BioVector? 50B11 條件永生化大鼠感覺神經(jīng)元細胞株文本版說明書

BioVector? 50B11 Conditionally Immortalized Rat Sensory Neuron 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? 50B11 條件永生化大鼠感覺神經(jīng)元細胞株 (BioVector? 50B11 Conditionally Immortalized Rat Sensory Neuron Cell Line)

• 常用別名 (Synonyms)：BioVector? 50B11，BioVector? 50B11 Rat DRG Line

• 生物學來源 (Organism Source)：大鼠 Rattus norvegicus（胚胎期第 14.5 天，E14.5）

• 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) (Peripheral Nervous System / Dorsal Root Ganglion, DRG)

• 生長特性 (Growth Properties)：

  • 增殖期/未分化 (Proliferating Phase)：貼壁生長，細胞呈圓形、卵圓形或短梭形的單層前體細胞狀態(tài)。(Adherent growth, presenting as a monolayer of round, ovoid, or short spindle-shaped neural progenitor cells.)

  • 分化期/成熟后 (Differentiated Phase)：有絲分裂完全停止，細胞雙向或多向延展出極長的神經(jīng)突觸，互相交織成極其復雜的神經(jīng)元互聯(lián)網(wǎng)絡。(Mitotic division completely ceases. Cells extend extremely long neurites bi-directionally or multi-directionally, intertwining into a highly complex, functional neuronal network.)

• 永生化工程機制 (Immortalization Mechanism)：細胞通過逆轉(zhuǎn)錄病毒轉(zhuǎn)導技術，在基因組中集成了受調(diào)控的調(diào)節(jié)型 v-Myc 癌基因。(Cells are engineered via retroviral transduction to integrate a regulated v-Myc oncogene within the host genome.)

• 核心科研價值 (Core Research Significance)：原代大鼠 DRG 神經(jīng)元極難成活且無法傳代，BioVector? 50B11 完美解決了這一瓶頸。它廣泛用于外周傷害性感受等研究，也常用于由化療藥物（如紫杉醇、順鉑）引起的周圍神經(jīng)病變（CIPN）的毒理學評價，以及靶向鎮(zhèn)痛藥與離子通道阻斷劑的高通量藥效篩選。(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 細胞生物學特征與核心標志物表型 / Cellular Properties and Biomarker Profiles

BioVector? 50B11 具備極其典型的“干性/未分化”與“成熟/感覺神經(jīng)元”雙向表型切換特征：The BioVector? 50B11 line manifests a highly distinct bidirectional phenotypic switch between the progenitor stem state and the mature sensory neuron state:

未分化增殖狀態(tài) (Proliferating State)

細胞分裂旺盛。在常規(guī)生長培養(yǎng)基維持時，v-Myc 保持活躍轉(zhuǎn)錄，驅(qū)動細胞快速擴增（倍增時間約為 24 到 36 小時）。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.

誘導分化成熟狀態(tài) (Differentiated State)

當向培養(yǎng)基中加入分化啟動劑后，細胞在 24 到 48 小時內(nèi)徹底退出細胞周期，停止一切有絲分裂，并迅速向外彈射出粗壯的神經(jīng)軸突。其分子標志物在數(shù)天內(nèi)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:

• 外周特異性標志物 (Peripheral Markers)：強陽性表達外周神經(jīng)特異性中間絲蛋白（外周蛋白 Peripherin），以及神經(jīng)元特異性核蛋白 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.)

• 痛覺關鍵離子通道 (Nociceptive Ion Channels)：在特定刺激下，細胞能夠穩(wěn)定表達并維持高水平的 TRPV1（辣椒素受體）、TRPA1 以及電壓門控鈉通道 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.)

