BioVector? SNU-410 Human Gallbladder Carcinoma Cell Line / SNU-410 人膽囊癌細(xì)胞系

一 產(chǎn)品基本信息與細(xì)胞生物學(xué)背景

• 細(xì)胞名稱：SNU-410（全稱 Seoul National University-410）。

• 物種與組織來(lái)源：人類（Homo sapiens），源自患有原發(fā)性膽囊癌（Primary Gallbladder Carcinoma）的 57 歲韓國(guó)女性的腫瘤組織。

• 細(xì)胞系建立背景：SNU-410 細(xì)胞系由韓國(guó)首爾大學(xué)醫(yī)學(xué)院（Seoul National University College of Medicine）著名的癌癥研究所（Cancer Research Institute）團(tuán)隊(duì)于 20 世紀(jì) 90 年代成功建立。該研究所（由 Jae-Gahb Park 教授團(tuán)隊(duì)領(lǐng)銜）系統(tǒng)性地從韓國(guó)本土癌癥患者中分離、建立了一系列“SNU”編號(hào)的消化系統(tǒng)腫瘤細(xì)胞株。膽囊癌由于臨床早期診斷困難、惡性程度極高且缺乏有效靶向藥物，SNU-410 的建立為研究亞洲高發(fā)的膽道系統(tǒng)惡性腫瘤（Biliary Tract Cancers, BTCs）提供了寶貴的人源模式底盤。

• 核心表型與細(xì)胞學(xué)特征：

  • 形態(tài)學(xué)特征：貼壁生長(zhǎng)，在顯微鏡下呈現(xiàn)典型的上皮樣（Epithelial-like）或多角形（Polygonal）形態(tài)。細(xì)胞常呈鋪路石狀密集緊湊排列，生長(zhǎng)邊界清晰，局部可見典型腺癌樣分化特征。

  • 基因組與標(biāo)志物特征：高表達(dá)上皮細(xì)胞標(biāo)志物（如 E-cadherin、Cytokeratins），并在特定膽道系統(tǒng)癌癥相關(guān)的基因通路（如 TP53 突變、KRAS 變異、SMAD4 改變或 p16/CDKN2A 異常等）中表現(xiàn)出獨(dú)特的臨床相關(guān)突變譜（具體特征常作為膽囊癌基因組測(cè)序研究的陽(yáng)性對(duì)照）。

• 生物安全級(jí)別：1級(jí)（BSL-1）。

二 核心科研價(jià)值與轉(zhuǎn)化醫(yī)學(xué)應(yīng)用

SNU-410 細(xì)胞株作為公認(rèn)的人源膽囊癌多中心標(biāo)準(zhǔn)對(duì)照，在消化道腫瘤轉(zhuǎn)化醫(yī)學(xué)領(lǐng)域具有明確的實(shí)驗(yàn)價(jià)值：

1. 膽道系統(tǒng)癌癥（BTC）分子發(fā)病機(jī)制探討：膽囊癌和膽管癌在生物學(xué)行為上具有獨(dú)特的異質(zhì)性。SNU-410 常被用作經(jīng)典的體外模型，深入解構(gòu)上皮-間充質(zhì)轉(zhuǎn)化（EMT）通路、Wnt/beta-catenin 信號(hào)級(jí)聯(lián)、以及 Notch 信號(hào)通路在膽囊上皮惡性轉(zhuǎn)化和早期局部浸潤(rùn)中的分子靶向機(jī)制。

2. 新型靶向藥、化療敏感性及免疫治療體外高通量篩選：針對(duì)臨床一線化療藥物（如吉西他濱 Gemcitabine、順鉑 Cisplatin）的耐藥機(jī)制是目前膽囊癌科研的熱點(diǎn)。SNU-410 被廣泛應(yīng)用于評(píng)價(jià)新型小分子激酶抑制劑（如 FGFR 抑制劑、EGFR/HER2 靶向藥、MEK 抑制劑等）的抗增殖與誘導(dǎo)凋亡效應(yīng)，以及聯(lián)合免疫檢查點(diǎn)阻斷（ICB）后的協(xié)同殺傷敏感性評(píng)估。

