F-11 BioVector® 条件永生化神经瘤细胞株

BioVector® F-11 条件永生化神经瘤细胞株文本版说明书

BioVector® F-11 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® F-11 条件永生化大鼠/小鼠杂交神经瘤细胞株 (BioVector® F-11 Conditionally Immortalized Rat/Mouse Hybrid Neuroblastoma Cell Line)

• 常用别名 (Synonyms)：BioVector® F-11，BioVector® F11 Cell

• 生物学来源 (Organism Source)：体细胞杂交株（大鼠 Rattus norvegicus 胚胎背根神经节神经元 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，细胞呈多边形、成纤维细胞样或未成熟神经母细胞样状态，分裂速度较快。(Adherent growth, presenting as a monolayer of polygonal, fibroblast-like, or immature neuroblast-like progenitor cells with relatively rapid division.)

  • 分化期/成熟后 (Differentiated Phase)：细胞停止有丝分裂，胞体皱缩并向外延伸出明显的神经元样突触，细胞间形成突触网状互联结构。(Mitotic division ceases. Cell bodies refract and extend prominent neuron-like neurites, developing an interconnected synaptosome-like network.)

• 杂交永生化机制 (Immortalization Mechanism)：通过聚乙二醇（PEG）介导技术，将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融合，通过 HAT 培养基筛选获得既能无限增殖又保留感觉神经元特征的杂交细胞系。(Engineered via Polyethylene Glycol mediated somatic cell fusion between primary rat embryonic dorsal root ganglion neurons and HPRT-deficient mouse N18TG2 neuroblastoma cells, isolated via HAT selection to secure an immortalized lineage retaining sensory hallmarks.)

• 核心科研价值 (Core Research Significance)：BioVector® F-11 是研究外周痛觉神经元机制最经典、最稳定的底盘细胞系之一。由于其同时具备神经瘤的易培养性与 DRG 神经元的功能活性，被广泛应用于热与机械痛觉感受性研究、阿片类受体与缓激肽受体信号通路分析、外周神经营养因子功能鉴定、以及痛觉过敏和镇痛小分子药物的体外高通量筛选。(BioVector® F-11 stands as one of the most classic and robust cellular chassis for exploring peripheral nociceptive pathways. Blending the culture hardiness of neuroblastomas with the functional receptors of DRG neurons, it is widely used for thermal/mechanical nociception modeling, opioid and bradykinin receptor signaling profiling, and high-throughput screening of analgesics.)

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

BioVector® F-11 杂交株在常规培养时表现为瘤样增殖，通过化学诱导可迅速向外周感觉神经元表型转化：The BioVector® F-11 hybrid line demonstrates tumor-like proliferation under base conditions, but converts rapidly toward a peripheral sensory neuron phenotype upon chemical induction:

未分化增殖状态 (Proliferating State)

细胞倍增时间通常在 20 到 30 小时之间。细胞形态相对扁平或呈梭形，汇合度高时会发生多层堆叠生长。The cell doubling time typically spans 20 to 30 hours. Cell morphology presents as relatively flat or spindle-shaped, exhibiting multi-layer stacking at high confluency.

诱导分化成熟状态 (Differentiated State)

当向培养基中加入低血清以及低浓度的 BioVector® Forskolin 或分化补剂后，细胞停止有丝分裂并伸展出长达数百微米的神经轴突。其功能标志物包括：Upon exposure to low-serum conditions coupled with BioVector® Forskolin or dedicated differentiation supplements, cells arrest mitosis and project neurites spanning hundreds of micrometers. Its functional biomarkers include:

• 特异性感觉受体 (Sensory Receptors)：高水平表达功能性 TRPV1（辣椒素受体），能对辣椒素刺激产生显著的阳性钙内流响应；同时高表达缓激肽 B2 受体（Bradykinin B2 Receptor）及 delta、mu 阿片受体。(Highly expresses functional TRPV1 capsaicin receptors, yielding robust calcium influx upon capsaicin challenge; co-expresses Bradykinin B2 receptors alongside delta and mu opioid receptors.)

