CN105154388B - method for separating and culturing skin keratinocytes - Google Patents
method for separating and culturing skin keratinocytes Download PDFInfo
- Publication number
- CN105154388B CN105154388B CN201510456338.2A CN201510456338A CN105154388B CN 105154388 B CN105154388 B CN 105154388B CN 201510456338 A CN201510456338 A CN 201510456338A CN 105154388 B CN105154388 B CN 105154388B
- Authority
- CN
- China
- Prior art keywords
- skin
- digestion
- keratinocytes
- culture
- tissue
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
aiming at the problems of low efficiency, rejection reaction and the like existing in the prior separation and culture of the keratinocyte, the invention provides a separation and culture method of the keratinocyte; adopts Trypsin/EDTA digestive juice and I-shaped collagenase to carry out two-step digestion, mainly extracts the keratinocytes with stronger proliferation capacity around the basal layer and the hair follicle, can obtain the single keratinocytes, has high activity, is easy to stick to the wall and is easy to proliferate.
Description
Technical Field
The invention relates to a method for separating and obtaining skin keratinocytes, in particular to a method for separating and culturing skin keratinocytes for culturing and obtaining tissue engineering epidermis.
Background
Skin keratinocyte cultures are widely used in scientific research, beauty products and medical treatment fields. The keratinocyte with proliferation ability extracted from the epidermis and partial dermis is amplified and cultured to establish the epidermal cell bank with higher activity.
In the medical field, when skin defect diseases such as leucoderma, burn, melanoma, scars, striae gravidarum and the like are treated, the traditional drug therapy or photochemotherapy is unstable after healing and needs continuous treatment. When the skin is taken from other parts of the body and transplanted, the skin at the skin-taking part is damaged or the skin source is in short supply. Skin defects can therefore be treated by keratinocyte cell cultures.
In addition, the current cosmetic product market is very broad, and the corresponding safety and efficacy become more important. Before being put on the market, the sensitivity test and the efficacy reflection can be carried out by replacing human skin with the skin keratinocyte cultures. This provides convenience for clinical trials while reducing the risk of trial failures, and human keratinocytes published by Reineckia carnea et al evaluate the cytotoxicity of cosmetics (toxicological impurities, 2008, 22 (1): 75-76).
There are many methods for isolating skin keratinocytes such as Tr method, Tr-C method, Dispase method, etc., but many problems are also encountered: easy to grow in a mixed way with other cells, can not survive for a long time, has low activity, is not easy to attach, needs special substrates and special culture media, and the like.
The culture method of skin keratinocyte includes nourishing layer culture method and non-nourishing layer and serum-free culture method. The most commonly used method is trophoblast culture, which uses the inactivation of fibroblast 3T3 cells of mouse embryonal tumor by C060 irradiation or mitomycin treatment as a substrate, and can promote cell adhesion and inhibit human fibroblast proliferation. However, 3T3 may produce harmful metabolites, and when applied to a human body, the product cultured in a culture medium containing bovine serum tends to cause rejection.
Disclosure of Invention
Aiming at the problems of low efficiency, rejection reaction and the like existing in the prior separation and culture of the keratinocyte, the invention provides a separation and culture method of the keratinocyte.
In a first aspect, the present invention provides a method for isolating keratinocytes, comprising:
Viable skin tissue removes the yellow fat layer, leaving the epidermis, and all or at least a portion of the dermis layer;
Placing the active skin tissue in Trypsin/EDTA digestive juice for cold digestion;
Adding pancreatin stop solution, and collecting epidermal tissue;
placing the collected epidermal tissue in collagenase type I for thermal digestion;
The cell suspension was collected.
In a second aspect, the present invention provides a method for culturing keratinocytes, comprising:
Viable skin tissue removes the yellow fat layer, leaving the epidermis, and all or at least a portion of the dermis layer;
Placing the active skin tissue in Trypsin/EDTA digestive juice for cold digestion;
Adding pancreatin stop solution, and collecting epidermal tissue;
Placing the collected epidermal tissue in collagenase type I for thermal digestion;
Collecting cell suspension, inoculating and culturing.
in the method of the present invention, the skin tissue may be skin of any part of a living body, for example, any one or more parts of the chest, abdomen, back, buttocks, and limbs.
In the method of the present invention, the living skin tissue may be further disrupted to form particles prior to cold digestion.