3. 異種移植小鼠成瘤模型構(gòu)建（Xenograft Models）：該細(xì)胞在免疫缺陷小鼠（如 BALB/c Nude 裸鼠、NOD-SCID 或 NSG 小鼠）皮下具備穩(wěn)定的成瘤能力。通過(guò)皮下構(gòu)建人源膽囊癌異種移植（CDX）模型，可用于精準(zhǔn)定量評(píng)價(jià)候選抗癌藥物在體內(nèi)的抑瘤率、藥代動(dòng)力學(xué)（PK）及安全性表征。

三 實(shí)驗(yàn)室細(xì)胞復(fù)蘇、貼壁常規(guī)培養(yǎng)、傳代與保存標(biāo)準(zhǔn)步驟

SNU-410 細(xì)胞對(duì)體外培養(yǎng)環(huán)境的機(jī)械剪切力和消化酶敏感度適中，屬于相對(duì)容易維持和傳代的貼壁細(xì)胞。但在日常培養(yǎng)中仍需警惕因局部過(guò)密而導(dǎo)致的細(xì)胞抱團(tuán)堆疊，進(jìn)而引起細(xì)胞表型退化或活性受損。

1. 培養(yǎng)基與化學(xué)試劑配置

• 基礎(chǔ)培養(yǎng)基（核心推薦）：RPMI-1640 基礎(chǔ)培養(yǎng)基。(注：首爾大學(xué)建立的絕大多數(shù) SNU 消化道腫瘤系列細(xì)胞株，均采用 RPMI-1640 作為最適配的標(biāo)準(zhǔn)生長(zhǎng)基質(zhì))。

• 完全培養(yǎng)基配方：RPMI-1640 基礎(chǔ)培養(yǎng)基 加 10% 優(yōu)質(zhì)胎牛血清（FBS） 加 1% 青霉素-鏈霉素雙抗（Penicillin-Streptomycin）。

• 細(xì)胞解離液：0.25% Trypsin-0.02% EDTA 消化液。

• 環(huán)境參數(shù)：37 攝氏度，5% 二氧化碳，飽和濕度孵箱。

2. 冷凍細(xì)胞復(fù)蘇步驟

1. 提前在無(wú)菌生物安全柜中配制好干凈的 T25 培養(yǎng)瓶，注入 5 - 6 mL 預(yù)熱至 37 攝氏度的完全培養(yǎng)基。

2. 從液氮罐或超低溫冰箱中取出 SNU-410 凍存管，立刻全量投入 37 攝氏度恒溫水浴箱中快速搖晃解凍，確保管內(nèi)凍塊在 1 分鐘內(nèi)完全融化，避免局部冰晶慢速重結(jié)晶對(duì)細(xì)胞膜造成二次撕裂。

3. 用 75% 酒精噴灑凍存管外壁進(jìn)行表面消毒，隨后移入生物安全柜內(nèi)。

4. 用無(wú)菌移液槍吸取融化的細(xì)胞懸液，緩慢滴加至盛有 4 mL 預(yù)熱完全培養(yǎng)基的 15 mL 離心管中，前后輕柔顛倒一次以稀釋化學(xué)保護(hù)劑。

5. 以 1000 rpm（約 200 g）室溫離心 4 - 5 分鐘，小心吸除含有二甲基亞砜（DMSO）的上清液。

6. 加入 1 - 2 mL 新鮮完全培養(yǎng)基重懸細(xì)胞沉淀，將其全量接種至準(zhǔn)備好的 T25 瓶中。前后輕柔十字晃動(dòng)混勻，置于孵箱中。

7. 復(fù)蘇次日（24 小時(shí)左右）常規(guī)在顯微鏡下觀察細(xì)胞貼壁和展弦狀態(tài)。此時(shí)應(yīng)進(jìn)行一次全量更換新鮮完全培養(yǎng)基的操作，以徹底清除復(fù)蘇過(guò)程中產(chǎn)生的死細(xì)胞碎屑及極微量殘留 DMSO。

