BioVector® ND7/23 条件永生化神经元细胞株文本版说明书

BioVector® ND7/23 Conditionally Immortalized Neuroblastoma Hybrid Cell Line Text-Based Datasheet

1 产品基本信息与遗传背景 / Product Identification and Genetic Background

• 产品名称 (Product Name)：BioVector® ND7/23 条件永生化小鼠/大鼠杂交感觉神经元细胞株 (BioVector® ND7/23 Conditionally Immortalized Mouse/Rat Hybrid Sensory Neuron Cell Line)

• 常用别名 (Synonyms)：BioVector® ND7/23，BioVector® ND7，BioVector® ND7-23 Cell

• 生物学来源 (Organism Source)：体细胞杂交株（大鼠 Rattus norvegicus 新生背根神经节神经元 融合 小鼠 Mus musculus N18Tg2 神经瘤细胞）

• 组织器官定位 (Tissue and Organ Site)：外周神经系统 / 背根神经节感觉神经元特性 (Peripheral Nervous System / Dorsal Root Ganglion Sensory Neuron Properties)

• 生长特性 (Growth Properties)：

  • 增殖期/未分化 (Proliferating Phase)：贴壁生长，细胞多呈圆球形、短梭形或折光度高的未分化成纤维细胞样形态，贴壁较轻，极易发生集聚生长。(Adherent growth, presenting as spherical, short spindle-shaped, or highly refractive undifferentiated fibroblast-like cells. They exhibit relatively light attachment and easily cluster together.)

  • 分化期/成熟后 (Differentiated Phase)：细胞彻底停止分裂，胞体收缩并变亮，迅速延展出极长的双极或多极神经轴突，突触间形成错综复杂的有功能的感觉神经网。(Mitotic division completely ceases. Cell bodies refract and contract, rapidly projecting extremely long bi-polar or multi-polar neurites that form an intricate, functional sensory neuronal network.)

• 杂交永生化机制 (Immortalization Mechanism)：利用聚乙二醇（PEG）技术，将新生大鼠原代背根神经节（DRG）感觉神经元与缺少 HPRT 酶的小鼠 N18Tg2 神经母细胞瘤细胞进行无性系杂交融合，通过 HAT 培养基筛选获得既具备无限增殖能力又完好保留 DRG 伤害感受器特性的靶向工具株。(Engineered via Polyethylene Glycol mediated clonal fusion between primary neonatal rat dorsal root ganglion neurons and HPRT-deficient mouse N18Tg2 neuroblastoma cells, followed by HAT selection to yield an immortalized tool line retaining classic DRG nociceptive traits.)

• 核心科研价值 (Core Research Significance)：BioVector® ND7/23 是全球疼痛机制研究与外周神经科学领域使用频率极高的标志性细胞系。与原代 DRG 神经元相比，它具有极高的稳定性和极低的操作难度。它被广泛用于机械痛、热痛、酸感外周敏化通路的研究，特别是机械敏感性离子通道（如 Piezo1 和 Piezo2）、温度敏感性 TRP 通道（如 TRPV1、TRPM8）以及电压门控钠通道（如 Nav1.7、Nav1.8）的表达与电生理功能解析，也是高通量筛选外周镇痛药的核心细胞底盘。(BioVector® ND7/23 stands as a globally recognized benchmark cell line in peripheral neuroscience and pain pathogenesis research. Offering superior robustness and simpler handling compared to primary DRG extractions, it is heavily used for mechanistic mapping of mechanical, thermal, and acid-evoked peripheral sensitization pathways. It is ideal for analyzing mechanosensitive channels (Piezo1/2), thermosensitive TRP channels (TRPV1/TRPM8), voltage-gated sodium currents (Nav1.7/Nav1.8), and serves as a premier platform for high-throughput screening of peripheral analgesics.)

