CN101392237A - Culture of tissue engineering skin and detection method of proliferation activity - Google Patents
Culture of tissue engineering skin and detection method of proliferation activity Download PDFInfo
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Abstract
The invention discloses a detection method of tissue engineering skin culture and proliferation activity, which comprises the following process: first three-dimensional host materials containing basilar membrane are prepared as the substitute of dermis, namely dermis tissue of human with epidermis being removed, and keratinocyte is cultivated; the keratinocyte is inoculated to the surface of the dermis tissue of human with epidermis being removed and coated with mixed tissue glue, sampling is carried out for the detection of the skin proliferation activity after the culture of air-liquid surface culture mode, the culture is stopped after the culture is continued for a week, and then the skin is picked out for skin proliferation activity detection. A fresh culture specimen picked is put into BrdU culture liquid and a culture box of 37 DEG C for breeding, immnohistochemical staining is carried out after 24 hours, proliferation cells are observed under a microscope, the quantity of proliferation cells is calculated by using a computer imaging system, and tissue engineering skin structure is observed by the method of histology simultaneously. The detection method of tissue engineering skin culture and proliferation activity can detect the impact of different culture conditions and relevant factors on the tissue engineering skin cell proliferation by quantization.
Description
Technical field
The present invention relates to the detection method of a kind of tissue engineering skin culture and proliferation activity.
Background technology
Organization engineering skin can be used for skin wound and changes skin and treating for skin disease that some are serious as the transplanting of back skins such as damage, burn, beauty treatment, have a wide range of applications and potential development space (Ehrenreich M, Ruszczak Z.Update on tissue-engineered biological dressings.Tissue Eng, 2006,12:2407-2424.).In the tissue engineering skin culture process, keeping the proliferation activity of seed cell is the key of skin development, also is to judge to transplant the important reference that can back skin survive.Therefore, in order to promote the proliferation activity of organization engineering skin cell, people constantly explore suitable culture condition and add various biotic components in substratum, as the Niu Chuiti extract, recombinant human epidermal growth factor (hEGF) etc., so that obtaining the skin of q.s, expection is used for clinical practice application (Poumay Y, Dupont F, Marcoux S, et al.A simple reconstructed humanepidermis:preparation of the culture model and utilization in vitro studies.Arch Dermatol Res, 2004,296 (5): 203-211.).For this reason, in the organization engineering skin cultivating process, grasp and understand the multiplication capacity and the vigor of organization engineering skin, for judging and with estimating tissue engineering skin culture condition and substratum nutritive ingredient, so that in time suitably intervene and adjustment has important application value and meaning.
At present the marker that is used for cell proliferation at biomedical sector mainly contains Ki67, PCNA (Proliferating cellnuclearantigen) and Brdu (5-bromo-2--deoxyuridine) etc.Shortcomings such as people often adopted isotopic labeling in RESEARCH ON CELL-BIOLOGY in the past, but there is radiocontamination in isotropic substance, and technical difficulty is big, and experimental period is long are restricted experimental applications.External Gibbs S etc. carry out observation (the Gibbs S of proliferation activity to the skin histology of vitro culture with Ki67, SilvaPinto AN, Murli S, et al.Epidermal growth factor and keratinocyte growth factordifferentially regulate epidermal migration, growth, and differentiation.WoundRepReg2000; 8:192-203).We also once used research (Lu Hongguang etc., the Chinese journal of dermatology 2005 that the skin histology of vitro culture is carried out proliferation activity with quadrat method; 38 (12): 738-741.).Can yet be regarded as a kind of nuclear antigen of clear marking of Ki67, but in actual applications, need to stop skin histology and cultivate, and then carry out the detection of Ki67.Brdu is the derivative of thymus pyrimidine, can be taken in by S phase cell, replace thymus pyrimidine to mix among the DNA, because of no endogenous Brdu existence in the biopsy cell in the DNA synthesis phase, so people Brdu commonly used is in conjunction with anti-Brdu monoclonal antibody marker detection biopsy cell, to judge its proliferation activity.Prepare anti-Brdu monoclonal antibody and mark detection technique is constantly improved raising from nineteen eighty-two Gratzer, the susceptibility of using the Brdu mark is similar to deuterium thymidine (3H-TdR).At present Brdu is as proliferative cell marker be used widely (Kormack DR, Pakic P.Cell proliferation without neurogenesis in adult primateneocortexScience.2001; 294:2127--30.).Yet, because Brdu is exogenous cell marker, if detect its proliferative behind the organization engineering skin adding Brdu to whole cultivation, can disturb and influence growing of skin histology, and the method that because living tissue BrdU mark may bring the organization engineering skin quality, and to be used for the security meeting that human body skin transplants afterwards influential, therefore find out a kind of ideal, can objective detection organization engineering skin proliferation activity influence skin growth again is very necessary.
