BioVector® HC-1 条件永生化集合管上皮细胞株文本版说明书

BioVector® HC-1 Conditionally Immortalized Cortical Collecting Duct Epithelial 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® HC-1 条件永生化小鼠皮质集合管上基质细胞株 (BioVector® HC-1 Conditionally Immortalized Mouse Renal Cortical Collecting Duct Epithelial Cell Line)

• 常用别名 (Synonyms)：BioVector® HC-1，BioVector® HC1 Cell

• 生物学来源 (Organism Source)：小鼠 Mus musculus（成年，转基因小鼠）

• 组织器官定位 (Tissue and Organ Site)：泌尿系统 / 肾脏皮质集合管 (Urinary System / Renal Cortical Collecting Duct, CCD)

• 生长特性 (Growth Properties)：贴壁生长，具有典型的上皮细胞鹅卵石样或铺路石样排列特征，汇合后形成紧密的细胞单层并展现出接触抑制。(Adherent growth, exhibiting classic cobblestone epithelial morphology. Upon reaching confluency, cells form a tight, polarized monolayer and manifest strict contact inhibition.)

• 永生化工程机制 (Immortalization Mechanism)：细胞衍生自带有受特殊启动子调控的 SV40 大 T 抗原（SV40 Large T antigen）转基因小鼠的肾皮质组织。该系统表现为温度敏感型或条件诱导型控制，使细胞在特定体外环境下能够无限扩增，而在切换环境后则快速进入终末功能分化状态。(Derived from the renal cortical tissues of a transgenic mouse line carrying the SV40 Large T antigen. This setup functions as a conditionally regulated engine, allowing the cells to divide continuously under permissive conditions and enter terminal functional differentiation upon non-permissive shifting.)

• 核心科研价值 (Core Research Significance)：肾脏皮质集合管（CCD）是机体调节水盐平衡、酸碱平衡以及对醛固酮和抗利尿激素（ADH）产生响应的关键部位。BioVector® HC-1 完好地保留了主细胞（Principal cells）和插班细胞（Intercalated cells）的关键转运体特征。它被广泛用于上皮钠通道（ENaC）、水通道蛋白（AQP2、AQP3）、尿素转运蛋白（UT-A1）的转运调控机制研究，也是解析高血压发病机制、醛固酮敏化通路以及利尿药靶向筛选的核心体外模型。(The renal cortical collecting duct is the structural hub regulating water-electrolyte balance, acid-base homeostasis, and systemic responses to aldosterone and antidiuretic hormone. BioVector® HC-1 beautifully preserves the transport infrastructure characteristic of principal and intercalated cell phenotypes. It is widely applied to map the regulatory pathways of the Epithelial Sodium Channel, Aquaporins, and Urea Transporters, serving as a premier platform for decoding hypertension pathogenesis and targeting novel diuretic pharmaceuticals.)

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

BioVector® HC-1 表现出高度的高度极性化和跨上皮转运能力，其表型状态可通过物理或化学环境精准调控：The BioVector® HC-1 line features deep cellular polarization and transepithelial transport properties, with its functional state tightly governed by environmental adjustments:

未分化增殖状态 (Permissive / Proliferating State)

在常规扩增条件下，SV40 大 T 抗原稳定表达，驱动上皮前体细胞快速进行有丝分裂，倍增时间约 24 到 36 小时。Under growth-permissive parameters, the continuous activation of the SV40 Large T antigen drives brisk mitotic expansion of epithelial progenitors, with doubling times ranging between 24 and 36 hours.

诱导分化成熟状态 (Non-Permissive / Differentiated State)

通过切换培养环境（如调整温度或加入分化促进剂）关闭大 T 抗原后，细胞完全停止有丝分裂，紧密贴合在一起，跨上皮电阻（TEER）显著上升，并高水平表达其功能性标志物：Shifting cultures to non-permissive tracks halts mitotic drives, forcing cells into an ultra-tight monolayer with high Transepithelial Electrical Resistance (TEER) readings and elevated functional biomarkers:

• 水与离子转运体 (Water and Ion Transporters)：高水平表达对血管加压素响应的水通道蛋白 2（AQP2）、上皮钠通道（ENaC）的所有亚基，以及调节酸碱平衡的质子泵（H-ATPase）和阴离子交换体（AE1）。(Robustly targets vasopressin-responsive Aquaporin-2, epithelial sodium channel subunits, alongside proton pumps and anion exchangers defining intercalated profiles.)

