hg8868皇冠国际的黑色素皮肤系统由正常的, human-derived epidermal keratinocytes (NHEK) and melanocytes (NHM) cultured to form a multilayered, 人表皮的高度分化模型. 共培养中的NHM发生黑色素形成，导致组织色素沉着. 黑种人 is produced using serum-free medium without artificial stimulators of melanogenesis such as TPA and IBMX. 培养物生长在空气-液体界面的组织培养插入物上, 允许局部应用皮肤美白剂或自晒黑剂. 黑种人 provides a useful 在体外 means to evaluate cosmetic and pharmaceutical agents designed to modulate skin pigmentation.
黑种人 exhibits in vivo-like morphological and ultrastructural characteristics which are uniform and highly reproducible. NHM localized in the basal cell layer of 黑种人 are dendritic and spontaneously produce melanin granules which progressively populate the layers of tissue. 培养3周, 黑种人 tissues become increasingly pigmented with retention of normal epithelial morphology. Tissues containing NHM derived from black donors show increased pigmentation versus those containing Caucasian-derived or Asian-derived NHM; all three types of cultures are distinctly darker than NHM-free tissues (表皮TM). The topical application of known inhibitors of melanogenesis significantly reduce melanin production and macroscopic darkening. 相反, 组织内的NHM会对已知的黑色素生成刺激物产生反应, 如α-促黑素细胞激素和β-成纤维细胞生长因子, 产生的组织比未经处理的组织变黑得更快. 黑种人’s straightforward assays has been incorporated as an integral testing strategy in laboratories for nearly 20 years. 有几十个技术参考资料可用, this growing body of data demonstrates that 黑种人 provides an inexpensive and effective means of assessing skin pigmentation while avoiding species extrapolation and the use of laboratory animals.
黑种人 provides an inexpensive and effective means of assessing skin pigmentation while avoiding species extrapolation and the use of laboratory animals. Simple protocols and the evaluation of early cellular endpoints allow scientists to acquire data in days, 不是几周或几个月.
皮肤亮
A quantitative assay to determine melanin content in the tissue is performed using the SOLVABLE™ melanin assay. This assay can be used to test the skin brightening efficacy of final cosmetic formulations.
色素的研究
黑种人已被用于研究皮肤色素沉着的生理机制.
组织:
工具: Standard 黑种人™ kit (MEL-300) consists of 24 individual tissues, each tissue 9 mm in diameter. (纸巾“包”包含纸巾, 少量培养基, and plasticware; contact hg8868皇冠国际 for specific kit contents.)
基质: Chemically modified, collagen-coated, 9 mm ID single well tissue culture plate inserts are used.
培养:在气液界面.
播种率: Nhm: nhek = 1/10. 可根据要求提供1/30的比例.
组织学: 8-12个细胞层(基底层、棘层和颗粒层).
角质层: 10-15层(基于TEM).
很多数字: 每个技术员每周生产的纸巾批号被分配到一个特定的批号. 通常，任何一周的纸巾生产都有多个批号. A letter of the alphabet is appended to the end of the lot number to differentiate between individual kits within a given lot of tissues. 所有组织试剂盒在细胞方面都是相同的, medium, 处理, 培养条件, 等.
装运: 在4°C培养基中，琼脂糖凝胶在24孔板中.
装运: 每星期一. 如有特殊要求，也可在星期四装运.
交付: 周二上午通过联邦快递优先服务(美国). 美国以外地区:周二至周三，视地点而定.
保质期: 包括运输时间在内，纸巾在使用前可在4°C下保存6天. 但是，除非必要，否则不建议延长储存期限. 除了, 如果组织在同一天连续使用，将获得最佳的再现性, e.g. 周二下午或之后在4°C下过夜(周三上午).
实验时长: Cultures can be continued for at least 2 weeks with good retention of normal epidermal morphology. 培养物必须每隔一天用5.0毫升新维护培养基(EPI-100-NMM-3)或epi - 100 nmm - 113. Cell culture inserts are placed hang top plates (HNG-TOP-12, provided) to allow the use of 5.0 mL.
替代组织:
MEL-300-A(或-B或-C)-EACH: 与MEL-300相同，除了只有1个组织，而不是24个组织.
MEL-300-A(或-B或-C) -FRZN-EA: 经过3次冻融循环的MEL-300-B组织. 设计用于黑色素测定的对照.
Mel-301 (-a， -b， -c): 比MEL-300少培养3天，比MEL-300分化少
Mel-312 (-a， -b， -c): 与MEL-300 (-A， -B， -C)相同，除了每个试剂盒12个组织而不是24个.
