CN114164164A - In-vitro culture kit for epidermal stem cells and application thereof - Google Patents
In-vitro culture kit for epidermal stem cells and application thereof Download PDFInfo
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- CN114164164A CN114164164A CN202111484594.4A CN202111484594A CN114164164A CN 114164164 A CN114164164 A CN 114164164A CN 202111484594 A CN202111484594 A CN 202111484594A CN 114164164 A CN114164164 A CN 114164164A
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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
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- C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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- C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/10—Growth factors
- C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
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- C12N2501/00—Active agents used in cell culture processes, e.g. differentation
- C12N2501/20—Cytokines; Chemokines
- C12N2501/23—Interleukins [IL]
- C12N2501/2303—Interleukin-3 (IL-3)
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/52—Fibronectin; Laminin
Abstract
The invention provides an in vitro culture kit of epidermal stem cells and application thereof, wherein the in vitro culture kit of epidermal stem cells comprises an adherence promoting culture medium and a transformation promoting culture medium; the adherence-promoting culture medium comprises a stem cell culture medium, a cell culture supplement, fetal bovine serum, adherence-promoting cytokines and IL-3, wherein the concentration of the IL-3 is 80-120 ng/mL; the transformation promoting culture medium comprises a stem cell culture medium, a cell culture supplement, a transformation promoting cytokine, fibronectin and IL-3, wherein the concentration of the IL-3 is 40-80 ng/mL. The invention also provides an in vitro culture method of the epidermal stem cells, the epidermal stem cells and an anti-wrinkle injection. According to the invention, by the mutual matching of the optimized culture medium and the culture method, a large number of epidermal stem cells with very high purity can be rapidly obtained, and the epidermal stem cells are used as components of the anti-wrinkle injection, and have obvious effects and higher value.
Description
Technical Field
The invention belongs to the technical field of cell biology, and particularly relates to an epidermal stem cell in-vitro culture kit and application thereof.
Background
Epidermal Stem Cells (ESCs) are a type of cell population that has the ability to proliferate and differentiate into various functional cells in the epidermis. It is mainly located in the epidermal basal layer, belongs to adult stem cells, has strong division and proliferation capacity, and can supplement aged and exfoliated keratinocytes.
The epidermal stem cells, as skin tissue-specific stem cells, not only maintain the daily metabolism of the epidermis, but also actively participate in the repair of damaged skin. The research on the proliferation, differentiation, migration and the like of the epidermal stem cells has important significance, so that the in vitro acquisition and culture of the epidermal stem cells are the basis of related research work. The conventional method is to carry out primary culture on epidermal tissues to obtain epidermal stem cells at present, but the method is complex to operate, needs cell sorting and has higher requirements on the operation level of technicians; the obtained epidermal stem cells need feeder cells during culture, are high in cost and are not beneficial to large-scale preparation.
How to obtain high-purity epidermal stem cells and maintain the phenotype of the epidermal stem cells and the in vitro proliferation capacity of the epidermal stem cells is a problem which needs to be solved urgently in the in vitro culture of the epidermal stem cells at present. Therefore, how to provide a method for obtaining and culturing epidermal stem cells, which is simple to operate and has low production cost, is a problem to be solved urgently.
Disclosure of Invention
Aiming at the defects and actual requirements of the prior art, the invention provides the in-vitro culture kit for the epidermal stem cells and the application thereof, the components of the culture medium are optimized, the adherence of the cells can be promoted, the transformation efficiency of the cells is improved, the prepared epidermal stem cells have high purity and strong in-vitro proliferation capacity, the operation is simple, and the flow type sorting is not needed.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect, the invention provides an in vitro culture kit for epidermal stem cells, which comprises an adherence promoting culture medium and a transformation promoting culture medium;
the anchorage-promoting culture medium comprises a stem cell culture medium, a cell culture supplement, fetal bovine serum, anchorage-promoting cytokines and IL-3, wherein the concentration of the IL-3 is 80-120 ng/mL, such as 80ng/mL, 85ng/mL, 90ng/mL, 95ng/mL, 100ng/mL, 105ng/mL, 110ng/mL, 115ng/mL or 120ng/mL, and other specific point values in the numerical range can be selected, and are not repeated;
the transformation promoting culture medium comprises a stem cell culture medium, a cell culture supplement, a transformation promoting cytokine, fibronectin and IL-3, wherein the concentration of the IL-3 is 40-80 ng/mL, for example, 40ng/mL, 45ng/mL, 50ng/mL, 55ng/mL, 60ng/mL, 65ng/mL, 70ng/mL, 75ng/mL or 80ng/mL, and other specific point values in the numerical range can be selected, and are not repeated.