2 细胞生物学特征与核心标志物表型 / Cellular Properties and Biomarker Profiles

BioVector® ND7/23 兼具神经瘤的快速有丝分裂特性与感觉神经元的分化潜能，其生理特征随培养基配方而发生精准改变：The BioVector® ND7/23 hybrid line balances the rapid mitotic drive of a neuroblastoma with the differentiation potential of a sensory neuron, showing precise phenotypic transitions dictated by media formulations:

未分化增殖状态 (Proliferating State)

细胞倍增速度快（约 20 到 24 小时）。细胞表面黏附力较弱，在普通塑料瓶中长满时极易聚集成团块，若受到剧烈震动会有部分细胞自发悬浮。Cells proliferate rapidly with a doubling time of approximately 20 to 24 hours. Cell-to-substrate adhesion is naturally modest; they easily form dense clusters at high confluency and may partially detach upon mechanical agitation.

诱导分化成熟状态 (Differentiated State)

在低血清联合 BioVector® Forskolin 或神经营养因子的协同诱导下，细胞在 48 到 72 小时内迅速展现出高度特异性的感觉神经元表型：Under low-serum restriction combined with BioVector® Forskolin or neurotrophic factors, cells rapidly switch into a highly specified sensory neuron phenotype within 48 to 72 hours:

• 特征性离子通道谱 (Ion Channel Repertoire)：稳定表达大鼠源性的电压门控钠通道 Nav1.7 和对河豚毒素耐受的 Nav1.8（TTX-resistant）电流；内源性表达机械敏感通道 Piezo1 和 Piezo2；在分化促进下可高效上调 TRPV1（辣椒素受体）的表达。(Stably channels rat-derived voltage-gated sodium currents including Nav1.7 and tetrodotoxin-resistant Nav1.8; endogenously expresses mechanosensitive Piezo1 and Piezo2 channels; upregulates functional TRPV1 capsaicin receptors under optimized induction.)

• 功能性表面受体 (Surface Receptors)：高水平表达经典的外周痛觉介导受体，包括嘌呤受体（P2X3）、缓激肽受体（B2）以及降钙素基因相关肽（CGRP）受体。(Robustly targets classic peripheral nociceptive signaling receptors, including purinergic P2X3, bradykinin B2, and CGRP receptors.)

• 骨架与标志物蛋白 (Structural Halmarks)：外周蛋白（Peripherin）呈现强阳性表达，神经元特异性核蛋白 NeuN 以及 Tuj1 表达显著上调。(Exhibits powerful positive expression of the peripheral filament Peripherin, alongside marked upregulation of the neuronal markers NeuN and Tuj1/Beta-III Tubulin.)

3 专用两阶段培养基配方规范 / Culturing Medium Formulations

警告 / Critical WarningBioVector® ND7/23 细胞贴壁性较弱，在日常扩增传代时，千万不可将细胞密度稀释得过低（如低于 1比10），否则细胞会因缺乏局部自分泌因子支持而停止生长或发生大量自溶死亡。BioVector® ND7/23 cells exhibit relatively weak substrate adherence. During routine expansion, never split the cultures too sparsely (e.g., exceeding a 1:10 ratio), as the absence of crucial local autocrine signaling will halt growth or induce extensive autolytic lysis.

A 阶段：增殖期完全生长培养基（用于常规细胞扩增与维持）

Phase A: Proliferating Complete Growth Medium (For Routine Expansion and Maintenance)

• 基础培养基 (Basal Medium)：BioVector® High-Glucose DMEM 高糖培养基（含 L-谷氨酰胺，含丙酮酸钠 / containing L-Glutamine and sodium pyruvate）。

• 血清添加 (Serum Supplement)：10% BioVector® Premium Fetal Bovine Serum 优质胎牛血清。

• 双抗补剂 (Antibiotics)：1% BioVector® Penicillin-Streptomycin Solution 青霉素-链霉素溶液。

B 阶段：神经功能分化培养基（实验前伤害感受器功能诱导）

Phase B: Neuro-Functional Differentiation Medium (For Pre-Experimental Nociceptor Induction)

