BioVector? NS-SV-AC Immortalized Human Astrocyte Cell Line / NS-SV-AC 人星形膠質永生化細胞系

一 產(chǎn)品基本信息與細胞生物學背景

• 細胞名稱：NS-SV-AC。

• 物種與組織來源：人類（Homo sapiens），源自人胎兒腦組織（腦皮質/中樞神經(jīng)系統(tǒng)前體細胞富集區(qū)的分離物）。

• 細胞系建立背景（人類星形膠質模式細胞）：

  傳統(tǒng)的原代人類星形膠質細胞體外壽命極短，難以通過多次傳代獲得均一穩(wěn)定的實驗批次。NS-SV-AC 細胞系是由科研團隊利用帶有猿猴病毒40大T抗原（SV40 Large T antigen）的復制缺陷型重組逆轉錄病毒/質粒載體，轉導原代人腦星形膠質細胞建立的永生化克隆株。SV40 Large T 抗原通過功能性結合并失活細胞內的宿主抑癌蛋白 p53 和視網(wǎng)膜母細胞瘤蛋白（pRb），從而繞過了正常的復制性衰老檢查點。該細胞系的成功建立，為研究人源中樞神經(jīng)系統(tǒng)（CNS）生理學、病理學及血腦屏障（BBB）提供了高度均一、可無限增殖的模型底盤。

• 核心表型與星形膠質細胞特征：

  • 標志物定性表達：經(jīng)免疫細胞化學和 Western Blot 驗證，NS-SV-AC 細胞強陽性表達星形膠質細胞的經(jīng)典特異性中間絲蛋白——膠質纖維酸性蛋白（Glial Fibrillary Acidic Protein, GFAP）。此外，細胞對 S100$\beta$ 蛋白 和 谷氨酰胺合成酶（Glutamine Synthetase, GS）亦表現(xiàn)出穩(wěn)定的陽性特征，維持了腦內中樞膠質系譜的核心生化和代謝屬性。

  • 形態(tài)學特征：貼壁生長。在常規(guī)低密度培養(yǎng)時，細胞呈現(xiàn)出原代星形膠質細胞特征性的多角形、扁平鋪展樣、或帶有數(shù)個粗短偽足的成纖維細胞樣形態(tài)。當細胞匯合度較高、融合成片時，胞體緊密靠攏，呈現(xiàn)典型的鋪路石狀膠質單層外觀。

• 生物安全級別：2級（BSL-2）。因其基因組內整合了 SV40 大 T 表達序列，盡管經(jīng)檢測無復制型傳染性病毒顆粒自發(fā)釋放，仍應在二級生物安全柜中按照 BSL-2 標準規(guī)程嚴謹操作。

二 核心科研價值與轉化醫(yī)學應用

NS-SV-AC 作為標桿性的人源膠質細胞株，被廣泛用于解構腦部生理和疾病演變的核心機制：

1. 中樞神經(jīng)系統(tǒng)神經(jīng)炎癥（Neuroinflammation）體外模型：

   星形膠質細胞是腦內天然免疫和炎性響應的核心放大器。NS-SV-AC 細胞對促炎細胞因子（如 TNF-$\alpha$, IL-1$\beta$, IFN-$\gamma$）以及脂多糖（LPS）高度敏感。在這些因子刺激下，該細胞能迅速發(fā)生膠質瘢痕化/反應性膠質增生（Reactive Astrogliosis）樣改變，豐度分泌 IL-6、IL-8、MCP-1（CCL2）等趨化因子及活性氧（ROS），是篩選新型中樞抗炎和神經(jīng)保護小分子藥物的優(yōu)異底盤。

2. 體外人源血腦屏障（BBB）共培養(yǎng)模型重構：

   在體內，星形膠質細胞的血管周足（End-feet）緊密包繞著腦毛細血管內皮細胞，是維持血腦屏障緊密連接和選擇性通透性的關鍵。NS-SV-AC 常與人腦微血管內皮細胞（如 hCMEC/D3）和人腦周細胞（Pericytes）聯(lián)合構建多細胞上下層 Transwell 共培養(yǎng)體系，用于體外定量測定中樞神經(jīng)系統(tǒng)候選藥物的跨上皮電阻（TEER）和屏障滲透率。

