BioVector® DMS 114 Human Small Cell Lung Cancer Cell Line / DMS 114 人小细胞肺癌细胞系

一 产品基本信息与细胞生物学背景

• 细胞名称：DMS 114（亦书写为 DMS-114）。

• 物种与组织来源：人类（Homo sapiens），源自一名患有小细胞肺癌（Small Cell Lung Cancer, SCLC）的成年的原发性肺部肿瘤组织（经手术切除或活检获取）。

• 细胞系建立背景：DMS 114 细胞系于 20 世纪 80 年代由达特茅斯医学院（Dartmouth Medical School，细胞系名称前缀“DMS”由此而来）的科研团队成功建立。小细胞肺癌属于神经内分泌癌，恶性程度极高、进展极快且早期易发生全身广泛转移。DMS 114 细胞系的成功建立与鉴定，为全球医学界深入探究非小细胞肺癌与小细胞肺癌之间的异质性差异、以及攻克顽固性肺部神经内分泌肿瘤提供了重要的人源细胞模式底盘。

• 核心表型与细胞学特征：

  • 形态学特征：DMS 114 属于典型的贴壁生长型小细胞肺癌细胞。在倒置显微镜下，细胞主要呈现上皮样（Epithelial-like）或多角形（Polygonal）形态。细胞体体积较小，核质比极高，常表现为紧密连接的多克隆集团或铺路石状成片贴壁生长。

  • 神经内分泌特征（Neuroendocrine Markers）：作为经典的小细胞肺癌模型，DMS 114 保留了肺部神经内分泌细胞的生化特征，能够特异性高表达神经内分泌标志物，如突触素（Synaptophysin, SYN）、嗜铬粒蛋白A（Chromogranin A, CgA）以及神经元特异性烯醇化酶（NSE），并具有合成与释放特定肽类激素的活性。

• 生物安全级别：1级（BSL-1）。

二 核心科研价值与转化医学应用

DMS 114 细胞因其明确的神经内分泌背景和稳定的贴壁增殖特性，在远端肺癌药理学中具有极高学术价值：

1. 小细胞肺癌（SCLC）靶向与化疗耐药机制研究：小细胞肺癌患者在临床初期对一线化疗（如依托泊苷 Etoposide 联合顺铂 Cisplatin 或卡铂 Carboplatin）表现出极高的敏感性，但几乎所有患者都会在短时间内产生多药耐药（MDR）。DMS 114 被广泛用作敏感株或用于体外阶梯诱导耐药模型，用于筛选新型拓扑异构酶抑制剂、DNA 损伤修复（DDR）通路靶向药（如 PARP 抑制剂、ATR/CHK1 抑制剂）的杀伤敏感性。

2. 神经内分泌分化与转录因子调控网络重塑：SCLC 的发生发展高度依赖于特定转录因子（如 ASCL1, NEUROD1, POU2F3 等）的驱动。DMS 114 常被整合入小细胞肺癌分子分型矩阵中，作为研究特定原癌基因扩增（如 MYC 家族、RB1/TP53 双突变失活背景）如何启动肿瘤神经内分泌分化级联反应的分子生物学工具。

3. 皮下与原位异种移植小鼠成瘤模型构建（CDX Models）：相较于许多呈悬浮抱团生长、在体内成瘤率不稳定且难以计数的 SCLC 细胞系（如 H69 等），贴壁型生长的 DMS 114 在免疫缺陷小鼠（如 BALB/c Nude 裸鼠、NOD-SCID 小鼠）皮下构建异种移植（CDX）模型时表现出极佳的稳定性、极高的成瘤率与可测性。它是评估候选抗癌新药体内肿瘤生长抑制率（TGI）和药代动力学（PK）的标杆底盘。

三 实验室细胞复苏、贴壁常规培养、传代与保存标准步骤

DMS 114 细胞体积较小、胞间黏附力极强，在融合成片后较难通过常规力度的吹打将其彻底分散为单细胞。在日常维护中需注意把控消化酶的作用时间，避免因消化不足导致细胞成片重叠堆叠、或因过度消化导致小体积细胞解离受损。

1. 培养基与化学试剂配置

• 基础培养基：Waymouth's MB 752/1 培养基（或遵照特定实验习惯与克隆来源，使用 RPMI-1640 基础培养基）。

• 完全培养基配方：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，饱合湿度环境。

2. 冷冻细胞复苏步骤

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

2. 从液氮罐中取出 DMS 114 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，确保在 1 分钟内令管内冰块完全融化。

