BioVector? DMS 114 Human Small Cell Lung Cancer Cell Line / DMS 114 人小細胞肺癌細胞系

一 產品基本信息與細胞生物學背景

• 細胞名稱：DMS 114（亦書寫為 DMS-114）。

• 物種與組織來源：人類（Homo sapiens），源自一名患有小細胞肺癌（Small Cell Lung Cancer, SCLC）的成年的原發(fā)性肺部腫瘤組織（經手術切除或活檢獲取）。

• 細胞系建立背景：DMS 114 細胞系于 20 世紀 80 年代由達特茅斯醫(yī)學院（Dartmouth Medical School，細胞系名稱前綴“DMS”由此而來）的科研團隊成功建立。小細胞肺癌屬于神經內分泌癌，惡性程度極高、進展極快且早期易發(fā)生全身廣泛轉移。DMS 114 細胞系的成功建立與鑒定，為全球醫(yī)學界深入探究非小細胞肺癌與小細胞肺癌之間的異質性差異、以及攻克頑固性肺部神經內分泌腫瘤提供了重要的人源細胞模式底盤。

• 核心表型與細胞學特征：

  • 形態(tài)學特征：DMS 114 屬于典型的貼壁生長型小細胞肺癌細胞。在倒置顯微鏡下，細胞主要呈現上皮樣（Epithelial-like）或多角形（Polygonal）形態(tài)。細胞體體積較小，核質比極高，常表現為緊密連接的多克隆集團或鋪路石狀成片貼壁生長。

  • 神經內分泌特征（Neuroendocrine Markers）：作為經典的小細胞肺癌模型，DMS 114 保留了肺部神經內分泌細胞的生化特征，能夠特異性高表達神經內分泌標志物，如突觸素（Synaptophysin, SYN）、嗜鉻粒蛋白A（Chromogranin A, CgA）以及神經元特異性烯醇化酶（NSE），并具有合成與釋放特定肽類激素的活性。

• 生物安全級別：1級（BSL-1）。

二 核心科研價值與轉化醫(yī)學應用

DMS 114 細胞因其明確的神經內分泌背景和穩(wěn)定的貼壁增殖特性，在遠端肺癌藥理學中具有極高學術價值：

1. 小細胞肺癌（SCLC）靶向與化療耐藥機制研究：小細胞肺癌患者在臨床初期對一線化療（如依托泊苷 Etoposide 聯合順鉑 Cisplatin 或卡鉑 Carboplatin）表現出極高的敏感性，但幾乎所有患者都會在短時間內產生多藥耐藥（MDR）。DMS 114 被廣泛用作敏感株或用于體外階梯誘導耐藥模型，用于篩選新型拓撲異構酶抑制劑、DNA 損傷修復（DDR）通路靶向藥（如 PARP 抑制劑、ATR/CHK1 抑制劑）的殺傷敏感性。

2. 神經內分泌分化與轉錄因子調控網絡重塑：SCLC 的發(fā)生發(fā)展高度依賴于特定轉錄因子（如 ASCL1, NEUROD1, POU2F3 等）的驅動。DMS 114 常被整合入小細胞肺癌分子分型矩陣中，作為研究特定原癌基因擴增（如 MYC 家族、RB1/TP53 雙突變失活背景）如何啟動腫瘤神經內分泌分化級聯反應的分子生物學工具。

3. 皮下與原位異種移植小鼠成瘤模型構建（CDX Models）：相較于許多呈懸浮抱團生長、在體內成瘤率不穩(wěn)定且難以計數的 SCLC 細胞系（如 H69 等），貼壁型生長的 DMS 114 在免疫缺陷小鼠（如 BALB/c Nude 裸鼠、NOD-SCID 小鼠）皮下構建異種移植（CDX）模型時表現出極佳的穩(wěn)定性、極高的成瘤率與可測性。它是評估候選抗癌新藥體內腫瘤生長抑制率（TGI）和藥代動力學（PK）的標桿底盤。

三 實驗室細胞復蘇、貼壁常規(guī)培養(yǎng)、傳代與保存標準步驟

DMS 114 細胞體積較小、胞間黏附力極強，在融合成片后較難通過常規(guī)力度的吹打將其徹底分散為單細胞。在日常維護中需注意把控消化酶的作用時間，避免因消化不足導致細胞成片重疊堆疊、或因過度消化導致小體積細胞解離受損。