Summary of the invention
Technical problem to be solved by this invention is, the detection method of a kind of tissue engineering skin culture and proliferation activity is provided, to overcome interference that prior art exists and the deficiency of growing that influences skin histology.
The general planning that the present invention solves the problems of the technologies described above is: at first make and contain the three dimensional matrix material of basement membrane components as dermal substitute, be the people remove epidermis dermis tissue (humande-epidermizedhumandermis, be called for short HDED), and cultivate keratinocyte; Then a certain amount of keratinocyte is inoculated in the HDED surface that the surface scribbles tissue glue, carries out organ culture by air-liquid level training method, cultured continuously with small biopsy device sampling, was carried out the skin proliferation vigor and is detected about 12-15 days.Surplus skin histology continues to cultivate, and continues to cultivate all backs and stops cultivating, and skin is taken out, and capable again skin proliferation vigor detects.The method that the skin proliferation vigor detects is: it is 100 μ g/ml BrdU nutrient solutions (lucifuge) that the fresh culture sample that takes off is placed final concentration, after hatching about 24 hours in 37 ℃ of incubators, the tissue that will take out with 5-bromodeoxyuridine nucleosides carries out the dyeing of immunohistochemical methods method, at the micro-proliferative cell of observing down, and, use Histological method's tissues observed engineering skin texture and proterties simultaneously with computer imaging system calculating proliferative cell quantity.
The epidermis dermis tissue is removed in making: the skin sample that rounds the shape surgical excision, routine disinfection, in Bechtop, it is thick to be trimmed to 2.5mm under dissecting microscope, biopsy device with 10mm is drawn materials, the sample that takes off is dipped among the PBS, place 56 ℃ water bath 30min then, after the taking-up epidermis is separated with corium, partly place under the plate room temperature about 1h with its airing corium, be positioned over then in the vinyon bottle, add a DMSO, the sealing bottle cap is inserted 5～7min in the liquid nitrogen (120 ℃～-160 ℃) rapidly, take out the back and place 30min, and then insert in the liquid nitrogen in room temperature.Continuous like this 10 circulations repeatedly obtain containing the three dimensional matrix material of basement membrane components, and promptly the people's removes epidermis dermis tissue (HDED).
Cultivate keratinocyte: get male sex's postoperative skin of peritomizing, be cut into about 2～5mm left and right sides flap, put in 0.25% the Dispase enzyme, 4 ℃ of digested overnight, take out after 16～18 hours, separate epidermis, shred the back, stop digestion with DMEM:F12 (3:1) nutrient solution that contains 10% foetal calf serum with digesting under the 0.25% trypsinase 0.01%EDTA room temperature.Skin histology nutrient solution re-suspended cell is 2～3 * 10 with density
5/ ml is inoculated in the culturing bottle that overlays the trophocyte.5%CO2 is hatched in 37 ℃ of cell culture incubators, changes liquid once in 2～3 days.Culturing cell reaches when 70%-80% merges state and goes down to posterity.
The cultivation of organization engineering skin and proliferation activity testing process: bracing frame is put into 6 hole culture dish, after HDED is flat on the bracing frame.Get 2～3 generation keratinocyte with (1.5) * 10
6The DED surface of/ml inoculation precoating mixed structure glue, add the skin histology nutrient solution: change liquid after submerged culture 5 days under the liquid level, and change gas one liquid level into and cultivate, changed liquid once in 2～3 days, to cultivate skin after 2 weeks takes out, biopsy device with 2mm is got small skin graft in the organization engineering skin edge, carries out the skin proliferation vigor and detects.Surplus skin histology continues to cultivate, and continues to cultivate all backs and stops cultivating, and skin is taken out, and capable again skin proliferation vigor detects.Skin proliferation vigor detection method is: the fresh culture sample in 100 μ g/ml BrdU culture dish (lucifuge), was hatched in 37 ℃ of incubators about 24 hours, and the back is taken out fixing.Cut into slices proliferative cell in observation of 5-bromodeoxyuridine nucleosides immunohistochemical staining and the computation organization behind the paraffin embedding.Use image analysis system analytical calculation skin proliferation cell density then.Its method is counting basal cell and contiguous spinose cell BrdU positive cell number/every millimeter basilar membrane length (mm)=cell proliferation density.