3. 神經(jīng)退行性疾?。ㄈ绨柎暮Ｄ?、ALS）與膠質毒性機制研究：

   星形膠質細胞的功能支持喪失或毒性功能獲得，直接驅動了神經(jīng)元的退行性病變。NS-SV-AC 被廣泛用于評估外源性 $\beta$-淀粉樣肽（A$\beta$）、Tau 蛋白寡聚體對膠質細胞代謝（如谷氨酸攝取、ATP 釋放）的破壞，探討其如何因谷氨酸轉運體（EAAT2/GLT-1）功能耗竭進而引發(fā)神經(jīng)元興奮性毒性。

三 實驗室細胞復蘇、貼壁常規(guī)培養(yǎng)、傳代與保存標準步驟

NS-SV-AC 細胞生長狀態(tài)強健，但在貼壁初期對外界剪切力有一定敏感性。在日常維護中需把控好消化酶活性，避免過度生長引發(fā)的細胞老齡化。

1. 專用培養(yǎng)基與核心成分配置

• 基礎培養(yǎng)基：高糖 DMEM 培養(yǎng)基（High-glucose DMEM）。

• 專用完全培養(yǎng)基配方：

  • 高糖 DMEM 基礎培養(yǎng)基

  • 加 10% 優(yōu)質胎牛血清（FBS）

  • 加 1% 青霉素-鏈霉素雙抗（Penicillin-Streptomycin）

• 細胞解離液：0.25% Trypsin-0.02% EDTA 消化液。

• 生長參數(shù)常數(shù)：37 攝氏度，5% 二氧化碳，飽和濕度。倍增時間通常為 24 - 30 小時（進入對數(shù)期后增殖平穩(wěn)迅速）。

2. 冷凍細胞復蘇步驟

1. 提前在無菌生物安全柜中配制好干凈的 T25 培養(yǎng)瓶，注入 5 - 6 mL 預熱至 37 攝氏度的完全培養(yǎng)基，并置于孵箱中預熱平衡。

2. 從液氮罐中取出 NS-SV-AC 凍存管，立刻全量投入 37 攝氏度恒溫水浴箱中快速搖晃解凍，確保管內冰塊在 1 分鐘內完全融化。

3. 用 75% 酒精噴灑消毒凍存管外壁，移入二級生物安全柜。

4. 用移液槍吸取全量解凍的細胞懸液，極其緩慢地逐滴滴入盛有 4 mL 預熱完全培養(yǎng)基的 15 mL 離心管中，輕柔顛倒一次混勻（務必輕柔，以防高滲透壓的 DMSO 對復蘇膜造成瞬間剪切物理損傷）。

5. 以 1000 rpm（約 200 g）室溫離心 5 分鐘，小心抽干含有 DMSO 的冷凍保護劑上清液。

6. 加入 1 mL 預熱的完全培養(yǎng)基，使用 P1000 移液槍輕輕重懸沉淀化開。

7. 將懸液全量接種至準備好的 T25 培養(yǎng)瓶中，輕柔十字晃動混勻，置于 37 攝氏度孵箱中培養(yǎng)。

8. 孵育 24 小時后，待細胞完全貼壁，全量更換一次新鮮的預熱完全培養(yǎng)基，以徹底清除死細胞碎屑及極微量殘留的 DMSO。

3. 日常貼壁常規(guī)傳代操作

• 傳代時機：當細胞匯合度（Confluency）達到 80% - 85% 時必須啟動傳代。NS-SV-AC 細胞若任其長滿至 100% 極度過密狀態(tài)，貼壁層極易自發(fā)增厚并堆疊，這會導致底層細胞由于缺乏營養(yǎng)和接觸抑制產(chǎn)生嚴重的空泡化（Vacuolation），從而惡化后續(xù)傳代時的貼壁效率。