• 基础骨架 (Basal Matrix)：BioVector® Low-Serum High-Glucose DMEM 减毒低血清培养基（或采用无血清 BioVector® Neurobasal Medium）。

• 降低后的血清 (Reduced Serum)：0.5% 到 1.0% BioVector® Premium Fetal Bovine Serum 优质胎牛血清（严格限制血清浓度以强迫细胞退出分裂周期）。

• 核心功能组件 (Core Supplement)：2% BioVector® B-27 Supplement (50X 标准型)。

• 分化与通道启动剂 (Core Differentiation Inducers)：

  • BioVector® Forskolin (福司高林补剂)：终浓度为 10 到 20 uM（必加成分，用于上调胞内 cAMP 以驱动轴突爆发延伸并活化 TRPV1 等通道 / Essential component; drives intracellular cAMP to propel explosive neurite branching and functionalize TRP channels）。

  • BioVector® Recombinant Rat NGF (大鼠重组神经生长因子，可选)：终浓度 50 ng/mL（联合 Forskolin 使用可显著增强 Nav1.8 等通道的电生理电流密度 / Optional; synergizes with Forskolin to substantially enhance the current density of voltage-gated Nav1.8 channels）。

4 物理环境与贴壁包被控制参数 / Environmental Controls and Coating Parameters

• 孵育温度 (Incubation Temperature)：37.0 摄氏度（Constant temperature at 37.0 degrees Celsius）。

• 气相环境 (Gas Phase)：5% 二氧化碳 (CO2)，加湿平衡空气 (5% Carbon Dioxide balanced with humidified atmospheric air)。

• 基质包被规范 (Matrix Coating Protocol - Critical)：

  • 增殖期 (Growth Phase)：日常传代扩增时，细胞可直接接种于未包被的普通组织培养塑料瓶（TC-treated）中，但操作时需轻拿轻放，避免剧烈摇晃导致细胞成片漂浮。(Cells can be expanded directly within uncoated standard tissue culture-treated flasks, but handle gently to prevent physical shaking from detaching the cluster sheets.)

  • 分化期 (Differentiation Phase)：在进行分化功能实验前，必须提前使用 BioVector® Poly-L-Lysine（聚左旋赖氨酸，PLL，0.01%）对孔板或盖玻片进行基础包被，随后加铺一层 BioVector® Laminin（层粘连蛋白，5 到 10 ug/mL）进行复合包被。只有复合包被底物才能牢固锚定长轴突，防止其在换液或加药刺激时脱落。(Prior to functional assays, experimental plates or coverslips must be pre-coated with BioVector® Poly-L-Lysine (PLL, 0.01%), followed by a secondary coating of BioVector® Laminin (5 to 10 ug/mL). This composite extracellular matrix is vital to anchor extended neurites, preventing culture shedding during fluid changes or drug testing.)

5 细胞传代与诱导分化操作规范 / Subculturing and Differentiation Protocols

增殖期常规传代操作（未分化状态）/ Routine Passaging (Proliferating State)

• 传代临界点 (Passaging Threshold)：当细胞密度达到 80% 汇合度时必须传代。切勿让 BioVector® ND7/23 完全长满并重叠堆积，否则会导致细胞大面积老化脱落，并不可逆地丧失向伤害感受器分化的潜能。(Passage promptly when cell density reaches 80% confluency. Allowing the cells to overgrow and pile up triggers widespread detachment and irreversible loss of nociceptive differentiation potential.)

• 建议传代稀释比例 (Splitting Ratio)：1:3 到 1:5（常规每 2 天传代一次）。

1. 完全抽干陈旧的生长培养基，使用无菌的 BioVector® PBS 缓冲液轻轻洗涤细胞单层 1 次。(Carefully aspirate spent medium and rinse the monolayer once with sterile BioVector® PBS.)

