3D Human skin (인공피부) 를 이용한 화장품의 피부 미백실험
MelanoDerm Tissue Model
Procedures for use of MEL-300-A, MEL-300-B, MEL-300-C
I. Shipping and Storage of MelanoDerm Tissues and Kit Components
a) Shipping: MelanoDerm is produced so that tissue is ready for shipment on Monday. Delivery into your lab will depend on your location and the mode of transport.
b) Storage: If experiments will not commence upon receipt, place the sealed 24-well plate containing the MEL-300 tissue samples and the maintenance medium into the refrigerator (4°C). The tissues and maintenance medium should be stored unopened at 4°C until experiments begin. If the MTT assay is to be run, please refer to the MTT ET-50 protocol for EpiDerm (EPI-200, EPI-100a) available from MatTek for storage conditions of the MTT toxicology kit (part # MTT-100) and assay procedures.
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II. Preparation of MelanoDerm
a) Pre-warm media: Pre-warm the maintenance medium (provided) to 37°C. Using sterile technique, pipet 0.9 ml of the maintenance medium into each well of the sterile 6-well plates (provided). Label the 6-well plates indicating treatment/ exposure conditions to be used.
b) Transfer MelanoDerm samples: 1 hour before treatment is to begin, remove the MEL-300 samples from the refrigerator. Under sterile conditions using sterile forceps, transfer the MEL-300 samples into the 6-well plates containing the pre-warmed maintenance medium. Note: Care should be taken to remove any adherent agarose sticking to the outside of the cell culture inserts containing the MEL-300 samples.
c) Pre-equilibration: Incubate the 6-well plates containing the MEL-300 samples in a humidified 37°C, 5% CO2 incubator for 1 hour prior to applying treatment.
III. Melanogenesis/whitening studies
a) Pre-screen treatment conditions: A good skin lightener should inhibit melanin synthesis but not cause cytotoxicity to the tissue. Cytotoxicity testing can be performed with the less expensive EpiDerm tissue (EPI-200). Follow procedures in the MTT ET-50 protocol except: i) apply 25 and 10 uL of each test article to duplicate (n=2) EpiDerm tissues, ii) use exposure times of 2 and 7 days, and iii) negative control tissues (n=4) should be treated with 25 uL of sterile, ultrapure water for 48 hours. After exposure is complete, the tissues are rinsed with PBS and the MTT assay is run. Calculate the % viability for each treatment from the formula:
% viability = 100 x [OD(sample)/OD(negative control)]
In order to avoid cytotoxicity in the skin lightening study, the viability should be >90% at 48 hours. If both the 25 and 10 uL dose are > 90% viability, use the 25 uL dose since the positive control (1% kojic acid) is run at 25 uL. Note: If the % viability for the 10 uL treatment is <90%, it is likely that such a material would be an irritant.
b) Treatment conditions: Liquid or lotion materials can be applied directly to the MEL-300 stratum corneum by pipetting with a positive displacement pipette into the cell culture insert which contains the MEL-300 tissue. 25 or 10 ml of liquid sample are applied (25 mg of solids) based on the results of the cytotoxicity pre-screen. Note: The 25 ml represents a dose of ~40 mg/cm2 – for many lotions (e.g. sunscreens are typically tested at 2 mg/cm2), this represents an exaggerated exposure and a lower, more in vivo-like dose may be desirable. Alternatively, dosing can be applied using the intended end-use application in mg/cm2; the surface area of the MEL-300 tissues is 0.6 cm2. If you are using the “Recommended whitening study design” described in Section V.d, 8 tissues per treatment condition will be required. ( 중략 )
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