BioVector® SNU-410 Human Gallbladder Carcinoma Cell Line / SNU-410 人胆囊癌细胞系

一 产品基本信息与细胞生物学背景

• 细胞名称：SNU-410（全称 Seoul National University-410）。

• 物种与组织来源：人类（Homo sapiens），源自患有原发性胆囊癌（Primary Gallbladder Carcinoma）的 57 岁韩国女性的肿瘤组织。

• 细胞系建立背景：SNU-410 细胞系由韩国首尔大学医学院（Seoul National University College of Medicine）著名的癌症研究所（Cancer Research Institute）团队于 20 世纪 90 年代成功建立。该研究所（由 Jae-Gahb Park 教授团队领衔）系统性地从韩国本土癌症患者中分离、建立了一系列“SNU”编号的消化系统肿瘤细胞株。胆囊癌由于临床早期诊断困难、恶性程度极高且缺乏有效靶向药物，SNU-410 的建立为研究亚洲高发的胆道系统恶性肿瘤（Biliary Tract Cancers, BTCs）提供了宝贵的人源模式底盘。

• 核心表型与细胞学特征：

  • 形态学特征：贴壁生长，在显微镜下呈现典型的上皮样（Epithelial-like）或多角形（Polygonal）形态。细胞常呈铺路石状密集紧凑排列，生长边界清晰，局部可见典型腺癌样分化特征。

  • 基因组与标志物特征：高表达上皮细胞标志物（如 E-cadherin、Cytokeratins），并在特定胆道系统癌症相关的基因通路（如 TP53 突变、KRAS 变异、SMAD4 改变或 p16/CDKN2A 异常等）中表现出独特的临床相关突变谱（具体特征常作为胆囊癌基因组测序研究的阳性对照）。

• 生物安全级别：1级（BSL-1）。

二 核心科研价值与转化医学应用

SNU-410 细胞株作为公认的人源胆囊癌多中心标准对照，在消化道肿瘤转化医学领域具有明确的实验价值：

1. 胆道系统癌症（BTC）分子发病机制探讨：胆囊癌和胆管癌在生物学行为上具有独特的异质性。SNU-410 常被用作经典的体外模型，深入解构上皮-间充质转化（EMT）通路、Wnt/beta-catenin 信号级联、以及 Notch 信号通路在胆囊上皮恶性转化和早期局部浸润中的分子靶向机制。

2. 新型靶向药、化疗敏感性及免疫治疗体外高通量筛选：针对临床一线化疗药物（如吉西他滨 Gemcitabine、顺铂 Cisplatin）的耐药机制是目前胆囊癌科研的热点。SNU-410 被广泛应用于评价新型小分子激酶抑制剂（如 FGFR 抑制剂、EGFR/HER2 靶向药、MEK 抑制剂等）的抗增殖与诱导凋亡效应，以及联合免疫检查点阻断（ICB）后的协同杀伤敏感性评估。

3. 异种移植小鼠成瘤模型构建（Xenograft Models）：该细胞在免疫缺陷小鼠（如 BALB/c Nude 裸鼠、NOD-SCID 或 NSG 小鼠）皮下具备稳定的成瘤能力。通过皮下构建人源胆囊癌异种移植（CDX）模型，可用于精准定量评价候选抗癌药物在体内的抑瘤率、药代动力学（PK）及安全性表征。

三 实验室细胞复苏、贴壁常规培养、传代与保存标准步骤

SNU-410 细胞对体外培养环境的机械剪切力和消化酶敏感度适中，属于相对容易维持和传代的贴壁细胞。但在日常培养中仍需警惕因局部过密而导致的细胞抱团堆叠，进而引起细胞表型退化或活性受损。

1. 培养基与化学试剂配置

• 基础培养基（核心推荐）：RPMI-1640 基础培养基。(注：首尔大学建立的绝大多数 SNU 消化道肿瘤系列细胞株，均采用 RPMI-1640 作为最适配的标准生长基质)。

• 完全培养基配方：RPMI-1640 基础培养基 加 10% 优质胎牛血清（FBS） 加 1% 青霉素-链霉素双抗（Penicillin-Streptomycin）。