• 操作流程：

  1. 吸除細胞瓶內的舊培養(yǎng)基，使用無菌的、不含鈣鎂離子的 PBS 緩沖液輕輕漂洗細胞表面 1 - 2 次，徹底洗凈血清殘余（血清中的大分子會顯著抑制胰酶的活性）。

  2. 加入適量 0.25% 胰酶-EDTA 消化液（T25 瓶常規(guī)加入 1 mL），全面覆蓋細胞層。置于 37 攝氏度孵箱中消化 1.5 - 3 分鐘。

  3. 在倒置顯微鏡下動態(tài)觀察。該細胞對胰酶較為敏感，當看到多角形或偽足狀的胞體快速收回變圓、胞間裂隙顯著增大、輕敲瓶壁細胞開始大面積整體滑動脫落時，立即加入 2 倍體積的含血清完全培養(yǎng)基終止消化。

  4. 用槍頭輕柔吹打瓶壁數(shù)次，將脫落的細胞全部收集至 15 mL 離心管中，1000 rpm 離心 5 分鐘。

  5. 棄上清，用預熱的完全培養(yǎng)基重懸，打散成單細胞懸液。按照 1 比 3 至 1 比 5 的稀釋比例接種入新的培養(yǎng)器皿中。通常每 2 - 3 天傳代一次。

4. 細胞長期保存標準

• 凍存液配方：90% 優(yōu)質完全培養(yǎng)基（或純胎牛血清） 加 10% 分析級二甲基亞砜（DMSO）。亦可使用市售高質免程序降溫的專用細胞凍存液。

• 冷凍規(guī)范：

  1. 必須收集處于連續(xù)傳代期間、處于對數(shù)生長最旺盛期（匯合度約 80%）、形態(tài)結構高度健康的 NS-SV-AC 細胞。

  2. 經(jīng)溫和消化、離心沉淀后，用配制好的冷凍液懸浮并調整密度至 每毫升 1,500,000 到 2,500,000 個細胞。

  3. 分裝至無菌凍存管中，立刻放入標準程序降溫盒（如 Mr. Frosty），并將其置于 零下 80 攝氏度超低溫冰箱內過夜，完成每分鐘穩(wěn)定降溫 1 攝氏度的穩(wěn)態(tài)梯度降溫。

  4. 次日，必須以極快速度將凍存管投遞至液氮罐（零下 196 攝氏度）的氣相或液相中鎖死長期保存。絕對禁止在 零下 80 度普通冰箱中存放超過 2 周，以防止長期的微小熱輻射導致細胞內部冰晶重組，進而導致 SV40 永生化表型及細胞活性的衰退。

Part 2 English Section

I General Information and Cell Biological Background

• Cell Line Name: NS-SV-AC.

• Organism and Tissue Extraction Origin: Homo sapiens (human); isolated from primary human fetal brain tissues (enriched micro-dissections of cerebral cortical pre-glial matrices).

• Cell Line Establishment Background (Human Astrocyte Model Chassis):

  Native primary human astrocytes isolated from neural biospecimens exhibit low in vitro proliferative limits and rapidly undergo replication senescence. To resolve this critical research bottleneck, the NS-SV-AC cell line was successfully generated via the integration of the Simian Virus 40 Large T antigen (SV40 Large T antigen) into primary fetal astrocytes using a replication-deficient retroviral/plasmid vector. The SV40 Large T antigen continuously complexes with and inactivates host tumor suppressor checkpoints—specifically p53 and the Retinoblastoma protein (pRb)—allowing the cultures to bypass replication boundaries. NS-SV-AC serves as an infinitely expanding, highly uniform human model to explore central nervous system (CNS) homeostasis, neuro-pathology, and blood-brain barrier (BBB) kinetics.