3. 日常貼壁常規(guī)傳代操作

• 傳代時(shí)機(jī)：當(dāng)細(xì)胞融合度達(dá)到 80% - 90%（即上皮樣細(xì)胞基本鋪滿瓶底，開始擠壓對(duì)接，但尚未完全重疊生長(zhǎng)）時(shí)必須進(jìn)行傳代。SNU-410 細(xì)胞若達(dá)到 100% 極度過(guò)密狀態(tài)，細(xì)胞會(huì)自發(fā)向上層疊堆積，導(dǎo)致下層細(xì)胞因缺氧缺營(yíng)養(yǎng)而自發(fā)大面積成片脫落。

• 操作流程：

  1. 吸除細(xì)胞瓶?jī)?nèi)的舊培養(yǎng)基，使用無(wú)菌的、不含鈣鎂離子的 PBS 緩沖液輕輕漂洗細(xì)胞表面 1 - 2 次，徹底洗去殘存的、會(huì)抑制胰酶活性的血清。

  2. 加入適量 0.25% 胰酶消化液（T25 瓶常規(guī)加入 1 mL），搖晃使其全面覆蓋細(xì)胞層。置于 37 攝氏度孵箱中消化 2 - 4 分鐘。

  3. 在倒置顯微鏡下進(jìn)行實(shí)時(shí)動(dòng)態(tài)觀察。當(dāng)發(fā)現(xiàn)多角形細(xì)胞體自發(fā)回縮、變圓、胞間間隙明顯增大、輕敲瓶壁可見上皮樣細(xì)胞成片滑落或分散移動(dòng)時(shí)，立刻加入 2 到 3 倍體積的含血清完全培養(yǎng)基以終止胰酶的解離反應(yīng)。

  4. 用移液槍在瓶壁輕輕吹打，使未脫落的細(xì)胞徹底剝離，并打散形成單細(xì)胞懸液。收集懸液入管，1000 rpm 離心 5 分鐘。

  5. 棄去上清，加入新鮮完全培養(yǎng)基。按照 1 比 3 至 1 比 5 的常規(guī)稀釋比例，接種至新的培養(yǎng)器皿中。

  6. 通常每 2 - 3 天傳代一次，期間根據(jù)液體消耗速度和顏色改變適度補(bǔ)充或更換培養(yǎng)基。

4. 細(xì)胞長(zhǎng)期保存標(biāo)準(zhǔn)

• 凍存液配方：90% 優(yōu)質(zhì)完全培養(yǎng)基（或純胎牛血清） 加 10% 分析級(jí)二甲基亞砜（DMSO）。

• 冷凍規(guī)范：

  1. 收集處于對(duì)數(shù)生長(zhǎng)最旺盛期、細(xì)胞密度在 80% 左右、健康且無(wú)空泡化表現(xiàn)的 SNU-410 細(xì)胞。

  2. 經(jīng)溫和消化、離心沉淀后，用配置好的凍存液調(diào)整細(xì)胞密度至 每毫升 1,000,000 到 2,000,000 個(gè)細(xì)胞。

  3. 分裝入無(wú)菌冷凍管中，立刻移入標(biāo)準(zhǔn)程序降溫盒（如 Mr. Frosty），并置于 零下 80 攝氏度冰箱中過(guò)夜梯度降溫（遵循約每分鐘降溫 1 攝氏度的穩(wěn)態(tài)速率）。

  4. 次日，必須迅速將凍存管轉(zhuǎn)移入液氮罐（零下 196 攝氏度）長(zhǎng)期保存。嚴(yán)禁在 零下 80 攝氏度普通冰箱內(nèi)長(zhǎng)期存放，以防長(zhǎng)期的微小熱幅射導(dǎo)致細(xì)胞內(nèi)部冰晶重塑，進(jìn)而嚴(yán)重惡化后續(xù)復(fù)蘇時(shí)的貼壁存活率與功能完整性。

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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