• 细胞解离液：0.25% Trypsin-0.02% EDTA 消化液。

• 环境参数：37 摄氏度，5% 二氧化碳，饱和湿度孵箱。

2. 冷冻细胞复苏步骤

1. 提前在无菌生物安全柜中配制好干净的 T25 培养瓶，注入 5 - 6 mL 预热至 37 摄氏度的完全培养基。

2. 从液氮罐或超低温冰箱中取出 SNU-410 冻存管，立刻全量投入 37 摄氏度恒温水浴箱中快速摇晃解冻，确保管内冻块在 1 分钟内完全融化，避免局部冰晶慢速重结晶对细胞膜造成二次撕裂。

3. 用 75% 酒精喷洒冻存管外壁进行表面消毒，随后移入生物安全柜内。

4. 用无菌移液枪吸取融化的细胞悬液，缓慢滴加至盛有 4 mL 预热完全培养基的 15 mL 离心管中，前后轻柔颠倒一次以稀释化学保护剂。

5. 以 1000 rpm（约 200 g）室温离心 4 - 5 分钟，小心吸除含有二甲基亚砜（DMSO）的上清液。

6. 加入 1 - 2 mL 新鲜完全培养基重悬细胞沉淀，将其全量接种至准备好的 T25 瓶中。前后轻柔十字晃动混匀，置于孵箱中。

7. 复苏次日（24 小时左右）常规在显微镜下观察细胞贴壁和展弦状态。此时应进行一次全量更换新鲜完全培养基的操作，以彻底清除复苏过程中产生的死细胞碎屑及极微量残留 DMSO。

3. 日常贴壁常规传代操作

• 传代时机：当细胞融合度达到 80% - 90%（即上皮样细胞基本铺满瓶底，开始挤压对接，但尚未完全重叠生长）时必须进行传代。SNU-410 细胞若达到 100% 极度过密状态，细胞会自发向上层叠堆积，导致下层细胞因缺氧缺营养而自发大面积成片脱落。

• 操作流程：

  1. 吸除细胞瓶内的旧培养基，使用无菌的、不含钙镁离子的 PBS 缓冲液轻轻漂洗细胞表面 1 - 2 次，彻底洗去残存的、会抑制胰酶活性的血清。

  2. 加入适量 0.25% 胰酶消化液（T25 瓶常规加入 1 mL），摇晃使其全面覆盖细胞层。置于 37 摄氏度孵箱中消化 2 - 4 分钟。

  3. 在倒置显微镜下进行实时动态观察。当发现多角形细胞体自发回缩、变圆、胞间间隙明显增大、轻敲瓶壁可见上皮样细胞成片滑落或分散移动时，立刻加入 2 到 3 倍体积的含血清完全培养基以终止胰酶的解离反应。

  4. 用移液枪在瓶壁轻轻吹打，使未脱落的细胞彻底剥离，并打散形成单细胞悬液。收集悬液入管，1000 rpm 离心 5 分钟。

  5. 弃去上清，加入新鮮完全培养基。按照 1 比 3 至 1 比 5 的常规稀释比例，接种至新的培养器皿中。

  6. 通常每 2 - 3 天传代一次，期间根据液体消耗速度和颜色改变适度补充或更换培养基。

4. 细胞长期保存标准

• 冻存液配方：90% 优质完全培养基（或纯胎牛血清） 加 10% 分析级二甲基亚砜（DMSO）。

• 冷冻规范：

  1. 收集处于对数生长最旺盛期、细胞密度在 80% 左右、健康且无空泡化表现的 SNU-410 细胞。

  2. 经温和消化、离心沉淀后，用配置好的冻存液调整细胞密度至 每毫升 1,000,000 到 2,000,000 个细胞。

  3. 分装入无菌冷冻管中，立刻移入标准程序降温盒（如 Mr. Frosty），并置于 零下 80 摄氏度冰箱中过夜梯度降温（遵循约每分钟降温 1 摄氏度的稳态速率）。

  4. 次日，必须迅速将冻存管转移入液氮罐（零下 196 摄氏度）长期保存。严禁在 零下 80 摄氏度普通冰箱内长期存放，以防长期的微小热幅射导致细胞内部冰晶重塑，进而严重恶化后续复苏时的贴壁存活率与功能完整性。

Part 2 English Section

I General Information and Cell Biological Background

• Cell Line Name: SNU-410 (Seoul National University-410).

