BioVector® 50B11 条件永生化大鼠感觉神经元细胞株文本版说明书

BioVector® 50B11 Conditionally Immortalized Rat Sensory Neuron 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® 50B11 条件永生化大鼠感觉神经元细胞株 (BioVector® 50B11 Conditionally Immortalized Rat Sensory Neuron Cell Line)

• 常用别名 (Synonyms)：BioVector® 50B11，BioVector® 50B11 Rat DRG Line

• 生物学来源 (Organism Source)：大鼠 Rattus norvegicus（胚胎期第 14.5 天，E14.5）

• 组织器官定位 (Tissue and Organ Site)：外周神经系统 / 背根神经节 (Peripheral Nervous System / Dorsal Root Ganglion, DRG)

• 生长特性 (Growth Properties)：

  • 增殖期/未分化 (Proliferating Phase)：贴壁生长，细胞呈圆形、卵圆形或短梭形的单层前体细胞状态。(Adherent growth, presenting as a monolayer of round, ovoid, or short spindle-shaped neural progenitor cells.)

  • 分化期/成熟后 (Differentiated Phase)：有丝分裂完全停止，细胞双向或多向延展出极长的神经突触，互相交织成极其复杂的神经元互联网络。(Mitotic division completely ceases. Cells extend extremely long neurites bi-directionally or multi-directionally, intertwining into a highly complex, functional neuronal network.)

• 永生化工程机制 (Immortalization Mechanism)：细胞通过逆转录病毒转导技术，在基因组中集成了受调控的调节型 v-Myc 癌基因。(Cells are engineered via retroviral transduction to integrate a regulated v-Myc oncogene within the host genome.)

• 核心科研价值 (Core Research Significance)：原代大鼠 DRG 神经元极难成活且无法传代，BioVector® 50B11 完美解决了这一瓶颈。它广泛用于外周伤害性感受等研究，也常用于由化疗药物（如紫杉醇、顺铂）引起的周围神经病变（CIPN）的毒理学评价，以及靶向镇痛药与离子通道阻断剂的高通量药效筛选。(Primary rat DRG neurons are notoriously fragile and unpassagable; the BioVector® 50B11 line perfectly bypasses this bottleneck. It is widely utilized for studying peripheral nociception, toxicological evaluation of chemotherapy-induced peripheral neuropathy (CIPN, such as Paclitaxel or Cisplatin injury), and high-throughput screening of targeted analgesics and ion-channel blockers.)

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

BioVector® 50B11 具备极其典型的“干性/未分化”与“成熟/感觉神经元”双向表型切换特征：The BioVector® 50B11 line manifests a highly distinct bidirectional phenotypic switch between the progenitor stem state and the mature sensory neuron state:

未分化增殖状态 (Proliferating State)

细胞分裂旺盛。在常规生长培养基维持时，v-Myc 保持活跃转录，驱动细胞快速扩增（倍增时间约为 24 到 36 小时）。Cells undergo active mitosis. When maintained in standard growth medium, the v-Myc oncogene remains transcriptionally active, driving rapid biomass expansion with a doubling time of approximately 24 to 36 hours.

诱导分化成熟状态 (Differentiated State)

当向培养基中加入分化启动剂后，细胞在 24 到 48 小时内彻底退出细胞周期，停止一切有丝分裂，并迅速向外弹射出粗壮的神经轴突。其分子标志物在数天内迅速重塑：Upon adding differentiation triggers, cells completely exit the cell cycle within 24 to 48 hours, halting all mitotic activities and rapidly projecting robust neurites. Its molecular biomarker spectrum is completely rewired within a few days:

• 外周特异性标志物 (Peripheral Markers)：强阳性表达外周神经特异性中间丝蛋白（外周蛋白 Peripherin），以及神经元特异性核蛋白 NeuN、beta-III 粘管蛋白（Tuj1 / Beta-III Tubulin）。(Robustly expresses the peripheral nerve-specific intermediate filament Peripherin, alongside pan-neuronal hallmarks NeuN and Tuj1/Beta-III Tubulin.)

• 痛觉关键离子通道 (Nociceptive Ion Channels)：在特定刺激下，细胞能够稳定表达并维持高水平的 TRPV1（辣椒素受体）、TRPA1 以及电压门控钠通道 Nav1.7、Nav1.8。(Under directed stimulation, cells stably express and maintain high levels of the functional TRPV1 capsaicin receptor, TRPA1, and voltage-gated sodium channels Nav1.7 and Nav1.8.)

• 神经肽谱系 (Neuropeptide Expression)：分化后的细胞可分泌经典的外周神经肽（P物质 Substance P 和降钙素基因相关肽 CGRP），表现出经典肽能感觉神经元（Peptidergic nociceptors）的功能特性。(Differentiated cells secrete classic peripheral neuropeptides, including Substance P and Calcitonin Gene-Related Peptide, capturing the physiological traits of peptidergic nociceptors.)