According to the invention, by optimizing the components of the culture medium, the adherence promoting effect of the adherence promoting culture medium on the cells is higher, the efficiency of the transformation promoting culture medium on the transformation promoting of the cells is higher, the number of epidermal stem cells prepared after the culture is more, the purity is better, the proliferation capacity is stronger, feeder layer cells are not needed in the culture process, the production cost is reduced, and the application prospect is wide.
Preferably, the volume fraction of the cell culture supplement in the adherence promoting medium is 1% to 10%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, etc., and other specific points in the value range can be selected, which is not described in detail herein.
Preferably, the volume fraction of the fetal bovine serum in the adherence promoting medium is 1% to 10%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, and other specific points in the range of values can be selected, which is not described herein again.
Preferably, the anchorage-promoting cytokines include rhGM-CSF, bFGF and SCF.
Preferably, the concentration of the rhGM-CSF in the adherence promoting culture medium is 800-1200 IU/mL, for example, 800IU/mL, 850IU/mL, 900IU/mL, 950IU/mL, 1000IU/mL, 1050IU/mL, 1100IU/mL, 1150IU/mL or 1200IU/mL, and the like, and other specific points in the numerical range can be selected, which is not described herein again.
Preferably, the concentration of bFGF in the adherence-promoting medium is 80-120 ng/mL, for example, 80ng/mL, 85ng/mL, 90ng/mL, 95ng/mL, 100ng/mL, 105ng/mL, 110ng/mL, 115ng/mL, or 120ng/mL, and other specific points within the range of values can be selected, which is not repeated herein.
Preferably, the concentration of the SCF in the adherence-promoting medium is 80-120 ng/mL, for example, 80ng/mL, 85ng/mL, 90ng/mL, 95ng/mL, 100ng/mL, 105ng/mL, 110ng/mL, 115ng/mL, or 120ng/mL, and other specific points within the range of values can be selected, which is not repeated herein.
Preferably, the volume fraction of the cell culture supplement in the transformation promoting medium is 1% to 10%, for example, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%, etc., and other specific points within the range of values can be selected, which is not described herein again.
Preferably, the concentration of the fibronectin in the transformation promoting medium is 40-80 μ g/mL, for example, 40 μ g/mL, 45 μ g/mL, 50 μ g/mL, 55 μ g/mL, 60 μ g/mL, 65 μ g/mL, 70 μ g/mL, 75 μ g/mL or 80 μ g/mL, and other specific points in the numerical range can be selected, which is not repeated herein.
Preferably, the transforming cytokine comprises EGF and BPE.
Preferably, the concentration of the EGF in the transformation promoting medium is 40-80 IU/mL, for example, 40IU/mL, 45IU/mL, 50IU/mL, 55IU/mL, 60IU/mL, 65IU/mL, 70IU/mL, 75IU/mL, or 80IU/mL, and other specific points in the numerical range may be selected, which is not described herein again.
Preferably, the concentration of the BPE in the transformation promoting medium is 40-80 μ g/mL, for example, 40 μ g/mL, 45 μ g/mL, 50 μ g/mL, 55 μ g/mL, 60 μ g/mL, 65 μ g/mL, 70 μ g/mL, 75 μ g/mL, or 80 μ g/mL, and the like, and other specific points in the numerical range can be selected, which is not repeated herein.