2. 加入少量的 BioVector® 0.25% Trypsin-EDTA 消化液（由于该细胞贴壁较轻，胰酶用量应少于常规细胞），在室温或 37 摄氏度孵箱中静置消化 30 秒 到 1 分钟。(Introduce a modest volume of BioVector® 0.25% Trypsin-EDTA; due to light attachment, use less volume than standard lines and incubate for 30 seconds to 1 minute.)

3. 镜下观察发现大部分细胞变圆，轻敲瓶壁见细胞呈细沙状快速滑落时，立刻加入双倍体积的含血清 BioVector® 增殖期完全培养基终止消化。(Once cells round up and slip away freely upon light tapping, immediately add double the volume of serum-containing Phase A medium to completely halt trypsinization.)

4. 用移液枪极其轻柔地吹打瓶壁，切勿用力过猛，将细胞配制成均匀的单细胞悬液。(Pipette very gently to break up remaining clusters into a uniform single-cell suspension without mechanical shear damage.)

5. 在 200乘以g（低速离心力）下离心 5 分钟，彻底弃去含有残留胰酶的上清液。(Centrifuge at 200 x g for 5 minutes and thoroughly vacuum away the spent supernatant.)

6. 加入新鲜的 BioVector® 增殖期完全生长培养基重悬，计数后接种至新的培养瓶中。(Resuspend in fresh Phase A growth medium, determine count, and allocate into fresh expansion vessels.)

实验前感觉神经元样分化流程 / Directed Sensory Differentiation Protocol

1. 将增殖期的 BioVector® ND7/23 细胞消化，以大约 3.0乘以10的4次方 cells/cm2 的中等密度接种于提前执行过 BioVector® PLL/Laminin 复合包被 的实验板中。(Digest cells and seed them at a moderate density of approx 3.0 x 10^4 cells/cm2 into multi-well plates pre-treated with the BioVector® PLL/Laminin composite matrix.)

2. 首先使用常规的 10% 血清完全培养基，让细胞贴壁恢复过夜（约 12 到 16 小时）。(Maintain initially in standard Phase A medium overnight for 12-16 hours to ensure secure initial anchoring.)

3. 次日，完全抽干原培养基，更换为含有 10 到 20 uM BioVector® Forskolin 的 B阶段：神经功能分化培养基。(The following day, thoroughly evacuate the wells and introduce low-serum Phase B Neuro-Functional Differentiation Medium fortified with 10 to 20 uM BioVector® Forskolin.)

4. 持续诱导 48 到 72 小时（2 到 3 天），细胞将长出极其复杂的网状互联突触，此时细胞表面的 Nav1.7、Nav1.8 钠通道和机械敏感 Piezo 通道功能达到表达巅峰。此时即可直接用于全细胞膜片钳电生理记录、机械划痕刺激或辣椒素刺激引发的钙成像荧光检测。(After 48 to 72 hours (2 to 3 days) of continuous induction, an intricate network of interconnected neurites will mature. At this juncture, the functional expression of surface Nav1.7, Nav1.8, and mechanosensitive Piezo channels peaks, rendering the cultures fully primed for patch-clamp electrophysiology, mechanical indentation mapping, or capsaicin-evoked Calcium Imaging assays.)

6 复苏与冻存恢复流线 / Thawing and Post-Cryo Recovery Workflow

1. 提前在标准 T25 培养瓶中注入 5.0 mL 预热的增殖期完全生长培养基，置于孵箱中平衡 pH 值与温度。(Pre-warm 5.0 mL of Phase A growth medium in a sterile T25 flask and equilibrate inside the incubator to stabilize pH levels and thermal baselines.)

2. 从液氮中取出 BioVector® ND7/23 冻存管，立刻全量投入 37 摄氏度恒温水浴箱中快速水平摇晃，在 60 秒内使其快速完全融化。(Retrieve the BioVector® ND7/23 cryovial from liquid nitrogen and plunge it immediately into a 37 degrees Celsius water bath, agitating horizontally to complete the melting sequence within 60 seconds.)