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

警告 / Critical WarningBioVector® 50B11 的分化需要依赖 cAMP 信号通路的激活。在常规扩增阶段，绝对不能接触福司高林（Forskolin），否则会导致感受态细胞提前分化并丧失传代能力。Differentiation of BioVector® 50B11 relies heavily on cAMP signaling activation. During routine expansion, cultures must never be exposed to Forskolin, as this triggers premature terminal differentiation and permanent loss of passaging capability.

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，不含丙酮酸钠 / containing L-Glutamine, without 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® Neurobasal Medium 神经元基础培养基。

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

• 分化与干性关闭核心启动剂 (Core Differentiation Inducers)：

  • BioVector® Forskolin (福司高林补剂)：终浓度为 10 到 50 uM（必加成分，用于上调胞内 cAMP，启动 TRPV1 等通道表达并驱动突触爆发式生长 / Essential component; drives intracellular cAMP levels to upregulate TRPV1 channels and propel explosive neurite outgrowth）。

  • BioVector® Doxycycline (多西环素补剂，视具体亚克隆配套)：终浓度 1.0 ug/mL（用以彻底关闭 v-Myc 不死性 / Final concentration at 1.0 ug/mL to suppress v-Myc-mediated immortalization）。

• 神经营养因子 (Neurotrophic Factors, 可选/增强型)：BioVector® Recombinant Rat NGF (大鼠重组神经生长因子)，终浓度 50 ng/mL。(BioVector® Recombinant Rat NGF at a final concentration of 50 ng/mL can be supplemented to further enhance electrophysiological maturation.)

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（温控误差应小于正负 0.3 摄氏度 / Constant temperature with variations within plus or minus 0.3 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)：日常扩增时，细胞可直接贴壁于标准的塑料培养瓶（TC-treated）中。(Cells can be seeded directly into standard tissue culture-treated plasticware during expansion.)

  • 分化期 (Differentiation Phase)：由于细胞分化后突触极其细长且张力极大，若直接接种在裸塑料板上，细胞会在突触回缩时成片脱落。在启动分化接种前，必须提前使用 BioVector® Poly-L-Lysine（聚左旋赖氨酸，PLL，0.01%）包被孔板，随后再加一层 BioVector® Laminin（层粘连蛋白，5 到 10 ug/mL）进行复合包被。(Because differentiated neurites exert high mechanical tension, cells will detach in sheets if seeded on bare plasticware. Prior to differentiation plating, vessels must be pre-coated with BioVector® Poly-L-Lysine (PLL, 0.01%), followed by a secondary layer of BioVector® Laminin (5 to 10 ug/mL) to construct a robust composite extracellular matrix.)

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)：当细胞单层密度达到 75% 至 85% 汇合度时必须传代。BioVector® 50B11 密度过高时容易自发聚集堆叠，导致底层细胞受压分化，因此严禁将细胞养得过满。(Passage promptly when cell density reaches 75% to 85% confluency. Allowing overgrowth triggers spontaneous cell-stacking, leading to contact-induced differentiation; never allow the monolayer to become 100% confluent.)

• 建议传代稀释比例 (Splitting Ratio)：1:4 到 1:6（常规每 2 到 3 天传代一次 / Typically passaged every 2 to 3 days）。

1. 完全吸除陈旧的生长培养基，使用不含钙镁离子的无菌 BioVector® PBS 缓冲液洗涤细胞单层 1 次。(Completely aspirate spent medium and wash the monolayer once with sterile, calcium-free and magnesium-free BioVector® PBS.)

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

3. 镜下观察发现细胞变圆、轻敲瓶身见细胞呈细沙状脱落时，立即加入等体积的含血清增殖期完全培养基终止消化。(Once microscopic inspection shows cells rounding up and dislodging upon gentle tapping, immediately add an equal volume of serum-containing proliferation growth medium to neutralize the trypsin enzyme.)

4. 用移液枪轻柔吹打贴壁面，将细胞彻底分散为均匀的单细胞悬液。(Gently pipette against the vessel culture surface to disperse cells into a homogeneous single-cell suspension.)

5. 将细胞悬液收集至离心管中，在 200乘以g（低速离心力）下离心 5 分钟，彻底弃去上清液。(Harvest the suspension into a conical tube, centrifuge at 200 x g for 5 minutes, and completely decant the spent supernatant.)

6. 加入新鲜的增殖期完全生长培养基重悬，按常规比例分装至新的培养瓶中。(Resuspend the cell pellet in fresh Phase A proliferation medium, perform cell counting, and distribute evenly into new flasks.)

