BioVector? HC-1 條件永生化集合管上皮細(xì)胞株文本版說(shuō)明書(shū)

BioVector? HC-1 Conditionally Immortalized Cortical Collecting Duct Epithelial Cell Line Text-Based Datasheet

1 產(chǎn)品基本信息與遺傳背景 / Product Identification and Genetic Background

• 產(chǎn)品名稱 (Product Name)：BioVector? HC-1 條件永生化小鼠皮質(zhì)集合管上基質(zhì)細(xì)胞株 (BioVector? HC-1 Conditionally Immortalized Mouse Renal Cortical Collecting Duct Epithelial Cell Line)

• 常用別名 (Synonyms)：BioVector? HC-1，BioVector? HC1 Cell

• 生物學(xué)來(lái)源 (Organism Source)：小鼠 Mus musculus（成年，轉(zhuǎn)基因小鼠）

• 組織器官定位 (Tissue and Organ Site)：泌尿系統(tǒng) / 腎臟皮質(zhì)集合管 (Urinary System / Renal Cortical Collecting Duct, CCD)

• 生長(zhǎng)特性 (Growth Properties)：貼壁生長(zhǎng)，具有典型的上皮細(xì)胞鵝卵石樣或鋪路石樣排列特征，匯合后形成緊密的細(xì)胞單層并展現(xiàn)出接觸抑制。(Adherent growth, exhibiting classic cobblestone epithelial morphology. Upon reaching confluency, cells form a tight, polarized monolayer and manifest strict contact inhibition.)

• 永生化工程機(jī)制 (Immortalization Mechanism)：細(xì)胞衍生自帶有受特殊啟動(dòng)子調(diào)控的 SV40 大 T 抗原（SV40 Large T antigen）轉(zhuǎn)基因小鼠的腎皮質(zhì)組織。該系統(tǒng)表現(xiàn)為溫度敏感型或條件誘導(dǎo)型控制，使細(xì)胞在特定體外環(huán)境下能夠無(wú)限擴(kuò)增，而在切換環(huán)境后則快速進(jìn)入終末功能分化狀態(tài)。(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.)

• 核心科研價(jià)值 (Core Research Significance)：腎臟皮質(zhì)集合管（CCD）是機(jī)體調(diào)節(jié)水鹽平衡、酸堿平衡以及對(duì)醛固酮和抗利尿激素（ADH）產(chǎn)生響應(yīng)的關(guān)鍵部位。BioVector? HC-1 完好地保留了主細(xì)胞（Principal cells）和插班細(xì)胞（Intercalated cells）的關(guān)鍵轉(zhuǎn)運(yùn)體特征。它被廣泛用于上皮鈉通道（ENaC）、水通道蛋白（AQP2、AQP3）、尿素轉(zhuǎn)運(yùn)蛋白（UT-A1）的轉(zhuǎn)運(yùn)調(diào)控機(jī)制研究，也是解析高血壓發(fā)病機(jī)制、醛固酮敏化通路以及利尿藥靶向篩選的核心體外模型。(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 細(xì)胞生物學(xué)特征與核心標(biāo)志物表型 / Cellular Properties and Biomarker Profiles

BioVector? HC-1 表現(xiàn)出高度的高度極性化和跨上皮轉(zhuǎn)運(yùn)能力，其表型狀態(tài)可通過(guò)物理或化學(xué)環(huán)境精準(zhǔn)調(diào)控：The BioVector? HC-1 line features deep cellular polarization and transepithelial transport properties, with its functional state tightly governed by environmental adjustments:

未分化增殖狀態(tài) (Permissive / Proliferating State)

在常規(guī)擴(kuò)增條件下，SV40 大 T 抗原穩(wěn)定表達(dá)，驅(qū)動(dòng)上皮前體細(xì)胞快速進(jìn)行有絲分裂，倍增時(shí)間約 24 到 36 小時(shí)。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.

誘導(dǎo)分化成熟狀態(tài) (Non-Permissive / Differentiated State)

通過(guò)切換培養(yǎng)環(huán)境（如調(diào)整溫度或加入分化促進(jìn)劑）關(guān)閉大 T 抗原后，細(xì)胞完全停止有絲分裂，緊密貼合在一起，跨上皮電阻（TEER）顯著上升，并高水平表達(dá)其功能性標(biāo)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:

• 水與離子轉(zhuǎn)運(yùn)體 (Water and Ion Transporters)：高水平表達(dá)對(duì)血管加壓素響應(yīng)的水通道蛋白 2（AQP2）、上皮鈉通道（ENaC）的所有亞基，以及調(diào)節(jié)酸堿平衡的質(zhì)子泵（H-ATPase）和陰離子交換體（AE1）。(Robustly targets vasopressin-responsive Aquaporin-2, epithelial sodium channel subunits, alongside proton pumps and anion exchangers defining intercalated profiles.)