In the method of the present invention, the thickness of the remaining dermal layer in the skin tissue is preferably 0.2 to 5mm, more preferably 0.5 to 3mm, and still more preferably 1 to 2 mm.
In the method of the invention, the concentration of the Trypsin/EDTA digestive juice is preferably 0.01-0.2 wt%, more preferably 0.02-0.1 wt%, more preferably 0.03-0.08 wt%, more preferably 0.04-0.07 wt%, and more preferably 0.05-0.06 wt%.
in the method of the present invention, the cold digestion temperature is in the range of 2 to 8 ℃, more preferably 3 to 6 ℃, and still more preferably 4 to 5 ℃.
In the method of the present invention, the cold digestion time is preferably 6 to 24 hours, more preferably 8 to 22 hours, more preferably 10 to 20 hours, more preferably 12 to 18 hours, and more preferably 14 to 16 hours.
In the method of the present invention, the concentration of collagenase type I is preferably 0.01% to 1%, more preferably 0.02% to 0.8%, more preferably 0.04% to 0.7%, more preferably 0.05% to 0.5%.
In the method of the present invention, the thermal digestion temperature is in the range of 35 to 38 ℃, more preferably 36 to 37.5 ℃, and still more preferably 36.5 to 37 ℃.
In the process according to the invention, the thermal digestion is preferably carried out for a period of time ranging from 1 to 10 hours, more preferably from 2 to 8 hours, even more preferably from 3 to 6 hours.
Wherein the thermal digestion is preferably carried out under stirring conditions.
in the method of the present invention, the culture medium for the inoculation culture can be a conventional culture medium, and in the present invention, a KGM-Gold or DKSFM culture medium is preferred.
Wherein, the culture medium can also be added with additional factors, such as transferrin, insulin, hormone, epidermal growth factor, such as one or more of hydrocortisone, progesterone, transferrin, and iron chelator.
Wherein, the additional factors which can be added into the KGM-Gold culture medium at least comprise any one or more of Hydrocortisone (Hydrocortisone), Transferrin (Transferrin), Epinephrine (Epinephrine), GA-1000, BPE, rhEGF and insulin.
Wherein, the DKSFM medium may contain at least one or more of EGF, Penicillin (Penicillin), Streptomycin (Streptomyces), amphotericin B (Ampotericin B).
In the method of the present invention, during the seeding process, the seeding cell density is preferably 103-.
In the method of the present invention, the culture is preferably performed without a trophoblast and without serum.
In a preferred embodiment of the method of the present invention, before the cold digestion, a step of pre-treating the active skin tissue is further included, wherein the pre-treating at least includes any one or more of cleaning and disinfection.
wherein, the disinfection is preferably carried out by using any one or more of iodine tincture and alcohol.
Wherein the cleaning is preferably at least DPBS cleaning.
Wherein the disinfection time is preferably 10s-5min, more preferably 20s-3min, more preferably 30s-1min, more preferably 35s-45 s.
The third aspect of the invention is to provide an epidermal tissue culture obtained by the culture method.
In a fourth aspect, the invention provides a use of the culture method or the epidermal tissue culture.
wherein the application may be selected from:
Establishing an epidermal cell bank;
Preparing a material for carrying out sensitivity test on a cosmetic product;
preparing the skin repairing material.
The skin repairing material can be used for any one or more of scar repairing, striae gravidarum removing, skin injury treating and skin disease treating.
Wherein the skin diseases include: pigmentary change and pigmented nevus, fungal infection, and virus infection, such as vitiligo, freckle, albinism, tinea versicolor, chloasma, purpura, pigmented nevus, nevus taitian, heat shock erythema, tinea manuum, tinea pedis, tinea corporis, herpes, wart, etc.
Wherein the skin injury comprises any one or more of operation wound, sunburn, burn, cold injury, abrasion, cut wound, ulcer, and dermal atrophy caused by trauma. The surgical wound surface is the wound surface after resection of tumor, birthmark and the like. The ulcer may comprise a metabolic disease such as a diabetic foot ulcer, a chronic ulcer of the skin following radiation and/or chemotherapy, a pressure ulcer, a venous ulcer, and the like.
wherein, the skin repair material is used for being implanted into a wound surface to replace or cover the damaged skin.
the invention has the following advantages:
1) the invention adopts a two-step digestion method, mainly extracts the keratinocytes with stronger proliferation capability around the basal layer and the hair follicle, can obtain the single keratinocytes, has high activity, is easy to stick to the wall and is easy to proliferate.
