BioVector® NCI-H2373 Human Malignant Mesothelioma Cell Line / NCI-H2373 人恶性胸膜间皮瘤细胞株

一 产品基本信息与细胞生物学背景

• 细胞名称：NCI-H2373（亦常写作 H2373）。

• 物种与人群来源：人类（Homo sapiens），源自一名高龄白人男性患者。

• 组织源起与病理背景：NCI-H2373 是一株源自人类恶性胸膜间皮瘤（Malignant Pleural Mesothelioma, MPM）组织块的恶性肿瘤细胞系。该细胞系由美国国家癌症研究所（NCI）成功建立并进行系统性鉴定。恶性胸膜间皮瘤是一种高度侵袭性、预后极差、且通常与长期接触石棉（Asbestos）密切相关的胸膜表面恶性肿瘤。由于 MPM 临床治疗手段匮乏且对常规化疗极易产生耐药性，NCI-H2373 细胞株成为全球研究间皮瘤恶性演变机制、肿瘤靶向异质性、以及临床新型靶向/免疫药物筛选的经典人类疾病模式底盘。

• 核心表型与细胞生物学特征：

  • 形态学表现：贴壁生长。在倒置显微镜下，NCI-H2373 细胞表现为明显的上皮样（Epithelial-like）与成纤维纺锤形突起混合的形态。细胞多呈多角形或不规则拉长状，胞质清晰，核仁明显。在细胞汇合度较高、密集成片时，细胞呈现出多层重叠生长或紧密交织的典型恶性肿瘤细胞排布特征。

  • 基因组与标志物图谱：经生化与分子生物学鉴定，该细胞系持续表达间皮瘤相关的核心诊断标志物（如 Calretinin 间皮素、WT1 等）。同时，作为间皮瘤特征，它常伴随有关键抑癌基因（如 NF2/Merlin、BAP1 或 CDKN2A/p16）的缺失或突变，这为其靶向合成致死（Synthetic lethality）药物的研究提供了精准的遗传背景。

• 生物安全级别：1级（BSL-1）。

二 核心科研价值与转化医学应用

NCI-H2373 细胞株在现代现代胸部肿瘤学、耐药分子机制及创新疗法开发中占有重要一席：

1. 构建间皮瘤异种移植肿瘤模型（CDX 模型）：通过将 NCI-H2373 细胞接种于免疫缺陷小鼠（如 BALB/c Nude 或 NOD/SCID）的皮下或胸膜腔内（原位移植），可成功诱导形成人类间皮瘤实体瘤病灶。这是评估新型抗癌药物在活体内（In vivo）靶向杀伤效能、药代动力学（PK）的标准化临床前生物台表。

2. 新型靶向治疗与“合成致死”药物高通量体外筛查：针对间皮瘤高频发生的 BAP1 或 NF2 基因缺陷，利用 NCI-H2373 细胞系体外评估诸如 EZH2 抑制剂、PARP 抑制剂或 Hippo 信号通路阻断剂等前沿靶向小分子药物的合成致死杀伤效应，助力挖掘突破性的二线/三线临床治疗方案。

3. 免疫检查点与肿瘤微环境交互（TME）探索：用于检测间皮瘤细胞表面 PD-L1 表达谱系的动态调控，以及通过将 NCI-H2373 与人类外周血单核细胞（PBMC）或 T 细胞在体外共培养，解构恶性间皮瘤如何通过分泌特殊细胞因子逃逸宿主免疫监视（Immune evasion）的分子病理机制。

三 实验室细胞复苏、贴壁培养、常规传代与保存标准步骤

1. 专用培养基配置与生长环境

• 基础培养基：经典的 RPMI-1640 基础培养基。

• 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-链霉素双抗溶液（100 U/mL 青霉素 + 100 $\mu$g/mL 链霉素）。

• 细胞解离液：0.25% Trypsin - 0.02% EDTA 消化液。

• 培养物理常数：标准 37 摄氏度，含 5% 二氧化碳（$CO_2$） 的恒温高湿度饱和无菌孵箱。

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 ℃ 的完全培养基。

2. 从液氮罐中取出 NCI-H2373 冻存管，立刻全量投入 37 ℃ 恒温水浴箱中，快速用力摇晃以期在 1 分钟内令管内冰块完全融化（切忌慢速解冻，否则融复过程中的冰晶二次重结晶会严重刺破肿瘤细胞膜，导致成活率剧烈崩塌）。

