BioVector® NS-SV-AC Immortalized Human Astrocyte Cell Line / NS-SV-AC 人星形胶质永生化细胞系

一 产品基本信息与细胞生物学背景

• 细胞名称：NS-SV-AC。

• 物种与组织来源：人类（Homo sapiens），源自人胎儿脑组织（脑皮质/中枢神经系统前体细胞富集区的分离物）。

• 细胞系建立背景（人类星形胶质模式细胞）：

  传统的原代人类星形胶质细胞体外寿命极短，难以通过多次传代获得均一稳定的实验批次。NS-SV-AC 细胞系是由科研团队利用带有猿猴病毒40大T抗原（SV40 Large T antigen）的复制缺陷型重组逆转录病毒/质粒载体，转导原代人脑星形胶质细胞建立的永生化克隆株。SV40 Large T 抗原通过功能性结合并失活细胞内的宿主抑癌蛋白 p53 和视网膜母细胞瘤蛋白（pRb），从而绕过了正常的复制性衰老检查点。该细胞系的成功建立，为研究人源中枢神经系统（CNS）生理学、病理学及血脑屏障（BBB）提供了高度均一、可无限增殖的模型底盘。

• 核心表型与星形胶质细胞特征：

  • 标志物定性表达：经免疫细胞化学和 Western Blot 验证，NS-SV-AC 细胞强阳性表达星形胶质细胞的经典特异性中间丝蛋白——胶质纤维酸性蛋白（Glial Fibrillary Acidic Protein, GFAP）。此外，细胞对 S100$\beta$ 蛋白 和 谷氨酰胺合成酶（Glutamine Synthetase, GS）亦表现出稳定的阳性特征，维持了脑内中枢胶质系谱的核心生化和代谢属性。

  • 形态学特征：贴壁生长。在常规低密度培养时，细胞呈现出原代星形胶质细胞特征性的多角形、扁平铺展样、或带有数个粗短伪足的成纤维细胞样形态。当细胞汇合度较高、融合成片时，胞体紧密靠拢，呈现典型的铺路石状胶质单层外观。

• 生物安全级别：2级（BSL-2）。因其基因组内整合了 SV40 大 T 表达序列，尽管经检测无复制型传染性病毒颗粒自发释放，仍应在二级生物安全柜中按照 BSL-2 标准规程严谨操作。

二 核心科研价值与转化医学应用

NS-SV-AC 作为标杆性的人源胶质细胞株，被广泛用于解构脑部生理和疾病演变的核心机制：

1. 中枢神经系统神经炎症（Neuroinflammation）体外模型：

   星形胶质细胞是脑内天然免疫和炎性响应的核心放大器。NS-SV-AC 细胞对促炎细胞因子（如 TNF-$\alpha$, IL-1$\beta$, IFN-$\gamma$）以及脂多糖（LPS）高度敏感。在这些因子刺激下，该细胞能迅速发生胶质瘢痕化/反应性胶质增生（Reactive Astrogliosis）样改变，丰度分泌 IL-6、IL-8、MCP-1（CCL2）等趋化因子及活性氧（ROS），是筛选新型中枢抗炎和神经保护小分子药物的优异底盘。

2. 体外人源血脑屏障（BBB）共培养模型重构：

   在体内，星形胶质细胞的血管周足（End-feet）紧密包绕着脑毛细血管内皮细胞，是维持血脑屏障紧密连接和选择性通透性的关键。NS-SV-AC 常与人脑微血管内皮细胞（如 hCMEC/D3）和人脑周细胞（Pericytes）联合构建多细胞上下层 Transwell 共培养体系，用于体外定量测定中枢神经系统候选药物的跨上皮电阻（TEER）和屏障渗透率。

3. 神经退行性疾病（如阿尔茨海默病、ALS）与胶质毒性机制研究：

   星形胶质细胞的功能支持丧失或毒性功能获得，直接驱动了神经元的退行性病变。NS-SV-AC 被广泛用于评估外源性 $\beta$-淀粉样肽（A$\beta$）、Tau 蛋白寡聚体对胶质细胞代谢（如谷氨酸摄取、ATP 释放）的破坏，探讨其如何因谷氨酸转运体（EAAT2/GLT-1）功能耗竭进而引发神经元兴奋性毒性。

