BioVector? F-11 條件永生化神經(jīng)瘤細(xì)胞株文本版說明書

BioVector? F-11 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 (Product Name)：BioVector? F-11 條件永生化大鼠/小鼠雜交神經(jīng)瘤細(xì)胞株 (BioVector? F-11 Conditionally Immortalized Rat/Mouse Hybrid Neuroblastoma Cell Line)

• 常用別名 (Synonyms)：BioVector? F-11，BioVector? F11 Cell

• 生物學(xué)來源 (Organism Source)：體細(xì)胞雜交株（大鼠 Rattus norvegicus 胚胎背根神經(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)感覺神經(jīng)元特性 (Peripheral Nervous System / Dorsal Root Ganglion Sensory Neuron Properties)

• 生長特性 (Growth Properties)：

  • 增殖期/未分化 (Proliferating Phase)：貼壁生長，細(xì)胞呈多邊形、成纖維細(xì)胞樣或未成熟神經(jīng)母細(xì)胞樣狀態(tài)，分裂速度較快。(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)：細(xì)胞停止有絲分裂，胞體皺縮并向外延伸出明顯的神經(jīng)元樣突觸，細(xì)胞間形成突觸網(wǎng)狀互聯(lián)結(jié)構(gòu)。(Mitotic division ceases. Cell bodies refract and extend prominent neuron-like neurites, developing an interconnected synaptosome-like network.)

• 雜交永生化機(jī)制 (Immortalization Mechanism)：通過聚乙二醇（PEG）介導(dǎo)技術(shù)，將大鼠原代背根神經(jīng)節(jié)（DRG）感覺神經(jīng)元與缺少 HPRT 酶的小鼠 N18TG2 神經(jīng)母細(xì)胞瘤細(xì)胞進(jìn)行無性系融合，通過 HAT 培養(yǎng)基篩選獲得既能無限增殖又保留感覺神經(jīng)元特征的雜交細(xì)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.)

• 核心科研價(jià)值 (Core Research Significance)：BioVector? F-11 是研究外周痛覺神經(jīng)元機(jī)制最經(jīng)典、最穩(wěn)定的底盤細(xì)胞系之一。由于其同時(shí)具備神經(jīng)瘤的易培養(yǎng)性與 DRG 神經(jīng)元的功能活性，被廣泛應(yīng)用于熱與機(jī)械痛覺感受性研究、阿片類受體與緩激肽受體信號(hào)通路分析、外周神經(jīng)營養(yǎng)因子功能鑒定、以及痛覺過敏和鎮(zhèn)痛小分子藥物的體外高通量篩選。(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 細(xì)胞生物學(xué)特征與核心標(biāo)志物表型 / Cellular Properties and Biomarker Profiles

BioVector? F-11 雜交株在常規(guī)培養(yǎng)時(shí)表現(xiàn)為瘤樣增殖，通過化學(xué)誘導(dǎo)可迅速向外周感覺神經(jīng)元表型轉(zhuǎn)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:

未分化增殖狀態(tài) (Proliferating State)

細(xì)胞倍增時(shí)間通常在 20 到 30 小時(shí)之間。細(xì)胞形態(tài)相對(duì)扁平或呈梭形，匯合度高時(shí)會(huì)發(fā)生多層堆疊生長。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.

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

當(dāng)向培養(yǎng)基中加入低血清以及低濃度的 BioVector? Forskolin 或分化補(bǔ)劑后，細(xì)胞停止有絲分裂并伸展出長達(dá)數(shù)百微米的神經(jīng)軸突。其功能標(biāo)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:

• 特異性感覺受體 (Sensory Receptors)：高水平表達(dá)功能性 TRPV1（辣椒素受體），能對(duì)辣椒素刺激產(chǎn)生顯著的陽性鈣內(nèi)流響應(yīng)；同時(shí)高表達(dá)緩激肽 B2 受體（Bradykinin B2 Receptor）及 delta、mu 阿片受體。(Highly expresses functional TRPV1 capsaicin receptors, yielding robust calcium influx upon capsaicin challenge; co-expresses Bradykinin B2 receptors alongside delta and mu opioid receptors.)