As a preferred technical scheme, the in vitro culture kit for the epidermal stem cells comprises an adherence promoting culture medium and a transformation promoting culture medium;
the adherence promoting culture medium comprises 1-10% of cell culture supplement, 1-10% of fetal bovine serum, 800-1200 IU/mL of rhGM-CSF, 80-120 ng/mL of bFGF, 80-120 ng/mL of SCF and 380-120 ng/mL of IL-380 by volume fraction, and the balance of stem cell culture medium;
the transformation promoting culture medium comprises 1-10% of cell culture supplement, 40-80 IU/mL of EGF, 40-80 mu g/mL of BPE, 40-80 mu g/mL of fibronectin, 340-80 ng/mL of IL-340 and the balance of stem cell culture medium in terms of volume fraction.
In a second aspect, the present invention provides a method for in vitro culturing of epidermal stem cells, the method comprising:
cleaning umbilical cord tissue, sterilizing, taking out HUATONG colloid, shearing, centrifuging, and collecting tissue blocks;
using the adherence promoting culture medium of the first aspect to culture cells for 3-6 days to promote adherence of the cells, wherein the culture time can be, for example, 3 days, 3.5 days, 4 days, 4.5 days, 5 days, 5.5 days, or 6 days, and other specific point values in the numerical range can be selected, and are not described in detail herein;
subculturing adherent cells for 4-8 days by using the transformation promoting medium of the first aspect, collecting the cells, and obtaining the epidermal stem cells, wherein the culture time may be, for example, 4 days, 4.5 days, 5 days, 5.5 days, 6 days, 6.5 days, 7 days, 7.5 days, or 8 days, and other specific point values within the numerical range may be selected, and are not described herein any more.
According to the invention, the culture method is simple to operate, flow sorting is not required, tissue cracking is not required, self tissue is not required to be obtained for primary culture, the requirement on the technical level of operators is low, and the culture efficiency is high; feeder cells are not needed in the culture process, and the cost is low.
Preferably, the period of promoting cell adherence further comprises the step of removing semi-adherent cells.
Preferably, before subculture, the cell adherence rate is not lower than 50%, for example, may be 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, and other specific points within the numerical range may be selected, which is not described herein again.
Preferably, the cell adherence rate during subculture is not lower than 80%, for example, 80%, 85%, 90%, or 95%, and other specific values within the range can be selected, which is not described herein again.
Preferably, the number of subcultures is 2 to 4, and may be 2, 3 or 4, for example.
Preferably, the step of quality control detection is further included before the collection of the cells.
As a preferred technical scheme, the method for culturing the epidermal stem cells in vitro comprises the following steps:
cleaning umbilical cord tissue, sterilizing, taking out HUATONG colloid, shearing, centrifuging, and collecting tissue blocks;
culturing the tissue block for 3-6 days by using the adherence promoting culture medium of the first aspect to promote adherence of cells and remove semi-adherence cells;
when the cell anchorage rate is not less than 50%, subculturing adherent cells for 4-8 days by using the transformation promoting culture medium of the first aspect, wherein the cell anchorage rate is not less than 80% during subculturing, subculturing for 2-4 times, and collecting cells after quality control detection to obtain the epidermal stem cells.
In a third aspect, the present invention provides an epidermal stem cell, which is cultured by the method for culturing an epidermal stem cell in vitro according to the second aspect.
In the invention, the epidermal stem cells have high purity, good proliferation capacity and wide application prospect.
In a fourth aspect, the present invention provides a kit for culturing epidermal stem cells in vitro as defined in the first aspect, a method for culturing epidermal stem cells in vitro as defined in the second aspect, or a combination of at least two of the epidermal stem cells as defined in the third aspect, for use in preparing an anti-wrinkle injection.
In a fifth aspect, the invention provides an anti-wrinkle injection, which contains the epidermal stem cells of the third aspect and an injection;
the injection comprises a sodium chloride solution, an astragalus injection, human serum albumin, a cell factor and recombinant protein;
the density of the epidermal stem cells in the anti-wrinkle injection is (1-5) x 108The volume/mL may be, for example, 1X 1081.5X 10 units/mL82X 10 units/mL82.5X 10 units/mL83X 10 pieces/mL83.5X 10 units/mL84X 10 units/mL8cell/mL, 4.5X 108one/mL or 5X 108one/mL, etc., and other specific point values within the numerical range can be selected, which are not described in detail herein.
