CN107267441B - Method for establishing three-dimensional skin model for anti-inflammatory and anti-allergy efficacy evaluation - Google Patents
Method for establishing three-dimensional skin model for anti-inflammatory and anti-allergy efficacy evaluation Download PDFInfo
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Abstract
The invention relates to a method for establishing a three-dimensional skin model, which comprises the following steps: 1) preparing an extracellular matrix polymer hydrogel solution; 2) mixing fibroblasts with an extracellular matrix polymer hydrogel solution, and placing the mixture into a culture chamber for culture; 3) inoculating keratinocyte, and culturing through an air-liquid interface to obtain a three-dimensional skin model containing a dermis layer, an epidermis layer and a horny layer. The method can effectively establish the three-dimensional skin model, has simple and convenient preparation method, can be used for screening anti-inflammatory and anti-allergy medicaments for the skin, and has good application prospect.
Description
Technical Field
The invention relates to a method for establishing a skin model, in particular to a method for establishing a novel three-dimensional skin model for evaluating anti-inflammatory and anti-allergy effects.
Background
The skin is used as a first barrier for covering and protecting important tissues and organs of the body surface and blocking interference of external substances, is easily damaged by factors such as trauma, burn, inflammation, ulcer, tumor after operation, congenital diseases and the like, and can defend through certain special reactions. The incidence rate of allergic skin diseases such as dermatitis, eczema and the like worldwide exceeds 10 percent. At present, the screening of skin anti-inflammatory and anti-allergy medicines or cosmetic raw materials is very inconvenient, the cost is high, and a mouse ear swelling caused by xylene, a rat foot sole swelling caused by egg white, a rat cotton ball granuloma chronic inflammation model and a mouse local lymph node sensitization experiment are common animal experiment models. However, as cosmetic laws in europe promulgate ban on animal experiments in cosmetic products, screening and development of anti-inflammatory and anti-allergy chemicals and cosmetic raw materials is more difficult to achieve. Meanwhile, due to the complexity of skin sensitization and inflammation mechanisms, the whole sensitization and inflammation causing process is difficult to be comprehensively reflected in the same in-vitro test system, so that more in-vitro experiments need to be developed to provide prediction and evaluation for skin anti-inflammatory and anti-allergy effects of chemicals and personal care products.
Disclosure of Invention
In order to solve the problems, the technical scheme of the invention is to provide a construction method of a three-dimensional skin model and application thereof.
The invention relates to a method for establishing a three-dimensional skin model, which comprises the following steps:
1) preparing an extracellular matrix polymer hydrogel solution;
2) mixing fibroblasts with an extracellular matrix polymer hydrogel solution, and placing the mixture into a culture chamber for culture;
3) inoculating keratinocyte, and culturing through an air-liquid interface to obtain a three-dimensional skin model containing a dermis layer, an epidermis layer and a horny layer.
In the step 1), the extracellular matrix polymer hydrogel solution is a mixed solution of hyaluronic acid, polyethylene glycol diacrylate and type I collagen, wherein the weight ratio of hyaluronic acid, polyethylene glycol diacrylate and type I collagen is 4:1:1, and preferably, the concentrations of hyaluronic acid, polyethylene glycol diacrylate and type I collagen are 6.67mg/ml, 1.67mg/ml and 1.67mg/ml respectively.
In the step 2), 1.25 × 10 is added while mixing the fibroblast and the extracellular matrix polymer hydrogel solution5～2×105Mast cells, mixed;
preferably, the mast cells are prepared as follows: taking scar tissue, removing subcutaneous fat, cutting into small pieces, adding digestive juice for digestion for 4h, shearing to obtain milky white suspension, filtering, and collecting filtrate; centrifuging the filtrate at 4 deg.C, removing supernatant, resuspending, centrifuging, removing supernatant, inoculating suspension cells into DMEM culture solution containing 10% FBS, and culturing to obtain mast cells; the digestive juice is a mixed solution of collagenase type I and hyaluronidase, wherein the concentration of the collagenase type I is 0.3mg/ml, and the concentration of the hyaluronidase is 100U/ml.