1. 培養(yǎng)基與化學試劑配置

• 基礎培養(yǎng)基：Waymouth's MB 752/1 培養(yǎng)基（或遵照特定實驗習慣與克隆來源，使用 RPMI-1640 基礎培養(yǎng)基）。

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

• 細胞解離液：0.25% Trypsin-0.02% EDTA 消化液。

• 環(huán)境參數：37 攝氏度，5% 二氧化碳，飽合濕度環(huán)境。

2. 冷凍細胞復蘇步驟

1. 提前在無菌生物安全柜中準備好干凈的 T25 培養(yǎng)瓶，注入 5 - 6 mL 預熱至 37 攝氏度的完全培養(yǎng)基。

2. 從液氮罐中取出 DMS 114 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，確保在 1 分鐘內令管內冰塊完全融化。

3. 用 75% 酒精噴灑凍存管外壁進行表面消毒，移入生物安全柜。

4. 用無菌移液槍吸取融化的細胞懸液，緩慢滴加至盛有 4 mL 預熱完全培養(yǎng)基的 15 mL 離心管中（操作務必輕柔，切勿劇烈吹打，防止對復蘇狀態(tài)下的小體積細胞造成剪切傷）。

5. 以 1000 rpm（約 200 g）離心 4 - 5 分鐘，小心吸干含有二甲基亞砜（DMSO）的冷凍保護劑上清。

6. 加入 1 mL 新鮮完全培養(yǎng)基，使用 P1000 移液槍頭將其輕輕重懸化開，隨后將其接種至準備好的 T25 瓶中。前后輕柔十字晃動混勻，置于孵箱中。

7. 復蘇 24 小時后，在顯微鏡下常規(guī)觀察細胞貼壁展弦狀態(tài)。全量更換一次新鮮的預熱完全培養(yǎng)基，以徹底清除在解凍過程中產生的死細胞碎屑及極微量殘留 DMSO。

3. 日常貼壁常規(guī)傳代操作

• 傳代時機：當細胞融合度達到 80% - 90%（即小上皮樣細胞密集對接，但尚未完全疊層擠壓）時必須進行傳代。DMS 114 細胞若達到 100% 極度過密狀態(tài)，細胞會自發(fā)產生強烈的接觸抑制，形成大面積致密的抱團“細胞橋”，此時再行消化極難將其打散，嚴重惡化后續(xù)的貼壁與分裂活性。

• 操作流程：

  1. 吸除細胞瓶內的舊培養(yǎng)基，使用無菌的、不含鈣鎂離子的 PBS 緩沖液輕輕漂洗細胞表面 1 - 2 次，徹底洗去殘存的、會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），輕搖使其完全覆蓋細胞層。置于 37 攝氏度孵箱中消化 2 - 4 分鐘。

  3. 在倒置顯微鏡下進行實時動態(tài)觀察。由于 DMS 114 胞間緊密連接豐度高，消化時當看到多角形細胞體邊緣自發(fā)回縮變圓、胞間裂隙增大、輕敲瓶壁細胞可見大面積成片移動時，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。

  4. 用移液槍在瓶壁輕輕吹打。由于細胞體積小且易抱團，可適當增加吹打次數，使成片的細胞團剝離并盡可能打散形成均勻的細胞懸液。收集入管，1000 rpm 離心 5 分鐘。

  5. 棄去上清，加入新鮮完全培養(yǎng)基重懸。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)瓶中。通常每 2 - 3 天傳代一次。

4. 細胞長期保存標準

• 凍存液配方：90% 優(yōu)質完全培養(yǎng)基（或純胎牛血清） 加 10% 分析級二甲基亞砜（DMSO）。

• 冷凍規(guī)范：

  1. 收集處于對數生長最旺盛期、健康指數高、融合度在 80% 左右、未發(fā)生空泡化衰老的 DMS 114 細胞。

  2. 經溫和消化、離心沉淀后，用配置好的凍存液懸浮，調整細胞密度至 每毫升 1,500,000 到 2,500,000 個細胞。

  3. 分裝入無菌凍存管中，立刻移入標準程序降溫盒（如 Mr. Frosty），并置于 零下 80 攝氏度冰箱中過夜梯度降溫（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移入液氮罐（零下 196 攝氏度）長期鎖死保存。絕對禁止在 零下 80 攝氏度普通冰箱內長期存放，以防微小的溫度震蕩導致細胞內部冰晶重組，進而嚴重惡化后續(xù)復蘇時的貼壁存活率與神經內分泌功能表型。

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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