Above-mentioned organization engineering skin making method, can cultivate at the air liquid level for making skin histology, 70 μ m mesh sieve cell filters (cell strainer) are trimmed to the cultivation bracing frame, its method is: the nylon membrane that cuts off the strainer periphery, four legs that keep are accomplished the 5mm height, put into the culture dish hole in 6 holes then, place HDED on it.Like this, nutrient solution can pass the hole sizer of cell filter unobstructedly, and ensures the liquid level growth of skin histology at ingress of air.
Tissue culture medium (TCM) is that DMEM:F12 is 3:1 in the above-mentioned organization engineering skin making method, Regular Insulin (5 μ g/ml), the fast phosphorus of gland (0.18mM), hydrocortisone (0.5 μ g/ml), vibrio cholera toxin (10-10M), Streptomycin sulphate (100 μ g/ml), penicillin (100units/ml), 1% non-essential amino acid, 10% foetal calf serum.
Need in the above-mentioned organization engineering skin making method to be applied to the HDED surface with mixed structure's glue, mixed structure's glue time spent prepares temporarily.Method is for to mix with zymoplasm collagen glue, tranexamic acid in the 1:1:1 ratio.
Compared with the prior art, the present invention can the not cutaneous again growth of objective detection organization engineering skin proliferation activity.Because proliferative cell marker Brdu mixes after the sampling, can not produce detrimentally affect to the whole organization engineering skin of cultivating.
Embodiment
Embodiments of the invention: general planning of the present invention is: at first make and contain the three dimensional matrix material of basement membrane components as dermal substitute, be the people remove epidermis dermis tissue (humande-epidermizedhumandermis, be called for short HDED), and cultivate keratinocyte; Then a certain amount of keratinocyte is inoculated in the HDED surface that the surface scribbles tissue glue, carries out organ culture by air-liquid level training method, cultured continuously with small biopsy device sampling, was carried out the skin proliferation vigor and is detected about 12-15 days.Surplus skin histology continues to cultivate, and continues to cultivate all backs and stops cultivating, and skin is taken out, and capable again skin proliferation vigor detects.Is in the 100 μ g/ml BrdU nutrient solutions (lucifuge) with the fresh culture sample that takes off in final concentration, 37? after hatching about 24 hours in the incubator, with 5-bromodeoxyuridine nucleosides immunohistochemical staining, at the micro-proliferative cell of observing down, and, use Histological method's tissues observed engineering skin texture and proterties simultaneously with computer imaging system calculating proliferative cell quantity.
The epidermis dermis tissue is removed in making: the skin sample that rounds the shape surgical excision, routine disinfection, in Bechtop, it is thick to be trimmed to 2.5mm under dissecting microscope, biopsy device with 10mm is drawn materials, the sample that takes off is dipped among the PBS, place 56 ℃ water bath 30min then, after the taking-up epidermis is separated with corium, partly place under the plate room temperature about 1h with its airing corium, be positioned over then in the vinyon bottle, add a DMSO, the sealing bottle cap is inserted 5～7min in the liquid nitrogen (120 ℃～-160 ℃) rapidly, take out the back and place 30min, and then insert in the liquid nitrogen in room temperature.Continuous like this 10 circulations repeatedly obtain containing the three dimensional matrix material of basement membrane components, and promptly the people's removes epidermis dermis tissue (HDED).
Make keratinocyte: get male sex's postoperative skin of peritomizing, be cut into about 2～5mm left and right sides skin graft, put in 0.25% the Dispase enzyme, 4 ℃ of digested overnight, take out after 16～18 hours, separate epidermis, shred the back, stop digestion with the DMEM nutrient solution that contains 10% foetal calf serum with digesting under the 0.25% trypsinase 0.01%EDTA room temperature.Keratinocyte substratum re-suspended cell is 2～3 * 10 with density
5/ ml is inoculated in the culturing bottle that overlays the trophocyte.5%CO2 under 37 ℃ of conditions, is hatched in the cell culture incubator, changes liquid once in 2～3 days.Culturing cell reaches when 70%-80% merges state and goes down to posterity.