In the invention, the anti-wrinkle injection contains epidermal stem cells and factors for promoting cell proliferation and division, and has high-efficiency anti-wrinkle effect; the anti-wrinkle injection can be directly injected for use, is convenient to use and has practical application value.
Preferably, the mass concentration of the sodium chloride solution is 0.7% to 0.9%, for example, 0.7%, 0.75%, 0.8%, 0.85%, or 0.9%, and other specific values within the numerical range may be selected, and are not described in detail herein.
Preferably, the volume fraction of the astragalus injection in the injection is 1% to 5%, for example, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, etc., and other specific points in the numerical range can be selected, which is not described herein again.
Preferably, the concentration of the human serum albumin in the injection is 0.01-0.1 g/mL, for example, 0.01g/mL, 0.02g/mL, 0.03g/mL, 0.04g/mL, 0.05g/mL, 0.06g/mL, 0.07g/mL, 0.08g/mL, 0.09g/mL or 0.1g/mL, and the like, and other specific points in the value range can be selected, which is not described herein again.
Preferably, the cytokine comprises SCF, and the concentration of the SCF in the injection is 5-9 ng/mL, for example, 5ng/mL, 5.5ng/mL, 6ng/mL, 6.5ng/mL, 7ng/mL, 7.5ng/mL, 8ng/mL, 8.5ng/mL, or 9ng/mL, and other specific points in the numerical range can be selected, which is not repeated herein.
Preferably, the recombinant protein comprises a Dnmt3a recombinant protein and a Dnmt3b recombinant protein, the concentration of the Dnmt3a recombinant protein and the concentration of the Dnmt3b recombinant protein in the injection are both 0.5-2 mug/mL, such as 0.5 mug/mL, 1 mug/mL, 1.5 mug/mL or 2 mug/mL, and the like, and other specific points in the value range can be selected, so that the description is omitted.
According to a preferable technical scheme, the injection comprises 1-5% of astragalus injection, 0.01-0.1 g/mL of human serum albumin, 5-9 ng/mL of SCF, 0.5-2 μ g/mL of Dnmt3a recombinant protein and 0.5-2 μ g/mL of Dnmt3b recombinant protein by volume fraction, and the balance of sodium chloride solution with the mass concentration of 0.7-0.9%.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, by optimizing the formulas of the adherence-promoting culture medium and the transformation-promoting culture medium, the adherence rate and the transformation efficiency of the cells are obviously improved, the number of epidermal stem cells which can be efficiently and rapidly prepared is more, and the proliferation capacity is stronger; according to the invention, the umbilical cord Wharton jelly mesenchymal stem cells are subjected to directional induction and transformation, so that the prepared epidermal stem cells have high purity, do not need flow type sorting and are simple and convenient to operate; the epidermal stem cells have good transformation efficiency and strong proliferation capacity, and can promote the proliferation of cells and the repair of skin injury; feeder cells are not needed during culture, and the cost is low; self tissues do not need to be obtained and the tissues are cracked, so that the requirements on the operation level of technicians are low, and the success rate is high; the anti-wrinkle injection contains epidermal stem cells and factors for promoting cell proliferation and division, can promote cell proliferation and division in the dermis layer of the skin after injection, has obvious anti-wrinkle effect, can be directly injected for use, is convenient to use, and has wide application prospect.
Detailed Description
To further illustrate the technical means and effects of the present invention, the present invention is further described with reference to the following examples. It is to be understood that the specific embodiments described herein are merely illustrative of the invention and are not limiting of the invention.
The examples do not show the specific techniques or conditions, according to the technical or conditions described in the literature in the field, or according to the product specifications. The reagents or apparatus used are conventional products commercially available from normal sources, not indicated by the manufacturer.
Materials:
stem cell culture medium and fetal bovine serum were purchased from seimer feishell science and technology (china) ltd;
cell culture supplements were purchased from Helios Bioscience;
IL-3, rhGM-CSF, bFGF, SCF, fibronectin, EGF, BPE, Dnmt3a recombinant protein, and Dnmt3b recombinant protein were purchased from HZbscience;
the radix astragali injection is purchased from Shenwei pharmaceutical industry group Co., Ltd;
human serum albumin was purchased from shanghai leshi blood products, inc;
CD49f + antibody and CD71+ antibody were purchased from BD (china).