In step 2), 5X 10 was added per 1ml of hydrogel solution5～8×105Placing fibroblast into the culture chamber, culturing in the culture plate for 30min to form hydrogel, and culturing for 5 days.
In step 3), the amount of keratinocytes seeded was: when 12-hole plate culture is adopted in the step 2), 2 multiplied by 10 is added into each hole5～4×105Keratinocytes are formed.
In the step 3), the method for culturing the gas-liquid interface comprises the following steps:
after inoculating keratinocytes, culturing for 5 days with the DMEM medium flooding the culture chamber, and then culturing for 14 days with the DMEM medium at the bottom of the culture chamber;
or inoculating keratinocyte, culturing for 1 day under the condition that DMEM culture medium submerges the culture chamber, adding immunocyte to the periphery of the culture chamber, continuously culturing for 4 days, and then culturing for 14 days under the condition that DMEM culture medium is at the bottom of the culture chamber to obtain the product; wherein the amount of keratinocytes seeded is: when 12-hole plate culture is adopted in the step 2), 2 multiplied by 10 is added into each hole5～5×105An immune cell, said immune cell beingMonocytes, dendritic cells, T lymphocytes or macrophages.
The fibroblasts were prepared as follows: taking dermal layer tissue of skin, cutting into small pieces, culturing with DMEM medium containing 10% FBS, changing culture solution every 2 days, culturing for 6 days, recovering cells, and screening fibroblasts by a flow cytometer;
the keratinocytes were prepared as follows: taking skin epidermal layer tissue, cutting into small pieces, culturing with DMEM medium containing 10% FBS, changing culture solution every 2 days, culturing for 6 days, recovering cells, and screening keratinocyte by flow cytometry;
the monocytes were prepared as follows: taking blood, treating by adopting lymphocyte layering liquid, centrifuging, taking a mononuclear cell layer, adhering to the wall by adopting an RPMI-1640 culture medium for 2 hours, removing cells which are not adhered to the wall or are not adhered firmly, and scraping off adherent cells to be mononuclear cells;
the granulocytes were prepared as follows: treating blood with lymphocyte layering solution, centrifuging, collecting granulocyte layer, washing, and culturing with RPMI-1640 culture medium;
the dendritic cells were prepared as follows: collecting blood, processing with lymphocyte layering solution, centrifuging, collecting mononuclear cell layer, adhering to wall with RPMI-1640 culture medium for 2 hr, removing nonadherent or nonadherent cells, collecting adherent cells, separating CD14+ mononuclear cells by immunomagnetic bead method, placing RPMI-1640 culture solution containing 10% FBS, 50ng/mL rhGM-CSF and 25ng/mL IL-4 at 37 deg.C and 5% CO2Culturing in an incubator for 5-6 days to obtain dendritic cells;
the T lymphocytes are prepared as follows: treating blood with lymphocyte layering solution, centrifuging, collecting mononuclear cell layer, adhering to wall with RPMI-1640 culture medium for 2 hr, collecting nonadherent cells, and adding RPMI-1640 culture solution containing 10% FBS to obtain T lymphocyte;
the macrophage cell was prepared as follows: treating blood with lymphocyte layering solution, centrifuging, collecting mononuclear cell layer, culturing in RPMI-1640 culture medium for 1-2 days, adding phorbol ester (PMA) RPMI-1640 culture medium with final concentration of 125ng/mL, performing cell differentiation, and culturing for 2-3 days to obtain macrophage.
The invention also provides a three-dimensional skin model established by the method.
The invention also provides application of the three-dimensional skin model in screening anti-inflammatory and anti-allergy skin drugs.
The invention also provides a method for screening anti-inflammatory and anti-allergy medicaments for treating skin, which comprises the following steps:
a. a three-dimensional skin model established according to the method;
b. applying the candidate to the three-dimensional skin model in step a;
c. a three-dimensional skin model was used to evaluate potential anti-inflammatory and anti-allergy drugs.
