BioVector? ND7/23 條件永生化神經(jīng)元細(xì)胞株文本版說(shuō)明書(shū)

BioVector? ND7/23 Conditionally Immortalized Neuroblastoma Hybrid 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(chēng) (Product Name)：BioVector? ND7/23 條件永生化小鼠/大鼠雜交感覺(jué)神經(jīng)元細(xì)胞株 (BioVector? ND7/23 Conditionally Immortalized Mouse/Rat Hybrid Sensory Neuron Cell Line)

• 常用別名 (Synonyms)：BioVector? ND7/23，BioVector? ND7，BioVector? ND7-23 Cell

• 生物學(xué)來(lái)源 (Organism Source)：體細(xì)胞雜交株（大鼠 Rattus norvegicus 新生背根神經(jīng)節(jié)神經(jīng)元 融合 小鼠 Mus musculus N18Tg2 神經(jīng)瘤細(xì)胞）

• 組織器官定位 (Tissue and Organ Site)：外周神經(jīng)系統(tǒng) / 背根神經(jīng)節(jié)感覺(jué)神經(jīng)元特性 (Peripheral Nervous System / Dorsal Root Ganglion Sensory Neuron Properties)

• 生長(zhǎng)特性 (Growth Properties)：

  • 增殖期/未分化 (Proliferating Phase)：貼壁生長(zhǎng)，細(xì)胞多呈圓球形、短梭形或折光度高的未分化成纖維細(xì)胞樣形態(tài)，貼壁較輕，極易發(fā)生集聚生長(zhǎng)。(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)：細(xì)胞徹底停止分裂，胞體收縮并變亮，迅速延展出極長(zhǎng)的雙極或多極神經(jīng)軸突，突觸間形成錯(cuò)綜復(fù)雜的有功能的感覺(jué)神經(jīng)網(wǎng)。(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.)

• 雜交永生化機(jī)制 (Immortalization Mechanism)：利用聚乙二醇（PEG）技術(shù)，將新生大鼠原代背根神經(jīng)節(jié)（DRG）感覺(jué)神經(jīng)元與缺少 HPRT 酶的小鼠 N18Tg2 神經(jīng)母細(xì)胞瘤細(xì)胞進(jìn)行無(wú)性系雜交融合，通過(guò) HAT 培養(yǎng)基篩選獲得既具備無(wú)限增殖能力又完好保留 DRG 傷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.)

• 核心科研價(jià)值 (Core Research Significance)：BioVector? ND7/23 是全球疼痛機(jī)制研究與外周神經(jīng)科學(xué)領(lǐng)域使用頻率極高的標(biāo)志性細(xì)胞系。與原代 DRG 神經(jīng)元相比，它具有極高的穩(wěn)定性和極低的操作難度。它被廣泛用于機(jī)械痛、熱痛、酸感外周敏化通路的研究，特別是機(jī)械敏感性離子通道（如 Piezo1 和 Piezo2）、溫度敏感性 TRP 通道（如 TRPV1、TRPM8）以及電壓門(mén)控鈉通道（如 Nav1.7、Nav1.8）的表達(dá)與電生理功能解析，也是高通量篩選外周鎮(zhèn)痛藥的核心細(xì)胞底盤(pán)。(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 細(xì)胞生物學(xué)特征與核心標(biāo)志物表型 / Cellular Properties and Biomarker Profiles

BioVector? ND7/23 兼具神經(jīng)瘤的快速有絲分裂特性與感覺(jué)神經(jīng)元的分化潛能，其生理特征隨培養(yǎng)基配方而發(fā)生精準(zhǔn)改變：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:

未分化增殖狀態(tài) (Proliferating State)

細(xì)胞倍增速度快（約 20 到 24 小時(shí)）。細(xì)胞表面黏附力較弱，在普通塑料瓶中長(zhǎng)滿(mǎn)時(shí)極易聚集成團(tuán)塊，若受到劇烈震動(dòng)會(huì)有部分細(xì)胞自發(fā)懸浮。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.

誘導(dǎo)分化成熟狀態(tài) (Differentiated State)

在低血清聯(lián)合 BioVector? Forskolin 或神經(jīng)營(yíng)養(yǎng)因子的協(xié)同誘導(dǎo)下，細(xì)胞在 48 到 72 小時(shí)內(nèi)迅速展現(xiàn)出高度特異性的感覺(jué)神經(jīng)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:

• 特征性離子通道譜 (Ion Channel Repertoire)：穩(wěn)定表達(dá)大鼠源性的電壓門(mén)控鈉通道 Nav1.7 和對(duì)河豚毒素耐受的 Nav1.8（TTX-resistant）電流；內(nèi)源性表達(dá)機(jī)械敏感通道 Piezo1 和 Piezo2；在分化促進(jìn)下可高效上調(diào) TRPV1（辣椒素受體）的表達(dá)。(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.)