3. 用 75% 酒精喷洒冻存管外壁进行表面消毒，移入生物安全柜。

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（操作务必轻柔，切勿剧烈吹打，防止对复苏状态下的小体积细胞造成剪切伤）。

5. 以 1000 rpm（约 200 g）离心 4 - 5 分钟，小心吸干含有二甲基亚砜（DMSO）的冷冻保护剂上清。

6. 加入 1 mL 新鲜完全培养基，使用 P1000 移液枪头将其轻轻重悬化开，随后将其接种至准备好的 T25 瓶中。前后轻柔十字晃动混匀，置于孵箱中。

7. 复苏 24 小时后，在显微镜下常规观察细胞贴壁展弦状态。全量更换一次新鲜的预热完全培养基，以彻底清除在解冻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%（即小上皮样细胞密集对接，但尚未完全叠层挤压）时必须进行传代。DMS 114 细胞若达到 100% 极度过密状态，细胞会自发产生强烈的接触抑制，形成大面积致密的抱团“细胞桥”，此时再行消化极难将其打散，严重恶化后续的贴壁与分裂活性。

• 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），轻摇使其完全覆盖细胞层。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。由于 DMS 114 胞间紧密连接丰度高，消化时当看到多角形细胞体边缘自发回缩变圆、胞间裂隙增大、轻敲瓶壁细胞可见大面积成片移动时，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。由于细胞体积小且易抱团，可适当增加吹打次数，使成片的细胞团剥离并尽可能打散形成均匀的细胞悬液。收集入管，1000 rpm 离心 5 分钟。

  5. 弃去上清，加入新鮮完全培养基重悬。按照 1 比 3 至 1 比 5 的常规稀释比例，接种至新的培养瓶中。通常每 2 - 3 天传代一次。

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、健康指数高、融合度在 80% 左右、未发生空泡化衰老的 DMS 114 细胞。

  2. 经温和消化、离心沉淀后，用配置好的冻存液悬浮，调整细胞密度至 每毫升 1,500,000 到 2,500,000 个细胞。

  3. 分装入无菌冻存管中，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）长期锁死保存。绝对禁止在 零下 80 摄氏度普通冰箱内长期存放，以防微小的温度震荡导致细胞内部冰晶重组，进而严重恶化后续复苏时的贴壁存活率与神经内分泌功能表型。

Part 2 English Section

I General Information and Cell Biological Background

• Cell Line Name: DMS 114 (Standardly referenced as DMS-114).

• Organism and Tissue Extraction Origin: Homo sapiens (human); derived from primary lung tumor tissue resected via surgery or biopsy from an adult donor diagnosed with Small Cell Lung Cancer (SCLC).

• Cell Line Establishment Background:The DMS 114 cell line was established in the 1980s by the research cohort at Dartmouth Medical School (the prefix "DMS" designates this academic origin). Small cell lung cancer represents a highly malignant, poorly differentiated neuroendocrine carcinoma characterized by rapid doubling kinetics and early systemic metastasis. The successful development and authentication of DMS 114 offered a valuable human-derived model to contrast the biological landscapes of SCLC and Non-Small Cell Lung Cancer (NSCLC), standing as an essential platform to study neuroendocrine malignancies.

• Core Morphological Phenotype and Cellular Traits:

  • Morphological Form: Characterized as an adherent growth variant of small cell lung cancer models. Under inverted phase-contrast microscopy, it presents a typical epithelial-like or polygonal morphology. The cells have small diameters and a high nuclear-to-cytoplasmic (N/C) ratio, proliferating as tight multi-clonal aggregates or cobblestone-like sheets.

  • Neuroendocrine Characteristics (Neuroendocrine Markers): Retains the phenotypic hallmarks of native pulmonary neuroendocrine cells. DMS 114 scores heavily positive for definitive diagnostic biomarkers including Synaptophysin (SYN), Chromogranin A (CgA), and Neuron-Specific Enolase (NSE), maintaining active synthesis and secretory pathways for specific peptide hormones.