• 激素受體系統(tǒng) (Hormone Receptor Framework)：穩(wěn)定維持鹽皮質(zhì)激素受體（MR）的表達(dá)，能對(duì)醛固酮（Aldosterone）刺激產(chǎn)生敏感的基因轉(zhuǎn)錄調(diào)控響應(yīng)。(Stably maintains Mineralocorticoid Receptor expressions, exhibiting high transcriptional sensitivity under Aldosterone provocation cascades.)

• 連接蛋白復(fù)合物 (Junction Complexes)：在分化單層中，緊密連接蛋白（Zonula Occludens-1, ZO-1）和閉合蛋白（Occludin）在細(xì)胞邊緣呈現(xiàn)完美的連續(xù)網(wǎng)格狀分布，證實(shí)其具備卓越屏障功能。(Within the differentiated monolayer, ZO-1 and Occludin reveal beautiful, unbroken grid-like framing around cell boundaries, validating superb epithelial barrier competencies.)

3 專用兩階段培養(yǎng)基配方規(guī)范 / Culturing Medium Formulations

警告 / Critical WarningBioVector? HC-1 屬于功能敏感型集合管上皮細(xì)胞，在常規(guī)擴(kuò)增傳代時(shí)，必須保證基礎(chǔ)培養(yǎng)基中添加了足量的上皮生長(zhǎng)因子和必需微量激素。如果缺少這些支持補(bǔ)劑，細(xì)胞上皮特征極易退化為非特異性的成纖維樣細(xì)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 階段：增殖期完全生長(zhǎng)培養(yǎng)基（用于常規(guī)細(xì)胞擴(kuò)增與維持）

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

• 基礎(chǔ)培養(yǎng)基 (Basal Medium)：BioVector? DMEM/F12 復(fù)合培養(yǎng)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 優(yōu)質(zhì)胎牛血清。

• 上皮專用微量激素雞尾酒補(bǔ)劑 (Epithelial Hormone Cocktail Supplement - Essential)：

  • BioVector? Insulin (胰島素)：終濃度 5 ug/mL。

  • BioVector? Transferrin (轉(zhuǎn)鐵蛋白)：終濃度 5 ug/mL。

  • BioVector? Selenium (硒酸鈉)：終濃度 5 ng/mL。

  • BioVector? Hydrocortisone (氫化可的松)：終濃度 50 nM（用于維持基礎(chǔ)離子泵轉(zhuǎn)運(yùn)活性）。

  • BioVector? Triiodothyronine (三碘甲狀腺原氨酸 T3)：終濃度 1 nM。

  • BioVector? EGF (人重組表皮生長(zhǎng)因子)：終濃度 10 ng/mL（強(qiáng)效驅(qū)動(dòng)增殖）。

• 雙抗補(bǔ)劑 (Antibiotics)：1% BioVector? Penicillin-Streptomycin Solution 青霉素-鏈霉素溶液。

B 階段：專性功能分化培養(yǎng)基（實(shí)驗(yàn)前屏障與轉(zhuǎn)運(yùn)功能誘導(dǎo)）

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

• 基礎(chǔ)骨架 (Basal Matrix)：BioVector? DMEM/F12 復(fù)合培養(yǎng)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 優(yōu)質(zhì)胎牛血清（或替換為 BioVector? Serum-Free Supplemental Matrix）。

• 激素維持與誘導(dǎo)物 (Hormone Triggers)：

  • 維持上述 Phase A 中的 Insulin-Transferrin-Selenium 組合，并可根據(jù)實(shí)驗(yàn)?zāi)康念~外加入 BioVector? Aldosterone（醛固酮補(bǔ)劑，10 到 100 nM）或 BioVector? Dexamethasone（地塞米松補(bǔ)劑）以誘導(dǎo) ENaC 鈉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 物理環(huán)境與膜片跨上皮轉(zhuǎn)運(yùn)控制參數(shù) / Environmental and TEER Parameters

• 增殖期擴(kuò)增溫度 (Growth Temperature)：通常在 33 到 37 攝氏度環(huán)境下進(jìn)行快速擴(kuò)增（具體視具體克隆的溫度敏感系統(tǒng)設(shè)計(jì)而定 / Dependent on the temperature-sensitive vector design of the specific clonal repository）。

• 分化期成熟溫度 (Differentiation Temperature)：通常調(diào)整至 37.0 攝氏度并撤除 EGF 因子，促使大 T 抗原失活并全面開(kāi)啟極性化分化。(Maintained strictly at 37.0 degrees Celsius under EGF starvation to yield polarized monolayer maturation.)