Example 1
The embodiment provides an in vitro culture kit for epidermal stem cells, which comprises an adherence promoting culture medium and a transformation promoting culture medium;
the adherence promoting culture medium comprises 8% of cell culture supplement, 8% of fetal bovine serum, 1000 IUGM-CSF/mL of rhGM-CSF, 100ng/mL of bFGF, 100ng/mL of SCF and 100ng/mL of IL-3100 ng/mL by volume fraction, and the balance is stem cell culture medium;
the transformation promoting medium comprises 8% of cell culture supplement, 70IU/mL of EGF, 70 mu g/mL of BPE, 70 mu g/mL of fibronectin and 370 ng/mL of IL-370, and the balance is stem cell culture medium by volume fraction.
Example 2
The embodiment provides an in vitro culture kit for epidermal stem cells, which comprises an adherence promoting culture medium and a transformation promoting culture medium;
the adherence promoting culture medium comprises 10% of cell culture supplement, 10% of fetal bovine serum, 800 IUGM-CSF/mL of rhGM-CSF, 80ng/mL of bFGF, 80ng/mL of SCF and 380 ng/mL of IL, and the balance of stem cell culture medium by volume fraction;
the transformation promoting medium comprises 10% of cell culture supplement, 40IU/mL of EGF, 40 mu g/mL of BPE, 40 mu g/mL of fibronectin and 340 ng/mL of IL-340 in volume fraction, and the balance of stem cell culture medium.
Example 3
The embodiment provides an in vitro culture kit for epidermal stem cells, which comprises an adherence promoting culture medium and a transformation promoting culture medium;
the adherence promoting culture medium comprises 1% of cell culture supplement, 1% of fetal bovine serum, 1200 IUGM-CSF/mL of rhGM-CSF, 120ng/mL of bFGF, 120ng/mL of SCF and IL-3120 ng/mL by volume fraction, and the balance is stem cell culture medium;
the transformation promoting medium comprises 1% of cell culture supplement, 80IU/mL of EGF, 80 mu g/mL of BPE, 80 mu g/mL of fibronectin and 380 ng/mL of IL-380, and the balance is stem cell culture medium.
Example 4
The present embodiment provides an epidermal stem cell, which is cultured by the following method:
(1) sterilizing an operation table, reagents and equipment before culture;
(2) cleaning and disinfecting umbilical cord tissues;
cleaning umbilical cord tissue with D-PBS solution for 2 times, soaking in 75% alcohol solution for 20s for sterilization; washing with D-PBS solution for 2 times, and soaking in 75% ethanol solution for 20 s; washing with D-PBS solution for 1 time, placing in a clean culture dish containing D-PBS solution, and cutting into 2cm pieces;
(3) taking out the Huatong colloid, shearing, and centrifugally collecting tissue blocks:
placing the umbilical cord in D-PBS solution with hemostatic forceps, cleaning blood, cutting small hole with surgical knife, tearing open umbilical cord, and extracting HUATONG colloid;
washing the Huatong colloid with D-PBS solution, and cutting into 2X 2cm2Transferring the small blocks into a centrifuge tube, centrifuging for 10min at the room temperature of 400g, and removing supernatant to obtain tissue blocks;
(4) promoting cell adherence:
inoculating the tissue block into a T75 cell culture bottle, adding 20mL of the adherence promoting culture medium in the embodiment 1, and culturing for 3 days at 37 ℃ and 5% carbon dioxide concentration without shaking the culture bottle to promote cell adherence;
slowly pouring out culture supernatant on the 4 th day, and removing semi-adherent cells; adding 20mL of adherence promoting culture medium, and continuously culturing at 37 ℃ and 5% of carbon dioxide concentration;
on day 6, the cells were observed under a microscope, the anchorage rate of the cells exceeded 50%, the culture supernatant was slowly poured out, the tissue mass was removed, 20mL of the transformation promoting medium of example 1 was added, and the cells were cultured at 37 ℃ and 5% carbon dioxide concentration;
(5) subculturing:
on the 8 th day, the cells are placed under a microscope for observation, the anchorage rate of the cells exceeds 80%, the digested cells are subjected to subculture, each bottle contains 20mL of transformation promoting culture medium, and the cells are cultured under the conditions of 37 ℃ and 5% of carbon dioxide concentration;
on the 10 th day, the cells are placed under a microscope for observation, the anchorage rate of the cells exceeds 80%, the digested cells are subjected to subculture, each bottle contains 20mL of transformation promoting culture medium, and the cells are cultured under the conditions of 37 ℃ and 5% of carbon dioxide concentration;
on the 12 th day, the cells are placed under a microscope for observation, the anchorage rate of the cells exceeds 80%, the digested cells are subjected to subculture, each bottle contains 20mL of transformation promoting culture medium, and the cells are cultured under the conditions of 37 ℃ and 5% of carbon dioxide concentration;
(6) collecting cells:
and on 14 days, digesting the cells, collecting the cells after quality control detection, and obtaining the epidermal stem cells.