• Organism and Tissue Extraction Origin: Homo sapiens (human); derived from primary gallbladder carcinoma tissue resected from a 57-year-old Korean female donor.

• Cell Line Establishment Background:The SNU-410 cell line was successfully established in the 1990s by the research team at the Cancer Research Institute of Seoul National University College of Medicine (led by Prof. Jae-Gahb Park). This pioneering cohort systematically isolated and validated a comprehensive bank of gastrointestinal and biliary malignancy models prefixed with "SNU". Given that primary gallbladder carcinoma typically presents at advanced clinical stages, carries an aggressive prognosis, and exhibits profound resistance to conventional targeted strategies, the validation of SNU-410 provided a high-fidelity human substrate to explore Biliary Tract Cancers (BTCs).

• Core Morphological Phenotype and Markings:

  • Morphological Form: Adherent growth; under inverted phase-contrast microscopy, it exhibits a classic epithelial-like or polygonal morphology. Cells proliferate in tight, cobblestone-like configurations with well-defined colonial boundaries, reflecting clear adenocarcinoma differentiation hallmarks.

  • Genomic and Expression Mapping: Displays robust expression profiles of standard epithelial architecture markers (such as E-cadherin and various Cytokeratins). The line harbors distinct mutation footprints common to biliary tract carcinomas (e.g., specific alterations within TP53, KRAS, SMAD4, or p16/CDKN2A networks), frequently integrated as positive controls in comprehensive oncology sequencing screens.

• Biosafety Matrix: Classified under Biosafety Level 1 (BSL-1) containment parameters.

II Strategic Research Value and Translational Fields

As an internationally recognized human gallbladder carcinoma reference lineage, SNU-410 offers substantial therapeutic evaluation value within translational oncology research:

1. Elucidating Biliary Tract Cancer (BTC) Molecular Pathogenesis:Gallbladder carcinomas exhibit intense inter- and intra-tumor heterogeneity. SNU-410 acts as a reliable model to decode the intracellular machinery driving Epithelial-Mesenchymal Transition (EMT), analyze Wnt/beta-catenin signaling cascades, and map the regulatory loops of the Notch pathway during primary mucosal transformation and early tissue invasion.

2. High-Throughput Screening of Targeted Agents, Chemosensitivity, and Immunotherapies:Overcoming resistance to first-line clinical chemotherapeutic regimens (e.g., Gemcitabine and Cisplatin) represents a critical mandate in gallbladder oncology. SNU-410 is deployed as a baseline platform to evaluate the anti-proliferative and apoptotic efficiency of novel small-molecule kinase inhibitors (such as FGFR inhibitors, EGFR/HER2 antagonists, and MEK cascade blockers), as well as synergistic dynamics when paired with Immune Checkpoint Blockade (ICB) platforms.

3. In Vivo Tumor Modeling via Syngeneic/Xenograft Interfacing:The cell line shows highly predictable tumor-take dynamics when inoculated subcutaneously into immunodeficient rodent recipients (e.g., BALB/c nude, NOD-SCID, or NSG mice). Developing cell line-derived xenograft (CDX) models via SNU-410 facilitates the quantitative in vivo characterization of novel anti-cancer agents, including empirical tracking of tumor growth inhibition (TGI) rates, pharmacokinetic (PK) modeling, and toxicological safety profiling.

III Laboratory Thawing, Cultivation, Passaging, and Cryopreservation Protocols

SNU-410 cells display moderate tolerance to mechanical shear stresses and enzymatic cleavage. While straightforward to cultivate, cultures require systematic monitoring to ensure monolayers do not achieve extreme hyper-confluency, which can induce physical overcrowding, piling, and subsequent phenotypic degradation.

1. Growth Medium & Chemical Reagent Formulations

• Basal Medium: RPMI-1640 growth medium. (Note: The vast majority of the SNU gastrointestinal cancer line bank was originally selected and optimized utilizing RPMI-1640 as the base substrate, which remains the definitive recommendation).

• Complete Growth Formulation: Basal RPMI-1640 medium enriched with 10% high-grade Fetal Bovine Serum (FBS) and fortified with 1% Penicillin-Streptomycin dual antibiotics.

• Cell Dissociation Enzyme: Standard 0.25% Trypsin-0.02% EDTA solution.

• Environmental Cultivation Constants: Incubate at 37 degrees Celsius inside a humidified atmosphere charged with 5% Carbon Dioxide.