The preparation of organization engineering skin: can under air-liquid level state, cultivate for making the organization engineering skin tissue, 70 μ m mesh sieve cell filters (cell strainer) are trimmed to the cultivation bracing frame, its method is: the nylon membrane that cuts off the strainer periphery, four legs that keep are accomplished the 5mm height, put into the culture dish hole in 6 holes then, can make nutrient solution can pass the hole sizer of cell filter so unobstructedly, and ensure the liquid level growth of skin histology at ingress of air.During inoculation, with (1.5) * 10
6/ ml keratinocyte is inoculated in the HDED surface of precoating mixed structure glue, and described mixed structure glue is to be that collagen glue, tranexamic acid and the zymoplasm of 1:1:1 is freshly prepared with volume ratio.Add the skin histology nutrient solution,, under 37 ℃ of conditions, hatch in the cell culture incubator at 5%CO2, at first under the liquid level submerged culture 5 days, change gas one liquid level behind the back into and cultivate, changed liquid once in 2～3 days, to carry out skin organ air one liquid level and cultivate, cultured continuously is about 3 weeks.Here used skin histology nutrient solution is that the volume ratio with DMEM:F12 is the ratio preparation of 3:1, wherein, content of insulin is 5 μ g/ml, and the content of the fast phosphorus of gland is 0.18mM, the content of hydrocortisone is 0.5 μ g/ml, and the content of vibrio cholera toxin is 10
-10M, the content of Streptomycin sulphate are 100 μ g/ml, and the content of penicillin is 100U/ml, and the content of non-essential amino acid is 1%, and the content of foetal calf serum is 10%.Because include better nutritivity and induce skin keratin to form the growth components of cell, this substratum is beneficial to the cultivation of epiderm skin tissue.According to experiment purpose, can in basic medium, add other composition.To cultivate skin after 2 weeks takes out, biopsy device with 2mm is got small skin graft in the organization engineering skin edge, then surplus skin histology is put back in the culture dish, add substratum, proceeding gas one liquid level according to above-mentioned cultural method cultivates, cultivate week back termination and cultivate, skin is taken out, the skin proliferation vigor of going again detects.
The organization engineering skin proliferation activity detects: 1) the small skin graft that takes off in the culturing process is positioned in the final concentration 100 μ g/mlBrdu culture dish (lucifuge), hatched in 37 ℃ of incubators about 24 hours, the back is taken out fixing.Cut into slices behind the paraffin embedding, 5-bromodeoxyuridine nucleosides immunohistochemical staining is observed and with image analysis system analytical calculation skin proliferation cell density; 2) organization engineering skin after will stopping cultivating is positioned in the final concentration 100 μ g/ml Brdu culture dish (lucifuge), hatches in 37 ℃ of incubators about 24 hours, and the back is taken out fixing.Cut into slices behind the paraffin embedding, 5-bromodeoxyuridine nucleosides immunohistochemical staining is observed and with image analysis system analytical calculation skin proliferation cell density
The organization engineering skin that makes through as above method has been carried out histological observation to the contriver and the skin proliferation vigor detects, and further detected the influence of recombinant human epidermal growth factor (hEGF) to organization engineering skin propagation density, and its method is as follows:
Experiment one, the cultivation of a kind of organization engineering skin of the present invention and skin proliferation vigor are detected.
1) the small skin graft that takes off in the culturing process being carried out histology and skin proliferation vigor detects.
Method is as follows: the organization engineering skin that will cultivate for 2 weeks takes out, and gets small skin graft with the biopsy device of 2mm in the organization engineering skin edge.Carry out the detection of skin histology structure and thin proliferation activity according to following method:
1, routine paraffin wax section: 4% formaldehyde solution fixed preparation, 4 ℃ are spent the night, gradient alcohol dehydration, dimethylbenzene is transparent, waxdip, embedding, paraffin serial section, 4 μ m, oven dry.
2, Hematorylin Yihong dyeing: tissue slice successively is dipped in dimethylbenzene, dewaxes in 100%, 95%, 70% alcohol.Hematorylin is contaminated 5min, the flowing water flushing, and Yihong alcohol dyes a moment, flowing water flushing, chip select, the differentiation of 70%, 95%, 100% alcohol, 5 minutes/time.Oven dry back mounting.
