MEL 526-luc BioVector® 稳定转荧光基因人类黑色素瘤细胞株

BioVector® MEL 526-luc 稳定转基因人类黑色素瘤细胞株——产品技术说明书

1 产品基本信息与转导背景产品名称：BioVector MEL 526-luc 稳定转基因人类黑色素瘤细胞株常用别名：MEL-526-luc，MEL526 Luciferase，MEL 526荧光素酶标记株生物学来源：人类 Homo sapiens组织器官：皮肤 / 黑色素瘤转移灶（源自人类恶性黑色素瘤患者的转移性淋巴结）生长特性：贴壁型上皮样或梭形单层细胞标记基因表达特征：通过慢病毒（Lentivirus）介导的转导技术，在亲本 MEL 526 细胞株的基因组中稳定集成了北美萤火虫荧光素酶（Firefly Luciferase）基因。该细胞株在体外和体内表现出极高的光子释放率，且发光强度与活细胞数量呈现高度完美的线性正相关。核心科研价值：主要用于活体动物体内成像技术（IVIS成像），在构建小鼠皮下黑色素瘤模型、肺转移模型、肿瘤免疫治疗评估（如 CAR-T 细胞体内杀伤效能监测）以及新型抗肿瘤小分子药物的体内高通量筛选中扮演核心工具株。

2 生物发光特性与分子标志物谱系MEL 526-luc 细胞株将恶性肿瘤的高侵袭表型与高灵敏度的生物发光检测系统进行了完美融合：

核心发光机制：细胞内的荧光素酶蛋白在氧气、三磷酸腺苷（ATP）和镁离子的参与下，能够催化底物 D-荧光素（D-Luciferin）发生氧化反应，释放出波长约为 560 纳米至 610 纳米的黄绿色至橙色光子。由于死细胞无法提供 ATP，因此该发光系统能够非常精准地定量反映活细胞的总量。筛选抗性标记：基因组携带有嘌呤霉素抗性基因（Puro），用于维持高纯度的转基因稳定表达。组织特异性标志物：细胞稳定维持人类黑色素瘤特异性标志物，包括 Melan-A（MART-1）、gp100、S100 蛋白及 Tyrosinase（酪氨酸酶）的表达。此外，该株细胞具有 HLA-A2 阳性单倍型，在肿瘤免疫、人工抗原提呈及 T 细胞过继回输实验中具有极高的应用权重。

3 专用培养基配方规范

为了维持 MEL 526-luc 细胞株的高活力以及荧光素酶基因的长期稳定表达，栽培过程中必须严格按照以下组分配置完全生长培养基：

标准完全生长培养基配方基础培养基：高糖 RPMI-1640 培养基（含 L-谷氨酰胺，不含丙酮酸钠）。血清添加：10% 优质胎牛血清 FBS。维持期选择性抗生素：嘌呤霉素（Puromycin），终浓度维持在 0.5 到 1.0 ug/mL（注意：在细胞刚刚复苏后的前 24 小时内不要添加 Puromycin；在日常传代和常规维持时需持续添加，以防止非发光细胞的自发性过表达淘汰）。双抗补剂：1% 青霉素-链霉素溶液（终浓度：100 U/mL 青霉素，100 ug/mL 链霉素）。

细胞冻存保护液标准配方：90% 完全生长培养基（RPMI-1640 + 10% FBS） + 10% 细胞级二甲基亚砜 DMSO。高回收率配方：45% 基础 RPMI-1640 + 45% 优质胎牛血清 + 10% DMSO。

4 物理环境控制参数孵育温度：37.0 ℃（恒温设置，温控误差需小于正负 0.5 ℃）气相环境：5% 二氧化碳（CO2），加湿平衡常压无菌空气相对湿度：大于 95% 恒湿环境物理外貌特征：细胞贴壁后呈拉长的梭形、多角形或星芒状上皮样，生长速度较快，在对数生长期会形成密集的贴壁网络。