• 神經(jīng)元結(jié)構(gòu)蛋白 (Structural Halmarks)：高表達(dá)外周神經(jīng)特異性中間絲蛋白（外周蛋白 Peripherin），以及常規(guī)神經(jīng)絲蛋白（Neurofilament）和 Tuj1 蛋白。(Demonstrates elevated expression of the peripheral nerve-specific intermediate filament Peripherin, alongside neurofilament structures and Tuj1 protein.)

• 電生理及神經(jīng)遞質(zhì)特征 (Electrophysiological Traits)：分化后的細(xì)胞具備對(duì)電刺激產(chǎn)生功能性全或無動(dòng)作電位的能力，并能合成和釋放與痛覺傳導(dǎo)密切相關(guān)的神經(jīng)遞質(zhì)。(Differentiated entries display the capability to generate functional all-or-none action potentials under clamp stimulation and synthesize neurotransmitters associated with pain transmission.)

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

警告 / Critical WarningBioVector? F-11 屬于融合雜交株，傳代密度過低會(huì)導(dǎo)致細(xì)胞喪失分泌因子支持而大面積死亡。分化誘導(dǎo)時(shí)必須降低血清濃度并加入分化補(bǔ)劑，否則細(xì)胞將維持瘤性增殖而不長突觸。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 階段：增殖期完全生長培養(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 4.5 g/L 葡萄糖，含 L-谷氨酰胺，含丙酮酸鈉 / 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 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 減毒低血清高糖培養(yǎng)基（或直接采用 BioVector? Neurobasal Medium 神經(jīng)元基礎(chǔ)培養(yǎng)基）。

• 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（低血清是退出細(xì)胞周期的核心原動(dòng)力）。

• 核心功能組件 (Core Supplement)：1% BioVector? B-27 Supplement (50X 標(biāo)準(zhǔn)型)。

• 分化與受體啟動(dòng)劑 (Core Differentiation Inducers)：

  • BioVector? Forskolin (福司高林補(bǔ)劑)：終濃度為 10 到 20 uM（用以激活腺苷酸環(huán)化酶，上調(diào) cAMP 信號(hào)通路，促使 TRPV1 通道和軸突表達(dá) / 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)：

  • 增殖期 (Growth Phase)：日常擴(kuò)增與細(xì)胞傳代時(shí)，細(xì)胞貼壁能力極強(qiáng)，可直接接種于未包被的普通塑料培養(yǎng)瓶中。(Cells exhibit high adherence and can be passaged into uncoated tissue culture-treated flasks during expansion.)

  • 分化期 (Differentiation Phase)：為了防止分化后期神經(jīng)軸突回縮拉扯導(dǎo)致胞體成片脫落，在進(jìn)行分化實(shí)驗(yàn)接種前，建議提前使用 BioVector? Poly-L-Lysine（聚左旋賴氨酸，PLL，0.01%）對(duì)孔板進(jìn)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 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)胞融合度達(dá)到 80% 到 85% 時(shí)必須及時(shí)傳代。如果細(xì)胞徹底長滿并發(fā)生多層重疊，接觸抑制會(huì)導(dǎo)致細(xì)胞自發(fā)老化并降低后續(xù)對(duì)辣椒素的響應(yīng)靈敏度。(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.)

• 建議傳代稀釋比例 (Splitting Ratio)：1:4 到 1:6（通常每 2 到 3 天需傳代一次）。

1. 完全抽干陳舊的培養(yǎng)基，使用無菌的 BioVector? PBS 緩沖液輕輕洗滌細(xì)胞表面 1 次。(Aspirate spent medium and rinse the monolayer once with sterile BioVector? PBS.)

2. 加入適量預(yù)熱的 BioVector? 0.25% Trypsin-EDTA 消化液，置于 37 攝氏度孵箱中消化 1 到 2 分鐘。(Introduce an appropriate volume of pre-warmed BioVector? 0.25% Trypsin-EDTA and incubate at 37 degrees Celsius for 1-2 minutes.)

