 24 hours.
Further, 6-well plates were used as the well plates, and 2ml of the medium was added to each well.
Furthermore, the epidermal stem cells were cultured in the well plates for 4 to 7 days, and the extracellular vesicles of the epidermal stem cells were cultured in a medium of fetal bovine serum from which the extracellular vesicles of bovine serum were removed for more than 48 hours.
Further, the centrifugation temperature of the above four times is 4 ℃; the pellet was washed in PBS to remove contaminating proteins.
Further, the extracellular vesicles of the epidermal stem cells are put into a 10-15% fetal bovine serum culture medium for growth; another medium of fetal calf serum with removed bovine extracellular vesicles was obtained by ultracentrifugation of fetal calf serum.
The exosome obtained by the method has Wnts protein for promoting the proliferation of epidermal stem cells, and has strong capacity of promoting the regeneration and growth activity of the epidermal stem cells; and the enzyme system is complete, so that the problem of fibrotic scars or damaged skin appendages caused by injury repair can be repaired. The epidermal stem cell exosome cultured by the invention can collect trace epidermal cells of a specific patient according to the requirement to culture the epidermal stem cells and prepare the epidermal stem cell exosome for the specific patient, and has high medical value. The exosome can be used for delaying skin aging and promoting the activity of epidermal stem cells caused by aging or inflammation, can be applied in the forms of subcutaneous injection, spray, dressing and the like, and has high medical and commercial values, wherein the forms of the exosome include but are not limited to treatment of healing-refractory skin wound healing, promotion of skin regeneration, anti-wrinkle and the like.
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FIG. 1 is a flow chart of epidermal stem cell culture in an example of the present invention.
FIG. 2 is a flow chart of the preparation of extracellular secretion in the example of the present invention.
FIGS. 3A-3B are graphs of proliferation markers and mean cell counts in examples of the invention.
Detailed Description
In order to make the aforementioned and other features and advantages of the invention more comprehensible, embodiments accompanied with figures are described in detail below.
The embodiment specifically discloses a preparation method of an epidermal stem cell exosome, which comprises the following operation steps:
1. epidermal stem cell culture, as shown in FIG. 1.
A skin tissue sample was removed of subcutaneous tissue and fat, and digested with 2.5mg/mL Dispase II (Cat #17150041, gibco) at 37 ℃ for 2 hours to obtain an epidermal sheet. The samples were then digested with 0.25% Trypsin/EDTA (Cat #25200072, gibco) pre-warmed for 10 minutes at 37 ℃ (in other embodiments any length of 8 to 15 minutes may be selected as desired), centrifuged at 1200 Xg (in other embodiments any length of 1000 Xg to 1500 Xg may be selected as desired) for 3 minutes (in other embodiments any length of 2.5 to 3.5 minutes may be selected as desired), and washed 3 times with PBS at 4 ℃ to allow the cells to be seeded into 6-well plates. 6-well plates were pre-coated with Matrigel (Cat #354230, corning) 24 hours prior to use.
Cells were inoculated onto 6-well plates plated with Matrigel gel, 2ml of culture medium was added to each well before inoculation, and the culture medium ratio was: advanced (Advanced) TM DMEM/F12 medium (Cat #12634, invitrogen) containing 1% of the non-essential amino acids NEAA (Cat #11140, gibco), 2% by weight TM Additive (Cat #17504, gibco), 1% advanced cell culture additive GlutaMAX TM (Cat #35050, gibco), 1% non-ionic ampholytic buffer HEPES (Cat #15630, gibco), 1mM N-acetylcysteine N-Ace (Cat # A9165, sigma), 50ng/mL recombinant human epidermal growth factor protein hEGF (Cat #236-EG, R&D) 2 μ M pathway inhibitor A83-01 (Cat # SML0788, sigma), 10 μ M eukaryotic adenylate cyclase activator Forskolin (Cat # S2449, selleck), 100U/L penicillin streptomycin double antibody solution (Cat # 01151463), 50ng/mL recombinant human Wnts protein (Cat #5036-WN R)&D) In that respect The above-mentioned concentration values of the respective components are preferred parameter values, and the various additives may be replaced by additives of similar composition or function, in practice, at concentrations in the interval of 50% to 200% given the optimum concentration (e.g. 0.5% to 2% of the non-essential amino acid NEAA, 1% to 4% B-27% TM Additive, 0.5% -2% advanced cell culture additive GlutaMAX TM 0.5-2% non-ionic amphoteric buffer HEPES, 0.5-2 mM N-acetylcysteine N-Ace, 25-100 ng/mL recombinant human epidermal growth factor protein hEGF, 1-4 MuM pathway inhibitor A83-01, 5-20 MuM eukaryotic adenylate cyclase activator Forskolin, 50-200U/L penicillin streptomycin double-antibody solution, 25-100 ng/mL recombinant human Wnts protein) can completely or basically obtain the final product of the invention, rootDetermined according to actual requirements. Epidermal stem cells were cultured in 6-well plates for 5 days.
2. Extracellular exosomes were isolated as shown in figure 2.
Extracellular vesicles were isolated from epidermal stem cell culture supernatant. The cells are cultured under the normal condition at 10-15 cm 2 The flask is supplemented with 10% -15% fetal calf serum culture medium for growth. Fetal calf serum was centrifuged at 110000g for 180 minutes by ultracentrifugation to prepare fetal calf serum from which the outer vesicles of bovine cells were removed. After reaching 70% -80% confluence, the cultured epidermal stem cells were cultured in a medium supplemented with extracellular vesicle-free fetal bovine serum for more than 48 hours. The medium was collected and centrifuged at 300g for 10 min at 4 ℃ and 2000g for 20 min at 4 ℃ to remove dead cells. The supernatant was further centrifuged at 10000g at 4 ℃ for 30 minutes to eliminate contaminating cell debris. Extracellular vesicles were precipitated from the supernatant by ultracentrifugation using an SW32 Ti rotor at 110000g for 70 min at 4 ℃. The pellet was washed in PBS to eliminate contaminating proteins. And carrying out another round of centrifugation at high speed to discard the supernatant, and finally obtaining the separated cell exosomes. The parameters of time, quality and the like in the separation process can be determined within the range of 50-200% according to actual requirements, and the determined parameters are preferred parameters.