• 神經(jīng)肽譜系 (Neuropeptide Expression)：分化后的細胞可分泌經(jīng)典的外周神經(jīng)肽（P物質(zhì) Substance P 和降鈣素基因相關肽 CGRP），表現(xiàn)出經(jīng)典肽能感覺神經(jīng)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 專用兩階段培養(yǎng)基配方規(guī)范 / Culturing Medium Formulations

警告 / Critical WarningBioVector? 50B11 的分化需要依賴 cAMP 信號通路的激活。在常規(guī)擴增階段，絕對不能接觸福司高林（Forskolin），否則會導致感受態(tài)細胞提前分化并喪失傳代能力。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 階段：增殖期完全生長培養(yǎng)基（用于常規(guī)細胞擴增與維持）

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

• 基礎培養(yǎng)基 (Basal Medium)：BioVector? High-Glucose DMEM 高糖培養(yǎng)基（含 L-谷氨酰胺，不含丙酮酸鈉 / containing L-Glutamine, without 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（實驗前傷害感受器功能誘導）

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

• 基礎骨架 (Basal Matrix)：BioVector? Neurobasal Medium 神經(jīng)元基礎培養(yǎng)基。

• 核心功能組件 (Core Supplement)：2% BioVector? B-27 Supplement (50X 標準型)。

• 分化與干性關閉核心啟動劑 (Core Differentiation Inducers)：

  • BioVector? Forskolin (福司高林補劑)：終濃度為 10 到 50 uM（必加成分，用于上調(diào)胞內(nèi) cAMP，啟動 TRPV1 等通道表達并驅(qū)動突觸爆發(fā)式生長 / Essential component; drives intracellular cAMP levels to upregulate TRPV1 channels and propel explosive neurite outgrowth）。

  • BioVector? Doxycycline (多西環(huán)素補劑，視具體亞克隆配套)：終濃度 1.0 ug/mL（用以徹底關閉 v-Myc 不死性 / Final concentration at 1.0 ug/mL to suppress v-Myc-mediated immortalization）。

• 神經(jīng)營養(yǎng)因子 (Neurotrophic Factors, 可選/增強型)：BioVector? Recombinant Rat NGF (大鼠重組神經(jīng)生長因子)，終濃度 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（溫控誤差應小于正負 0.3 攝氏度 / Constant temperature with variations within plus or minus 0.3 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)：日常擴增時，細胞可直接貼壁于標準的塑料培養(yǎng)瓶（TC-treated）中。(Cells can be seeded directly into standard tissue culture-treated plasticware during expansion.)

  • 分化期 (Differentiation Phase)：由于細胞分化后突觸極其細長且張力極大，若直接接種在裸塑料板上，細胞會在突觸回縮時成片脫落。在啟動分化接種前，必須提前使用 BioVector? Poly-L-Lysine（聚左旋賴氨酸，PLL，0.01%）包被孔板，隨后再加一層 BioVector? Laminin（層粘連蛋白，5 到 10 ug/mL）進行復合包被。(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 細胞傳代與誘導分化操作規(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 (Passaging Threshold)：當細胞單層密度達到 75% 至 85% 匯合度時必須傳代。BioVector? 50B11 密度過高時容易自發(fā)聚集堆疊，導致底層細胞受壓分化，因此嚴禁將細胞養(yǎng)得過滿。(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.)

• 建議傳代稀釋比例 (Splitting Ratio)：1:4 到 1:6（常規(guī)每 2 到 3 天傳代一次 / Typically passaged every 2 to 3 days）。

1. 完全吸除陳舊的生長培養(yǎng)基，使用不含鈣鎂離子的無菌 BioVector? PBS 緩沖液洗滌細胞單層 1 次。(Completely aspirate spent medium and wash the monolayer once with sterile, calcium-free and magnesium-free BioVector? PBS.)

2. 加入適量預熱的 BioVector? 0.25% Trypsin-EDTA 消化液，在 37 攝氏度孵箱中靜置消化 1 到 2 分鐘。(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. 鏡下觀察發(fā)現(xiàn)細胞變圓、輕敲瓶身見細胞呈細沙狀脫落時，立即加入等體積的含血清增殖期完全培養(yǎng)基終止消化。(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. 用移液槍輕柔吹打貼壁面，將細胞徹底分散為均勻的單細胞懸液。(Gently pipette against the vessel culture surface to disperse cells into a homogeneous single-cell suspension.)

5. 將細胞懸液收集至離心管中，在 200乘以g（低速離心力）下離心 5 分鐘，徹底棄去上清液。(Harvest the suspension into a conical tube, centrifuge at 200 x g for 5 minutes, and completely decant the spent supernatant.)