• 激素受体系统 (Hormone Receptor Framework)：稳定维持盐皮质激素受体（MR）的表达，能对醛固酮（Aldosterone）刺激产生敏感的基因转录调控响应。(Stably maintains Mineralocorticoid Receptor expressions, exhibiting high transcriptional sensitivity under Aldosterone provocation cascades.)

• 连接蛋白复合物 (Junction Complexes)：在分化单层中，紧密连接蛋白（Zonula Occludens-1, ZO-1）和闭合蛋白（Occludin）在细胞边缘呈现完美的连续网格状分布，证实其具备卓越屏障功能。(Within the differentiated monolayer, ZO-1 and Occludin reveal beautiful, unbroken grid-like framing around cell boundaries, validating superb epithelial barrier competencies.)

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

警告 / Critical WarningBioVector® HC-1 属于功能敏感型集合管上皮细胞，在常规扩增传代时，必须保证基础培养基中添加了足量的上皮生长因子和必需微量激素。如果缺少这些支持补剂，细胞上皮特征极易退化为非特异性的成纤维样细胞。BioVector® HC-1 represents a highly specialized collecting duct epithelial model. During expansion, it is mandatory to supplement the basal feed with adequate epithelial growth factors and hormonal trace elements. Deprivation of these supplements will trigger irreversible drift into non-specific fibroblastic profiles.

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® DMEM/F12 复合培养基（1比1比例混合，含 L-谷氨酰胺 / 1:1 mixture with L-Glutamine）。

• 血清添加 (Serum Supplement)：5% 到 10% BioVector® Premium Fetal Bovine Serum 优质胎牛血清。

• 上皮专用微量激素鸡尾酒补剂 (Epithelial Hormone Cocktail Supplement - Essential)：

  • BioVector® Insulin (胰岛素)：终浓度 5 ug/mL。

  • BioVector® Transferrin (转铁蛋白)：终浓度 5 ug/mL。

  • BioVector® Selenium (硒酸钠)：终浓度 5 ng/mL。

  • BioVector® Hydrocortisone (氢化可的松)：终浓度 50 nM（用于维持基础离子泵转运活性）。

  • BioVector® Triiodothyronine (三碘甲状腺原氨酸 T3)：终浓度 1 nM。

  • BioVector® EGF (人重组表皮生长因子)：终浓度 10 ng/mL（强效驱动增殖）。

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

B 阶段：专性功能分化培养基（实验前屏障与转运功能诱导）

Phase B: Functional Differentiation Medium (For Pre-Experimental Barrier and Transport Induction)

• 基础骨架 (Basal Matrix)：BioVector® DMEM/F12 复合培养基（不含 EGF 因子 / stripped of EGF to halt proliferation drive）。

• 降低后的血清 (Reduced Serum)：1% 到 2% BioVector® Premium Fetal Bovine Serum 优质胎牛血清（或替换为 BioVector® Serum-Free Supplemental Matrix）。

• 激素维持与诱导物 (Hormone Triggers)：

  • 维持上述 Phase A 中的 Insulin-Transferrin-Selenium 组合，并可根据实验目的额外加入 BioVector® Aldosterone（醛固酮补剂，10 到 100 nM）或 BioVector® Dexamethasone（地塞米松补剂）以诱导 ENaC 钠通道或水通道蛋白的高峰值活化。(Retain the base Insulin-Transferrin-Selenium core from Phase A, and introduce BioVector® Aldosterone at 10 to 100 nM to trigger localized upregulation of functional ENaC or AQP2 networks.)

4 物理环境与膜片跨上皮转运控制参数 / Environmental and TEER Parameters

• 增殖期扩增温度 (Growth Temperature)：通常在 33 到 37 摄氏度环境下进行快速扩增（具体视具体克隆的温度敏感系统设计而定 / Dependent on the temperature-sensitive vector design of the specific clonal repository）。

• 分化期成熟温度 (Differentiation Temperature)：通常调整至 37.0 摄氏度并撤除 EGF 因子，促使大 T 抗原失活并全面开启极性化分化。(Maintained strictly at 37.0 degrees Celsius under EGF starvation to yield polarized monolayer maturation.)