3,5-bromodeoxyuridine nucleosides immunohistochemical methods mark: the fresh culture sample is placed 100 μ g/ml Brdu substratum, hatched in 37 ℃ of incubators about 24 hours, take out the back and use the PBS rinsing, use 4% formalin fixed.Paraffin embedding, section, dewaxing, PBS rinsing, citrate buffer, microwave oven 5 minutes, the PBS rinsing places 0.6% hydrogen peroxide liquid, darkroom 15min, PBS rinsing, 10% horse serum blocking antibody, anti-Brdu monoclonal antibody 4oC refrigerator overnight, the PBS rinsing, the anti-mouse IgG of biotin labeled horse (1:200) room temperature 30 minutes, the ABC method is hatched, the PBS rinsing, DAB colour developing, PBS rinsing, haematoxylin dyeing, washing, sample is fixed.Replace an anti-blank of doing with PBS.Knot really judges: proliferating cell nuclear is dyed the positive cell of red-brown
Histology and immunohistochemical methods detected result are as follows:
Hematorylin Yihong dyeing observes that organization engineering skin is made up of stratum basale, stratum spinosum epidermidis, granulosa cell, keratinocyte layer and corium is formed.With normal human skin tissues structure basically identical.
5-bromodeoxyuridine nucleosides immunohistochemical methods display organization engineering skin is in growth conditions, and proliferative cell is mainly at basal layer of epidermis and stratum spinosum epidermidis.
The organization engineering skin structural integrity of cultivating with this method more than is described, 5-bromodeoxyuridine nucleosides is exempted from immunohistochemical staining can count the organization engineering skin proliferative cell, can further assess the proliferation activity of organization engineering skin.
2) organization engineering skin after will stopping cultivating carries out histology and the skin proliferation vigor detects.
Method is with experiment one.
Histology and immunohistochemical methods detected result are as follows:
Hematorylin Yihong dyeing observes that organization engineering skin is made up of stratum basale, stratum spinosum epidermidis, granulosa cell, keratinocyte layer and corium is formed.Compare with the organization engineering skin of cultivating for 2 weeks, stratum corneum is grown ripe.
5-bromodeoxyuridine nucleosides immunohistochemical methods display organization engineering skin is in growth conditions, at the stratum basale and the visible 5-bromodeoxyuridine of the stratum spinosum epidermidis nucleosides male proliferative cell of epidermis.
Organization engineering skin structural integrity with these 3 weeks of method cultured continuously more than is described, and maintains proliferation activity, do not influence the growth of organization engineering skin in the culturing process with the biopsy device sampling of 2mm.
Experiment two, recombinant human epidermal growth factor (hEGF) are observed the influence of organization engineering skin propagation density.
Method is that experiment is divided into two groups: recombinant human epidermal growth factor group (adding recombinant human epidermal growth factor in the skin histology substratum, to make its final concentration be 10 μ g/ml, is solvent with the dimethyl sulfoxide (DMSO)) and solvent dimethyl sulfoxide (DMSO) group (dimethyl sulfoxide (DMSO) that adds equivalent in substratum).Every group of 6 organization engineering skins carry out organ culture, cultured continuously 18 days.The recombinant human epidermal growth factor group: adding recombinant human epidermal growth factor in substratum, to make its final concentration be 10 Therewith/ml, at 5%CO2, under 37 ℃ of conditions, hatch in the cell culture incubator, changed liquid once in 2～3 days, at the 10th day that cultivates, respectively the organization engineering skin of cultivating was taken a sample in the 18th day, sample is put in the 100 μ g/mlBrdu substratum, hatched in 37 ℃ of incubators about 24 hours, take out back Brdu immunohistochemical staining, with computer image analysis system analytical calculation skin proliferation cell density; Dimethyl sulfoxide (DMSO) group control group: the dimethyl sulfoxide (DMSO) of in substratum, adding equivalent, the 10th day of cultivating of continuity, organization engineering skin sampling to cultivating respectively in the 18th day, it is in the 100 μ g/mlBrdu nutrient solutions that sample is put in final concentration, hatched in 37 ℃ of incubators about 24 hours, take out back Brdu immunohistochemical staining, calculate the skin proliferation cell density with the computer image analysis system.The result shows cultivation the 10th day, and recombinant human epidermal growth factor group skin proliferation cell density is 43.8 ± 7.5, and the dimethyl sulfoxide (DMSO) group is respectively 28.5 ± 6.38, and two class means relatively have significant difference (P＜0.05); Cultivating the 18th day recombinant human epidermal growth factor cell proliferation density is 39.35 ± 7.17, and the dimethyl sulfoxide (DMSO) group is that 19.81 ± 5.50, two class means compare, and significant difference (P＜0.05) is arranged.