• 神经元结构蛋白 (Structural Halmarks)：高表达外周神经特异性中间丝蛋白（外周蛋白 Peripherin），以及常规神经丝蛋白（Neurofilament）和 Tuj1 蛋白。(Demonstrates elevated expression of the peripheral nerve-specific intermediate filament Peripherin, alongside neurofilament structures and Tuj1 protein.)

• 电生理及神经递质特征 (Electrophysiological Traits)：分化后的细胞具备对电刺激产生功能性全或无动作电位的能力，并能合成和释放与痛觉传导密切相关的神经递质。(Differentiated entries display the capability to generate functional all-or-none action potentials under clamp stimulation and synthesize neurotransmitters associated with pain transmission.)

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

警告 / Critical WarningBioVector® F-11 属于融合杂交株，传代密度过低会导致细胞丧失分泌因子支持而大面积死亡。分化诱导时必须降低血清浓度并加入分化补剂，否则细胞将维持瘤性增殖而不长突触。BioVector® F-11 is a hybrid line; seeding cells at excessively low densities deprives them of autocrine support, leading to widespread cell death. During differentiation induction, serum metrics must be down-regulated, otherwise cells persist in tumorous expansion without neurite projection.

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 4.5 g/L 葡萄糖，含 L-谷氨酰胺，含丙酮酸钠 / containing 4.5 grams per liter Glucose, with 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 减毒低血清高糖培养基（或直接采用 BioVector® Neurobasal Medium 神经元基础培养基）。

• 降低后的血清 (Reduced Serum)：0.5% 到 1.0% BioVector® Premium Fetal Bovine Serum 优质胎牛血清（低血清是退出细胞周期的核心原动力）。

• 核心功能组件 (Core Supplement)：1% BioVector® B-27 Supplement (50X 标准型)。

• 分化与受体启动剂 (Core Differentiation Inducers)：

  • BioVector® Forskolin (福司高林补剂)：终浓度为 10 到 20 uM（用以激活腺苷酸环化酶，上调 cAMP 信号通路，促使 TRPV1 通道和轴突表达 / Applied at 10 to 20 uM final concentration to stimulate adenylate cyclase and upregulate cAMP pathways for TRPV1 channel and axon expression）。

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)：

  • 增殖期 (Growth Phase)：日常扩增与细胞传代时，细胞贴壁能力极强，可直接接种于未包被的普通塑料培养瓶中。(Cells exhibit high adherence and can be passaged into uncoated tissue culture-treated flasks during expansion.)

  • 分化期 (Differentiation Phase)：为了防止分化后期神经轴突回缩拉扯导致胞体成片脱落，在进行分化实验接种前，建议提前使用 BioVector® Poly-L-Lysine（聚左旋赖氨酸，PLL，0.01%）对孔板进行底物包被。(To prevent long neurite tension from pulling cell bodies into floating clusters, it is highly recommended to pre-coat experimental plates with BioVector® Poly-L-Lysine (PLL, 0.01%) prior to differentiation seeding.)

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)：当细胞融合度达到 80% 到 85% 时必须及时传代。如果细胞彻底长满并发生多层重叠，接触抑制会导致细胞自发老化并降低后续对辣椒素的响应灵敏度。(Passage precisely when confluency hits 80% to 85%. Allowing overgrowth or multi-layer over-stacking induces contact-mediated senescence, diminishing downstream functional responsiveness to capsaicin.)

• 建议传代稀释比例 (Splitting Ratio)：1:4 到 1:6（通常每 2 到 3 天需传代一次）。

1. 完全抽干陈旧的培养基，使用无菌的 BioVector® PBS 缓冲液轻轻洗涤细胞表面 1 次。(Aspirate spent medium and rinse the monolayer once with sterile BioVector® PBS.)

2. 加入适量预热的 BioVector® 0.25% Trypsin-EDTA 消化液，置于 37 摄氏度孵箱中消化 1 到 2 分钟。(Introduce an appropriate volume of pre-warmed BioVector® 0.25% Trypsin-EDTA and incubate at 37 degrees Celsius for 1-2 minutes.)

3. 镜下观察到大部分细胞变圆变亮，轻敲培养瓶四周见细胞成片脱落时，立即加入双倍体积的含血清增殖期完全培养基终止胰酶活性。(Once cells round up and release upon light tapping, immediately add double the volume of serum-fortified Phase A growth medium to halt trypsinization.)