Example 5
This example provides epidermal stem cells cultured using the in vitro epidermal stem cell culture kit of example 2, in the same manner as in example 4.
Example 6
This example provides epidermal stem cells cultured using the in vitro epidermal stem cell culture kit of example 3, in the same manner as in example 4.
The flow detection is carried out on the epidermal stem cells prepared in the embodiments 4-6, and the steps are as follows:
1. placing the culture flask in a biological safety cabinet, digesting with pancreatin until the cells fall off completely, stopping digestion with D-PBS, collecting the cells, centrifuging at 400g for 5min at room temperature, and then re-suspending the cells with 10mL of D-PBS;
2. placing about 100 ten thousand (about 200 μ L) cells in an EP tube, adding 800 μ L D-PBS, mixing, centrifuging at 400g at room temperature for 5min, and removing supernatant to obtain cells;
3. cells were mixed well with 200. mu.L of 5% FBS-containing D-PBS, and 200. mu.L of the resuspension was placed in 1 new flow tube;
4. adding 0.5 μ L of CD49f + antibody and 0.5 μ L of CD71+ antibody to the flow tube in sequence;
5. placing in vortex oscillator, mixing for 20s, and dyeing at 4 deg.C in dark for 20 min;
6. operating the computer and storing data;
7. the data is analyzed.
The results are shown in Table 1.
TABLE 1
As can be seen from Table 1, the cells prepared in examples 4 to 6 all have good transformation efficiency, and the transformation efficiency of the high concentration factor combination can be achieved by using a lower concentration of the transformation promoting factor combination. Comparing example 6 with examples 4 and 5, it can be seen that the lower concentration factor combination has a more efficient conversion efficiency.
Example 7
This example provides an anti-wrinkle injection comprising the epidermal stem cells cultured in example 4 and an injection;
the density of the epidermal stem cells in the anti-wrinkle injection is 1 x 108Per mL;
the injection comprises 3 percent of astragalus injection, 0.08g/mL of human serum albumin, 7ng/mL of SCF, 1.5 mu g/mL of Dnmt3a recombinant protein and 1.5 mu g/mL of Dnmt3b recombinant protein by volume fraction, and the balance is 0.8 percent of sodium chloride solution by mass concentration.
Example 8
This example provides an anti-wrinkle injection comprising the epidermal stem cells cultured in example 5 and an injection;
the density of the epidermal stem cells in the anti-wrinkle injection is 1 x 108Per mL;
the injection comprises 5% of astragalus injection, 0.1g/mL of human serum albumin, 9ng/mL of SCF, 2 mug/mL of Dnmt3a recombinant protein and 2 mug/mL of Dnmt3b recombinant protein by volume fraction, and the balance is 0.7% of sodium chloride solution by mass concentration.
Example 9
This example provides an anti-wrinkle injection comprising the epidermal stem cells cultured in example 6 and an injection;
the density of the epidermal stem cells in the anti-wrinkle injection is 1 x 108Per mL;
the injection comprises 1% of astragalus injection, 0.01g/mL of human serum albumin, 5ng/mL of SCF, 0.5 mu g/mL of Dnmt3a recombinant protein and 0.5 mu g/mL of Dnmt3b recombinant protein by volume fraction, and the balance is 0.9% of sodium chloride solution by mass concentration.