三 实验室细胞复苏、贴壁常规培养、传代与保存标准步骤

NS-SV-AC 细胞生长状态强健，但在贴壁初期对外界剪切力有一定敏感性。在日常维护中需把控好消化酶活性，避免过度生长引发的细胞老龄化。

1. 专用培养基与核心成分配置

• 基础培养基：高糖 DMEM 培养基（High-glucose DMEM）。

• 专用完全培养基配方：

  • 高糖 DMEM 基础培养基

  • 加 10% 优质胎牛血清（FBS）

  • 加 1% 青霉素-链霉素双抗（Penicillin-Streptomycin）

• 细胞解离液：0.25% Trypsin-0.02% EDTA 消化液。

• 生长参数常数：37 摄氏度，5% 二氧化碳，饱和湿度。倍增时间通常为 24 - 30 小时（进入对数期后增殖平稳迅速）。

2. 冷冻细胞复苏步骤

1. 提前在无菌生物安全柜中配制好干净的 T25 培养瓶，注入 5 - 6 mL 预热至 37 摄氏度的完全培养基，并置于孵箱中预热平衡。

2. 从液氮罐中取出 NS-SV-AC 冻存管，立刻全量投入 37 摄氏度恒温水浴箱中快速摇晃解冻，确保管内冰块在 1 分钟内完全融化。

3. 用 75% 酒精喷洒消毒冻存管外壁，移入二级生物安全柜。

4. 用移液枪吸取全量解冻的细胞悬液，极其缓慢地逐滴滴入盛有 4 mL 预热完全培养基的 15 mL 离心管中，轻柔颠倒一次混匀（务必轻柔，以防高渗透压的 DMSO 对复苏膜造成瞬间剪切物理损伤）。

5. 以 1000 rpm（约 200 g）室温离心 5 分钟，小心抽干含有 DMSO 的冷冻保护剂上清液。

6. 加入 1 mL 预热的完全培养基，使用 P1000 移液枪轻轻重悬沉淀化开。

7. 将悬液全量接种至准备好的 T25 培养瓶中，轻柔十字晃动混匀，置于 37 摄氏度孵箱中培养。

8. 孵育 24 小时后，待细胞完全贴壁，全量更换一次新鲜的预热完全培养基，以彻底清除死细胞碎屑及极微量残留的 DMSO。

3. 日常贴壁常规传代操作

• 传代时机：当细胞汇合度（Confluency）达到 80% - 85% 时必须启动传代。NS-SV-AC 细胞若任其长满至 100% 极度过密状态，贴壁层极易自发增厚并堆叠，这会导致底层细胞由于缺乏营养和接触抑制产生严重的空泡化（Vacuolation），从而恶化后续传代时的贴壁效率。

• 操作流程：

  1. 吸除细胞瓶内的旧培养基，使用无菌的、不含钙镁离子的 PBS 缓冲液轻轻漂洗细胞表面 1 - 2 次，彻底洗净血清残余（血清中的大分子会显著抑制胰酶的活性）。

  2. 加入适量 0.25% 胰酶-EDTA 消化液（T25 瓶常规加入 1 mL），全面覆盖细胞层。置于 37 摄氏度孵箱中消化 1.5 - 3 分钟。

  3. 在倒置显微镜下动态观察。该细胞对胰酶较为敏感，当看到多角形或伪足状的胞体快速收回变圆、胞间裂隙显著增大、轻敲瓶壁细胞开始大面积整体滑动脱落时，立即加入 2 倍体积的含血清完全培养基终止消化。

  4. 用枪头轻柔吹打瓶壁数次，将脱落的细胞全部收集至 15 mL 离心管中，1000 rpm 离心 5 分钟。

  5. 弃上清，用预热的完全培养基重悬，打散成单细胞悬液。按照 1 比 3 至 1 比 5 的稀释比例接种入新的培养器皿中。通常每 2 - 3 天传代一次。

4. 细胞长期保存标准

• 冻存液配方：90% 优质完全培养基（或纯胎牛血清） 加 10% 分析级二甲基亚砜（DMSO）。亦可使用市售高质免程序降温的专用细胞冻存液。

• 冷冻规范：

  1. 必须收集处于连续传代期间、处于对数生长最旺盛期（汇合度约 80%）、形态结构高度健康的 NS-SV-AC 细胞。

  2. 经温和消化、离心沉淀后，用配制好的冷冻液悬浮并调整密度至 每毫升 1,500,000 到 2,500,000 个细胞。

  3. 分装至无菌冻存管中，立刻放入标准程序降温盒（如 Mr. Frosty），并将其置于 零下 80 摄氏度超低温冰箱内过夜，完成每分钟稳定降温 1 摄氏度的稳态梯度降温。

  4. 次日，必须以极快速度将冻存管投递至液氮罐（零下 196 摄氏度）的气相或液相中锁死长期保存。绝对禁止在 零下 80 度普通冰箱中存放超过 2 周，以防止长期的微小热辐射导致细胞内部冰晶重组，进而导致 SV40 永生化表型及细胞活性的衰退。

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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