BioVector® TS603 人源 IDH1 突变型少突胶质瘤细胞系

BioVector® TS603 Human IDH1-Mutated Oligodendroglioma Cell Line Manual

第一部分 中文说明

一 产品基本信息与遗传学背景

• 细胞名称：TS603 人源 IDH1 突变型少突胶质瘤/胶质瘤细胞系

• 物种来源：人源（Human），建立自临床一名患有间变性少突胶质瘤（Anaplastic Oligodendroglioma, WHO Grade III）患者的脑部肿瘤组织。该细胞系最初由纪念斯隆-凯特琳癌症中心（MSKCC）分离、鉴定并确立。

• 核心遗传学与分子标志：

  • 内源性 IDH1 R132H 杂合突变：TS603 细胞系在其内源性 IDH1 基因的 Arg132 位点携带经典的单核苷酸位点突变（c.395G>A; p.Arg132His），基因组呈现杂合突变状态。

  • 致癌代谢物 D-2HG 高分泌（功能获得性表型）：R132H 突变改变了 IDH1 酶的催化活性，使其获得了全新的逆向催化功能（Gain-of-function），将 alpha-酮戊二酸（alpha-KG）逆向还原为“癌性代谢物”——D-2-羟基戊二酸（D-2-hydroxyglutarate, D-2HG）。TS603 在体外培养中能够持续稳定地大量分泌 D-2HG。

  • 染色体 1p/19q 联合缺失（Codel 状态）：本细胞系完美保留了人类少突胶质瘤最具特征性的分子病理标志，即染色体 1p 臂与 19q 臂的完全联合缺失（1p/19q co-deleted），是研究该亚型胶质瘤极具代表性的标准化体外模型。

• 生长特性：主要呈现三维（3D）干细胞样肿瘤球（Tumorspheres / Neurospheres）悬浮生长状态，也可在基质胶包被的培养皿表面呈贴壁状态生长。

• 生物安全级别：2级（BSL-2）。本细胞系源于人类恶性脑肿瘤组织，具有高度活跃的代谢重编程特性，所有涉及活细胞的实验、传代、分装操作均须在二级生物安全实验室的生物安全柜内进行。

二 核心科研价值与转化医学应用

普通的胶质瘤细胞系（如 U87、U251 等）缺乏真实的内源性 IDH 突变。TS603 细胞能够在长期的体外传代中，极其罕见地同时稳定维持内源性 IDH1 突变与 1p/19q 双缺失表型：

1. 胶质瘤表观遗传学（G-CIMP 现象）研究：用于揭示胞内高浓度 D-2HG 蓄积如何特异性抑制 DNA 去甲基化酶和组蛋白去甲基化酶，进而启动全基因组 CpG 岛恶性超甲基化（G-CIMP 阳性表型）并阻断神经胶质细胞正常分化的精细机制。

2. 突变型 IDH1 靶向小分子药物筛选：作为测试突变型 IDH1 专属抑制剂（如 AGI-5198、Vorasidenib）及表观遗传修复药物（如地西他滨 / DAC）效能的标准阳性对照模型，用以观察 D-2HG 滴度下降、表观标记重塑以及诱导星形胶质细胞分化（GFAP 表达上调）的动力学曲线。

3. 合成致死与代谢靶点挖掘：利用 TS603 的特殊代谢缺陷，在大规模文库中筛查能够与 IDH 突变产生协同杀伤效应的代谢阻断剂（如谷胱甘肽 GSH 代谢抑制剂、细胞周期阻断剂 Zotiraciclib 等）。

三 实验室细胞复苏、悬浮球培养传代与冷冻保存标准步骤

1. 专用无血清神经球培养基配置（NBE 培养基）

为了维持 TS603 细胞的干细胞样肿瘤球空间构象并防止其发生自发性分化，必须使用完全无血清的定向增殖体系：

• 标准完全培养基配方：

  • Neurobasal 基础培养基（或 50% DMEM/F12 混合基础培养基）

  • 补充添加：1% B27 或 N2 细胞生长添加剂、20 ng/mL 重组人表皮生长因子（EGF）、20 ng/mL 重组人碱性成纤维细胞生长因子（bFGF）、2 micrograms/mL 肝素钠（Heparin Sodium），以及 1% 灭菌双抗（青霉素-链霉素）。