2) the method of the invention is suitable for all human normal skin tissues, including hair-bearing sites.
3) The method of the invention does not need a trophoblast and bovine serum, can be directly paved in a pretreated culture dish or bottle for culture, is similar to the condition culture in a physiological state, and eliminates the influence of foreign proteins.
4) the culture medium used in the method only aims at the culture of the skin keratinocyte, and avoids the pollution of other cells.
5) The skin keratinocyte culture obtained by the method has extremely wide application in scientific research, beauty products and medical treatment fields.
Drawings
FIG. 1 shows the results of Tr-method keratinocyte culture (A is the result after 3 days, B is the result after 5 days);
FIG. 2 shows the results of the two-step digestion method of the present invention for culturing keratinocytes (A is the result after 3 days, B is the result after 5 days);
FIG. 3 shows the results of the present invention using breast skin to culture keratinocytes (A is the result after 2 days, B is the result after 5 days);
FIG. 4 shows the results of the present invention for culturing keratinocytes using foreskin skin (A is the result after 2 days, B is the result after 5 days).
Detailed Description
The present invention will be described in further detail with reference to examples.
Example 1: successfully separating and expanding human epidermal cells from adult normal breast skin
(1) Materials and methods
Sample preparation: adult normal chest skin (including epidermis and dermis) discarded from hospital surgery, about 3cm 2.
Cell culture medium: KGM-Gold.
(2) Cell isolation and culture
a) Removing hair from the skin part, removing hair from the part with hair, removing cuticle in advance when the cuticle is too thick, sterilizing with 75% alcohol cotton ball, and collecting tissue slice.
b) fresh skin samples were placed in HBSS/DPBS/DMEM, stored at 4 ℃ and transported to the laboratory and area measured.
c) The samples were sterilized in iodine and 75% alcohol, respectively, and washed with DPBS.
d) The yellow fat layer and most of the white dermis layer in the tissue sheet are removed, the epidermis and the dermis layer with the thickness of 0.2-2mm are remained, and the skin sheet is decomposed into tiny particles.
e) the skin particles are placed in 0.05 percent of Trypsin and EDTA solution and are placed for 12 to 18 hours at the temperature of 4 ℃.
f) Adding pancreatin stop solution.
g) Epidermal tissue was collected by filtration through a 100um cell filter.
h) adding type I collagenase with concentration of 0.05% -0.5% into epidermal tissue, and magnetically stirring for 2-6 hours at 37 ℃.
i) The cell suspension was collected by filtration, and the cell vial was washed with DMEM (GIBCO), and the cell suspension was collected by filtration.
j) The cell pellet was centrifuged and washed, counted and seeded at a seeding density of 105cells/mL onto culture vessels.
k) If the residue is remained, the cell can be continuously digested by 0.05 percent of Trypsin and EDTA for cell recovery.
The cells were isolated in a large number of hours with aggregates (fig. 3A), gradually attached to the wall the next day, and microscopically visible keratinocytes clones, which grew predominantly with increasing days of culture, pushing away other contaminating cells, and which gradually proliferated and connected into sheets, typically "paving stones", as shown in fig. 3B. The cells were substantially free of fibroblast contamination. The good state and the proliferation capacity can be still maintained after passage and frozen storage.
example 2: successfully separating and expanding human epidermal cells from adult normal breast skin
(1) Materials and methods
Sample preparation: adult normal chest skin (including epidermis and dermis) discarded from hospital surgery, about 3cm 2.
Cell culture medium: DKSFM.
(2) Cell isolation and culture
a) Removing hair from the skin part, removing hair from the part with hair, removing cuticle in advance when the cuticle is too thick, sterilizing with 75% alcohol cotton ball, and collecting tissue slice.
b) Fresh skin samples were placed in HBSS/DPBS/DMEM, stored at 4 ℃ and transported to the laboratory and area measured.
c) The samples were sterilized in iodine and 75% alcohol, respectively, and washed with DPBS.
d) the yellow fat layer and most of the white dermis layer in the tissue sheet are removed, the epidermis and the dermis layer with the thickness of 0.2-2mm are remained, and the skin sheet is decomposed into tiny particles.
e) The skin particles are placed in 0.05 percent of Trypsin and EDTA solution and are placed for 12 to 18 hours at the temperature of 4 ℃.
f) Adding pancreatin stop solution.
g) Epidermal tissue was collected by filtration through a 100um cell filter.
h) Adding type I collagenase with concentration of 0.05% -0.5% into epidermal tissue, and magnetically stirring for 2-6 hours at 37 ℃.
i) The cell suspension was collected by filtration, and the cell vial was washed with DMEM (GIBCO), and the cell suspension was collected by filtration.
j) The cell pellet was centrifuged and washed, counted and seeded at a seeding density of 105cells/mL onto culture vessels.