3. 用 75% 酒精喷洒消毒外壁，移入生物安全柜。

4. 吸取细胞悬液，缓慢滴加至盛有 4 - 5 mL 预热完全培养基的 15 mL 离心管中，轻柔颠倒一次以稀释残留的 DMSO。

5. 以 1000 rpm（约 200 g）室温温和离心 5 分钟，小心抽干含有 DMSO 的上清液。

6. 加入 1 mL 新鲜完全培养基，使用 P1000 移液枪极其轻柔地吹打重悬细胞沉淀。

7. 将悬液接种至准备好的 T25 瓶中，轻柔“十字形”摇匀以防止细胞聚集堆叠。

8. 拧松瓶盖，放入 37 ℃、5% $CO_2$ 孵箱中静置暗培养。

9. 24 小时后必须观察贴壁状态，并全量更换一次新鲜培养基，彻底清除未贴壁的死细胞和残余微量碎屑。

3. 日常贴壁常规传代操作

• 传代时机：当混合型上皮样/纺锤形的细胞密集成片，汇合度（Confluency）达到 80% - 85% 时必须传代。NCI-H2373 细胞在密度过高时会出现自发多层重叠堆积，导致下层细胞因缺氧缺营养发生局部老化脱落，传代频率通常为每 3 - 4 天一次。

• 操作流程：

  1. 吸除旧培养基，使用无菌、无钙镁离子的 PBS 缓冲液轻轻漂洗细胞表面 1 次，彻底洗净残余血清蛋白（残存的血清会强力中和并失活胰酶的催化活性）。

  2. 加入适量 0.25% Trypsin-EDTA 消化液（T25 瓶常规加入 1 mL），使其完全覆盖细胞层，随后放入 37 ℃ 孵箱中温和消化。

  3. 镜下动态观察：间皮瘤细胞的胞间连接和基底膜锚定较紧，通常在 37 ℃ 下消化 2 - 4 分钟。当在倒置显微镜下动态观察到原本拉长的纺锤形细胞胞体收缩变圆、多角形边缘变松、且轻敲培养瓶一侧时有成片细胞自发滑动，说明消化达标。

  4. 立刻向瓶内倒入 2 倍体积的含血清完全培养基，终止胰酶的酶解剪切。

  5. 用移液枪或移液管轻柔冲洗瓶壁，将细胞完全洗脱下来，吹打 3 - 5 次将其调理成均匀的单细胞悬液（如有未打散的团块容易导致后期成片局部堆积）。

  6. 将悬液收集至离心管中，1000 rpm 离心 5 分钟，弃去酶解上清。

  7. 加入新鲜完全培养基重悬沉淀，按照 1:2 至 1:4 的常规传代比例接种至新的培养瓶中，补足完全培养基，放回孵箱中继续扩增。

4. 细胞长期保存标准

• 冻存液配方：90% 优质完全培养基（或 80% 基础 RPMI-1640 + 10% FBS）加 10% 最高分析级二甲基亚砜（DMSO）。亦可直接使用商用无血清型细胞高级冻存液。

• 冷冻降温规范：

  1. 收集形态健康、处于对数生长最旺盛期的 NCI-H2373 细胞，离心收集细胞沉淀。

  2. 用配制好的冷冻液悬浮，调整细胞终密度至 每毫升 1,000,000 到 3,000,000 个活细胞。

  3. 分装入无菌冻存管，立刻移入标准程序降温盒（如 Mr. Frosty）。

  4. 将降温盒投入 -80 ℃ 超低温冰箱中过夜梯度降温（确保达到 $-1\text{ }^\circ\text{C/min}$ 的标称降温速率）。

  5. 24 小时内，迅速将冻存管转移入液氮罐（-196 ℃）中锁死长期保存。严禁在 -80 ℃ 冰箱中无限期搁置，以防微小的温度震荡导致胞内冰晶融化破坏间皮瘤细胞的超微膜结构及特定的基因突变表型。

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

BioVector NTCC质粒载体菌株细胞蛋白抗体基因保藏中心

电话:400-800-2947

工作QQ/微信同号:1843439339

网址http://www.biovector.net