• 气相环境 (Gas Phase)：5% 二氧化碳 (CO2)，加湿常压平衡空气 (5% Carbon Dioxide in a fully humidified incubator atmosphere)。

• 多孔透气支撑膜应用 (Permeable Support Application - Highly Recommended)：

  • 在进行跨上皮电位（TEER）测量、水通量测定或钠离子跨膜转运实验时，强烈建议将 BioVector® HC-1 细胞接种于经过 BioVector® Collagen（胶原蛋白，I型或IV型）包被的 Transwell 多孔聚碳酸酯透气膜上。相较于传统的塑料硬底孔板，透气膜生长环境能让细胞实现真正的基底侧与管腔侧双向极性分化，使其离子转运特性提升数倍。(For evaluating TEER currents or water-flux metrics, it is highly recommended to seed BioVector® HC-1 onto Transwell permeable inserts pre-coated with BioVector® Collagen. This allows authentic basolateral-to-apical polar scaling, maximizing transport capacities over hard plastic dishes.)

5 细胞传代与功能分化操作规范 / Subculturing and Differentiation Protocols

增殖期常规传代操作 / Routine Passaging (Proliferating State)

• 传代临界点 (Passaging Threshold)：当细胞单层融合度达到 85% 到 90% 时必须进行传代。由于上皮细胞贴壁紧密且具有接触抑制，一旦让其长得过满（100% 融合）并保持数日，细胞会自发进入分化轨道而导致后续传代时难以被胰酶消化分散。(Passage when cultures reach 85% to 90% confluency. Avoid keeping a 100% saturated monolayer for extended periods, as extensive extracellular matrix deposition makes subsequent trypsinization difficult.)

• 建议传代稀释比例 (Splitting Ratio)：1:3 到 1:4（常规每 3 到 4 天传代一次）。

1. 完全吸除旧的生长培养基，使用无菌且不含钙镁离子的 BioVector® PBS 缓冲液充分洗涤细胞表面 2 次，以彻底去除抑制胰酶的血清成分。(Evacuate spent medium and wash twice with sterile, calcium-free and magnesium-free BioVector® PBS to ensure complete neutralization of serum elements.)

2. 加入适量预热的 BioVector® 0.25% Trypsin-EDTA 强效消化液，放入 37 摄氏度孵箱中消化 3 到 5 分钟。(Introduce pre-warmed BioVector® 0.25% Trypsin-EDTA and incubate at 37 degrees Celsius for 3 to 5 minutes.)

3. 镜下观察发现细胞间隙明显增大、细胞单层开始碎裂并变圆时，立即加入双倍体积的含血清 BioVector® 增殖期完全培养基终止消化。(Once microscopic inspection confirms widening intercellular clefts and rounding of sheet matrices, immediately add double the volume of serum-containing Phase A growth medium to halt trypsin activity.)

4. 用移液枪对贴壁面进行适度吹打，将紧密相连的上皮细胞团块彻底打散为单细胞悬液。(Pipette with moderate force against the substrate to completely disaggregate cohesive epithelial sheet clusters into a single-cell suspension.)

5. 在 250乘以g 下离心 5 分钟，彻底弃去含有上清的残液。(Centrifuge at 250 x g for 5 minutes and thoroughly vacuum away the supernatant.)

6. 加入新鲜的 BioVector® 增殖期生长培养基重悬，分装至新培养瓶中。(Resuspend into fresh Phase A growth media and dispense into fresh expansion vessels.)

实验前跨上皮转运功能诱导流程 / Polarized Transport Induction Protocol

1. 将增殖期的 BioVector® HC-1 细胞消化，以大约 5.0乘以10的4次方 cells/cm2 的高密度接种于经过 BioVector® 胶原蛋白包被 的 Transwell 小室上盖膜中。(Digest expanding cells and seed them at a high density of approx 5.0 x 10^4 cells/cm2 into Transwell inserts pre-coated with BioVector® Collagen.)

