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CN111500527A - Separation culture method of epidermal stem cells - Google Patents

Separation culture method of epidermal stem cells Download PDF

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Publication number
CN111500527A
CN111500527A CN202010607136.4A CN202010607136A CN111500527A CN 111500527 A CN111500527 A CN 111500527A CN 202010607136 A CN202010607136 A CN 202010607136A CN 111500527 A CN111500527 A CN 111500527A
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epidermal stem
stem cells
digesting
culture method
cells according
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张晓南
吴芳春
谷涌泉
侍晓云
张斌
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Beijing Yulong Shengshi Biotechnology Co ltd
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Beijing Yulong Shengshi Biotechnology Co ltd
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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    • C12N2500/10—Metals; Metal chelators
    • C12N2500/20—Transition metals
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
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Abstract

The invention discloses a separation culture method of epidermal stem cells, which comprises the following steps: (1) washing human skin, digesting with protease, and separating epidermis and dermis; (2) digesting the epidermis; (3) terminating digestion, and centrifugally resuspending to obtain epidermal stem cell single cell suspension; (4) inoculating and culturing in an incubator; (5) after removing trophoblast cells, digesting and passaging; (6) and (6) detecting. The method for separating and culturing the epidermal stem cells is an in-vitro culture method capable of improving the activity and the proliferation capacity of the epidermal stem cells, and is simple, convenient to operate, time-saving and labor-saving.