实验前神经元短周期诱导分化流程 / Short-Cycle Directed Differentiation Protocol

BioVector® 50B11 具有极其迅猛的轴突生长能力，属于典型的高效短周期实验模型：The BioVector® 50B11 line demonstrates exceptionally rapid neurite outgrowth dynamics, serving as a highly efficient short-cycle experimental system:

1. 将增殖期的 BioVector® 50B11 细胞消化，以中等稀释密度（约 3.0乘以10的4次方 cells/cm2）接种至提前进行过 BioVector® PLL/Laminin 复合包被 的实验板中。(Digest expanding cells and seed them at a moderate density of approximately 3.0 x 10^4 cells/cm2 into culture plates pre-coated with the BioVector® PLL/Laminin composite matrix.)

2. 接种时使用常规的增殖培养基，让细胞在孵箱中贴壁过夜（约 12 到 18 小时）以恢复状态。(Utilize standard Phase A proliferation medium during seeding to allow the cells to attach firmly and recover overnight for 12-18 hours.)

3. 次日，彻底吸干旧培养基，更换为含有 10 到 50 uM BioVector® Forskolin 的 B阶段：神经功能分化培养基。(The following day, thoroughly aspirate the medium and replace it with Phase B Neuro-Functional Differentiation Medium fortified with 10-50 uM BioVector® Forskolin.)

4. 更换培养基 12 小时后，即可在显微镜下观察到大量细胞伸出两极轴突。(Extensive bi-polar neurite extensions can be observed visually under the microscope within 12 hours post-induction.)

5. 诱导 48 到 72 小时（2 到 3 天） 时，突触生长达到巅峰并互相连接，此时细胞内的 TRPV1、Nav1.7 离子通道大量功能化开放。此时即可直接用于辣椒素（Capsaicin）刺激引发的钙成像（Calcium Imaging）检测，或加入化疗药物进行神经轴突毒性断裂动力学研究。(Incubate continuously for 48 to 72 hours (2 to 3 days) until neurite networks intersect fully and peak channel functionalization is achieved. At this stage, functional TRPV1 and Nav1.7 channels are highly operational, rendering the culture ready for capsaicin-induced Calcium Imaging assays, or chemotherapy-mediated axonal degeneration kinetics evaluation.)

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。(Pre-warm 5.0 mL of Phase A proliferation growth medium in a sterile T25 flask and equilibrate inside the 37 degrees Celsius incubator to stabilize the gas phase and pH baseline.)

2. 从液氮中取出 BioVector® 50B11 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® 50B11 cryovial from liquid nitrogen and plunge it immediately into a 37 degrees Celsius water bath, agitating horizontally to complete the thawing 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, transfer to the biosafety hood, and dropwise introduce the cell suspension into a 15 mL conical tube packed with 4.0 mL of pre-warmed proliferation 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 aspirate the supernatant to eradicate toxic DMSO residues.)

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, gently tap the tube base to release the pellet, and transfer the entire volume 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 complete medium change 24 hours post-thaw to discard unattached cell debris and eliminate trace elements.)

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 或 BSL-2（视各国家/研究机构对逆转录病毒工程株的定义而定）。属于标准的工程化啮齿类动物细胞系，日常操作需在无菌操作台内进行，废弃物执行常规高压灭菌程序。(BSL-1 or BSL-2 depending on institutional guidelines governing retrovirally engineered rodent lines. Standard sterile laboratory barriers must be maintained; dispose of spent materials via biohazard autoclaving protocols.)

• 超低温长期保存 (Long-Term Banking)：冻存保质配方为 90% 增殖期完全生长培养基 + 10% 细胞级 DMSO（或直接采用 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 摄氏度普通冰箱内长期存放，否则复苏后的分裂活力会发生严重衰退。(The standard freezing formula consists of 90% Phase A proliferation growth medium + 10% analytical grade DMSO, or directly utilize BioVector® Serum-Free Cryopreservation Medium. Cryovials must reside permanently within liquid nitrogen vapor or liquid phase (minus 150 to minus 196 degrees Celsius). Storage in mechanical minus 80 degrees Celsius freezers for extended periods is strictly prohibited, as it compromises post-thaw mitotic viability and post-induction neurite kinetic performance.)

• TRPV1 响应性定期抽检 / Functional Quality Control Threshold：随着传代次数的增多（连续传代超过 20 到 25 代后），BioVector® 50B11 细胞容易发生表型漂移，表现为：在加入 BioVector® Forskolin 诱导后虽然能长出突触，但对辣椒素（Capsaicin）刺激不再产生强烈的钙流信号。因此，每隔 5 到 10 代，应抽取一孔分化后的细胞加入 1 到 10 uM 的 Capsaicin，通过荧光染料验证其钙内流响应。若响应信号微弱，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 passage numbers (exceeding 20 to 25 continuous subcultures) can trigger phenotypic drift, wherein cells retain neurite outgrowth capabilities under Forskolin induction but lose functional Calcium flux signaling in response to Capsaicin stimulation. To safeguard quality control, evaluate a differentiated well every 5 to 10 passages by challenging with 1 to 10 uM Capsaicin via fluorescent calcium indicators. If signaling responses drop precipitously, the line has drifted; discard the active culture immediately and re-thaw a lower-passage master vial from the seed bank.)

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