2. 双侧均加满 BioVector® 增殖期完全培养基，在增殖温度下培养 24 小时使其完全铺满贴牢。(Fill both apical and basolateral compartments with Phase A growth media, incubating for 24 hours to ensure standard coverage.)

3. 当检测到单层细胞彻底融合后，双侧同步抽干培养基，更换为全新的 B阶段：专性功能分化培养基（撤除 EGF，限制血清，并根据需要加入醛固酮刺激剂）。(Once full coverage is verified, evacuate both compartments and introduce Phase B Functional Differentiation Medium.)

4. 持续极性化诱导 5 到 7 天，期间每隔 2 天进行一次双侧全量换液。在此期间，使用上皮电压表（如 EVOM）可观察到单层跨上皮电阻（TEER）逐日稳步攀升，并在第 6 天左右达到平台期。此时单层紧密连接闭合完全，水通道与钠通道在顶端膜（Apical membrane）定向极化定位完毕，即可正式用于跨膜离子电流记录、水通量渗透压刺激实验或药物阻断动力学分析。(Maintain polarized induction for 5 to 7 days, executing total media renewal every 2 days. Transepithelial Electrical Resistance (TEER) metrics will rise steadily, stabilizing around day 6, indicating tight junction closure and apical channel localization. The system is then ready for trans-membrane current clamp assays or solute-flux kinetics.)

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 值与气体浓度。(Pre-warm 5.0 mL of Phase A growth medium in a T25 flask and balance inside the incubator to settle pH and gas variables.)

2. 从液氮中取出 BioVector® HC-1 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® HC-1 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 15 mL 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并补充新鲜的激素微量组分。(Incubate at designated conditions. Execute a mandatory complete fluid change 24 hours post-thaw to eliminate dead cell drift and replenish micro-hormone concentrations.)

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-2。由于该细胞是通过转导或转基因方式集成了 SV40 大 T 抗原核心序列，属于二级生物安全管控范畴。日常操作必须在二级生物安全柜（BSC-2）内开展，所有接触过该细胞的液体、枪头及培养皿必须执行 121 摄氏度高压蒸汽灭菌 30 分钟后方可作废弃处理。(BSL-2 classification due to the genomic integration of the SV40 Large T antigen sequence. All handling must be conducted within a Class II Biosafety Cabinet; spent liquids, pipettes, and culture vessels must undergo autoclaving at 121 degrees Celsius for 30 minutes prior to disposal.)

• 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® Premium 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）。切勿在零下 80 摄氏度普通冰箱内长期搁置超过 1 个月，否则上皮细胞的极性分化潜能和复苏存活率会发生不可逆的退化。(The standard freezing blend consists of 90% Phase A growth medium + 10% analytical grade DMSO, or BioVector® Premium Serum-Free Cryopreservation Medium. Vials must reside permanently inside liquid nitrogen repositories (minus 150 to minus 196 degrees Celsius). Storage in mechanical minus 80 degrees Celsius freezers for extended spans is prohibited, as it compromises polar differentiation metrics and viability.)

• 跨上皮电阻与通道响应质控红线 / Epithelial Barrier Quality Control Threshold：随着传代代次的增加（连续传代超过 20 到 25 代以上），BioVector® HC-1 细胞容易发生去分化表型漂移。质控表现为：将细胞接种于 Transwell 小室并在分化培养基中诱导 7 天后，如果测得的单层跨上皮电阻（TEER）无法突破 150 欧姆乘以平方厘米，或者加入阿米洛利（Amiloride，ENaC 通道特异性阻断剂）后无法引起明显的短路电流下降，则证实该批次细胞已经发生去分化退化。必须立即停止实验使用，并重新复苏更低代次的种子库进行恢复。(Extended passaging (exceeding 20 to 25 continuous subcultures) can trigger dedifferentiation drift in epithelial platforms. For strict quality control, define a test insert after 7 days of polarized induction: if the recorded TEER value fails to pass 150 ohms x cm2, or if exposure to Amiloride (a specific ENaC channel blocker) fails to provoke a significant drop in short-circuit currents, the lineage has lost its identity. Cease further experimental use immediately and re-thaw a lower-passage master stock from the cryo-vault.)

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