Mel-606 (-a， -b， -c): 与MEL-300 (a)相同, -B, -C)除了每包6张纸巾, 每个组织在直径较大的细胞培养插入物(ID = 22 mm)中培养.
mel -300英尺(-a， -b， -c): Full-thickness human skin tissue model composed of fibroblast-containing collagen matrix (dermis); normal human epidermal keratinocytes, 正常的人类黑素细胞(表皮). 每包24张纸巾.
细胞:
类型: a) Normal human epidermal keratinocytes (NHEK); b) Normal human melanocytes (NHM).
基因组成: NHEK和NHM分别来自不同的捐赠者. 含有匹配的NHEK和NHM的组织可根据特殊要求提供.
民族起源: 含NHM的白种人黑色素皮(MEL-300-C), 非裔美国人(梅尔- 300 b), 以及亚洲(MEL-300-A)捐助者.
来自: Neonatal-foreskin组织.
选择: 有限数量的NHEK菌株 & NHM来自成人乳房组织.
筛选: 艾滋病毒，乙型肝炎和丙型肝炎.
介质:
基础培养基: Dulbecco 's Modified Eagle 's Medium (DMEM).
生长因子/激素: 表皮生长因子, 胰岛素, 氢化可的松, 以及其他专有的表皮分化刺激物.
血清: 没有一个.
抗生素: 庆大霉素5µg/mL(为庆大霉素正常水平的10%).
抗真菌剂: 两性霉素B.25µg / mL.
酸碱指示剂: 酚红.
其他添加剂: 用于增强表皮屏障形成的脂质前体(专有).
选择: 无酚红(MEL-300-B-PRF), 不需抗生素(梅尔- 300 b -沛富), anti-fungal-free(梅尔- 300 b -等于off), 不含氢化可的松的培养基和组织(MEL-300-B-HCF)可用. 代理商至少在发货前3天被移除.
维护中: 建议使用三种维护介质.
对于长期的实验，其中色素沉着和组织学是重要的, EPI-100-NMM-3的使用, epi - 100 nmm - 113, 或epi - 100 - llmm - 3所示.
EPI-100-NMM-3 (New Maintenance Medium) contains KGF which preserves histology but does not contain any factors that directly promote pigmentation.
epi - 100 nmm - 113含有KGF, βfgf, 和α-MSH，促进色素沉着和组织保持.
长寿命保养介质, epi - 100 - llmm - 3所示, 含有高水平的α-MSH和βfgf, 也含有与EPI-100-NMM-3和EPI-100-NNM-113相同水平的KGF. Tissues will darken most rapidly in LLMM-3, less rapidly in NMM-113, and least rapidly in NMM-3.
当添加5时，组织保留效果最好.每48小时0 mL培养基.
质量控制和无菌:
目视检查: 所有组织都经过目视检查，如果发现物理缺陷, 纸巾被拒绝装运. Select tissues are examined microscopically to ensure that melanocytes are visible (for MEL-300-B and MEL-300-A tissues) and that they are evenly distributed throughout the tissue.
最终用途测试: 组织继续培养，以确保肉眼组织变暗. 将组织暴露在1% Triton X-100中5小时. The viability at 5 hrs must exceed 35% as determined by the MTT assay (See hg8868皇冠国际 表皮™ ET-50 protocol) for each lot of tissue.
不育: 在整个生产过程中使用的所有介质都要进行无菌检查. Maintenance medium is incubated with and without antibiotics for 1 week and checked for sterility. The agarose gel from the 24-well plate used for shipping is also incubated for 1 week and checked for any sign of contamination.
病原体筛查: 所有细胞都经过筛选，使用PCR检测HIV、乙肝和丙肝呈阴性. However, no known test method can offer complete assurance that the cells are pathogen-free. 因此, these products and all human-derived products should be handled at BSL-2 levels (biosafety level 2) or higher as recommended in the CDC-NIH manual, 微生物和生物医学实验室的生物安全,” 1998. For further assistance, please contact your site Safety Officer or hg8868皇冠国际 technical service.
不合格批件通知: 如果纸巾没有通过我们的质量检验或无菌检验, the customer will be notified and the tissues will be replaced without charge when appropriate. 因为我们的QC和无菌检测是在出货后完成的, 在一般情况下，我们会尽快发出通知, 可行性失败将在周三下午5点前通知.m.; sterility failures will be notified within 8 days of shipment).
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