An integrated test system based on skin sensitization inflammation experiment is established to provide an integrated detection strategy, and a skin model containing immune cells for anti-inflammatory and anti-allergy efficacy detection is developed. The established skin model is used for establishing a standardized, safe and efficient in-vitro skin care health product three-dimensional skin evaluation system, the in-vitro evaluation system is used as one of methods for replacing traditional animal experiments, the in-vitro evaluation method for detecting the safety and efficacy of a single chemical and a product is verified, the experimental stability, repeatability and feasibility of the in-vitro evaluation system as a replacement method are verified, important references are provided for three-dimensional skin research, prediction and evaluation are provided for skin anti-allergy and anti-inflammatory effects of the chemical and a personal care product, and a basis with reference significance is provided for China in the aspect of replacing animal experiments.
Three-dimensional Skin (3D-Skin) is a perfect tissue engineered Skin, and is an artificial Skin prepared by mixing dermal fibroblasts, epidermal keratinocytes and extracellular matrix substitutes. Besides clinical use, tissue engineering skin also promotes development of non-clinical research work due to high stability and simple operation. In the fields of skin biology research, skin aging, light protection, animal replacement test experiments and the like, the three-dimensional skin constructed in vitro has high application value. The three-dimensional skin model can meet the interaction between different cells and can provide an important bridge between in vitro and in vivo.
The method can effectively establish the three-dimensional skin model, has simple and convenient preparation method, can be used for screening anti-inflammatory and anti-allergy medicaments for the skin, and has good application prospect.
According to the above-mentioned aspects of the present invention, many other modifications, substitutions, or alterations can be made without departing from the basic technical concept of the present invention as defined by the general technical knowledge and common practice in the field relevant to the present invention.
Drawings
FIG. 1A, C fibroblasts with poor growth; B. well-grown fibroblasts
FIG. 2A, C shows poorly growing keratinocytes; B. well-growing keratinocytes
FIG. 3A HyStemTM (thiol-modified hyaluronic acid HA) alone; ExtralinkTM (polyethylene glycol diacrylate, PEGDA) alone; type I collagen alone; d hyaluronic acid-collagen hydrogel of the invention
FIG. 4 three-dimensional skin model and HE staining
FIG. 5 is a three-dimensional skin model simulation
FIG. 6 schematic diagram of the construction of the full-thickness skin model
FIG. 7 peripheral blood-derived immunocyte culture
FIG. 8 three-dimensional skin model culture containing primary peripheral blood-derived immune cells (monocytes, lymphocytes, granulocytes, etc.)
FIG. 9 contains a three-dimensional skin model of mast cells such as
FIG. 10 three-dimensional skin model containing mast cells
FIG. 11 inflammatory factor assay (solid phase chip)
FIG. 12HE staining and scanning Electron microscopy
FIG. 13 cell viability after being subjected to test substance treatment
Detailed Description
Example 1 preparation of a three-dimensional skin model of the invention
The method for constructing the skin with the structural shape similar to that of the normal skin by compounding all types of cells of the whole skin layer including immune cells (lymphocytes, dendritic cells, granulocytes, monocytes, macrophages and the like) and biological scaffold materials in vitro.
1 the method for constructing the three-dimensional skin model of the invention
1.1 Source and preparation of various cells
1) Obtaining fibroblasts and keratinocytes:
skin tissue of normal human is taken, and the tissue is sterilized to separate small pieces (3 mm. times.3 mm) of dermal layer tissue and epidermal layer tissue. The tissue pieces were placed in sterile petri dishes and medium (DMEM medium with 10% FBS) was added. The tissue small blocks adhere to the veneering part of the culture dish, a part of cells can be adhered to the surface of the culture dish to grow, the culture solution is replaced every 2 days, the growth condition of the cells is observed through a microscope, the cells are recovered after 6 days of culture, and the cells are screened through a flow cytometer to obtain fibroblasts and keratinocytes. The method comprises culturing cells at 37 deg.C with 5% CO2Cultured in an incubator
Growth patterns of fibroblasts and keratinocytes were obtained as shown in fig. 1B, D, fig. 2B, D.
