BioVector? MEL 526-luc 穩(wěn)定轉(zhuǎn)基因人類黑色素瘤細(xì)胞株——產(chǎn)品技術(shù)說明書

1 產(chǎn)品基本信息與轉(zhuǎn)導(dǎo)背景產(chǎn)品名稱：BioVector MEL 526-luc 穩(wěn)定轉(zhuǎn)基因人類黑色素瘤細(xì)胞株常用別名：MEL-526-luc，MEL526 Luciferase，MEL 526熒光素酶標(biāo)記株生物學(xué)來源：人類 Homo sapiens組織器官：皮膚 / 黑色素瘤轉(zhuǎn)移灶（源自人類惡性黑色素瘤患者的轉(zhuǎn)移性淋巴結(jié)）生長特性：貼壁型上皮樣或梭形單層細(xì)胞標(biāo)記基因表達特征：通過慢病毒（Lentivirus）介導(dǎo)的轉(zhuǎn)導(dǎo)技術(shù)，在親本 MEL 526 細(xì)胞株的基因組中穩(wěn)定集成了北美螢火蟲熒光素酶（Firefly Luciferase）基因。該細(xì)胞株在體外和體內(nèi)表現(xiàn)出極高的光子釋放率，且發(fā)光強度與活細(xì)胞數(shù)量呈現(xiàn)高度完美的線性正相關(guān)。核心科研價值：主要用于活體動物體內(nèi)成像技術(shù)（IVIS成像），在構(gòu)建小鼠皮下黑色素瘤模型、肺轉(zhuǎn)移模型、腫瘤免疫治療評估（如 CAR-T 細(xì)胞體內(nèi)殺傷效能監(jiān)測）以及新型抗腫瘤小分子藥物的體內(nèi)高通量篩選中扮演核心工具株。

2 生物發(fā)光特性與分子標(biāo)志物譜系MEL 526-luc 細(xì)胞株將惡性腫瘤的高侵襲表型與高靈敏度的生物發(fā)光檢測系統(tǒng)進行了完美融合：

核心發(fā)光機制：細(xì)胞內(nèi)的熒光素酶蛋白在氧氣、三磷酸腺苷（ATP）和鎂離子的參與下，能夠催化底物 D-熒光素（D-Luciferin）發(fā)生氧化反應(yīng)，釋放出波長約為 560 納米至 610 納米的黃綠色至橙色光子。由于死細(xì)胞無法提供 ATP，因此該發(fā)光系統(tǒng)能夠非常精準(zhǔn)地定量反映活細(xì)胞的總量。篩選抗性標(biāo)記：基因組攜帶有嘌呤霉素抗性基因（Puro），用于維持高純度的轉(zhuǎn)基因穩(wěn)定表達。組織特異性標(biāo)志物：細(xì)胞穩(wěn)定維持人類黑色素瘤特異性標(biāo)志物，包括 Melan-A（MART-1）、gp100、S100 蛋白及 Tyrosinase（酪氨酸酶）的表達。此外，該株細(xì)胞具有 HLA-A2 陽性單倍型，在腫瘤免疫、人工抗原提呈及 T 細(xì)胞過繼回輸實驗中具有極高的應(yīng)用權(quán)重。

3 專用培養(yǎng)基配方規(guī)范

為了維持 MEL 526-luc 細(xì)胞株的高活力以及熒光素酶基因的長期穩(wěn)定表達，栽培過程中必須嚴(yán)格按照以下組分配置完全生長培養(yǎng)基：

標(biāo)準(zhǔn)完全生長培養(yǎng)基配方基礎(chǔ)培養(yǎng)基：高糖 RPMI-1640 培養(yǎng)基（含 L-谷氨酰胺，不含丙酮酸鈉）。血清添加：10% 優(yōu)質(zhì)胎牛血清 FBS。維持期選擇性抗生素：嘌呤霉素（Puromycin），終濃度維持在 0.5 到 1.0 ug/mL（注意：在細(xì)胞剛剛復(fù)蘇后的前 24 小時內(nèi)不要添加 Puromycin；在日常傳代和常規(guī)維持時需持續(xù)添加，以防止非發(fā)光細(xì)胞的自發(fā)性過表達淘汰）。雙抗補劑：1% 青霉素-鏈霉素溶液（終濃度：100 U/mL 青霉素，100 ug/mL 鏈霉素）。

細(xì)胞凍存保護液標(biāo)準(zhǔn)配方：90% 完全生長培養(yǎng)基（RPMI-1640 + 10% FBS） + 10% 細(xì)胞級二甲基亞砜 DMSO。高回收率配方：45% 基礎(chǔ) RPMI-1640 + 45% 優(yōu)質(zhì)胎牛血清 + 10% DMSO。

4 物理環(huán)境控制參數(shù)孵育溫度：37.0 ℃（恒溫設(shè)置，溫控誤差需小于正負(fù) 0.5 ℃）氣相環(huán)境：5% 二氧化碳（CO2），加濕平衡常壓無菌空氣相對濕度：大于 95% 恒濕環(huán)境物理外貌特征：細(xì)胞貼壁后呈拉長的梭形、多角形或星芒狀上皮樣，生長速度較快，在對數(shù)生長期會形成密集的貼壁網(wǎng)絡(luò)。