3. 酒精表面消毒后移入超净台，用移液枪吸出胞悬液，缓慢逐滴注入盛有 4.0 mL 预热增殖培养基的 15 mL 离心管中。(Sanitize the vial exterior with 75% ethanol and dropwise introduce the suspension into a 15 mL conical tube packed with 4.0 mL of pre-warmed growth medium.)

4. 在 200乘以g 下低速离心 5 分钟，彻底吸干含有毒 DMSO 的上清液。(Centrifuge at 200 x g for 5 minutes and thoroughly vacuum away the toxic DMSO-laden supernatant.)

5. 加入 1.5 mL 新鲜增殖培养基轻弹悬起细胞沉淀，随后全量接种到预热平衡的 T25 瓶中。(Add 1.5 mL of fresh proliferation medium, tap gently to scatter the cell pellet, and transfer the pool into the pre-stabilized T25 flask.)

6. 置于 37 摄氏度孵箱栽培。24 小时后必须进行一次全量换液，以清除未贴壁的死细胞碎片和残留毒性成分。(Incubate at 37 degrees Celsius, 5% CO2. Perform a mandatory total medium replenishment 24 hours post-thaw to eliminate dead cell drift and trace chemical impurities.)

7 生物安全、长期保存与质控规范 / Biosafety, Storage and Quality Controls

• 生物安全级别 (Biosafety Level)：BSL-1。属于安全的工程化啮齿类动物杂交系，无已知对人类具致病性的病毒成分，实验废弃物执行常规高压灭菌消毒程序。(BSL-1 classification. Represents a safe rodent engineered hybrid line free of known human viral threats; manage spent culture items via standard institutional biohazard autoclaving disinfection streams.)

• 超低温长期保存 (Long-Term Banking)：冻存保质配方为 90% BioVector® 增殖期完全生长培养基 + 10% 细胞级 DMSO（或采用 BioVector® Serum-Free Cryopreservation Medium 无血清通用型冻存液）。冻存管必须永久存放于液氮环境中（零下 150 摄氏度至零下 196 摄氏度）。严禁在零下 80 摄氏度机械冰箱中长期搁置超过 2 周，否则该细胞株的复苏存活率以及分化成长突触的电生理活性会发生断崖式下跌。(The standard freezing blend consists of 90% Phase A growth medium + 10% analytical grade DMSO, or BioVector® Serum-Free Cryopreservation Medium. Vials must reside permanently inside liquid nitrogen vapor or liquid phase (minus 150 to minus 196 degrees Celsius). Storage in mechanical minus 80 degrees Celsius freezers for extended spans is strictly prohibited, as it compromises post-thaw survival rates and electrophysiological current induction.)

• Nav/Piezo 功能质控红线 / Functional Quality Control Threshold：随着传代次数的不断增加（连续传代超过 25 代以上），BioVector® ND7/23 杂交株容易发生严重的核型不稳或染色体丢失，表现为：在加入 BioVector® Forskolin 诱导后虽然能变长，但全细胞膜片钳下记录不到任何 TTX 耐受性的 Nav1.8 钠电流，或者对机械压力刺激失去了 Piezo 介导的快速失活内流。因此，建议每隔 5 到 10 代，对分化后的细胞进行一次通道表达抽检（如通过实时定量 PCR 或特异性阻断剂响应测试）。若关键通道功能丧失，证实该代次细胞已退化，需立即废弃并重新复苏低代次种子库。(Extended passaging (exceeding 25 continuous subcultures) can trigger karyotypic instability or chromosomal loss in hybridomas, where cells project extensions under induction but fail to generate functional TTX-resistant Nav1.8 sodium currents or Piezo-mediated mechanosensitive influx. To preserve functional consistency, check channel expression every 5 to 10 passages (via RT-qPCR or targeted antagonist challenge assays). If key channel activity vanishes, the culture has drifted; discard immediately and re-thaw a lower-passage seed vial from the master bank.)

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