• 功能性表面受體 (Surface Receptors)：高水平表達(dá)經(jīng)典的外周痛覺(jué)介導(dǎo)受體，包括嘌呤受體（P2X3）、緩激肽受體（B2）以及降鈣素基因相關(guān)肽（CGRP）受體。(Robustly targets classic peripheral nociceptive signaling receptors, including purinergic P2X3, bradykinin B2, and CGRP receptors.)

• 骨架與標(biāo)志物蛋白 (Structural Halmarks)：外周蛋白（Peripherin）呈現(xiàn)強(qiáng)陽(yáng)性表達(dá)，神經(jīng)元特異性核蛋白 NeuN 以及 Tuj1 表達(dá)顯著上調(diào)。(Exhibits powerful positive expression of the peripheral filament Peripherin, alongside marked upregulation of the neuronal markers NeuN and Tuj1/Beta-III Tubulin.)

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

警告 / Critical WarningBioVector? ND7/23 細(xì)胞貼壁性較弱，在日常擴(kuò)增傳代時(shí)，千萬(wàn)不可將細(xì)胞密度稀釋得過(guò)低（如低于 1比10），否則細(xì)胞會(huì)因缺乏局部自分泌因子支持而停止生長(zhǎng)或發(fā)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 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? High-Glucose DMEM 高糖培養(yǎng)基（含 L-谷氨酰胺，含丙酮酸鈉 / containing L-Glutamine and sodium pyruvate）。

• 血清添加 (Serum Supplement)：10% BioVector? Premium Fetal Bovine Serum 優(yōu)質(zhì)胎牛血清。

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

B 階段：神經(jīng)功能分化培養(yǎng)基（實(shí)驗(yàn)前傷害感受器功能誘導(dǎo)）

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

• 基礎(chǔ)骨架 (Basal Matrix)：BioVector? Low-Serum High-Glucose DMEM 減毒低血清培養(yǎng)基（或采用無(wú)血清 BioVector? Neurobasal Medium）。

• 降低后的血清 (Reduced Serum)：0.5% 到 1.0% BioVector? Premium Fetal Bovine Serum 優(yōu)質(zhì)胎牛血清（嚴(yán)格限制血清濃度以強(qiáng)迫細(xì)胞退出分裂周期）。

• 核心功能組件 (Core Supplement)：2% BioVector? B-27 Supplement (50X 標(biāo)準(zhǔn)型)。

• 分化與通道啟動(dòng)劑 (Core Differentiation Inducers)：

  • BioVector? Forskolin (福司高林補(bǔ)劑)：終濃度為 10 到 20 uM（必加成分，用于上調(diào)胞內(nèi) cAMP 以驅(qū)動(dòng)軸突爆發(fā)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 (大鼠重組神經(jīng)生長(zhǎng)因子，可選)：終濃度 50 ng/mL（聯(lián)合 Forskolin 使用可顯著增強(qiáng) Nav1.8 等通道的電生理電流密度 / Optional; synergizes with Forskolin to substantially enhance the current density of voltage-gated Nav1.8 channels）。

4 物理環(huán)境與貼壁包被控制參數(shù) / Environmental Controls and Coating Parameters

• 孵育溫度 (Incubation Temperature)：37.0 攝氏度（Constant temperature at 37.0 degrees Celsius）。

• 氣相環(huán)境 (Gas Phase)：5% 二氧化碳 (CO2)，加濕平衡空氣 (5% Carbon Dioxide balanced with humidified atmospheric air)。

• 基質(zhì)包被規(guī)范 (Matrix Coating Protocol - Critical)：

  • 增殖期 (Growth Phase)：日常傳代擴(kuò)增時(shí)，細(xì)胞可直接接種于未包被的普通組織培養(yǎng)塑料瓶（TC-treated）中，但操作時(shí)需輕拿輕放，避免劇烈搖晃導(dǎo)致細(xì)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)：在進(jìn)行分化功能實(shí)驗(yàn)前，必須提前使用 BioVector? Poly-L-Lysine（聚左旋賴(lài)氨酸，PLL，0.01%）對(duì)孔板或蓋玻片進(jìn)行基礎(chǔ)包被，隨后加鋪一層 BioVector? Laminin（層粘連蛋白，5 到 10 ug/mL）進(jìn)行復(fù)合包被。只有復(fù)合包被底物才能牢固錨定長(zhǎng)軸突，防止其在換液或加藥刺激時(shí)脫落。(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 細(xì)胞傳代與誘導(dǎo)分化操作規(guī)范 / Subculturing and Differentiation Protocols

增殖期常規(guī)傳代操作（未分化狀態(tài)）/ Routine Passaging (Proliferating State)

• 傳代臨界點(diǎn) (Passaging Threshold)：當(dāng)細(xì)胞密度達(dá)到 80% 匯合度時(shí)必須傳代。切勿讓 BioVector? ND7/23 完全長(zhǎng)滿(mǎn)并重疊堆積，否則會(huì)導(dǎo)致細(xì)胞大面積老化脫落，并不可逆地喪失向傷害感受器分化的潛能。(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.)