Example 10
In the present embodiment, the anti-wrinkle injection prepared in examples 7 to 9 is injected, and the steps are as follows:
(1) preparing iodine tincture, cotton swab, cotton ball, etc., spreading disposable sterile surgical sheet, performing ultraviolet ozone sterilization for 30min in advance for the surgical room, purifying room air with ventilation device, and treating for 10 min.
(2) The surface of the wrinkle is wiped and disinfected by iodine tincture without anesthesia, then 75% medical alcohol is further wiped and disinfected, the head and the tail of the wrinkle are marked by a disposable sterile surgical marker pen, and the marks are made every 4mm between the head and the tail to be used as injection points;
rotating the disposable microinjector filled with the anti-wrinkle injection to enable the scales to face upwards, enabling the needle head port to face downwards, uniformly mixing the anti-wrinkle injection again, evacuating air in the microinjector, enabling the needle head port to face downwards to be tightly attached to a skin injection point, slightly pricking the anti-wrinkle injection into the skin surface layer at a depth of about 2mm at an angle of about 45 degrees, injecting 1 mu L of the anti-wrinkle injection into the skin surface layer, and sequentially and directionally and quantitatively injecting the anti-wrinkle injection into all the mark points.
(3) After the injection is finished, washing the face, touching, pressing the injection point and making up are prohibited within 48 h.
By instrument analysis and detection, after the anti-wrinkle injection prepared in examples 7-9 is injected for 1 week, the wrinkles on the face have the signs of lightening, the injection part has natural luster, the wrinkles on the back are obviously reduced after 2 weeks, and the injection part has natural luster and ruddy. In addition, adverse reactions such as red swelling, pruritus, immunological rejection and the like do not occur, the safety is ensured, and the method has a practical application value.
In conclusion, the epidermal stem cell in-vitro culture kit provided by the invention has the advantages that by optimizing the components of the culture medium and matching with the epidermal stem cell in-vitro culture method provided by the invention, the epidermal stem cells prepared by high-efficiency and rapid transformation have good proliferation and division capabilities, the purity is high, flow sorting and feeder layer cell auxiliary culture are not required, and the operation is simple; after being mixed with the injection and injected into the skin, the composition can be quickly split, has obvious anti-wrinkle effect, is convenient to use and has wide application prospect.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (10)
1. An in vitro culture kit for epidermal stem cells is characterized by comprising an adherence promoting culture medium and a transformation promoting culture medium;
the adherence-promoting culture medium comprises a stem cell culture medium, a cell culture supplement, fetal bovine serum, adherence-promoting cytokines and IL-3, wherein the concentration of the IL-3 is 80-120 ng/mL;
the transformation promoting culture medium comprises a stem cell culture medium, a cell culture supplement, a transformation promoting cytokine, fibronectin and IL-3, wherein the concentration of the IL-3 is 40-80 ng/mL.
2. The epidermal stem cell in vitro culture kit of claim 1, wherein the volume fraction of the cell culture supplement in the adherence promoting medium is 1-10%;
preferably, the volume fraction of the fetal calf serum in the adherence promoting culture medium is 1-10%;
preferably, the anchorage-promoting cytokines include rhGM-CSF, bFGF and SCF;
preferably, the concentration of the rhGM-CSF in the adherence promoting culture medium is 800-1200 IU/mL;
preferably, the concentration of the bFGF in the adherence promoting culture medium is 80-120 ng/mL;
preferably, the concentration of the SCF in the adherence promoting culture medium is 80-120 ng/mL;
preferably, the volume fraction of the cell culture supplement in the transformation promoting medium is 1% to 10%;
preferably, the concentration of the fibronectin in the transformation promoting culture medium is 40-80 mug/mL;
preferably, the transforming cytokine comprises EGF and BPE;
preferably, the concentration of the EGF in the transformation promoting culture medium is 40-80 IU/mL;
preferably, the concentration of the BPE in the transformation promoting medium is 40-80 mu g/mL.