The cells are separated out for a plurality of hours, the cells are clustered, the cells gradually adhere to the wall in the next day, the keratinocyte clones can be seen under a microscope, with the increase of the culture days, the keratinocyte growth is dominant, other foreign cells are squeezed away, the clones are continuously proliferated and gradually connected into slices, and the slices are in a typical paving stone shape, as shown in figure 3. The cells were substantially free of fibroblast contamination. The good state and the proliferation capacity can be still maintained after passage and frozen storage.
Example 2: preparation of epidermal cells from healthy male prepuce
(1) Materials and methods
Sample preparation: taking foreskin skin after human healthy male circumcision
(2) Cell separation and culture: the procedure was the same as in example 1.
(3) as a result: the separated cells are easy to adhere to the wall, have more clones, can be quickly connected into a sheet and are in a paving stone shape, as shown in figure 4. The cells are pure, and can still maintain good state and proliferation capacity after passage and frozen storage.
Comparative example
Since the Dispase method acts mainly on the basal layer to separate the epidermal layer from the dermal layer, and keratinocytes having high proliferation ability are mainly present in the basal layer and around the hair follicles, the Dispase method damages these keratinocytes and cannot efficiently obtain high-activity keratinocytes.
As shown in FIG. 1, the control experiment was performed by isolating the same sample by the Tr method and the two-step digestion method, respectively, and the results showed that the Tr method showed very few small clones at the third day, the cloning efficiency was about 5% (FIG. 1A), the two-step digestion method showed a significant cloning efficiency of about 20% (FIG. 1B), and the Tr method showed a slow clone proliferation at the fifth day (FIG. 2A), and the two-step digestion method showed a significantly enhanced clone proliferation ability, and was localized (FIG. 2B).
The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.
Claims (7)
1. A method for isolating keratinocytes, comprising:
Removing the yellow fat layer from the living skin tissue, and retaining the epidermis and all or at least part of the dermis layer, wherein the thickness of the retained dermis layer in the skin tissue is 0.2-2 mm;
Placing the active skin tissue in Trypsin/EDTA digestive juice for cold digestion, wherein the concentration of the Trypsin/EDTA digestive juice is 0.05%, the cold digestion temperature is within the range of 2-8 ℃, and the cold digestion time is 6-24 hours;
Adding pancreatin stop solution, and collecting epidermal tissue;
Placing the collected epidermal tissue in collagenase type I for thermal digestion, wherein the concentration of the collagenase type I is 0.05-0.5%, the thermal digestion temperature is within the range of 35-38 ℃, and the thermal digestion time is 1-10 hours; the cell suspension was collected.
2. the method of claim 1, wherein the viable skin tissue is fragmented to form particles prior to cold digestion.
3. The method of claim 1, further comprising a step of pre-treating the viable skin tissue prior to said cold digestion, said pre-treating comprising at least any one or more of washing and disinfecting.
4. A method for culturing keratinocytes, comprising:
The method for separating keratinocytes according to claim 1, wherein the cell suspension is collected and cultured by inoculation.