5 貼壁傳代操作規(guī)范與紅線傳代臨界匯合度：當(dāng)細(xì)胞單層密度達到 80% - 90% 匯合度時必須立即進行傳代。切勿讓細(xì)胞長期處于 100% 滿瓶飽和狀態(tài)，否則會導(dǎo)致細(xì)胞發(fā)生局部自發(fā)性成片脫落、接觸抑制衰亡以及發(fā)光信號強度的不均一性衰減。建議傳代稀釋比例：1:3 到 1:6（通常每 2-3 天需要傳代一次）。

標(biāo)準(zhǔn)消化分散步驟：1 完全吸除器皿中的陳舊生長培養(yǎng)基。2 使用無菌、不含鈣鎂離子的 PBS 緩沖液輕柔洗滌細(xì)胞單層 1 次，以徹底清除殘余血清中的胰酶抑制物。3 加入預(yù)熱的 0.25% Trypsin - 0.02% EDTA 消化液（以能完全覆蓋細(xì)胞單層為準(zhǔn)，T25 瓶常規(guī)加入 1.0 mL；T75 瓶加入 2.0 到 3.0 mL）。4 將培養(yǎng)瓶置于 37 ℃ 孵箱中靜置消化 2 到 4 分鐘。通過倒置顯微鏡觀察，當(dāng)發(fā)現(xiàn)拉長的梭形細(xì)胞回縮變圓、細(xì)胞間隙明顯增大、輕敲瓶身見細(xì)胞呈沙狀滑落時，立即加入等體積的含血清完全生長培養(yǎng)基終止胰酶活性。5 用移液槍輕柔吹打貼壁面，將細(xì)胞徹底分散為均勻的單細(xì)胞懸液。切忌大力劇烈吹打，防止對黑色素瘤細(xì)胞膜造成機械損傷。6 將胞懸液收集至離心管中，在 300 x g（約 1000 rpm）下低速離心 5 分鐘。7 徹底抽干棄去上清液，加入含有 0.5-1.0 ug/mL 嘌呤霉素的新鮮完全生長培養(yǎng)基重懸，按預(yù)定傳代比例分裝至新的培養(yǎng)瓶中，放回 37 ℃ 孵箱栽培。

6 復(fù)蘇與凍存后恢復(fù)流線1 提前在無菌 T25 培養(yǎng)瓶中注入 5.0 mL 不含嘌呤霉素的完全生長培養(yǎng)基，置于 37 ℃、5% CO2 孵箱中進行 20 分鐘的氣相與 pH 平衡。2 從液氮罐中取出冷凍的 MEL 526-luc 凍存管，立刻全量投入 37 ℃ 恒溫水浴箱中快速水平搖晃。3 必須在 60 至 90 秒內(nèi)使其完全融化，直至管內(nèi)只剩最后一絲微小冰芯（快融原則，確保管帽 O 型圈不接觸水面防止污染）。4 用 75% 酒精消毒管壁后移入超凈臺，用移液槍吸出融化后的胞懸液，緩慢逐滴注入盛有 4.0 mL 預(yù)熱完全培養(yǎng)基（不含 Puromycin）的 15 mL 離心管中，輕柔混勻。5 在 300 x g 下離心 5 分鐘，徹底吸干含有毒 DMSO 的上清液。6 加入 1.5 mL 新鮮的不含 Puromycin 完全培養(yǎng)基輕彈懸起細(xì)胞，隨后全量接種到已完成預(yù)熱平衡的 T25 瓶中。7 置于 37 ℃ 孵箱栽培。接種 24 小時后，必須進行一次全量換液以清除未貼壁的細(xì)胞廢渣，并在本次換液時開始正式加入 0.5-1.0 ug/mL 的嘌呤霉素進行抗性維持。

7 超低溫長期保存與質(zhì)控規(guī)范生物安全級別：BSL-2（2級生物安全柜操作）。由于細(xì)胞包含慢病毒轉(zhuǎn)導(dǎo)的基因片段且為人類來源，必須嚴(yán)格執(zhí)行無菌防護屏障和廢棄物高壓滅菌規(guī)范。長期封存溫控：凍存管必須永久存放于液氮蒸汽或液相（-150 ℃ 至 -196 ℃）中。嚴(yán)禁在機械式 -80 ℃ 普通冰箱中存放超過 1 個月，溫度劇烈波動會導(dǎo)致熒光素酶表達元件發(fā)生自發(fā)性基因沉默或細(xì)胞復(fù)蘇率斷崖式下跌。體外發(fā)光活性復(fù)核：在將該細(xì)胞注入小鼠體內(nèi)構(gòu)建荷瘤模型前，應(yīng)抽取部分傳代細(xì)胞進行體外發(fā)光滴定。在 96 孔板中梯度稀釋細(xì)胞，每孔加入 150 ug/mL 的 D-Luciferin 工作液，使用多功能微孔板閱讀器檢測化學(xué)發(fā)光（Luminescence）。要求發(fā)光值（RLU）與活細(xì)胞數(shù)量之間的線性相關(guān)系數(shù) R2 大于 0.99，方可證實其作為分子成像追蹤底盤的合格性。

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.