4. 用移液枪轻柔吹打贴壁表面，配置成均匀无团块的单细胞悬液。(Pipette gently to disperse the mixture into a uniform single-cell suspension.)

5. 将悬液收集至离心管，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 discard the supernatant.)

6. 加入新鲜的 BioVector® 增殖期完全生长培养基重悬，调整接种密度并分装至新培养瓶中。(Resuspend in fresh growth medium and allocate at correct densities into fresh flasks.)

实验前感觉神经元样短周期分化流程 / Directed Short-Cycle Differentiation Protocol

1. 将增殖期的 BioVector® F-11 细胞消化重悬，以大约 2.5乘以10的4次方 cells/cm2 的密度接种于提前使用 BioVector® PLL 包被 的实验板中。(Digest cells and seed them at a density of approx 2.5 x 10^4 cells/cm2 into experimental plates pre-coated with BioVector® PLL.)

2. 使用常规的 10% 血清完全培养基让细胞贴壁恢复过夜（约 16 小时）。(Maintain in Phase A growth medium overnight for approx 16 hours to facilitate initial anchoring.)

3. 次日，完全吸干孔板内的培养基，更换为含有 10 到 20 uM BioVector® Forskolin 的 B阶段：神经功能分化培养基（血清降至 1.0% 或以下）。(The following day, completely evacuate the wells and introduce low-serum Phase B Neuro-Functional Differentiation Medium fortified with 10 to 20 uM BioVector® Forskolin.)

4. 诱导 48 到 96 小时（2 到 4 天） 期间，细胞将完全退出有丝分裂，并长出长而清晰的交织神经突触。此时细胞表面的 TRPV1 离子通道与阿片受体功能达到表达峰值，可直接用于辣椒素或缓激肽刺激引发的钙成像荧光检测、全细胞电生理膜片钳分析以及疼痛信号转导通路阻断实验。(Incubate continuously for 48 to 96 hours (2 to 4 days) until mitotic expansion ceases and clear neurite webs form. At this stage, operational TRPV1 channels and opioid receptors peak in expression, making the system ideal for capsaicin/bradykinin-triggered Calcium Imaging, patch clamp diagnostics, or nociceptive pain transduction pathway 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 37 摄氏度孵箱中平衡 pH 值。(Pre-warm 5.0 mL of Phase A growth medium in a T25 flask and balance inside the incubator to stabilize pH levels.)

2. 从液氮中取出 BioVector® F-11 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® F-11 cryovial and plunge it immediately into a 37 degrees Celsius water bath, shaking horizontally to melt completely 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 conical tube carrying 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 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。属于常规的啮齿类动物工程化杂交系，无已知人类病原体传染性，实验废弃物按常规医疗废弃物高压灭菌处理即可。(BSL-1 classification. Represents a standard rodent engineered hybrid line with no known viral threats to humans; manage spent items via institutional biohazard waste 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）。切勿长期搁置在零下 80 摄氏度机械冰箱中，否则细胞株的复苏存活率以及分化成长突触的活性会随着时间推移发生大幅衰退。(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 prohibited, as it compromises post-thaw survival metrics and neurite projection vigor.)

• TRPV1 功能质控红线 / Functional Quality Control Threshold：随着传代次数的增加（连续传代超过 30 代以上），杂交瘤细胞容易发生染色体丢失或表型退化，表现为：在加入 BioVector® Forskolin 诱导后虽然能变长，但对辣椒素（Capsaicin）刺激不再产生任何钙流内流信号。因此，建议每隔 5 到 10 代，挑取一孔分化后的细胞加入 1 uM 的 Capsaicin，通过钙荧光染料验证其受体功能性。若功能性消失，需立即废弃该代次细胞，并重新复苏低代次种子库。(Extended passaging (exceeding 30 continuous subcultures) can trigger chromosomal dropouts or phenotypic drifts in hybridomas, where cells project extensions under induction but fail to generate calcium flux signals upon Capsaicin challenge. To preserve functional consistency, challenge a differentiated well every 5 to 10 passages with 1 uM Capsaicin via calcium-sensitive indicators. If specific signaling diminishes, the culture has drifted; discard immediately and re-thaw a lower-passage seed vial.)

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