6. 加入新鮮的增殖期完全生長培養(yǎng)基重懸，按常規(guī)比例分裝至新的培養(yǎng)瓶中。(Resuspend the cell pellet in fresh Phase A proliferation medium, perform cell counting, and distribute evenly into new flasks.)

實驗前神經(jīng)元短周期誘導分化流程 / Short-Cycle Directed Differentiation Protocol

BioVector? 50B11 具有極其迅猛的軸突生長能力，屬于典型的高效短周期實驗模型：The BioVector? 50B11 line demonstrates exceptionally rapid neurite outgrowth dynamics, serving as a highly efficient short-cycle experimental system:

1. 將增殖期的 BioVector? 50B11 細胞消化，以中等稀釋密度（約 3.0乘以10的4次方 cells/cm2）接種至提前進行過 BioVector? PLL/Laminin 復合包被 的實驗板中。(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. 接種時使用常規(guī)的增殖培養(yǎng)基，讓細胞在孵箱中貼壁過夜（約 12 到 18 小時）以恢復狀態(tài)。(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，徹底吸干舊培養(yǎng)基，更換為含有 10 到 50 uM BioVector? Forskolin 的 B階段：神經(jīng)功能分化培養(yǎng)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. 更換培養(yǎng)基 12 小時后，即可在顯微鏡下觀察到大量細胞伸出兩極軸突。(Extensive bi-polar neurite extensions can be observed visually under the microscope within 12 hours post-induction.)

5. 誘導 48 到 72 小時（2 到 3 天） 時，突觸生長達到巔峰并互相連接，此時細胞內(nèi)的 TRPV1、Nav1.7 離子通道大量功能化開放。此時即可直接用于辣椒素（Capsaicin）刺激引發(fā)的鈣成像（Calcium Imaging）檢測，或加入化療藥物進行神經(jīng)軸突毒性斷裂動力學研究。(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 復蘇與凍存恢復流線 / Thawing and Post-Cryo Recovery Workflow

1. 提前在標準 T25 培養(yǎng)瓶中注入 5.0 mL 預熱的增殖期完全生長培養(yǎng)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. 從液氮中取出 BioVector? 50B11 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? 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凈臺，用移液槍吸出胞懸液，緩慢逐滴注入盛有 4.0 mL 預熱增殖培養(yǎng)基的 15 mL 離心管中。(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 aspirate the supernatant to eradicate toxic DMSO residues.)

5. 加入 1.5 mL 新鮮增殖培養(yǎng)基輕彈懸起細胞沉淀，隨后全量接種到預熱平衡的 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 攝氏度孵箱栽培。24 小時后必須進行一次全量換液，以清除未貼壁的死細胞和殘留毒性成分。(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、長期保存與質(zhì)控規(guī)范 / Biosafety, Storage and Quality Controls

• 生物安全級別 (Biosafety Level)：BSL-1 或 BSL-2（視各國家/研究機構對逆轉(zhuǎn)錄病毒工程株的定義而定）。屬于標準的工程化嚙齒類動物細胞系，日常操作需在無菌操作臺內(nèi)進行，廢棄物執(zhí)行常規(guī)高壓滅菌程序。(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.)

• 超低溫長期保存 (Long-Term Banking)：凍存保質(zhì)配方為 90% 增殖期完全生長培養(yǎng)基 + 10% 細胞級 DMSO（或直接采用 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）。嚴禁在零下 80 攝氏度普通冰箱內(nèi)長期存放，否則復蘇后的分裂活力會發(fā)生嚴重衰退。(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 響應性定期抽檢 / Functional Quality Control Threshold：隨著傳代次數(shù)的增多（連續(xù)傳代超過 20 到 25 代后），BioVector? 50B11 細胞容易發(fā)生表型漂移，表現(xiàn)為：在加入 BioVector? Forskolin 誘導后雖然能長出突觸，但對辣椒素（Capsaicin）刺激不再產(chǎn)生強烈的鈣流信號。因此，每隔 5 到 10 代，應抽取一孔分化后的細胞加入 1 到 10 uM 的 Capsaicin，通過熒光染料驗證其鈣內(nèi)流響應。若響應信號微弱，證實該代次細胞已退化，需立即廢棄并重新復蘇低代次種子庫。(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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