2) Obtaining primary peripheral blood cells:
aseptically collecting 6ml venous blood, injecting into a heparin anticoagulation tube, shaking up gently, adding PBS or Hank, s solution with the same volume at room temperature, and blowing, beating and mixing evenly. Placing 6ml of lymphocyte layering liquid into a centrifuge tube, centrifuging the diluted blood sample for 30min at 18-20 ℃ and 2000rpm above the separation liquid surface, and dividing the blood sample into four layers from the tube bottom to the liquid surface after centrifugation, namely a red blood cell and granulocyte layer, a layering liquid layer, a mononuclear cell layer and a plasma layer in sequence.
The culture method of the mononuclear cells comprises the following steps: the upper yellowish diluted plasma was removed, the opalescent mononuclear cell layer was gently aspirated, placed in a new centrifuge tube, washed twice with PBS, and mononuclear cells were cultured with RPMI-1640 medium (10% FBS).
The monocyte culture method comprises the following steps: transferring the mononuclear cells into a culture bottle for culture (RPMI-1640 culture medium), adsorbing for 2h, removing nonadherent cells by suction, washing the nonadherent cells, scraping the adherent cells to obtain mononuclear cells, and adding the RPMI-1640 culture medium (containing 10% FBS) to culture the mononuclear cells.
The granulocyte culture method comprises the following steps: the upper layer of pale yellow diluted plasma was removed, the opalescent mononuclear cell layer was removed, the separation layer was removed, the granulocytes were gently aspirated and placed into a new centrifuge tube, washed twice with PBS, and the granulocytes were cultured with RPMI-1640 medium (containing 10% FBS).
The dendritic cell culture method comprises the following steps: culturing the above mononuclear cells in culture flask (RPMI-1640 culture medium), attaching for 2 hr, removing nonadherent cells, collecting adherent mononuclear cells, separating CD14+ mononuclear cells by immunomagnetic bead method, culturing with RPMI-1640 culture solution containing 10% FBS, 50ng/mL rhGM-CSF and 25ng/mL IL-4 at 37 deg.C and 5% CO2Culturing in an incubator, and differentiating the cells into dendritic cells after 5-6 days.
The T lymphocyte separation method comprises the following steps: and (3) transferring the mononuclear cells into a culture bottle for culture, attaching the mononuclear cells to the wall for 2 hours, collecting nonadherent cells, and adding an RPMI-1640 culture solution containing 10% FBS to obtain the T lymphocytes.
The culture method of the macrophage comprises the following steps: transferring the above monocyte into culture flask, culturing for 1-2 days, adding phorbol ester (PMA) RPMI-1640 culture medium with final concentration of 125ng/mL, performing cell differentiation, and culturing for 2-3 days to obtain macrophage.
The peripheral blood-derived immunocyte culture is shown in FIG. 7.
3) Obtaining of Primary mast cells
Removing subcutaneous fat from scar tissue of human, cleaning skin with Tyrode liquid, and cutting into 1mm pieces2Adding digestive juice (0.3mg/ml collagenase type I and 100U/ml hyaluronidase) into small pieces, digesting for 4h, shearing to obtain milky white suspension, filtering, and collecting filtrate. Centrifuging the filtrate at 4 deg.C, removing supernatant, resuspending the precipitate with cold Tyrode solution, centrifuging, removing supernatant, inoculating suspension cells into DMEM culture solution containing 10% FBS,to obtain the mast cells.
1.2 modeling method
1) Preparing an extracellular matrix polymer hydrogel solution:
hyaluronic acid-collagen hydrogel: HyStem kit (Sigma) is internally lyophilized solid HyStemTM (sulfhydryl modified hyaluronic acid HA) and ExtralinkTM (polyethylene glycol diacrylate, PEGDA) are respectively dissolved by deionized water to obtain 10mg/mL stock solution, and type I collagen (Sigma) is prepared into 10mg/mL stock solution with the pH value of 7.2. HA. The PEGDA and the type I collagen (4:1:1) are mixed evenly to obtain the hydrogel solution.