5 贴壁传代操作规范与红线传代临界汇合度：当细胞单层密度达到 80% - 90% 汇合度时必须立即进行传代。切勿让细胞长期处于 100% 满瓶饱和状态，否则会导致细胞发生局部自发性成片脱落、接触抑制衰亡以及发光信号强度的不均一性衰减。建议传代稀释比例：1:3 到 1:6（通常每 2-3 天需要传代一次）。

标准消化分散步骤：1 完全吸除器皿中的陈旧生长培养基。2 使用无菌、不含钙镁离子的 PBS 缓冲液轻柔洗涤细胞单层 1 次，以彻底清除残余血清中的胰酶抑制物。3 加入预热的 0.25% Trypsin - 0.02% EDTA 消化液（以能完全覆盖细胞单层为准，T25 瓶常规加入 1.0 mL；T75 瓶加入 2.0 到 3.0 mL）。4 将培养瓶置于 37 ℃ 孵箱中静置消化 2 到 4 分钟。通过倒置显微镜观察，当发现拉长的梭形细胞回缩变圆、细胞间隙明显增大、轻敲瓶身见细胞呈沙状滑落时，立即加入等体积的含血清完全生长培养基终止胰酶活性。5 用移液枪轻柔吹打贴壁面，将细胞彻底分散为均匀的单细胞悬液。切忌大力剧烈吹打，防止对黑色素瘤细胞膜造成机械损伤。6 将胞悬液收集至离心管中，在 300 x g（约 1000 rpm）下低速离心 5 分钟。7 彻底抽干弃去上清液，加入含有 0.5-1.0 ug/mL 嘌呤霉素的新鲜完全生长培养基重悬，按预定传代比例分装至新的培养瓶中，放回 37 ℃ 孵箱栽培。

6 复苏与冻存后恢复流线1 提前在无菌 T25 培养瓶中注入 5.0 mL 不含嘌呤霉素的完全生长培养基，置于 37 ℃、5% CO2 孵箱中进行 20 分钟的气相与 pH 平衡。2 从液氮罐中取出冷冻的 MEL 526-luc 冻存管，立刻全量投入 37 ℃ 恒温水浴箱中快速水平摇晃。3 必须在 60 至 90 秒内使其完全融化，直至管内只剩最后一丝微小冰芯（快融原则，确保管帽 O 型圈不接触水面防止污染）。4 用 75% 酒精消毒管壁后移入超净台，用移液枪吸出融化后的胞悬液，缓慢逐滴注入盛有 4.0 mL 预热完全培养基（不含 Puromycin）的 15 mL 离心管中，轻柔混匀。5 在 300 x g 下离心 5 分钟，彻底吸干含有毒 DMSO 的上清液。6 加入 1.5 mL 新鲜的不含 Puromycin 完全培养基轻弹悬起细胞，随后全量接种到已完成预热平衡的 T25 瓶中。7 置于 37 ℃ 孵箱栽培。接种 24 小时后，必须进行一次全量换液以清除未贴壁的细胞废渣，并在本次换液时开始正式加入 0.5-1.0 ug/mL 的嘌呤霉素进行抗性维持。

7 超低温长期保存与质控规范生物安全级别：BSL-2（2级生物安全柜操作）。由于细胞包含慢病毒转导的基因片段且为人类来源，必须严格执行无菌防护屏障和废弃物高压灭菌规范。长期封存温控：冻存管必须永久存放于液氮蒸汽或液相（-150 ℃ 至 -196 ℃）中。严禁在机械式 -80 ℃ 普通冰箱中存放超过 1 个月，温度剧烈波动会导致荧光素酶表达元件发生自发性基因沉默或细胞复苏率断崖式下跌。体外发光活性复核：在将该细胞注入小鼠体内构建荷瘤模型前，应抽取部分传代细胞进行体外发光滴定。在 96 孔板中梯度稀释细胞，每孔加入 150 ug/mL 的 D-Luciferin 工作液，使用多功能微孔板阅读器检测化学发光（Luminescence）。要求发光值（RLU）与活细胞数量之间的线性相关系数 R2 大于 0.99，方可证实其作为分子成像追踪底盘的合格性。