2. Cryovial Thawing and Recovery Sequence

1. Pre-incubate a pristine T25 tissue culture flask filled with 5 - 6 mL of fresh complete growth medium at 37 degrees Celsius inside the Class II Biosafety Cabinet.

2. Retrieve the SNU-410 cryovial from liquid nitrogen containment and submerge it instantly into a 37 degrees Celsius constant-temperature water bath. Shake rapidly and continuously to secure absolute thawing within 60 seconds, protecting the cells from slow ice-recrystallization-induced membrane lysis.

3. Spray the exterior tube casing with 75% ethanol before transferring it into the biosafety station.

4. Using a sterile pipettor, smoothly extract the thawed suspension and deliver it dropwise into a 15 mL conical tube packed with 4 mL of pre-warmed complete growth medium, gently inverting once to equalize osmotic pressures.

5. Sediment the cells via centrifugation at 1000 rpm (approximately 200 g) for 4 - 5 minutes at room temperature, then carefully decant the DMSO-laden supernatant.

6. Resuspend the cell pellet in 1 - 2 mL of fresh complete medium, transfer the entire volume into the prepared T25 flask, rock smoothly to balance seeding distribution, and incubate under standard atmospheric constants.

7. Inspect the adherent status approximately 24 hours post-thaw. Perform a complete medium replacement to eliminate non-adherent cell remnants and residual DMSO fractions.

3. Routine Adherent Passaging Mechanics and Maintenance

• Confluency Control Window: Subculturing routines must be initiated when monolayers achieve an optimal 80% - 90% confluency scale (where the polygonal cells line the entire flask plane but have not yet begun stratifying). Allowing SNU-410 cultures to reach 100% saturation forces cells to layer vertically on top of each other, resulting in widespread sheet detachment due to underlying localized hypoxia and nutrient depletion.

• Passaging Execution Steps:

  1. Aspirate the spent growth matrix and gently rinse the cell layer 1 - 2 times with sterile, calcium/magnesium-free PBS to remove all remaining serum proteins that could deactivate the trypsin enzyme.

  2. Administer a suitable volume of 0.25% Trypsin-EDTA enzyme (typically 1 mL for a T25 flask format), tilt the flask to ensure total monolayer coverage, and place inside the 37 degrees Celsius incubator for 2 - 4 minutes.

  3. Monitor cell detachment kinetics under an inverted microscope. As the polygonal cells round up, separate from neighbors, and slide upon gentle physical tapping of the flask wall, immediately add 2 to 3 volumes of serum-fortified complete growth medium to arrest enzymatic cleavage.

  4. Gently pipette the solution against the flask walls to rinse down any remaining cells and dissociate clusters into a single-cell suspension. Transfer the suspension into a conical tube and centrifuge at 1000 rpm for 5 minutes.

  5. Discard the supernatant, resuspend the cell pellet in fresh complete growth medium, and inoculate into new flasks utilizing standard split ratios of 1:3 to 1:5.

  6. Execute subculturing every 2 - 3 days, adjusting media parameters depending on metabolic consumption and colorimetric shifts.

4. Long-Term Cryopreservation Standards

• Cryoprotectant Preservation Matrix: 90% premium complete growth medium (or pure FBS) supplemented with 10% analytical-grade Dimethyl Sulfoxide (DMSO).

• Freezing Protocol Validation:

  1. Exclusively harvest healthy, log-phase cultures showing an optimal confluency of approximately 80% without signs of intracellular vacuolization.

  2. Post-enzymatic treatment and centrifugation, adjust the cell concentration inside the formulated cryoprotectant matrix to a target range of 1,000,000 to 2,000,000 cells per milliliter.

  3. Dispense the suspension into sterile cryovials, insert them immediately into a controlled-rate freezing device (e.g., Mr. Frosty), and place into a minus 80 degrees Celsius freezer overnight to achieve steady gradient cooling (approximately 1 degree Celsius per minute).

  4. The following day, swiftly transfer the frozen cryovials into liquid nitrogen storage tanks (minus 196 degrees Celsius) for definitive preservation. Do not store vials indefinitely inside a minus 80 degrees Celsius freezer; minor temperature oscillations over extended periods can compromise post-thaw recovery viability and structural cell survival rates.

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