• 建議傳代稀釋比例 (Splitting Ratio)：1:3 到 1:5（常規(guī)每 2 天傳代一次）。

1. 完全抽干陳舊的生長(zhǎng)培養(yǎng)基，使用無(wú)菌的 BioVector? PBS 緩沖液輕輕洗滌細(xì)胞單層 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（由于該細(xì)胞貼壁較輕，胰酶用量應(yīng)少于常規(guī)細(xì)胞），在室溫或 37 攝氏度孵箱中靜置消化 30 秒 到 1 分鐘。(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. 鏡下觀(guān)察發(fā)現(xiàn)大部分細(xì)胞變圓，輕敲瓶壁見(jiàn)細(xì)胞呈細(xì)沙狀快速滑落時(shí)，立刻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 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. 用移液槍極其輕柔地吹打瓶壁，切勿用力過(guò)猛，將細(xì)胞配制成均勻的單細(xì)胞懸液。(Pipette very gently to break up remaining clusters into a uniform single-cell suspension without mechanical shear damage.)

5. 在 200乘以g（低速離心力）下離心 5 分鐘，徹底棄去含有殘留胰酶的上清液。(Centrifuge at 200 x g for 5 minutes and thoroughly vacuum away the spent supernatant.)

6. 加入新鮮的 BioVector? 增殖期完全生長(zhǎng)培養(yǎng)基重懸，計(jì)數(shù)后接種至新的培養(yǎng)瓶中。(Resuspend in fresh Phase A growth medium, determine count, and allocate into fresh expansion vessels.)

實(shí)驗(yàn)前感覺(jué)神經(jīng)元樣分化流程 / Directed Sensory Differentiation Protocol

1. 將增殖期的 BioVector? ND7/23 細(xì)胞消化，以大約 3.0乘以10的4次方 cells/cm2 的中等密度接種于提前執(zhí)行過(guò) BioVector? PLL/Laminin 復(fù)合包被 的實(shí)驗(yàn)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. 首先使用常規(guī)的 10% 血清完全培養(yǎng)基，讓細(xì)胞貼壁恢復(fù)過(guò)夜（約 12 到 16 小時(shí)）。(Maintain initially in standard Phase A medium overnight for 12-16 hours to ensure secure initial anchoring.)

3. 次日，完全抽干原培養(yǎng)基，更換為含有 10 到 20 uM BioVector? Forskolin 的 B階段：神經(jīng)功能分化培養(yǎng)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. 持續(xù)誘導(dǎo) 48 到 72 小時(shí)（2 到 3 天），細(xì)胞將長(zhǎng)出極其復(fù)雜的網(wǎng)狀互聯(lián)突觸，此時(shí)細(xì)胞表面的 Nav1.7、Nav1.8 鈉通道和機(jī)械敏感 Piezo 通道功能達(dá)到表達(dá)巔峰。此時(shí)即可直接用于全細(xì)胞膜片鉗電生理記錄、機(jī)械劃痕刺激或辣椒素刺激引發(fā)的鈣成像熒光檢測(cè)。(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 復(fù)蘇與凍存恢復(fù)流線(xiàn) / 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，置于孵箱中平衡 pH 值與溫度。(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. 從液氮中取出 BioVector? ND7/23 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? 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 packed with 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 37 攝氏度孵箱栽培。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)胞碎片和殘留毒性成分。(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、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-1。屬于安全的工程化嚙齒類(lèi)動(dòng)物雜交系，無(wú)已知對(duì)人類(lèi)具致病性的病毒成分，實(shí)驗(yàn)廢棄物執(zhí)行常規(guī)高壓滅菌消毒程序。(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? Serum-Free Cryopreservation Medium 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）。嚴(yán)禁在零下 80 攝氏度機(jī)械冰箱中長(zhǎng)期擱置超過(guò) 2 周，否則該細(xì)胞株的復(fù)蘇存活率以及分化成長(zhǎng)突觸的電生理活性會(huì)發(fā)生斷崖式下跌。(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 功能質(zhì)控紅線(xiàn) / Functional Quality Control Threshold：隨著傳代次數(shù)的不斷增加（連續(xù)傳代超過(guò) 25 代以上），BioVector? ND7/23 雜交株容易發(fā)生嚴(yán)重的核型不穩(wěn)或染色體丟失，表現(xiàn)為：在加入 BioVector? Forskolin 誘導(dǎo)后雖然能變長(zhǎng)，但全細(xì)胞膜片鉗下記錄不到任何 TTX 耐受性的 Nav1.8 鈉電流，或者對(duì)機(jī)械壓力刺激失去了 Piezo 介導(dǎo)的快速失活內(nèi)流。因此，建議每隔 5 到 10 代，對(duì)分化后的細(xì)胞進(jìn)行一次通道表達(dá)抽檢（如通過(guò)實(shí)時(shí)定量 PCR 或特異性阻斷劑響應(yīng)測(cè)試）。若關(guān)鍵通道功能喪失，證實(shí)該代次細(xì)胞已退化，需立即廢棄并重新復(fù)蘇低代次種子庫(kù)。(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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