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

II Strategic Research Value and Translational Fields

DMS 114 cells provide substantial analytical value across several key oncological fields due to their neuroendocrine heritage and consistent adherent growth:

1. Unraveling Multidrug Chemoresistance Networks in SCLC:While small cell lung cancer initially responds well to first-line therapies (e.g., Etoposide paired with Cisplatin or Carboplatin), patients almost universally develop rapid, fatal Multidrug Resistance (MDR). DMS 114 is integrated as a sensitive parental model or utilized to generate acquired drug-resistant clones, evaluating the cytotoxicity profiles of novel topoisomerase inhibitors and DNA Damage Repair (DDR) inhibitors (such as PARP, ATR, or CHK1 antagonists).

2. Mapping Neuroendocrine Differentiation and Transcriptional Circuitries:SCLC progression is heavily governed by a specialized matrix of transcription factors (e.g., ASCL1, NEUROD1, POU2F3). DMS 114 serves as a reference chassis within molecular classification studies, helping chart how key oncogenic gene amplifications (such as MYC alterations alongside the inactivation of RB1 and TP53) orchestrate neuroendocrine differentiation and tumor behavior.

3. Predictable In Vivo Tumor Modeling via CDX Interfacing:Unlike many standard SCLC lines (such as NCI-H69) that grow in floating, irregular clusters—making cell counts difficult and tumor-take unpredictable—the adherent nature of DMS 114 ensures high consistency when constructing Cell Line-Derived Xenograft (CDX) models. Inoculated subcutaneously into athymic nude, NOD-SCID, or advanced immunodeficient mice, it establishes solid tumors with high reproducibility, making it a reliable choice for quantifying Tumor Growth Inhibition (TGI) rates and validating preclinical pharmacokinetic (PK) endpoints.

III Laboratory Thawing, Cultivation, Passaging, and Cryopreservation Protocols

DMS 114 cells are small and form strong paracellular attachments, making them resistant to single-cell dissociation via minor mechanical agitation once confluent. Daily subculturing requires careful regulation of enzymatic cleavage windows to avoid leaving persistent, stacked multi-cellular aggregates or causing structural cell stress through over-digestion.

1. Growth Medium & Chemical Reagent Formulations

• Basal Medium: Waymouth's MB 752/1 medium (or adjusted to standard laboratory protocols using modified RPMI-1640 growth matrix).

• Complete Growth Formulation: Basal growth medium enriched with 10% premium Fetal Bovine Serum (FBS) and fortified with 1% standard 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 DMS 114 cryovial from liquid nitrogen storage and submerge it instantly into a 37 degrees Celsius constant-temperature water bath. Shake rapidly and continuously to secure absolute thawing within 60 seconds.

3. Decontaminate the exterior casing with 75% ethanol before transfer into the biosafety cabinet.

4. Using a sterile pipettor, extract the thawed slurry and deliver it slowly, dropwise into a 15 mL conical tube packed with 4 mL of pre-warmed complete growth medium. Handle with care; avoid rapid pipetting to safeguard small-diameter cells from mechanical shear stress post-thaw.

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

6. Dispense 1 mL of fresh complete medium onto the cell pellet and resuspend gently using a P1000 micro-pipette tip. Transfer the entire suspension into the prepared T25 flask, cross-shake smoothly to optimize seeding distribution, and incubate under standard atmospheric constants.

7. Inspect the adherent status approximately 24 hours post-thaw. Perform a complete medium change to remove non-adherent cell debris and trace fractions of residual DMSO.

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 polygonal cell clusters interlock but have not yet crowded or stratified). Allowing DMS 114 cultures to reach absolute 100% saturation triggers strong contact inhibition, organizing dense cell aggregates that are highly resistant to trypsinization, which can degrade post-passage attachment and division metrics.

• Passaging Execution Steps:

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

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

  3. Monitor cell detachment kinetics under an inverted microscope. Due to the high paracellular adhesion of DMS 114, look for the polygonal edges to retract, round up, and slide upon firm physical tapping of the flask wall. At this point, 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 remaining cells. Because these small cells are prone to clumping, perform systematic, uniform pipetting to dissociate aggregates into a single-cell suspension. Transfer the fluid 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. Subculture every 2 - 3 days.

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 vacuolar degradation or aging.

  2. Post-enzymatic treatment and centrifugation, adjust the cell concentration inside the formulated cryoprotectant matrix to a target range of 1,500,000 to 2,500,000 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 ensure a steady gradient cooling rate of 1 degree Celsius per minute.

  4. The following day, swiftly transfer the frozen cryovials into liquid nitrogen storage tanks (minus 196 degrees Celsius) for long-term 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 lead to the degradation of neuroendocrine traits.

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