• 氣相環(huán)境 (Gas Phase)：5% 二氧化碳 (CO2)，加濕常壓平衡空氣 (5% Carbon Dioxide in a fully humidified incubator atmosphere)。

• 多孔透氣支撐膜應(yīng)用 (Permeable Support Application - Highly Recommended)：

  • 在進(jìn)行跨上皮電位（TEER）測(cè)量、水通量測(cè)定或鈉離子跨膜轉(zhuǎn)運(yùn)實(shí)驗(yàn)時(shí)，強(qiáng)烈建議將 BioVector? HC-1 細(xì)胞接種于經(jīng)過(guò) BioVector? Collagen（膠原蛋白，I型或IV型）包被的 Transwell 多孔聚碳酸酯透氣膜上。相較于傳統(tǒng)的塑料硬底孔板，透氣膜生長(zhǎng)環(huán)境能讓細(xì)胞實(shí)現(xiàn)真正的基底側(cè)與管腔側(cè)雙向極性分化，使其離子轉(zhuǎn)運(yùn)特性提升數(shù)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 細(xì)胞傳代與功能分化操作規(guī)范 / Subculturing and Differentiation Protocols

增殖期常規(guī)傳代操作 / Routine Passaging (Proliferating State)

• 傳代臨界點(diǎn) (Passaging Threshold)：當(dāng)細(xì)胞單層融合度達(dá)到 85% 到 90% 時(shí)必須進(jìn)行傳代。由于上皮細(xì)胞貼壁緊密且具有接觸抑制，一旦讓其長(zhǎng)得過(guò)滿（100% 融合）并保持?jǐn)?shù)日，細(xì)胞會(huì)自發(fā)進(jìn)入分化軌道而導(dǎo)致后續(xù)傳代時(shí)難以被胰酶消化分散。(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.)

• 建議傳代稀釋比例 (Splitting Ratio)：1:3 到 1:4（常規(guī)每 3 到 4 天傳代一次）。

1. 完全吸除舊的生長(zhǎng)培養(yǎng)基，使用無(wú)菌且不含鈣鎂離子的 BioVector? PBS 緩沖液充分洗滌細(xì)胞表面 2 次，以徹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. 加入適量預(yù)熱的 BioVector? 0.25% Trypsin-EDTA 強(qiáng)效消化液，放入 37 攝氏度孵箱中消化 3 到 5 分鐘。(Introduce pre-warmed BioVector? 0.25% Trypsin-EDTA and incubate at 37 degrees Celsius for 3 to 5 minutes.)

3. 鏡下觀察發(fā)現(xiàn)細(xì)胞間隙明顯增大、細(xì)胞單層開(kāi)始碎裂并變圓時(shí)，立即加入雙倍體積的含血清 BioVector? 增殖期完全培養(yǎng)基終止消化。(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. 用移液槍對(duì)貼壁面進(jìn)行適度吹打，將緊密相連的上皮細(xì)胞團(tuán)塊徹底打散為單細(xì)胞懸液。(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 下離心 5 分鐘，徹底棄去含有上清的殘液。(Centrifuge at 250 x g for 5 minutes and thoroughly vacuum away the supernatant.)

6. 加入新鮮的 BioVector? 增殖期生長(zhǎng)培養(yǎng)基重懸，分裝至新培養(yǎng)瓶中。(Resuspend into fresh Phase A growth media and dispense into fresh expansion vessels.)

實(shí)驗(yàn)前跨上皮轉(zhuǎn)運(yùn)功能誘導(dǎo)流程 / Polarized Transport Induction Protocol

1. 將增殖期的 BioVector? HC-1 細(xì)胞消化，以大約 5.0乘以10的4次方 cells/cm2 的高密度接種于經(jīng)過(guò) BioVector? 膠原蛋白包被 的 Transwell 小室上蓋膜中。(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. 雙側(cè)均加滿 BioVector? 增殖期完全培養(yǎng)基，在增殖溫度下培養(yǎng) 24 小時(shí)使其完全鋪滿貼牢。(Fill both apical and basolateral compartments with Phase A growth media, incubating for 24 hours to ensure standard coverage.)