The hydrogel solution is solidified to obtain the hydrogel three-dimensional scaffold, and the schematic diagram is shown in fig. 3D.
2) Constructing three-dimensional skin models
a. The first method comprises the following steps: full skin model
The construction scheme is shown in FIG. 6:
fibroblast cells were seeded into hydrogel solutions at 5X 10 per 1ml of hydrogel solution5～8×105Gently placing each cell into a culture chamber (transwell), transferring into a 12-well culture plate, culturing in an incubator, solidifying the hydrogel solution to form hydrogel after 30min to obtain a scaffold containing fibroblasts, adding DMEM culture medium to submerge the culture chamber, culturing to construct dermis, culturing for 5 days, and inoculating keratinocyte (2 × 10 cells per well) on the surface of the scaffold5～4×105) Formation of the epidermis by gas-liquid interfacial culture: specifically, the culture medium was cultured in the DMEM medium in such a manner that the culture chamber was submerged for 5 days, and then cultured in the DMEM medium at the bottom of the culture chamber for 14 days, thereby preparing the culture medium.
After the skin model is processed by paraffin embedding, HE staining is carried out, morphological comparison with normal human skin is carried out, and the culture condition is judged. The three-dimensional skin model and HE staining are shown in fig. 4; the three-dimensional skin model simulation diagram is shown in fig. 5.
b. And the second method comprises the following steps: three-dimensional skin model containing peripheral blood-derived immune cells
The construction scheme is shown in FIG. 8:
fibroblast cells were seeded into hydrogel solutions at 5X 10 per 1ml of hydrogel solution5～8×105Gently placing each cell into a culture chamber (transwell), transferring into a 12-well culture plate, culturing in an incubator, solidifying the hydrogel solution to form hydrogel after 30min to obtain a scaffold containing fibroblasts, adding DMEM culture medium to submerge the culture chamber, culturing to construct dermis, culturing for 5 days, and inoculating keratinocyte (2 × 10 cells per well) on the surface of the scaffold5～4×105Individual cells), which form the epidermis by gas-liquid interfacial culture: specifically, the culture is performed by immersing the culture chamber in DMEM medium for 1 day, adding immune cells (monocytes, dendritic cells, T lymphocytes or macrophages) to the periphery of the culture chamber, and adding 2 × 10 cells per well5～5×105The cells were cultured for 4 days and then for 14 days with DMEM medium at the bottom of the culture chamber, to prepare the cells.
c. And the third is that: three-dimensional skin model containing mast cells
The construction scheme is shown in FIG. 9:
inoculating fibroblast and mast cell into hydrogel solution, and adding 5 × 10 per 1ml hydrogel solution5～8×105Single fibroblast and 1.25X 105～2×105Gently placing mast cells into culture chamber (transwell), transferring into 12-well culture plate, culturing in incubator, solidifying hydrogel solution to form hydrogel after 30min to obtain scaffold containing fibroblast, adding DMEM culture medium to submerge culture chamber, culturing to construct dermis, culturing for 5 days, inoculating keratinocyte (adding 2 × 10 cells per well), and culturing5～4×105) Formation of the epidermis by gas-liquid interfacial culture: specifically, the culture medium is prepared by culturing the cells for 5 days in the condition that the culture chamber is submerged in the DMEM medium, and then culturing the cells for 14 days in the condition that the DMEM medium is positioned at the bottom of the culture chamber.
2 model detection
1) The tissue engineering skin constructed by the HE staining and detecting method is used for observing the constructed skin structure and the formation condition of various immune cells;
a) three-dimensional skin model containing primary peripheral blood-derived immune cells (monocytes, lymphocytes, granulocytes, etc.)
Culturing for 24-72h to separate the skin model from the immune fine cells, washing the surface of the skin model with normal saline, and preparing for HE staining; the separated immune cells are used for detecting the expression of cell surface markers; the culture supernatant is used for detecting inflammatory factors.