Description

Separation culture method of epidermal stem cells
Technical Field
The invention relates to the technical field of biology, in particular to a separation culture method of epidermal stem cells.
Background
The skin is the largest organ of the human body, plays an important role in resisting invasion of microorganisms and ultraviolet radiation, preventing loss of water and regulating body temperature, and is also one of the components of the immune system. In addition to these biological functions, the skin plays a very important role in maintaining the appearance of a human. The epidermis is a typical self-renewing tissue, and 60% of the differentiated tissues in the mammalian body, which are powerful, secrete bioactive substances, absorb nutrients, and maintain the integrity of the tissue. The epidermis of the outer layer of the skin continuously renews itself for life, stem cells at the base of the skin continuously proliferate and differentiate to replace the terminally differentiated cells at the outer layer, so that the tissue structure is renewed, and the death and shedding of the cells at the outer layer and the division of the stem cells at the base maintain a certain balance, which is the basic requirement for maintaining normal tissue structure and intracellular homeostasis.
An epidermal stem cell is a cell that has the potential to produce at least more than one highly differentiated daughter cell. From the mechanism of occurrence, stem cells do not differentiate directly to give terminally differentiated cells, but instead differentiate first into transiently expanded cells, which are committed progenitors because of their ability to give rise to committed differentiation into certain terminally differentiated cells.
However, the in vitro culture of epidermal stem cells is not yet mature. Therefore, the search for an in vitro culture method capable of improving the activity and proliferation capacity of epidermal stem cells has become a technical problem to be solved urgently by those skilled in the art.
Disclosure of Invention
Aiming at the problems in the prior art, the invention provides the isolated culture method of the epidermal stem cells, which is suitable for in vitro culture of the epidermal stem cells, has the advantages of high culture speed, short time consumption and simple operation, and provides a brand-new culture method for the scientific research field.
In order to achieve the purpose, the invention adopts the technical scheme that: an isolated culture method of epidermal stem cells comprises the following steps:
(1) washing human skin, digesting with protease, and separating epidermis and dermis;
(2) digesting the epidermis;
(3) terminating digestion, and centrifugally resuspending to obtain epidermal stem cell single cell suspension;
(4) inoculating and culturing in an incubator;
(5) after removing trophoblast cells, digesting and passaging;
(6) and (6) detecting.
Preferably, the area of the human skin sheet in the step (1) is 1-4 cm2About, the thickness is 0.5 cm.
In any of the above schemes, preferably, in the step (1), the human skin pieces are repeatedly washed with calcium-magnesium-free PBS containing 200 mg/L of penicillin, streptomycin and 2.5 mg/L of penicillin.
In any of the above embodiments, preferably, the protease in step (1) is 0.25% neutral protease II, and the digestion time is 30 min.
Preferably, in any of the above embodiments, the step (2) is performed by digesting the epidermis with a solution of trypsin and EDTA for 30min or overnight at 4 ℃.
Preferably, in any of the above schemes, when the digestion is terminated in step (3), the whole culture medium containing 10% FBS is used, a pipetting gun is used for blowing and beating for 3-5 times, the centrifugation is carried out for 1000 rpm, the time is 8-10 minutes, the supernatant is discarded, and the resuspension is carried out for 8-10 minutes at 1000 rpm, so as to obtain the epidermal stem cell single cell suspension.
Preferably, in any of the above schemes, the whole culture medium comprises DMEM and HamF-12, wherein the proportion of DMEM to HamF-12 is 2-4:0.5-1.5, 5% -15% FBS, 15-25ng/ml EGF, 4-8ug/ml transferrin, 1.5 × 10-4-2.0×10-4mol/L adenine, 3-6ug/ml insulin, 8-10-12-10mol/L cholera mycin, 3-6ug/ml hydrocortisone.
Preferably, in any of the above embodiments, the whole culture medium comprises DMEM and HamF-12, wherein the proportion of DMEM to HamF-12 is 4:1, 5% FBS, 15ng/ml EGF, 4ug/ml transferrin, 1.5 × 10-4mol/L adenine, 3ug/ml insulin, 8-10mol/L cholera mycin, 3ug/ml hydrocortisone.
Preferably, in any of the above embodiments, the whole culture medium comprises DMEM and HamF-12, wherein the proportion of DMEM to HamF-12 is 3:1, 10% FBS, 20ng/ml EGF, 5ug/ml transferrin, 1.8 × 10-4mol/L adenine, 5ug/ml insulin, 10-10mol/L cholera mycin, 4ug/ml hydrocortisone.
Preferably, in any of the above embodiments, the whole culture medium comprises DMEM and HamF-12, wherein the proportion of DMEM to HamF-12 is 4:1.5, 15% FBS, 25ng/ml EGF, 8ug/ml transferrin, 2.0 × 10-4mol/L adenine, 6ug/ml insulin, 12-10mol/L cholera mycin, 6ug/ml hydrocortisone.
Preferably, in any of the above embodiments, the step (4) comprises inoculating the single cell suspension of epidermal stem cells on mitomycin C-treated NIH3T3 cytotrophoblasts at 37 ℃ and 5% CO2Culturing in an incubator, wherein the primary culture is generally attached to the wall within 48 hours.
In any of the above embodiments, preferably, when the cells in step (5) grow to 80% confluency, after removing trophoblast cells with 0.02% EDTA, the cells are digested with 0.25% trypsin and 0.01% EDTA solution for passage.
In any of the above embodiments, preferably, the step (6) detects the specific molecular marker by immunofluorescence, immunohistochemistry and RT-PCR.
Preferably in any of the above embodiments, the specific molecular marker in step (6) comprises at least one of β integrin, keratin and K19.
The invention has the beneficial effects that: the invention provides a separation culture method of epidermal stem cells, which comprises the following steps:
(1) washing human skin, digesting with protease, and separating epidermis and dermis;
(2) digesting the epidermis;
(3) terminating digestion, and centrifugally resuspending to obtain epidermal stem cell single cell suspension;
(4) inoculating and culturing in an incubator;
(5) after removing trophoblast cells, digesting and passaging;
(6) and (6) detecting.
The invention also provides a complete culture medium which comprises DMEM and HamF-12, wherein the proportion of DMEM to HamF-12 is 2-4:0.5-1.5, 5% -15% FBS, 15-25ng/ml EGF, 4-8ug/ml transferrin, 1.5 × 10-4-2.0×10- 4mol/L adenine, 3-6ug/ml insulin, 8-10-12-10mol/L cholera mycin, 3-6ug/ml hydrocortisone.
DMEM and HamFl2 medium have particular utility when used in epithelial tissue culture. Can promote epithelial cell adhesion.
Transferrin is the major transferrin protein in plasma, providing the iron required for cellular internalization and cellular metabolism. All cells were found to grow without transferrin. Not only does the maintenance of normal cellular metabolism depend on iron in a biologically active form, but iron is also a cofactor for enzymes such as RNA polymerase, DNA synthase. And is also an important component of hemoglobin.