• 注意：在维持生长期间切勿加入任何常规动物血清（如 FBS），血清因子会诱导 TS603 发生不可逆的贴壁分化改变并丧失原有的干细胞特性。

2. 细胞复苏（Thawing Protocol）

1. 从液氮罐中取出 TS603 冻存管，立即完全浸入 37 摄氏度恒温水浴箱中快速摇晃。

2. 在 1 到 2 分钟内令其急速融化至仅剩微小冰芯。复苏必须迅速，以减少 DMSO 在融化状态下对脆弱干细胞的细胞毒性。

3. 用 70% 酒精对冻存管外部进行消毒后移入生物安全柜，用移液管将细胞悬液转移至 15 mL 干净离心管中。

4. 吸取 9 mL 预热的完全无血清 NBE 培养基，以滴加的方式（Dropwise）缓慢加入离心管中，期间轻轻转动离心管以防止渗透压休克。

5. 300 x g 离心 3 到 5 分钟，小心吸除含有 DMSO 的上清液。

6. 加入 5 mL 新鲜的完全无血清 NBE 培养基，用移液枪极轻柔地吹打 1-2 次重悬细胞，接种于超低贴壁（Ultra-low attachment）培养瓶或培养皿中，置于 37 摄氏度、5% CO2 孵箱中培养，促进其自发聚集形成肿瘤球。

3. 肿瘤球消化与传代（Passaging / Tumorsphere Dissociation）

• 传代时机：TS603 细胞生长相对缓慢（群体倍增时间通常长达 7 天左右）。当显微镜下观察到大量肿瘤球直径增大（达到 150 至 200 微米以上），或部分球体中心开始发黑（提示中心营养匮乏或发生坏死）时，必须执行消化传代。

• 传代步骤：

  1. 用移液管将包含所有悬浮肿瘤球的细胞悬液全部收集至 15 mL 离心管中。

  2. 250 x g 离心 3 分钟使肿瘤球沉淀，轻轻吸除旧培养基。

  3. 向沉淀中加入 1 至 2 mL 预热的 Accutase（阿库酶）或 TrypLE Express 消化液，确保覆盖细胞球。

  4. 置于 37 摄氏度孵箱中消化 5 至 8 分钟。期间每隔 2-3 分钟用 p1000 移液器对其进行适度的机械吹打，直至肉眼观察到大块的肿瘤球完全分散成单个细胞悬液。

  5. 加入 5 倍体积的完全 NBE 培养基终止消化，300 x g 离心 4 分钟弃上清。

  6. 进行细胞计数并评估活率，按照 50,000 至 100,000 活细胞/mL 的密度重新接种至新的超低贴壁培养瓶中。

4. 细胞冷冻保存（Cryopreservation）

• 冻存液配方：90% 新鲜完全无血清 NBE 培养基 + 10% 医用级高纯度 DMSO，或直接使用市售经证实的无血清无蛋白干细胞专属冻存液。

• 冻存操作：收集处于对数生长旺盛期且直径适中的肿瘤球，通过上述步骤消化分散为单细胞悬液。离心弃上清，调整细胞密度至每支冻存管包含至少 2,000,000 个活细胞。加入冻存液重悬后迅速分装，立即投入标准程序降温盒（异丙醇梯度降温盒，1 摄氏度/分钟），置于 -80 摄氏度过夜。次日必须迅速转移至 -196 摄氏度液氮罐中进行无限期冷冻长期保存。

Part 2 English Section

I General Information and Genetic Architecture

• Cell Line Name: TS603 Human Patient-Derived Glioma / Oligodendroglioma Cell Line.

• Species Origin: Human. Established from brain tumor biopsy tissues from a patient diagnosed with anaplastic oligodendroglioma (WHO Grade III). The cell line was originally isolated and authenticated by the Memorial Sloan Kettering Cancer Center (MSKCC) and Dr. Timothy Chan's laboratory.

• Core Genetic and Molecular Signatures:

  • Endogenous Heterozygous IDH1 R132H Mutation: TS603 is an internationally validated patient-derived cancer cell model that retains a stable, endogenous single-nucleotide point mutation at the Arg132 locus of the IDH1 gene (c.395G>A; p.Arg132His), resulting in a heterozygous variant.

  • Neomorphic Oncometabolite D-2HG Production: The R132H structural alteration shifts the enzymatic activity of IDH1. Instead of processing isocitrate to alpha-ketoglutarate (alpha-KG), it acts via an oncogenic gain-of-function mechanism to reduce alpha-KG directly into the prominent oncometabolite D-2-hydroxyglutarate (D-2HG). TS603 continuously aggregates and releases substantial baseline levels of D-2HG.

  • Chromosome 1p/19q Co-deletion (Codel Status): It exhibits the classical genetic signature characteristic of human oligodendrogliomas, harboring a complete heterozygous co-deletion of chromosome arms 1p and 19q. This specific genetic profile correlates with distinctive clinical vulnerabilities.

• Growth Topology: Primarily cultivated as three-dimensional (3D) stem-like tumorspheres / neurospheres in suspension, or as adherent sheets under specialized matrix coatings.

• Biosafety Matrix: BSL-2 containment infrastructure is mandated. The cell line is derived from human brain tissue and exhibits malignant metabolic activity. All expansion protocols require biological safety cabinets and certified chemical disinfection configurations to protect personnel.