5. The method according to claim 4, wherein the medium of the seed culture is selected from KGM-Gold or DKSFM medium.
6. The method as claimed in claim 4, wherein the seeding cell density is 103-107cells/ml during the seeding process.
7. The method of claim 4, wherein the culturing is performed without a trophoblast and without serum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510456338.2A CN105154388B (en) | 2015-07-29 | 2015-07-29 | method for separating and culturing skin keratinocytes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510456338.2A CN105154388B (en) | 2015-07-29 | 2015-07-29 | method for separating and culturing skin keratinocytes |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105154388A CN105154388A (en) | 2015-12-16 |
| CN105154388B true CN105154388B (en) | 2019-12-06 |
Family
ID=54795436
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510456338.2A Active CN105154388B (en) | 2015-07-29 | 2015-07-29 | method for separating and culturing skin keratinocytes |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105154388B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107287153B (en) * | 2017-06-18 | 2023-11-07 | 广东博溪生物科技有限公司 | Differential digestion method of human epidermal cells |
| CN107335096A (en) * | 2017-07-11 | 2017-11-10 | 上海白衣缘生物工程有限公司 | A kind of oral cavity sticking patch preparation method of compound oral mucosa epithelial cell |
| CN110170072A (en) * | 2019-05-31 | 2019-08-27 | 北京添易医学研究院 | The preparation of cell fibronectin and the method for repairing dermal tissue |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1528253A (en) * | 2003-10-17 | 2004-09-15 | 上海第二医科大学附属第九人民医院 | Tissue engineered composite skin and preparing method thereof |
| CN102086451A (en) * | 2009-12-07 | 2011-06-08 | 韩春茂 | Method for amplifying seed cells of skin tissue engineering |
| CN104099289A (en) * | 2013-04-08 | 2014-10-15 | 瑞田投资有限公司 | Epidermal tissue culturing method, and prepared epidermal tissue culture and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130071927A1 (en) * | 2010-05-05 | 2013-03-21 | Sydney Ivf Limited | Media and methods for cell culture |
-
2015
- 2015-07-29 CN CN201510456338.2A patent/CN105154388B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1528253A (en) * | 2003-10-17 | 2004-09-15 | 上海第二医科大学附属第九人民医院 | Tissue engineered composite skin and preparing method thereof |
| CN102086451A (en) * | 2009-12-07 | 2011-06-08 | 韩春茂 | Method for amplifying seed cells of skin tissue engineering |
| CN104099289A (en) * | 2013-04-08 | 2014-10-15 | 瑞田投资有限公司 | Epidermal tissue culturing method, and prepared epidermal tissue culture and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105154388A (en) | 2015-12-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US5866167A (en) | Non-viable total keratinocyte lysate for promoting wound healing | |
| CN105132358B (en) | Method for obtaining tissue engineering epidermis by culture and application thereof | |
| Gauthier et al. | Non-cultured epidermal suspension in vitiligo: from laboratory to clinic | |
| US7419661B2 (en) | Dermal sheath tissue in wound healing | |
| CN104087551B (en) | The method of a kind of Isolation and culture people's epidermal cell | |
| Stoner et al. | The treatment of hypopigmented lesions with cultured epithelial autograft | |
| CN105132357B (en) | serum-free culture system for epidermal cell culture | |
| US9592257B2 (en) | Complete human skin organ generated from culture-expanded cells | |
| CN104814980A (en) | Production method and applications of human embryo fibroblasts | |
| KR101293762B1 (en) | Culture media having enhanced wound healing activity and a method for preparing the same | |
| JPH02174848A (en) | Cuticle sheet;and production, storage and usage thereof | |
| EP1709160B1 (en) | Preparation of fibroblast-populated connective tissue substitute | |
| CN102335459A (en) | Hair follicle reconstruction method based on hair follicle stem cells and fibroblast | |
| CN105154388B (en) | method for separating and culturing skin keratinocytes | |
| CN111778206B (en) | Preparation method of deer antler stem cell conditioned medium for resisting skin aging | |
| CN102233144A (en) | Method for implementing subcutaneous tissue regeneration by using tissue engineering technology | |
| Cervelli et al. | Treatment of stable vitiligo by ReCell system | |
| CN106701670A (en) | Methods for enhancing bioactive factor secretion capacity of mesenchymal stem cells and extracting active factors in culture solution | |
| US20010006813A1 (en) | Methods and compositions for the preparation of cell transplants | |
| EP0980270B1 (en) | Dermal sheath tissue in wound healing | |
| WO2013191531A1 (en) | Autologous tissue-engineered human skin construct and a method for producing thereof | |
| US6585969B1 (en) | Non-viable keratinocyte cell composition or lysate for promoting wound healing | |
| WO2014117597A2 (en) | Rapid tissue engineering method for skin construction and repair of skin damage | |
| CN105925524A (en) | Method for transforming human primary fibroblast into epidermic cell | |
| CN102580165A (en) | Repairing agent for skin defect and preparation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20191029 Address after: Room 605, B1 building, bio nano Park, 218 Xinghu street, Suzhou Industrial Park, Suzhou, Jiangsu Applicant after: Suzhou nuopu Regenerative Medicine Co. Ltd. Address before: 214092, Jiangsu, Wuxi Province, Binhu District, Mashan Mei Lu Road, No. 130, -3 Applicant before: H&B HEALTH GROUP Applicant before: Liu Hong |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |