BioVector? NCI-H2373 Human Malignant Mesothelioma Cell Line / NCI-H2373 人惡性胸膜間皮瘤細(xì)胞株

一 產(chǎn)品基本信息與細(xì)胞生物學(xué)背景

• 細(xì)胞名稱：NCI-H2373（亦常寫作 H2373）。

• 物種與人群來源：人類（Homo sapiens），源自一名高齡白人男性患者。

• 組織源起與病理背景：NCI-H2373 是一株源自人類惡性胸膜間皮瘤（Malignant Pleural Mesothelioma, MPM）組織塊的惡性腫瘤細(xì)胞系。該細(xì)胞系由美國國家癌癥研究所（NCI）成功建立并進(jìn)行系統(tǒng)性鑒定。惡性胸膜間皮瘤是一種高度侵襲性、預(yù)后極差、且通常與長期接觸石棉（Asbestos）密切相關(guān)的胸膜表面惡性腫瘤。由于 MPM 臨床治療手段匱乏且對常規(guī)化療極易產(chǎn)生耐藥性，NCI-H2373 細(xì)胞株成為全球研究間皮瘤惡性演變機(jī)制、腫瘤靶向異質(zhì)性、以及臨床新型靶向/免疫藥物篩選的經(jīng)典人類疾病模式底盤。

• 核心表型與細(xì)胞生物學(xué)特征：

  • 形態(tài)學(xué)表現(xiàn)：貼壁生長。在倒置顯微鏡下，NCI-H2373 細(xì)胞表現(xiàn)為明顯的上皮樣（Epithelial-like）與成纖維紡錘形突起混合的形態(tài)。細(xì)胞多呈多角形或不規(guī)則拉長狀，胞質(zhì)清晰，核仁明顯。在細(xì)胞匯合度較高、密集成片時，細(xì)胞呈現(xiàn)出多層重疊生長或緊密交織的典型惡性腫瘤細(xì)胞排布特征。

  • 基因組與標(biāo)志物圖譜：經(jīng)生化與分子生物學(xué)鑒定，該細(xì)胞系持續(xù)表達(dá)間皮瘤相關(guān)的核心診斷標(biāo)志物（如 Calretinin 間皮素、WT1 等）。同時，作為間皮瘤特征，它常伴隨有關(guān)鍵抑癌基因（如 NF2/Merlin、BAP1 或 CDKN2A/p16）的缺失或突變，這為其靶向合成致死（Synthetic lethality）藥物的研究提供了精準(zhǔn)的遺傳背景。

• 生物安全級別：1級（BSL-1）。

二 核心科研價值與轉(zhuǎn)化醫(yī)學(xué)應(yīng)用

NCI-H2373 細(xì)胞株在現(xiàn)代現(xiàn)代胸部腫瘤學(xué)、耐藥分子機(jī)制及創(chuàng)新療法開發(fā)中占有重要一席：

1. 構(gòu)建間皮瘤異種移植腫瘤模型（CDX 模型）：通過將 NCI-H2373 細(xì)胞接種于免疫缺陷小鼠（如 BALB/c Nude 或 NOD/SCID）的皮下或胸膜腔內(nèi)（原位移植），可成功誘導(dǎo)形成人類間皮瘤實(shí)體瘤病灶。這是評估新型抗癌藥物在活體內(nèi)（In vivo）靶向殺傷效能、藥代動力學(xué)（PK）的標(biāo)準(zhǔn)化臨床前生物臺表。

2. 新型靶向治療與“合成致死”藥物高通量體外篩查：針對間皮瘤高頻發(fā)生的 BAP1 或 NF2 基因缺陷，利用 NCI-H2373 細(xì)胞系體外評估諸如 EZH2 抑制劑、PARP 抑制劑或 Hippo 信號通路阻斷劑等前沿靶向小分子藥物的合成致死殺傷效應(yīng)，助力挖掘突破性的二線/三線臨床治療方案。

3. 免疫檢查點(diǎn)與腫瘤微環(huán)境交互（TME）探索：用于檢測間皮瘤細(xì)胞表面 PD-L1 表達(dá)譜系的動態(tài)調(diào)控，以及通過將 NCI-H2373 與人類外周血單核細(xì)胞（PBMC）或 T 細(xì)胞在體外共培養(yǎng)，解構(gòu)惡性間皮瘤如何通過分泌特殊細(xì)胞因子逃逸宿主免疫監(jiān)視（Immune evasion）的分子病理機(jī)制。

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

1. 專用培養(yǎng)基配置與生長環(huán)境

• 基礎(chǔ)培養(yǎng)基：經(jīng)典的 RPMI-1640 基礎(chǔ)培養(yǎng)基。

• 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-鏈霉素雙抗溶液（100 U/mL 青霉素 + 100 $\mu$g/mL 鏈霉素）。

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

• 培養(yǎng)物理常數(shù)：標(biāo)準(zhǔn) 37 攝氏度，含 5% 二氧化碳（$CO_2$） 的恒溫高濕度飽和無菌孵箱。

2. 冷凍細(xì)胞復(fù)蘇步驟

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

2. 從液氮罐中取出 NCI-H2373 凍存管，立刻全量投入 37 ℃ 恒溫水浴箱中，快速用力搖晃以期在 1 分鐘內(nèi)令管內(nèi)冰塊完全融化（切忌慢速解凍，否則融復(fù)過程中的冰晶二次重結(jié)晶會嚴(yán)重刺破腫瘤細(xì)胞膜，導(dǎo)致成活率劇烈崩塌）。

3. 用 75% 酒精噴灑消毒外壁，移入生物安全柜。

4. 吸取細(xì)胞懸液，緩慢滴加至盛有 4 - 5 mL 預(yù)熱完全培養(yǎng)基的 15 mL 離心管中，輕柔顛倒一次以稀釋殘留的 DMSO。

5. 以 1000 rpm（約 200 g）室溫溫和離心 5 分鐘，小心抽干含有 DMSO 的上清液。

6. 加入 1 mL 新鮮完全培養(yǎng)基，使用 P1000 移液槍極其輕柔地吹打重懸細(xì)胞沉淀。

7. 將懸液接種至準(zhǔn)備好的 T25 瓶中，輕柔“十字形”搖勻以防止細(xì)胞聚集堆疊。

8. 擰松瓶蓋，放入 37 ℃、5% $CO_2$ 孵箱中靜置暗培養(yǎng)。

9. 24 小時后必須觀察貼壁狀態(tài)，并全量更換一次新鮮培養(yǎng)基，徹底清除未貼壁的死細(xì)胞和殘余微量碎屑。

3. 日常貼壁常規(guī)傳代操作

• 傳代時機(jī)：當(dāng)混合型上皮樣/紡錘形的細(xì)胞密集成片，匯合度（Confluency）達(dá)到 80% - 85% 時必須傳代。NCI-H2373 細(xì)胞在密度過高時會出現(xiàn)自發(fā)多層重疊堆積，導(dǎo)致下層細(xì)胞因缺氧缺營養(yǎng)發(fā)生局部老化脫落，傳代頻率通常為每 3 - 4 天一次。

• 操作流程：

  1. 吸除舊培養(yǎng)基，使用無菌、無鈣鎂離子的 PBS 緩沖液輕輕漂洗細(xì)胞表面 1 次，徹底洗凈殘余血清蛋白（殘存的血清會強(qiáng)力中和并失活胰酶的催化活性）。

  2. 加入適量 0.25% Trypsin-EDTA 消化液（T25 瓶常規(guī)加入 1 mL），使其完全覆蓋細(xì)胞層，隨后放入 37 ℃ 孵箱中溫和消化。

  3. 鏡下動態(tài)觀察：間皮瘤細(xì)胞的胞間連接和基底膜錨定較緊，通常在 37 ℃ 下消化 2 - 4 分鐘。當(dāng)在倒置顯微鏡下動態(tài)觀察到原本拉長的紡錘形細(xì)胞胞體收縮變圓、多角形邊緣變松、且輕敲培養(yǎng)瓶一側(cè)時有成片細(xì)胞自發(fā)滑動，說明消化達(dá)標(biāo)。

  4. 立刻向瓶內(nèi)倒入 2 倍體積的含血清完全培養(yǎng)基，終止胰酶的酶解剪切。

  5. 用移液槍或移液管輕柔沖洗瓶壁，將細(xì)胞完全洗脫下來，吹打 3 - 5 次將其調(diào)理成均勻的單細(xì)胞懸液（如有未打散的團(tuán)塊容易導(dǎo)致后期成片局部堆積）。

  6. 將懸液收集至離心管中，1000 rpm 離心 5 分鐘，棄去酶解上清。

  7. 加入新鮮完全培養(yǎng)基重懸沉淀，按照 1:2 至 1:4 的常規(guī)傳代比例接種至新的培養(yǎng)瓶中，補(bǔ)足完全培養(yǎng)基，放回孵箱中繼續(xù)擴(kuò)增。

4. 細(xì)胞長期保存標(biāo)準(zhǔn)

• 凍存液配方：90% 優(yōu)質(zhì)完全培養(yǎng)基（或 80% 基礎(chǔ) RPMI-1640 + 10% FBS）加 10% 最高分析級二甲基亞砜（DMSO）。亦可直接使用商用無血清型細(xì)胞高級凍存液。

• 冷凍降溫規(guī)范：

  1. 收集形態(tài)健康、處于對數(shù)生長最旺盛期的 NCI-H2373 細(xì)胞，離心收集細(xì)胞沉淀。

  2. 用配制好的冷凍液懸浮，調(diào)整細(xì)胞終密度至 每毫升 1,000,000 到 3,000,000 個活細(xì)胞。

  3. 分裝入無菌凍存管，立刻移入標(biāo)準(zhǔn)程序降溫盒（如 Mr. Frosty）。

  4. 將降溫盒投入 -80 ℃ 超低溫冰箱中過夜梯度降溫（確保達(dá)到 $-1\text{ }^\circ\text{C/min}$ 的標(biāo)稱降溫速率）。

  5. 24 小時內(nèi)，迅速將凍存管轉(zhuǎn)移入液氮罐（-196 ℃）中鎖死長期保存。嚴(yán)禁在 -80 ℃ 冰箱中無限期擱置，以防微小的溫度震蕩導(dǎo)致胞內(nèi)冰晶融化破壞間皮瘤細(xì)胞的超微膜結(jié)構(gòu)及特定的基因突變表型。

Part 2 English Section

I General Information and Cell Biological Background

• Cell Line Name: NCI-H2373 (also alternative standard nomenclature variations include H2373 or NCIH2373).

• Organism and Patient Demographics: Human (Homo sapiens); derived from an elderly Caucasian male patient.

• Tissue Extract and Pathological Framework:NCI-H2373 is a human malignant cell line isolated from a patient diagnosed with Malignant Pleural Mesothelioma (MPM). The cell line was successfully established and characterized by the National Cancer Institute (NCI).Malignant pleural mesothelioma is an aggressive, highly lethal neoplasm arising from the mesothelial linings of the pleural cavity, standardly linked to chronic occupational exposure to asbestos fibers. Because clinical MPM is refractory to conventional cytoreductive chemotherapies, the NCI-H2373 platform serves as a vital model utilized globally to map mesothelioma progression mechanisms, investigate clonal heterogeneity, and screen candidate targeted or immunotherapeutic compounds.

• Core Morphological Phenotype and Identity Characterization:

  • Morphological Structure: Adherent growth matrix. Under phase-contrast inverted profiling, NCI-H2373 presents a mixed topology comprising epithelioid-like elements and elongated fibroblast-like spindle structures. Cells manifest as irregular polygonal or stretched units featuring clear cytoplasm and prominent nucleoli. Upon achieving elevated density thresholds, they demonstrate standard oncogenic traits, including multilayered overlapping patterns and lost contact inhibition.

  • Genomic and Expression Profiles: Biomolecular validation confirms that this line stably retains core diagnostic markers associated with mesothelioma pathology, including Calretinin and WT1 (Wilms Tumor 1). In addition, it harbors genetic signatures characteristic of aggressive MPM, such as frequent functional deletions or loss-of-function mutations in critical tumor suppressors (e.g.,NF2/Merlin,BAP1, or CDKN2A/p16), presenting an ideal platform for exploring synthetic lethality screening cascades.

• Biosafety Threshold: Rated at Biosafety Level 1 (BSL-1).

II Strategic Research Value and Translational Oncology Applications

The NCI-H2373 line acts as a solid pre-clinical foundation for exploring mesothelioma vulnerability networks and evaluating novel candidate therapies:

1. Assembling Human Mesothelioma Xenograft Models (CDX Matrices):By engrafting NCI-H2373 cells subcutaneously or orthotopically within the pleural spaces of immunodeficient rodent hosts (e.g., BALB/c Nude or NOD/SCID mice), investigators can establish robust solid tumor models. These in vivo setups are required to validate the therapeutic efficacy, tumor-homing kinetics, and pharmacokinetics (PK) of novel anti-cancer agents.

2. High-Throughput Synthetic Lethality Drug Screening:Exploiting the endogenous tumor suppressor mutations (such as BAP1 or NF2 deficits) within NCI-H2373, this line is standardly deployed to evaluate the therapeutic potency of next-generation small-molecule inhibitors, including EZH2 blockers, PARP inhibitors, or Hippo signaling pathway modulators, accelerating the discovery of novel second- and third-line clinical therapeutic strategies.

3. Deconstructing Immuno-Oncology Cascades and Tumor Microenvironment Dynamics:Utilized to measure the expression kinetics of surface PD-L1 and investigate immune evasion pathways. Co-culturing NCI-H2373 with human peripheral blood mononuclear cells (PBMCs) or cytotoxic T cells helps clarify how pleural mesothelioma targets deploy specific immunosuppressive secretomes to deactivate host immune cells.