The invention combines the centrifugation with specific temperature, different duration and different rotating speed to carry out a series of combination and treatment, and finally the extracellular vesicles are precipitated from the supernatant. The pellet was washed in PBS to eliminate contaminating proteins. Finally, the extracellular vesicles are separated from the culture supernatant of the human epithelial stem cells, so that the extracellular exosomes are obtained. The method belongs to a mild method, the survival rate of the extracted epidermal stem cells is up to more than 98%, and the dry marker of the epidermal stem cells is up to more than 95%. The culture has strong proliferation capacity and rapid amplification rate (see FIGS. 3A-3B). The cultured stem cells have perfect functions and complete intercellular connection, receptor adhesion and capacity of secreting microenvironment components of the epidermal stem cells. Can be used as seed cells to be transplanted to a natural bracket to construct tissue engineering skin with functional polarity, and directly promote the wound healing of the skin; the source of the exosome-derived cells involved in the present invention is therefore epidermal stem cells close to human physiological conditions.
Although the present invention has been described with reference to the above embodiments, it should be understood that various changes, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
Claims (10)
1. A preparation method of an epidermal stem cell exosome is characterized by comprising the following steps:
removing subcutaneous tissues and fat from a skin tissue sample, digesting by using Dispase II to obtain an epidermal layer, and digesting by using Trypsin/EDTA (ethylene diamine tetraacetic acid) prepared from Trypsin and EDTA to obtain epidermal stem cells respectively;
preparing a culture medium which is Advanced TM DMEM/F12 culture medium or RPMI-1640 cell culture medium, wherein the components of the culture medium contain recombinant human Wnts protein;
pre-coating the well plate by using Matrigel, and adding a culture medium into each well of the well plate after waiting for a period of time; inoculating the epidermal stem cells into a pore plate containing a culture medium for culturing;
separating the extracellular vesicles of the epidermal stem cells from the supernatant of the cultured epidermal stem cells, putting the extracellular vesicles into a fetal bovine serum culture medium for growth, taking out the extracellular vesicles after 70-80% of the extracellular vesicles are fused, and adding the extracellular vesicles into another fetal bovine serum culture medium without bovine extracellular vesicles for culture for a period of time;
centrifuging the culture medium for the first time to remove dead cells; centrifuging the supernatant for the second time to eliminate contaminated cell debris; carrying out third centrifugal precipitation to obtain extracellular vesicles, and washing and precipitating to remove polluted proteins; and centrifuging for the fourth time to remove the supernatant, and separating the extracellular secretion.
2. The method of claim 1, wherein the digestion with Dispase II is performed under conditions of: the tissue was digested with Dispase II at 37 ℃ for several hours.
3. The method of claim 1, wherein the conditions for digestion with Trypsin/EDTA are: the epidermal layer is digested with preheated Trypsin/EDTA at 37 deg.C for 8-15 min, centrifuged at 1000 Xg-1500 Xg for 2.5-3.5 min, and washed several times with PBS at 4 deg.C.
4. The method of claim 1, wherein Advanced TM Components of the DMEM/F12 medium include non-essential amino acids NEAA and B-27 in addition to the recombinant human Wnts protein TM Additive, advanced cell culture additive GlutaMAX TM The anti-tumor protein polypeptide comprises a non-ionic amphoteric buffer solution HEPES, N-acetylcysteine N-Ace, recombinant human epidermal growth factor protein hEGF, a pathway inhibitor A83-01, a eukaryotic adenylate cyclase activator Forskolin and a penicillin streptomycin double-resistant solution.
5. The method of claim 4, wherein Advanced is performed TM Preferred compositions of the DMEM/F12 medium comprise 1% of the non-essential amino acids NEAA, 2% TM Additive, 1% advanced cell culture additive GlutaMAX TM 1% non-ionic amphoteric buffer HEPES, 1mM N-acetylcysteine N-Ace, 50ng/mL recombinant human epidermal growth factor protein hEGF, 2 mu M pathway inhibitor A83-01, 10 mu M eukaryotic adenylate cyclase activator Forskolin, 100U/L penicillin streptomycin double-antibody solution and 50ng/mL recombinant human Wnts protein.
6. The method of claim 1, wherein the well plate is pre-coated with Matrigel and is used after waiting 24 hours.
7. The method of claim 1, wherein the well plate is a 6-well plate, and 2ml of culture medium is added per well.
8. The method according to claim 1, wherein the epidermal stem cells are cultured in the well plate for 4 to 7 days, and the extracellular vesicles of the epidermal stem cells are cultured in a medium of fetal bovine serum from which the extracellular vesicles of bovine serum have been removed, for more than 48 hours.
9. The method of claim 1, wherein the centrifugation temperature of four times is 4 ℃; the pellet was washed in PBS to remove contaminating proteins.
10. The method of claim 1, wherein the extracellular vesicles of epidermal stem cells are grown in 10% to 15% fetal bovine serum medium; another medium of fetal calf serum with removed bovine extracellular vesicles was obtained by ultracentrifugation of fetal calf serum.
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| CN115521914A (en) * | 2022-10-12 | 2022-12-27 | 西北工业大学 | Human primary natural killer cell in-vitro amplification system and method |
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