HE staining: the specimen fixed by neutral formaldehyde is embedded by normal paraffin, and is continuously sliced on a paraffin slicer, and is mounted on a glass slide treated by APES, the slice is baked in a constant temperature oven at 60 ℃, sealed after HE dyeing, and observed by a light lens.
Parameters for flow cytometry detection of peripheral blood-derived immune cells (monocytes, lymphocytes, granulocytes, etc.): the cells were removed from the cell culture plate, transferred into respective EP tubes per well, harvested by centrifugation (250g, 5min, 4 ℃) using a centrifuge, washed once with FACS buffer (PBS + 0.1% BSA) and resuspended. Then adding a diluent containing the monoclonal antibody, carrying out a dark reaction at room temperature for 20 min, centrifuging to remove a supernatant, adding PBS (phosphate buffer solution) for dilution and mixing uniformly, and then carrying out detection on a machine, wherein FACS-Calibur (a product of BD company in America) is adopted, argon ion laser is adopted, the power is 15mw, and the excitation light wavelength is 488 nm.
And (3) detection results:
neutrophils: gr-1, cD11b
Mature T cells (CD3+), T helper induced cells (CD3+/CD4+), and,
Monocyte CD14+
Granulocyte CD 15+
Dendritic cell: CD11c +, MHCII +
Macrophage: CD14+, CD11b +
b) Three-dimensional skin model containing mast cells
HE staining: the specimen fixed by neutral formaldehyde is embedded by normal paraffin, and is continuously sliced on a paraffin slicer, and is mounted on a glass slide treated by APES, the slice is baked in a constant temperature oven at 60 ℃, sealed after HE dyeing, and observed by a light lens.
Mast cell (toluidine blue) staining: dewaxing the paraffin section to water conventionally, washing the paraffin section with water after 30min in toluidine blue dye solution, differentiating the paraffin section in glacial acetic acid solution, drying the paraffin section after washing with distilled water, enabling the paraffin section to be transparent, sealing neutral gum and observing the paraffin section under a light mirror.
The mast cell three-dimensional skin model is shown in fig. 10.
2) Changes in biomarker expression are detected using known stimulus detection models.
Control group of stimuli: lipopolysaccharide (LPS), 2, 4-nonadiene
Control group of the protectors: minoxidil
Negative control: double distilled water
Placing the pre-incubated skin model (second: three-dimensional skin model containing peripheral blood-derived immune cells) in a 12-well cell culture plate; smearing a sample test substance on the surface of a skin model, incubating for 24 hours in an incubator, and performing 3 groups of parallel tests on each group of samples; after 24 hours, washing a skin model surface test object by using normal saline, preparing for MTT test, observing by using a scanning electron microscope, carrying out HE dyeing, and detecting related index change by using flow cytometry; the supernatant was examined for the expression of the relevant inflammatory factors using the solid phase chip protein Profiler Human Cytokine Array Kit (BD). The inflammatory factor assay is shown in FIG. 11.
The results are shown in FIGS. 12 to 13:
the skin anti-inflammatory and anti-allergy efficacy of chemicals or personal care products is judged by monitoring the cell viability, morphological changes, cytokine expression, cell surface marker expression and relative fluorescence intensity after exposure to the test substance.
The three-dimensional skin model was incubated with 500uM of 2, 4-nonadienal or 2ug/ml LPS for 6 hours, then the culture medium was removed, and the culture medium was added again for 48 hours, and then the three-dimensional skin model was sliced and observed for imaging. Compared with the untreated control group, more skin cavities due to allergy and inflammation were observed on the longitudinal section; on the cross-section, more scaly lesions were observed to form on the skin surface. LPS and 2, 4-nonadienal induce the damage of the whole skin of the three-dimensional skin, 0.1mM minoxidil is added to the damaged three-dimensional skin model, and the skin cavity and the surface scale skin damage are effectively repaired after the upper side culture for 6 hours.