III Laboratory Thawing, Cultivation, Maintenance, and Subculturing Protocols

1. Basal Media Formulation and Atmospheric Environment Variables

• Basal Medium Base: Standard RPMI-1640 medium matrix.

• Complete Growth Matrix Formulation:

  • Basal RPMI-1640 medium template

  • Supplemented with 10% premium Fetal Bovine Serum (FBS)

  • Fortified with 1% standard Penicillin-Streptomycin dual antibiotic cocktail (100 U/mL Penicillin + 100 $\mu$g/mL Streptomycin).

• Cell Dissociation Solution: Standard 0.25% Trypsin - 0.02% EDTA mix.

• Physical Environmental Settings: Calibrate the incubator strictly to 37 °C, packed with 5% Carbon Dioxide ($CO_2$) under continuous humified saturation conditions.

2. Cryovial Thawing and Monolayer Recovery Protocol

1. Pre-warm a sterile T25 culture flask filled with 5 - 6 mL of complete growth medium to 37 °C inside the biosafety workstation.

2. Retrieve the NCI-H2373 cryovial from liquid nitrogen and submerge it instantly inside a 37 °C water bath. Agitate the vial continuously to melt the internal matrix within 60 seconds.Never allow slow thawing, as ice recrystallization during slow warming cycles can compromise cell membranes and decrease viability.

3. Mist the exterior shell with 75% ethanol before transfer into the biosafety station.

4. Draw up the fluid and transfer it slowly into a 15 mL conical tube containing 4 - 5 mL of pre-warmed complete growth medium to dilute the toxic DMSO footprint.

5. Centrifuge the suspension at 1000 rpm (~200 g) at room temperature for 5 minutes, then aspirate the chemical-laden supernatant.

6. Administer 1 mL of fresh complete growth medium onto the pellet and resuspend very gently using a P1000 micro-pipette.

7. Dispense the cells evenly into the prepared T25 flask, mix gently in a cross pattern to prevent localized cell crowding, and place into the 37 °C, 5% $CO_2$ incubator.

8. Perform a complete medium replacement 24 hours post-thaw to clear residual dead cell fragments and debris.

3. Routine Adherent Subculturing and Passaging Routines

• Confluency Assessment Control: Subculturing mechanics must be executed when the mixed epithelioid/spindle monolayers reach 80% - 85% confluency. Allowing NCI-H2373 cells to become overly dense prompts focus-formation and vertical stacking, causing the lower structural layers to detach due to localized nutrient deprivation. Expect a standard passaging schedule every 3 - 4 days.

• Passaging Execution Steps:

  1. Aspirate the spent growth fluid and wash the cell sheet once with sterile, calcium/magnesium-free PBS to remove any residual serum proteins that could inactivate the trypsin.

  2. Administer an appropriate thin layer of 0.25% Trypsin-EDTA solution (typically 1 mL for a T25 format) and incubate at 37 °C.

  3. Microscopic Tracking: Mesothelioma cells exhibit tight intercellular junctions and strong focal adhesions; trypsinization typically completes within 2 - 4 minutes at 37 °C. Monitor under an inverted microscope until the spindle shapes contract, round up, and begin to slide off the surface upon gentle tapping.

  4. Immediately add 2 volumes of serum-fortified complete growth medium to stop the enzymatic cleavage.

  5. Gently pipette the suspension against the flask wall to break up clusters, producing a homogenous single-cell suspension.

  6. Spin the cells down at 1000 rpm for 5 minutes, discard the trypsin-laden fluid, and resuspend in fresh complete growth medium.

  7. Seed the cells into new flasks utilizing standard split ratios of 1:2 to 1:4, top off with complete medium, and return to the incubator.

4. Long-Term Cryopreservation Parameters

• Cryoprotectant Preservation Formula: 90% fresh complete growth medium (or 80% basal RPMI-1640 + 10% premium FBS) packed with 10% analytical-grade Dimethyl Sulfoxide (DMSO).

• Controlled Gradient Freezing Protocol:

  1. Harvest healthy, log-phase NCI-H2373 monolayers showing typical mixed morphology. Centrifuge and isolate the pellet.

  2. Resuspend the cells in the chilled cryoprotectant matrix to achieve a target cell density of 1,000,000 to 3,000,000 cells per milliliter.

  3. Transfer the solution into sterile cryovials and place them immediately into a standard controlled-rate cooling container (e.g., Mr. Frosty).

  4. Deposit the cooling container inside a -80 °C ultra-low freezer overnight to execute a steady cooling rate of -1 °C/minute.

  5. Within 24 hours, quickly transfer the vials into liquid nitrogen storage tanks (-196 °C) for long-term preservation. Do not store vials indefinitely inside a -80 °C freezer, as minor temperature variations can compromise membrane integrity and degrade specific mutational stability phenotypes

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