II Strategic Research Value and Translational Fields

Unlike standard immortalized cancer lines (such as U87 or U251) that lack genuine IDH modifications, TS603 preserves endogenous IDH1 mutations alongside a 1p/19q co-deletion profile over extensive passage intervals:

1. Epigenetic Hypermethylation and G-CIMP Dynamics: Serves as a model for investigating how chronic oncometabolite D-2HG pooling inhibits alpha-KG-dependent dioxygenases and histone demethylases, establishing the Glioma CpG Island Methylator Phenotype (G-CIMP).

2. Targeted Small-Molecule Inhibitor Characterization: Deployed as a primary positive screening platform for evaluating mutant-IDH1 targeted inhibitors (such as AGI-5198 or Vorasidenib) and epigenetic therapies (such as Decitabine / DAC), assessing their ability to decrease D-2HG concentration, reverse epigenetic locks, and prompt astroglial differentiation.

3. High-Throughput Vulnerability Profiling: Deployed in combinatorial automated screening matrices to identify target mechanisms that exploit the unique metabolic liabilities of IDH-mutant cells, including dependencies on glutathione (GSH) metabolism and vulnerabilities to cell-cycle regulators like Zotiraciclib.

III Thawing, Proliferation, Passaging, and Cryopreservation Routines

1. Formulating the Complete Serum-Free Neurobasal Medium (NBE Framework)

To maintain its stem-like tumorsphere architecture and prevent spontaneous differentiation, TS603 must be cultivated in a defined serum-free system:

• Standard Growth Matrix Formulation:

  • Neurobasal Medium (or 50:50 DMEM/F12 alternative base)

  • Supplemented with: 1% B27 or N2 Growth Supplement, 20 ng/mL Recombinant Human EGF, 20 ng/mL Recombinant Human bFGF, 2 micrograms/mL Heparin Sodium Salt, and 1% Penicillin-Streptomycin cocktail.

• Note: Avoid adding standard Fetal Bovine Serum (FBS) unless executing specific differentiation assays, as serum factors trigger immediate morphological changes and irreversible loss of the stem-like phenotype.

2. Cryovial Thawing Routine

1. Retrieve the TS603 cryovial from liquid nitrogen and submerge it instantly into a 37 degree Celsius water bath with gentle agitation.

2. Complete the thawing cycle within 1 to 2 minutes until only a small ice crystal remains. Rapid processing is required to limit prolonged exposure to liquefied DMSO.

3. Swab the vial with 70% ethanol, transfer to a biosafety cabinet, and pipette the cell suspension into a sterile 15 mL conical tube.

4. Add 9 mL of pre-warmed, serum-free NBE medium dropwise into the tube while rotating it to minimize osmotic shock.

5. Centrifuge at 300 x g for 3 to 5 minutes, aspirate the DMSO-containing supernatant, and gently resuspend the cell pellet in 5 mL of fresh complete NBE media.

6. Transfer the cell suspension into an ultra-low attachment flask or dish to encourage spherical assembly, and incubate at 37 degrees Celsius under 5% CO2.

3. Subculturing and Tumorsphere Dissociation

• Passaging Interval: TS603 neurospheres expand slowly compared to conventional cancer lines (with population doubling times often tracking around 7 days). Perform subculturing when spheres grow large (exceeding 150 to 200 micrometers in diameter) or show dark, necrotic cores.

• Step-by-Step Harvesting:

  1. Collect the entire suspension containing the tumorspheres into a sterile 15 mL conical tube.

  2. Centrifuge at 250 x g for 3 minutes to sediment the clusters, then aspirate the spent medium.

  3. Add 1 to 2 mL of pre-warmed Accutase or TrypLE Express dissociation solution directly onto the pellet.

  4. Incubate at 37 degrees Celsius for 5 to 8 minutes. Gently pipette the suspension up and down with a p1000 micropipette every few minutes to assist mechanical disruption into a single-cell suspension.

  5. Quench the enzyme by adding 5 volumes of complete NBE medium. Centrifuge again to clear enzymatic residues.

  6. Count viable cells via a hemocytometer and re-seed into ultra-low attachment culture flasks at a density of approximately 50,000 to 100,000 viable cells/mL.

4. Cryopreservation Protocol

• Freezing Medium Matrix: 90% Complete NBE Growth Medium combined with 10% high-purity, tissue-culture grade DMSO, or a validated commercial serum-free cryopreservation solution.

• Freezing Routine: Dissociate healthy, mid-sized tumorspheres into a single-cell suspension during active growth. Spin down and adjust the cell concentration to a minimum of 2,000,000 viable cells per vial. Aliquot into cryovials and place inside a standardized isopropyl alcohol freezing container. Keep at -80 degrees Celsius overnight before shifting the tubes into liquid nitrogen (-196 degrees Celsius) for long-term storage.

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