Insulin, an essential component of most synthetic media, can promote cell proliferation, and stimulates the uptake of uridine and glucose by cells to synthesize RNA, proteins and lipids. Insulin can also be combined with insulin receptors on cell membranes to regulate various metabolic pathways in cells, increase the synthesis of fatty acid and glucose, and play an important role in cell growth. Transferrin is a glycoprotein which binds iron ions and its effect on cell growth is related to its property of binding iron ions, e.g. promoting lymphocyte proliferation, promoting antibody synthesis and secretion.
Hydrocortisone has the effect of promoting the proliferation and growth of epidermal epithelial cells and mammary epithelial cells, and can improve the clone formation rate of glial cells and fibroblasts.
The method for separating and culturing the epidermal stem cells is an in-vitro culture method capable of improving the activity and the proliferation capacity of the epidermal stem cells, and is simple, convenient to operate, time-saving and labor-saving.
Detailed Description
The present invention is further described below.
Example 1
In order to achieve the purpose, the invention adopts the technical scheme that:
a method for separating and culturing epidermal stem cells specifically comprises the following steps:
(1) taking a healthy skin piece under the aseptic condition, wherein the area size of the skin piece is 1-4 cm2About 0.5cm thick, repeatedly washed with 200 mg/L penicillin, streptomycin and 2.5 mg/L calcium-magnesium-free PBS for 3-5 times.
(2) 0.25% neutral protease II, the ratio of neutral protease II to tissue is greater than 2: 1, digesting for 30min, and separating epidermis from dermis.
(3) After soaking and digesting the tissue, the epidermal layer and the dermal layer are separated obviously, the epidermal layer and the dermal layer can be separated easily by forceps, after the epidermal layer and the dermal layer are separated, the epidermal layer and the dermal layer are digested by 0.25% trypsin and 0.01% EDTA solution, the ratio of the 0.25% trypsin to the 0.01% EDTA solution is more than 2: 1, the epidermis is digested at 37 ℃ for 30min or at 4 ℃ overnight.
(4) Digestion was stopped with whole medium containing 10% FBS. Blowing and scattering for 3-5 times by using a liquid transfer gun, centrifuging for 1000 r/min for 8-10 min, discarding supernatant, and resuspending cells for 8-10 min at 1000 r/min to obtain the epidermal stem cell single cell suspension.
(5) Inoculation into mitomycin-treated NIH3T35% CO at 37 ℃ on cell trophoblasts2Culturing in an incubator. The primary culture adhered within 48 h.
(6) The culture medium was changed every 3 days, and after the first change, growth halo which grew outward with single cells as the division center was observed with an inverted fluorescence microscope (olympus CKX 41), the cell morphology was dominated by oblate form, and then a confluent cell layer was gradually formed.
(7) Cell growth was observed under the microscope except for the central blank of cell helical growth, and when the cells grew to 80% confluency, trophoblast cells were removed with 0.02% EDTA, and then digested with 0.25% trypsin and 0.01% EDTA solution for passage. Specifically, washing was performed 2 times with PBS, followed by digestion with 0.25% trypsin and 0.01% EDTA solution for 30-40 seconds, and digestion was stopped with whole medium containing 10% FBS according to a 1: and 3, carrying out passage in proportion.
(8) The specific molecular markers of β integrin, keratin, K19 and the like are detected by immunofluorescence, immunohistochemistry and RT-PCR methods.
In this example, the whole medium comprises DMEM and HamF-12 at a ratio of 3:1, 10% FBS, 20ng/ml EGF, 5ug/ml transferrin, 1.8 × 10-4mol/L adenine, 5ug/ml insulin, 10-10mol/L cholera mycin, 4ug/ml hydrocortisone.
Example 2:
an epidermal stem cell isolation and culture method similar to that of example 1 except that the whole culture medium comprises DMEM and HamF-12 at a ratio of 4:1, 5% FBS, 15ng/ml EGF, 4ug/ml transferrin, 1.5 × 10-4mol/L adenine, 3ug/ml insulin, 8-10mol/L cholera mycin, 3ug/ml hydrocortisone.
Example 3:
an epidermal stem cell isolation and culture method similar to that of example 1 except that the whole culture medium comprises DMEM and HamF-12 at a ratio of 4:1.5, 15% FBS, 25ng/ml EGF, 8ug/ml transferrin, 2.0 × 10-4mol/L adenine, 6ug/ml insulin, 12-10mol/L cholera mycin, 6ug/ml hydrocortisone.
Comparative example 1
An epidermal stem cell isolation and culture method similar to that of example 1 except that the composition of the medium comprises DMEM and HamF-12 at a ratio of 0.5:1, 8% FBS, 20ng/ml EGF, 3ug/ml transferrin, 1.8 × 10-4mol/L adenine, 5ug/ml insulin, 10-10mol/L cholera mycin, 2ug/ml hydrocortisone.
Comparative example 2
An epidermal stem cell isolation and culture method similar to that of example 1 except that the medium comprises DMEM and HamF-12 at a ratio of 1:1, 15% FBS, 20ng/ml EGF, 2ug/ml transferrin, 1.8 × 10-4mol/L adenine, 5ug/ml insulin, 10-10mol/L cholera mycin, 2ug/ml hydrocortisone.
Comparative example 3
An epidermal stem cell isolation and culture method similar to that of example 1 except that the medium comprises DMEM and HamF-12 at a ratio of 3:1, 10% FBS, 20ng/ml EGF, 5ug/ml transferrin, 1.8 × 10-4mol/L adenine, 1ug/ml insulin, 10-10mol/L cholera mycin, 4ug/ml hydrocortisone.
Performance detection and effect evaluation:
detection of cell viability by MTT method
The epidermal stem cells obtained in each example and comparative example were tested by the MTT method, and their activities are shown in the following table:
TABLE 1 epidermal stem cell viability assay results
Figure DEST_PATH_IMAGE001
Wherein the viability of the epidermal stem cells prepared in examples 1-3 was significantly higher than that of comparative examples 1 and 2.
Immunofluorescence identifies the epidermal stem cell surface marker, and the detection result is shown in table 2:
TABLE 2 detection results of positive expression level
Figure DEST_PATH_IMAGE002
Flow cytometry detection of epidermal stem cell surface markers
Wherein, the flow cytometry analysis of the detection results of the epidermal stem cells prepared in the embodiments 1-3 shows that the positive expression level of CK19 protein in the epidermal stem cells in the embodiment 1 is 96.62%; example 2 the positive expression level of CK19 protein in epidermal stem cells was 95.19%; example 3 the positive expression level of CK19 protein in epidermal stem cells is 94.82%, the detection result is consistent with the detection result of immunofluorescence assay of epidermal stem cell surface markers, which further indicates that the epidermal stem cell separation and culture method of the present application is an in vitro culture method capable of improving the activity and proliferation capacity of epidermal stem cells, and the method is simple, convenient to operate, time-saving and labor-saving.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope of the present invention are included in the scope of the present invention.