• Core Morphological Phenotype and Lineage Characterization:

  • Lineage Expression Blueprint: Immunocytochemical and Western blot profiling confirm intense, stable expression of the definitive astrocyte intermediate filament protein—Glial Fibrillary Acidic Protein (GFAP). The line also maintains baseline validation for S100$\beta$ protein and Glutamine Synthetase (GS), confirming the preservation of central astrocytic metabolic networks.

  • Morphological Form: Adherent growth; under low-density subconfluent propagation, cells present a classic polygonal, broad, flattened, or fibroblast-like structure equipped with several short pseudopodia. Upon reaching high-density confluency, the cellular margins interlock, organizing into a uniform, cobblestone-like glial monolayer sheet.

• Biosafety Matrix: Classified under Biosafety Level 2 (BSL-2) criteria. Because the genomic DNA contains integrated SV40 Large T antigen open reading frames, all wet-lab manipulations must be strictly handled within certified Class II Biosafety Cabinets adhering to BSL-2 guidelines, despite zero baseline shedding of replication-competent viral particles.

II Strategic Research Value and Translational Fields

As a representative human astrocyte model, NS-SV-AC is widely utilized across several key neurobiological research fields:

1. Modeling CNS Neuroinflammation Cascades:

   Astrocytes serve as critical sensors and structural amplification nodes for innate immunity within the brain. NS-SV-AC cells are highly sensitive to pro-inflammatory cytokine exposure (e.g., TNF-$\alpha$, IL-1$\beta$, IFN-$\gamma$) and Lipopolysaccharide (LPS) challenges. Upon induction, the cells undergo reactive astrogliosis-like transformations, releasing significant chemokine profiles (IL-6, IL-8, MCP-1/CCL2) along with reactive oxygen species (ROS). This renders the line an optimal screening engine for evaluating novel central anti-inflammatory and neuroprotective small molecules.

2. Reconstituting In Vitro Human Blood-Brain Barrier (BBB) Matrices:

   In vivo, astrocytic end-feet wrap around brain capillary endothelial networks, regulating tight junction integrity and select transport permeability. NS-SV-AC is standardly co-cultured inside Transwell systems alongside human brain microvascular endothelial cells (such as the hCMEC/D3 framework) and human brain pericytes to assess Trans-Epithelial Electrical Resistance (TEER) metrics and clear trans-barrier chemical permeation constants.

3. Investigating Glial Toxicity in Neurodegenerative Progression (AD & ALS):

   The loss of physiological astrocytic support combined with the gain of neurotoxic phenotypes directly accelerates synaptic degradation. NS-SV-AC functions as a baseline substrate to evaluate cellular stress driven by exogenous $\beta$-amyloid (A$\beta$) and tau oligomers, mapping how the loss of glutamate transporters (such as EAAT2/GLT-1) leads to impaired clearance dynamics and secondary neuronal excitotoxicity.

III Laboratory Thawing, Cultivation, Passaging, and Cryopreservation Protocols

NS-SV-AC cells exhibit a highly robust growth profile but require careful monitoring of enzymatic cleavage kinetics during dissociation and strict confluency management to maintain standard phenotype lines.

1. Growth Medium & Nutrient Component Formulations

• Basal Medium: High-glucose DMEM medium.

• Complete Growth Matrix Formulation:

  • High-glucose DMEM basal medium

  • Enriched with 10% premium Fetal Bovine Serum (FBS)

  • Supplemented with 1% standard Penicillin-Streptomycin dual antibiotics.

• Cell Dissociation Enzyme: Standard 0.25% Trypsin-0.02% EDTA solution.

• Environmental Cultivation Constants: Incubate at 37 degrees Celsius inside a humidified atmosphere charged with 5% Carbon Dioxide. Population doubling times hover at a clean 24 to 30 hours during steady log-phase expansion.

2. Cryovial Thawing and Recovery Sequence

1. Pre-warm a sterile T25 tissue culture flask filled with 5 - 6 mL of complete growth medium inside a 37 degrees Celsius incubator to equilibrate the pH.