3. 當(dāng)檢測(cè)到單層細(xì)胞徹底融合后，雙側(cè)同步抽干培養(yǎng)基，更換為全新的 B階段：專性功能分化培養(yǎng)基（撤除 EGF，限制血清，并根據(jù)需要加入醛固酮刺激劑）。(Once full coverage is verified, evacuate both compartments and introduce Phase B Functional Differentiation Medium.)

4. 持續(xù)極性化誘導(dǎo) 5 到 7 天，期間每隔 2 天進(jìn)行一次雙側(cè)全量換液。在此期間，使用上皮電壓表（如 EVOM）可觀察到單層跨上皮電阻（TEER）逐日穩(wěn)步攀升，并在第 6 天左右達(dá)到平臺(tái)期。此時(shí)單層緊密連接閉合完全，水通道與鈉通道在頂端膜（Apical membrane）定向極化定位完畢，即可正式用于跨膜離子電流記錄、水通量滲透壓刺激實(shí)驗(yàn)或藥物阻斷動(dòng)力學(xué)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 復(fù)蘇與凍存恢復(fù)流線 / Thawing and Post-Cryo Recovery Workflow

1. 提前在標(biāo)準(zhǔn) T25 培養(yǎng)瓶中注入 5.0 mL 預(yù)熱的增殖期完全生長(zhǎng)培養(yǎng)基，置于 37 攝氏度孵箱中平衡 pH 值與氣體濃度。(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. 從液氮中取出 BioVector? HC-1 凍存管，立刻全量投入 37 攝氏度恒溫水浴箱中快速水平搖晃，在 60 秒內(nèi)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凈臺(tái)，用移液槍吸出胞懸液，緩慢逐滴注入盛有 4.0 mL 預(yù)熱增殖培養(yǎng)基的 15 mL 離心管中。(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 下低速離心 5 分鐘，徹底吸干含有毒 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 新鮮增殖培養(yǎng)基輕彈懸起細(xì)胞沉淀，隨后全量接種到預(yù)熱平衡的 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 小時(shí)后必須進(jìn)行一次全量換液，以清除未貼壁的死細(xì)胞碎片并補(bǔ)充新鮮的激素微量組分。(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、長(zhǎng)期保存與質(zhì)控規(guī)范 / Biosafety, Storage and Quality Controls

• 生物安全級(jí)別 (Biosafety Level)：BSL-2。由于該細(xì)胞是通過(guò)轉(zhuǎn)導(dǎo)或轉(zhuǎn)基因方式集成了 SV40 大 T 抗原核心序列，屬于二級(jí)生物安全管控范疇。日常操作必須在二級(jí)生物安全柜（BSC-2）內(nèi)開(kāi)展，所有接觸過(guò)該細(xì)胞的液體、槍頭及培養(yǎng)皿必須執(zhí)行 121 攝氏度高壓蒸汽滅菌 30 分鐘后方可作廢棄處理。(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.)

• 超低溫長(zhǎng)期保存 (Long-Term Banking)：凍存保質(zhì)配方為 90% BioVector? 增殖期完全生長(zhǎng)培養(yǎng)基 + 10% 細(xì)胞級(jí) DMSO（或采用 BioVector? Premium 無(wú)血清通用型凍存液）。凍存管必須永久存放于液氮環(huán)境中（零下 150 攝氏度至零下 196 攝氏度）。切勿在零下 80 攝氏度普通冰箱內(nèi)長(zhǎng)期擱置超過(guò) 1 個(gè)月，否則上皮細(xì)胞的極性分化潛能和復(fù)蘇存活率會(huì)發(fā)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.)

• 跨上皮電阻與通道響應(yīng)質(zhì)控紅線 / Epithelial Barrier Quality Control Threshold：隨著傳代代次的增加（連續(xù)傳代超過(guò) 20 到 25 代以上），BioVector? HC-1 細(xì)胞容易發(fā)生去分化表型漂移。質(zhì)控表現(xiàn)為：將細(xì)胞接種于 Transwell 小室并在分化培養(yǎng)基中誘導(dǎo) 7 天后，如果測(cè)得的單層跨上皮電阻（TEER）無(wú)法突破 150 歐姆乘以平方厘米，或者加入阿米洛利（Amiloride，ENaC 通道特異性阻斷劑）后無(wú)法引起明顯的短路電流下降，則證實(shí)該批次細(xì)胞已經(jīng)發(fā)生去分化退化。必須立即停止實(shí)驗(yàn)使用，并重新復(fù)蘇更低代次的種子庫(kù)進(jìn)行恢復(fù)。(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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