3. The in vitro culture kit for the epidermal stem cells according to claim 1 or 2, wherein the in vitro culture kit for the epidermal stem cells comprises an adherence-promoting medium and a transformation-promoting medium;
the adherence promoting culture medium comprises 1-10% of cell culture supplement, 1-10% of fetal bovine serum, 800-1200 IU/mL of rhGM-CSF, 80-120 ng/mL of bFGF, 80-120 ng/mL of SCF and 380-120 ng/mL of IL-380 by volume fraction, and the balance of stem cell culture medium;
the transformation promoting culture medium comprises 1-10% of cell culture supplement, 40-80 IU/mL of EGF, 40-80 mu g/mL of BPE, 40-80 mu g/mL of fibronectin, 340-80 ng/mL of IL-340 and the balance of stem cell culture medium in terms of volume fraction.
4. A method of in vitro culturing of epidermal stem cells, comprising:
cleaning umbilical cord tissue, sterilizing, taking out HUATONG colloid, shearing, centrifuging, and collecting tissue blocks;
culturing the tissue block for 3 to 6 days by using the adherence promoting culture medium as described in any one of claims 1 to 3 to promote cell adherence;
subculturing adherent cells for 4 to 8 days using the transformation promoting medium according to any one of claims 1 to 3, and collecting the cells to obtain the epidermal stem cells.
5. The method for in vitro culturing of epidermal stem cells according to claim 4, wherein said period of promoting cell adhesion further comprises the step of removing semi-adherent cells;
preferably, before subculture, the cell adherence rate is not less than 50%;
preferably, the cell adherence rate is not lower than 80% during subculture;
preferably, the number of subcultures is 2-4;
preferably, the step of quality control detection is further included before the collection of the cells.
6. The method of in vitro culturing of epidermal stem cells according to claim 4 or 5, characterized in that it comprises:
cleaning umbilical cord tissue, sterilizing, taking out HUATONG colloid, shearing, centrifuging, and collecting tissue blocks;
culturing the tissue block for 3 to 6 days by using the adherence promoting culture medium as described in any one of claims 1 to 3, promoting cell adherence, and removing semi-adherent cells;
when the cell adherence rate is not less than 50%, subculturing adherent cells for 4-8 days by using the transformation promoting medium as defined in any one of claims 1-3, wherein the cell adherence rate is not less than 80% during subculturing, subculturing for 2-4 times, and collecting cells after quality control detection to obtain the epidermal stem cells.
7. An epidermal stem cell obtained by culturing the epidermal stem cell according to any one of claims 4 to 6 in vitro.
8. Use of any one or a combination of at least two of the kit for in vitro culturing of the epidermal stem cell of any one of claims 1 to 3, the method for in vitro culturing of the epidermal stem cell of any one of claims 4 to 6, or the epidermal stem cell of claim 7 in the preparation of an anti-wrinkle injection.
9. An anti-wrinkle injection comprising the epidermal stem cell of claim 7 and an injection;
the injection comprises a sodium chloride solution, an astragalus injection, human serum albumin, a cell factor and recombinant protein;
the density of the epidermal stem cells in the anti-wrinkle injection is (1-5) x 108one/mL.
10. The anti-wrinkle injection solution according to claim 9, wherein the mass concentration of the sodium chloride solution is 0.7-0.9%;
preferably, the volume fraction of the astragalus injection in the injection is 1-5%;
preferably, the concentration of the human serum albumin in the injection is 0.01-0.1 g/mL;
preferably, the cytokine comprises SCF, and the concentration of the SCF in the injection is 5-9 ng/mL;
preferably, the recombinant protein comprises Dnmt3a recombinant protein and Dnmt3b recombinant protein, and the concentration of the Dnmt3a recombinant protein and the concentration of the Dnmt3b recombinant protein in the injection are both 0.5-2 mug/mL;
preferably, the injection comprises 1-5% of astragalus injection, 0.01-0.1 g/mL of human serum albumin, 5-9 ng/mL of SCF, 0.5-2 μ g/mL of Dnmt3a recombinant protein and 0.5-2 μ g/mL of Dnmt3b recombinant protein by volume fraction, and the balance is 0.7-0.9% of sodium chloride solution by mass concentration.
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