2. Retrieve the NS-SV-AC cryovial from liquid nitrogen storage and submerge it immediately within a 37 degrees Celsius constant-temperature water bath. Shake rapidly and continuously to secure absolute liquefaction within 60 seconds.

3. Decontaminate the exterior casing with 75% ethanol before transferring the vial into the Class II Biosafety Cabinet.

4. Extract the thawed cell slurry using a pipettor and deliver it extremely slowly, dropwise into a 15 mL conical tube packed with 4 mL of pre-warmed complete growth medium. Handle gently; a gradual addition minimizes acute osmotic shock mediated by the cryoprotectant matrix.

5. Centrifuge the suspension at 1000 rpm (approximately 200 g) for 5 minutes at room temperature, then carefully decant the DMSO-laden supernatant.

6. Dispense 1 mL of pre-warmed complete growth medium onto the pellet and resuspend gently using a P1000 micro-pipette. Transfer the cell suspension into the prepared T25 flask, cross-shake smoothly to optimize seeding distribution, and incubate under standard atmospheric constants.

7. Allow cells to attach securely for 24 hours. Perform a complete medium change using pre-warmed complete growth medium to clear non-adherent fragments and trace fractions of residual DMSO.

3. Routine Adherent Passaging Mechanics and Maintenance

• Confluency Control Window: Subculturing routines must be initiated when monolayers achieve an optimal 80% - 85% confluency range. Never allow NS-SV-AC sheets to reach absolute 100% full saturation or overgrowth. Overcrowded monolayers pile up and stratify, causing widespread baseline vacuolation due to localized nutrient deprivation, which degrades post-passage attachment efficiency.

• Passaging Execution Steps:

  1. Aspirate the spent growth matrix and gently rinse the cell sheet 1 - 2 times using sterile, calcium/magnesium-free PBS to remove all remaining serum proteins that could deactivate the trypsin.

  2. Administer a suitable volume of 0.25% Trypsin-EDTA enzyme (typically 1 mL for a T25 flask format), slide the fluid to cover the monolayer completely, and place inside the 37 degrees Celsius incubator for 1.5 - 3 minutes.

  3. Monitor cell detachment kinetics under an inverted microscope. As the polygonal cells retract their pseudopodia, round up, and slide freely upon gentle physical tapping of the flask wall, immediately add 2 volumes of serum-fortified complete growth medium to arrest enzymatic cleavage.

  4. Gently pipette the suspension against the flask interior surfaces to clear remaining clusters, collect the fluid into a conical tube, and centrifuge at 1000 rpm for 5 minutes.

  5. Discard the supernatant, resuspend the cell pellet in fresh, pre-warmed complete growth medium, and inoculate into new flasks utilizing standard split ratios of 1:3 to 1:5. Subculture every 2 - 3 days.

4. Long-Term Cryopreservation Standards

• Cryoprotectant Preservation Matrix: 90% premium complete growth medium (or pure FBS) supplemented with 10% analytical-grade Dimethyl Sulfoxide (DMSO) (or validated commercial serum-free preservation media).

• Freezing Protocol Validation:

  1. Exclusively harvest healthy, log-phase cultures showing an optimal confluency of approximately 80% under standard maintenance parameters.

  2. Post-enzymatic treatment and centrifugation, adjust the cell concentration inside the formulated cryoprotectant matrix to a target range of 1,500,000 to 2,500,000 cells per milliliter.

  3. Dispense the suspension into sterile cryovials, insert them immediately into a controlled-rate freezing device (e.g., Mr. Frosty), and place into a minus 80 degrees Celsius freezer overnight to ensure a steady gradient cooling rate of 1 degree Celsius per minute.

  4. The following day, swiftly transfer the frozen cryovials into liquid nitrogen storage tanks (minus 196 degrees Celsius) for definitive long-term preservation. Do not store vials indefinitely inside a minus 80 degrees Celsius freezer; minor temperature oscillations can lead to cryogenic matrix degradation, compromising the SV40 immortalized phenotype and lowering post-thaw recovery rates.

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