Claims (9)

1. An isolated culture method of epidermal stem cells comprises the following steps:
(1) washing human skin, digesting with protease, and separating epidermis and dermis;
(2) digesting the epidermis;
(3) terminating digestion with a complete culture medium, and centrifugally resuspending to obtain a single cell suspension of epidermal stem cells, wherein the complete culture medium comprises DMEM and HamF-12, the proportion of DMEM to HamF-12 is 2-4:0.5-1.5, 5% -15% FBS, 15-25ng/ml EGF, 4-8ug/ml transferrin, and 1.5 × 10-4-2.0×10-4mol/L adenine, 3-6ug/ml insulin, 8-10-12- 10mol/L cholera mycin, 3-6ug/ml hydrocortisone;
(4) inoculating and culturing in an incubator;
(5) after removing trophoblast cells, digesting and passaging;
(6) and (6) detecting.
2. The isolated culture method of epidermal stem cells according to claim 1, wherein the area of the human skin sheet in step (1) is 1-4 cm2About, the thickness is 0.5 cm.
3. The isolated culture method of epidermal stem cells according to claim 1, wherein in step (1), the human dermal sheet is repeatedly washed with calcium-magnesium-free PBS containing 200 mg/L of penicillin, streptomycin and 2.5 mg/L of penicillin.
4. The isolated culture method of epidermal stem cells according to claim 1, wherein the protease in step (1) is 0.25% neutral protease II, and the digestion time is 30 min.
5. The isolated culture method of epidermal stem cells according to claim 1, wherein said step (2) comprises digesting the epidermis with trypsin and EDTA solution for 30min or overnight at 4 ℃.
6. The isolated culture method of epidermal stem cells according to claim 1, wherein the digestion in step (3) is terminated by using a whole culture medium containing 10% FBS, blowing the whole culture medium with a pipette gun for 3-5 times, centrifuging for 1000 rpm for 8-10 minutes, discarding the supernatant, and resuspending for 8-10 minutes at 1000 rpm to obtain the epidermal stem cell single cell suspension.
7. The method for isolated culture of epidermal stem cells according to claim 1, wherein the step (4) comprises inoculating the single cell suspension of epidermal stem cells into the mitomycin-treated NIH3T35% CO at 37 ℃ on cell trophoblasts2Culturing in an incubator, wherein the primary culture is generally attached to the wall within 48 hours.
8. The method for isolated culture of epidermal stem cells according to claim 1, wherein when the cells of step (5) grow to 80% confluency, after removing trophoblast cells with 0.02% EDTA, the cells are digested with 0.25% trypsin and 0.01% EDTA solution for passage.
9. The method for isolated culture of epidermal stem cells according to claim 1, wherein the step (6) comprises detecting the specific molecular marker by immunofluorescence, immunohistochemistry and RT-PCR.
CN202010607136.4A 2020-06-30 2020-06-30 Separation culture method of epidermal stem cells Pending CN111500527A (en)

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