3. 鏡下觀察到大部分細(xì)胞變圓變亮，輕敲培養(yǎng)瓶四周見細(xì)胞成片脫落時(shí)，立即加入雙倍體積的含血清增殖期完全培養(yǎng)基終止胰酶活性。(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. 用移液槍輕柔吹打貼壁表面，配置成均勻無團(tuán)塊的單細(xì)胞懸液。(Pipette gently to disperse the mixture into a uniform single-cell suspension.)

5. 將懸液收集至離心管，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 discard the supernatant.)

6. 加入新鮮的 BioVector? 增殖期完全生長培養(yǎng)基重懸，調(diào)整接種密度并分裝至新培養(yǎng)瓶中。(Resuspend in fresh growth medium and allocate at correct densities into fresh flasks.)

實(shí)驗(yàn)前感覺神經(jīng)元樣短周期分化流程 / Directed Short-Cycle Differentiation Protocol

1. 將增殖期的 BioVector? F-11 細(xì)胞消化重懸，以大約 2.5乘以10的4次方 cells/cm2 的密度接種于提前使用 BioVector? PLL 包被 的實(shí)驗(yàn)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. 使用常規(guī)的 10% 血清完全培養(yǎng)基讓細(xì)胞貼壁恢復(fù)過夜（約 16 小時(shí)）。(Maintain in Phase A growth medium overnight for approx 16 hours to facilitate initial anchoring.)

3. 次日，完全吸干孔板內(nèi)的培養(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（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. 誘導(dǎo) 48 到 96 小時(shí)（2 到 4 天） 期間，細(xì)胞將完全退出有絲分裂，并長出長而清晰的交織神經(jīng)突觸。此時(shí)細(xì)胞表面的 TRPV1 離子通道與阿片受體功能達(dá)到表達(dá)峰值，可直接用于辣椒素或緩激肽刺激引發(fā)的鈣成像熒光檢測(cè)、全細(xì)胞電生理膜片鉗分析以及疼痛信號(hào)轉(zhuǎn)導(dǎo)通路阻斷實(shí)驗(yàn)。(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 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培養(yǎng)基，置于 37 攝氏度孵箱中平衡 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. 從液氮中取出 BioVector? F-11 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? 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 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 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 impurities.)

7 生物安全、長期保存與質(zhì)控規(guī)范 / Biosafety, Storage and Quality Controls

• 生物安全級(jí)別 (Biosafety Level)：BSL-1。屬于常規(guī)的嚙齒類動(dòng)物工程化雜交系，無已知人類病原體傳染性，實(shí)驗(yàn)廢棄物按常規(guī)醫(yī)療廢棄物高壓滅菌處理即可。(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.)

• 超低溫長期保存 (Long-Term Banking)：凍存保質(zhì)配方為 90% BioVector? 增殖期完全生長培養(yǎng)基 + 10% 細(xì)胞級(jí) DMSO（或采用 BioVector? Serum-Free Cryopreservation Medium 無血清通用型凍存液）。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）。切勿長期擱置在零下 80 攝氏度機(jī)械冰箱中，否則細(xì)胞株的復(fù)蘇存活率以及分化成長突觸的活性會(huì)隨著時(shí)間推移發(fā)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 功能質(zhì)控紅線 / Functional Quality Control Threshold：隨著傳代次數(shù)的增加（連續(xù)傳代超過 30 代以上），雜交瘤細(xì)胞容易發(fā)生染色體丟失或表型退化，表現(xiàn)為：在加入 BioVector? Forskolin 誘導(dǎo)后雖然能變長，但對(duì)辣椒素（Capsaicin）刺激不再產(chǎn)生任何鈣流內(nèi)流信號(hào)。因此，建議每隔 5 到 10 代，挑取一孔分化后的細(xì)胞加入 1 uM 的 Capsaicin，通過鈣熒光染料驗(yàn)證其受體功能性。若功能性消失，需立即廢棄該代次細(xì)胞，并重新復(fù)蘇低代次種子庫。(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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