BioVector MEL 526-luc Stably Transfected Human Melanoma Cell Line Product Datasheet

1 Product and Identification General InformationProduct Name: BioVector MEL 526-luc Stably Transfected Human Melanoma Cell LineSynonyms: MEL-526-luc, MEL526 Luciferase, MEL 526 Luciferase-Tagged LineOrganism Source: Human Homo sapiensTissue/Organ Site: Skin / Melanoma Metastasis (Derived from metastatic lymph node of a patient with malignant melanoma)Growth Properties: Adherent Epithelial-like or Spindle-shaped MonolayerMarker Gene Expression Profile: The firefly luciferase gene was stably integrated into the genome of the parental MEL 526 cell line using lentiviral transduction technology. This modified line exhibits an exceptionally high photon emission rate both in vitro and in vivo, showing a robust linear relationship between living cell number and bioluminescent signal intensity.Core Research Significance: Primarily used as a critical cellular tool for in vivo bioluminescence imaging (IVIS). It is widely utilized to establish subcutaneous melanoma models, lung metastasis models, in vivo evaluation of cancer immunotherapies (such as monitoring CAR-T cell cytotoxicity), and high-throughput screening of novel anti-tumor small molecules.

2 Bioluminescence Properties and Biomarker ProfileThe MEL 526-luc cell line integrates the highly aggressive phenotype of malignant tumors with a highly sensitive bioluminescence reporting system:

Core Bioluminescent Mechanism: The intracellular firefly luciferase enzyme catalyzes the oxidation of its substrate D-Luciferin in the presence of oxygen, ATP, and magnesium ions, releasing photons with a peak wavelength between 560 nm and 610 nm. Because dead cells lack ATP, this reporting system precisely and quantitatively reflects the total biomass of living cells.Selection Resistance Marker: The genome contains a puromycin resistance gene (Puro) used to maintain high purity and stable transgene expression during propagation.Tissue-Specific Antigen Spectrum: Stably retains classic human melanoma biomarkers including Melan-A (MART-1), gp100, S100 protein, and Tyrosinase. Furthermore, this line possesses an HLA-A2 positive haplotype, making it an invaluable tool for tumor immunology, artificial antigen presentation assays, and adoptively transferred T-cell killing evaluations.

3 Dedicated Culturing Medium and Formulations

To preserve phenotypic stability and maintain uniform expression of the firefly luciferase reporter, the complete growth medium must be formulated precisely as outlined below:

Standard Complete Growth Medium FormulationBasal Medium Base: High-Glucose RPMI-1640 Medium (with L-glutamine, without sodium pyruvate).Serum Supplement: 10% premium Fetal Bovine Serum FBS.Maintenance Selective Antibiotic: Puromycin (Final concentration maintained at 0.5 to 1.0 ug/mL. Note: Do not add puromycin during the first 24 hours post-thaw; apply continuously during routine passaging and long-term maintenance to prevent non-bioluminescent cells from overgrowing the culture).Antibiotic Cocktail: 1% Penicillin-Streptomycin Solution (Final concentration: 100 U/mL Penicillin, 100 ug/mL Streptomycin).

Cryopreservation Freezing Medium FormulationStandard Freezing Formula: 90% Complete Growth Medium (RPMI-1640 with 10% FBS) + 10% Analytical Grade DMSO (Dimethyl Sulfoxide).Alternative High-Recovery Formula: 45% Basal RPMI-1640 + 45% Premium FBS + 10% DMSO.

4 Controlled Culture Environmental ConditionsIncubator Temperature: 37.0 ℃ (constant temperature, variations should be strictly restricted within plus or minus 0.5 ℃)Gas Composition (CO2): 5% Carbon Dioxide balanced with humidified atmospheric airRelative Humidity: Greater than 95% relative humidityCellular Morphology: Upon adherence, cells exhibit an elongated spindle-shaped, polygonal, or stellate epithelial-like morphology, growing rapidly into a dense interconnected monolayer meshwork during log phase.