Conclusion: with the present invention can be more different intuitively the proliferation activity of culture condition undertissue engineering skin, and can require timing sampling to carry out the observation and the quantitative Analysis of cell proliferation density according to cultivation, using method is easy, is convenient to operation, good reproducibility.The present invention can quantize to detect influence and the regulation and control to organization engineering skin cell proliferation of various culture condition and correlative factor.
Claims (7)
- Cultivation of [claim 1] a kind of organization engineering skin and proliferation activity detection method, it is characterized in that: it comprises following process: at first make the three dimensional matrix material contain basement membrane components as dermal substitute be the people remove epidermis dermis tissue, cultivation keratinocyte; Then keratinocyte is inoculated in the surface scribble mixed structure's glue the people remove the epidermis dermis tissue surface, after air-the liquid level training method is cultivated,, carry out the skin proliferation vigor and detect with small biopsy device sampling; Take out again after surplus skin continues to cultivate and carry out the detection of skin proliferation vigor; Place BrdU nutrient solution, 37 ℃ of incubators to hatch the fresh culture sample that takes off, carry out the dyeing of immunohistochemical methods method, at the micro-skin proliferation cell of observing down, and, use Histological method's tissues observed engineering skin texture and proterties simultaneously with computer imaging system calculating proliferative cell quantity.
- Cultivation of [claim 2] organization engineering skin according to claim 1 and proliferation activity detection method is characterized in that: sampling is first carried out time that the skin proliferation vigor detects between cultivate by air-liquid level the 12nd～15 day.
- Cultivation of [claim 3] organization engineering skin according to claim 1 and proliferation activity detection method is characterized in that: surplus skin continues to cultivate week back termination to be cultivated, and skin is taken out, and the skin proliferation vigor of going again detects.
- Cultivation of [claim 4] organization engineering skin according to claim 1 and proliferation activity detection method is characterized in that: the stainless steel biopsy device that described small biopsy device is diameter 2mm.
- Is cultivation of [claim 5] organization engineering skin according to claim 1 and proliferation activity detection method is characterized in that: it the BrdU nutrient solution, 37 of 100 μ g/ml that the fresh culture sample that takes off is placed final concentration? hatched in the incubator 24 hours.
- Cultivation of [claim 6] organization engineering skin according to claim 1 and proliferation activity detection method is characterized in that: carrying out immunohistochemical methods method dyeing employing concentration is the 5-bromodeoxyuridine nucleosides nutrient solution of 100 μ g/ml.
- Cultivation of [claim 7] organization engineering skin according to claim 1 and proliferation activity detection method is characterized in that: described mixed structure glue is to be that collagen glue, tranexamic acid and the zymoplasm of 1:1:1 is freshly prepared with volume ratio.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955908A (en) * | 2010-07-22 | 2011-01-26 | 中国医学科学院皮肤病研究所 | Artificial epidermis in-vitro model for detecting sensitization of material and screening medicament |
| CN102220416A (en) * | 2011-04-20 | 2011-10-19 | 北京师范大学 | Method for monitoring number of cell division, and application thereof |
| CN109055303A (en) * | 2018-09-12 | 2018-12-21 | 山东麦德克斯生物科技有限公司 | A kind of construction method of skin histology |
| CN113015904A (en) * | 2018-11-29 | 2021-06-22 | 宝洁公司 | Method for screening personal care products |
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2008
- 2008-11-12 CN CNA2008103054991A patent/CN101392237A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955908A (en) * | 2010-07-22 | 2011-01-26 | 中国医学科学院皮肤病研究所 | Artificial epidermis in-vitro model for detecting sensitization of material and screening medicament |
| CN101955908B (en) * | 2010-07-22 | 2013-01-23 | 中国医学科学院皮肤病研究所 | Artificial epidermis in-vitro model for detecting sensitization of material and screening medicament |
| CN102220416A (en) * | 2011-04-20 | 2011-10-19 | 北京师范大学 | Method for monitoring number of cell division, and application thereof |
| CN109055303A (en) * | 2018-09-12 | 2018-12-21 | 山东麦德克斯生物科技有限公司 | A kind of construction method of skin histology |
| CN113015904A (en) * | 2018-11-29 | 2021-06-22 | 宝洁公司 | Method for screening personal care products |
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