When the first full-skin model and the third three-dimensional skin model containing mast cells are detected by the method, LPS and 2, 4-nonadienal can induce damage of three-dimensional skin, 0.1mM minoxidil is added into the damaged three-dimensional skin model, and the skin cavity and surface scale skin damage are effectively repaired after the damaged three-dimensional skin model is cultured for 6 hours.
The experimental result shows that the three-dimensional skin model is successfully established and can be used for screening anti-inflammatory and anti-allergy medicaments for the skin.
The experiment proves that the invention establishes a three-dimensional skin model, can be used for screening anti-inflammatory and anti-allergy medicaments for skin, and has good application prospect.
Claims (9)
1. A method for establishing a three-dimensional skin model for screening anti-inflammatory and anti-allergic drugs for treating skin is characterized by comprising the following steps of: the method comprises the following steps:
1) preparing an extracellular matrix polymer hydrogel solution; the extracellular matrix polymer hydrogel solution is a mixed solution of hyaluronic acid, polyethylene glycol diacrylate and type I collagen, wherein the weight ratio of the hyaluronic acid to the polyethylene glycol diacrylate to the type I collagen is 4:1:1, and the concentrations of the hyaluronic acid to the polyethylene glycol diacrylate to the type I collagen are respectively 6.67mg/ml, 1.67mg/ml and 1.67 mg/ml;
2) mixing fibroblasts with an extracellular matrix polymer hydrogel solution, and placing the mixture into a culture chamber for culture;
3) inoculating keratinocyte, and culturing through an air-liquid interface to obtain a three-dimensional skin model containing a dermis layer, an epidermis layer and a cuticle layer; the method for culturing the gas-liquid interface comprises the following steps: after inoculating keratinocyte, firstly culturing for 1 day under the condition that a DMEM culture medium submerges a culture chamber, adding immune cells to the periphery of the culture chamber, continuously culturing for 4 days, and then culturing for 14 days under the condition that the DMEM culture medium is positioned at the bottom of the culture chamber to obtain the keratinocyte-containing culture medium; the amount of immune cells inoculated was: when 12-hole plate culture is adopted in the step 2), 2 multiplied by 10 is added into each hole5~5×105An immune cell that is a monocyte, dendritic cell, T lymphocyte, or macrophage.
2. The method of claim 1, wherein: in the step 2), 1.25 × 10 is added while mixing the fibroblast and the extracellular matrix polymer hydrogel solution5~2×105Mast cells, mixed.
3. The method of claim 2, wherein: the mast cells were prepared as follows: taking scar tissue, removing subcutaneous fat, cutting into small pieces, adding digestive juice for digestion for 4h, shearing to obtain milky white suspension, filtering, and collecting filtrate; centrifuging the filtrate at 4 deg.C, removing supernatant, resuspending, centrifuging, removing supernatant, inoculating suspension cells into DMEM culture solution containing 10% FBS, and culturing to obtain mast cells; the digestive juice is a mixed solution of collagenase type I and hyaluronidase, wherein the concentration of the collagenase type I is 0.3mg/ml, and the concentration of the hyaluronidase is 100U/ml.
4. The method according to any one of claims 1 to 3, wherein: in step 2), 5X 10 was added per 1ml of hydrogel solution5~8×105Placing fibroblast into the culture chamber, culturing in the culture plate for 30min to form hydrogel, and culturing for 5 days.
5. The method of claim 1, wherein: in step 3), the amount of keratinocytes seeded was: when 12-hole plate culture is adopted in the step 2), 2 multiplied by 10 is added into each hole5~4×105Keratinocytes are formed.