5 Subculturing Passaging Protocol and MetricsOptimal Passaging Confluency: Subculturing must be performed promptly when the monolayer achieves 80% to 90% confluency. Never allow the cells to remain at a 100% overcrowded saturation state, which triggers sheets of cells detaching spontaneously, contact inhibition, and non-uniform degradation of bioluminescent intensity.Subcultivation Splitting Ratio: 1:3 to 1:6 (typically passaged every 2 to 3 days).

Step-by-Step Dissociation Method:1 Aspirate the spent growth fluid completely from the vessel.2 Rinse the cell monolayer gently with sterile, Calcium/Magnesium-free PBS once to eliminate trace serum which inhibits tryptic activity.3 Apply an appropriate volume of pre-warmed 0.25% Trypsin - 0.02% EDTA Solution (sufficient to fully submerge the monolayer; typically 1.0 mL for a T25 flask, 2.0-3.0 mL for a T75 flask).4 Incubate the vessel at 37 ℃ for 2 to 4 minutes. Monitor under an inverted microscope. Once the elongated cells round up and loosen from the plastic, immediately add an equal volume of serum-fortified complete growth medium to neutralize enzymatic activity.5 Gently pipette the suspension against the vessel wall to break down multi-cellular aggregates into a uniform single-cell suspension. Avoid excessive shear forces to prevent membrane damage.6 Transfer the suspension to a conical tube and centrifuge at 300 x g for 5 minutes.7 Discard the supernatant completely, resuspend the pellet in fresh complete growth medium containing 0.5-1.0 ug/mL Puromycin, and allocate target fractions into new culture vessels.

6 Thawing and Post-Cryo Recovery Workflow1 Pre-warm 5.0 mL of puromycin-free complete growth medium in a sterile T25 flask and balance it within the 37 ℃, 5% CO2 incubator for 20 minutes to stabilize the gas phase and pH value.2 Retrieve the MEL 526-luc cryovial from liquid nitrogen storage.3 Plunge the vial instantly into a 37 ℃ water bath and rapidly shake horizontally (keep the cap seal above the water line). Achieve complete thawing within 60 to 90 seconds to prevent intracellular ice recrystallization.4 Sanitize the vial exterior with 75% ethanol before transferring it into the biosafety cabinet.5 Transfer the thawed cell solution slowly and dropwise into a 15 mL sterile conical tube containing 4.0 mL of pre-warmed puromycin-free complete medium.6 Centrifuge at 300 x g for 5 minutes to form a clean cell pellet.7 Aspirate the supernatant completely to eliminate toxic DMSO residues.8 Add 1.5 mL of fresh puromycin-free complete medium, gently tap the tube to loosen the pellet, and seed the entire suspension into the pre-stabilized T25 flask.9 Incubate at 37 ℃, 5% CO2. Perform a complete medium exchange 24 hours post-thaw to clear dead non-adherent cell debris, and officially initiate puromycin selection at 0.5-1.0 ug/mL from this first fluid change.

7 Biosafety, Storage and Quality Control StandardsBiosafety Level: BSL-2 (Class II Biosafety Cabinet Operations). This cell line contains lentiviral genetic components and is of human tissue origin; standard institutional biosafety protocols and autoclaving containment measures must be strictly followed.Long-Term Storage Parameters: Cryovials must be stored permanently within the vapor or liquid phase of liquid nitrogen (-150 ℃ to -196 ℃). Short- or long-term storage in mechanical -80 ℃ freezers beyond 1 month is prohibited, as it causes irreversible drops in post-thaw survival rates and risks gene silencing of the luciferase expression cassette.In Vitro Luminescence Verification QC: Prior to inoculating the cells into mice for tumor modeling, an in vitro bioluminescence assay should be performed. Seed serial dilutions of the cells into a 96-well plate, add D-Luciferin working solution at a final concentration of 150 ug/mL, and measure the bioluminescence profile using a multimode plate reader. The coefficient of determination (R2) between living cell count and RLU value must exceed 0.99 to validate the line's performance for molecular imaging and tracking.