6. The method of claim 1, wherein: the fibroblasts were prepared as follows: taking dermal layer tissue of skin, cutting into small pieces, culturing with DMEM medium containing 10% FBS, changing culture solution every 2 days, culturing for 6 days, recovering cells, and screening fibroblasts by a flow cytometer;
the keratinocytes were prepared as follows: taking skin epidermal layer tissue, cutting into small pieces, culturing with DMEM medium containing 10% FBS, changing culture solution every 2 days, culturing for 6 days, recovering cells, and screening keratinocyte by flow cytometry;
the monocytes were prepared as follows: taking blood, treating by adopting lymphocyte layering liquid, centrifuging, taking a mononuclear cell layer, adhering to the wall by adopting an RPMI-1640 culture medium for 2 hours, removing cells which are not adhered to the wall or are not adhered firmly, and scraping off adherent cells to be mononuclear cells;
the granulocytes were prepared as follows: treating blood with lymphocyte layering solution, centrifuging, collecting granulocyte layer, washing, and culturing with RPMI-1640 culture medium;
the dendritic cells were prepared as follows: collecting blood, processing with lymphocyte layering solution, centrifuging, collecting mononuclear cell layer, adhering to wall with RPMI-1640 culture medium for 2 hr, removing nonadherent or nonadherent cells, collecting adherent cells, separating CD14+ mononuclear cells by immunomagnetic bead method, placing RPMI-1640 culture solution containing 10% FBS, 50ng/mL rhGM-CSF and 25ng/mL IL-4 at 37 deg.C and 5% CO2Culturing in an incubator for 5-6 days to obtain dendritic cells;
the T lymphocytes are prepared as follows: treating blood with lymphocyte layering solution, centrifuging, collecting mononuclear cell layer, adhering to wall with RPMI-1640 culture medium for 2 hr, collecting nonadherent cells, and adding RPMI-1640 culture solution containing 10% FBS to obtain T lymphocyte;
the macrophage cell was prepared as follows: treating blood with lymphocyte layering solution, centrifuging, collecting mononuclear cell layer, culturing in RPMI-1640 culture medium for 1-2 days, adding phorbol ester (PMA) RPMI-1640 culture medium with final concentration of 125ng/mL, performing cell differentiation, and culturing for 2-3 days to obtain macrophage.
7. A three-dimensional skin model created by the method of any one of claims 1 to 6.
8. Use of the three-dimensional skin model of claim 7 for screening anti-inflammatory and anti-allergy drugs for skin.
9. A method for screening anti-inflammatory and anti-allergy drugs for treating skin is characterized by comprising the following steps: the method comprises the following steps:
a. a three-dimensional skin model created according to the method of any one of claims 1 to 6;
b. applying the candidate to the three-dimensional skin model in step a;
c. a three-dimensional skin model was used to evaluate potential anti-inflammatory and anti-allergy drugs.
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| CN110940813A (en) * | 2019-10-31 | 2020-03-31 | 广州市华代生物科技有限公司 | In-vitro method for evaluating repair function by adopting reconstructed normal human body three-dimensional skin model |
| CN111073844A (en) * | 2019-11-29 | 2020-04-28 | 广州市华代生物科技有限公司 | Preparation method of long-term culture model of skin single-organ chip |
| CN113699202A (en) * | 2020-05-21 | 2021-11-26 | 华子昂 | Method for preparing collagen by using mixed cell and artificial cell culture nest |
| CN113564114B (en) * | 2021-01-16 | 2023-04-07 | 浙江工商大学 | Construction method and application of three-dimensional cell model of CMT93-DC-T cell |
| CN112961899B (en) * | 2021-02-23 | 2023-06-09 | 云南贝泰妮生物科技集团股份有限公司 | Anti-inflammatory efficacy screening method for cosmetic raw material in-vitro macrophages combined with 3D skin model |
| CN113549670A (en) * | 2021-07-20 | 2021-10-26 | 云南贝泰妮生物科技集团股份有限公司 | Cosmetic sensitization evaluation method for in-vitro cell and 3D epidermis model co-culture |
| CN113652392B (en) * | 2021-08-17 | 2023-09-26 | 云南贝泰妮生物科技集团股份有限公司 | Cosmetic anti-inflammatory efficacy assessment method based on in-vitro macrophage and 3D skin model co-culture |
| CN116162585A (en) * | 2021-11-24 | 2023-05-26 | 青岛蔚蓝生物集团有限公司 | Skin cell ultraviolet injury model constructed by HaCaT cells |
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