BioVector? TS603 人源 IDH1 突變型少突膠質(zhì)瘤細胞系

BioVector? TS603 Human IDH1-Mutated Oligodendroglioma Cell Line Manual

第一部分 中文說明

一 產(chǎn)品基本信息與遺傳學背景

• 細胞名稱：TS603 人源 IDH1 突變型少突膠質(zhì)瘤/膠質(zhì)瘤細胞系

• 物種來源：人源（Human），建立自臨床一名患有間變性少突膠質(zhì)瘤（Anaplastic Oligodendroglioma, WHO Grade III）患者的腦部腫瘤組織。該細胞系最初由紀念斯隆-凱特琳癌癥中心（MSKCC）分離、鑒定并確立。

• 核心遺傳學與分子標志：

  • 內(nèi)源性 IDH1 R132H 雜合突變：TS603 細胞系在其內(nèi)源性 IDH1 基因的 Arg132 位點攜帶經(jīng)典的單核苷酸位點突變（c.395G>A; p.Arg132His），基因組呈現(xiàn)雜合突變狀態(tài)。

  • 致癌代謝物 D-2HG 高分泌（功能獲得性表型）：R132H 突變改變了 IDH1 酶的催化活性，使其獲得了全新的逆向催化功能（Gain-of-function），將 alpha-酮戊二酸（alpha-KG）逆向還原為“癌性代謝物”——D-2-羥基戊二酸（D-2-hydroxyglutarate, D-2HG）。TS603 在體外培養(yǎng)中能夠持續(xù)穩(wěn)定地大量分泌 D-2HG。

  • 染色體 1p/19q 聯(lián)合缺失（Codel 狀態(tài)）：本細胞系完美保留了人類少突膠質(zhì)瘤最具特征性的分子病理標志，即染色體 1p 臂與 19q 臂的完全聯(lián)合缺失（1p/19q co-deleted），是研究該亞型膠質(zhì)瘤極具代表性的標準化體外模型。

• 生長特性：主要呈現(xiàn)三維（3D）干細胞樣腫瘤球（Tumorspheres / Neurospheres）懸浮生長狀態(tài)，也可在基質(zhì)膠包被的培養(yǎng)皿表面呈貼壁狀態(tài)生長。

• 生物安全級別：2級（BSL-2）。本細胞系源于人類惡性腦腫瘤組織，具有高度活躍的代謝重編程特性，所有涉及活細胞的實驗、傳代、分裝操作均須在二級生物安全實驗室的生物安全柜內(nèi)進行。

二 核心科研價值與轉(zhuǎn)化醫(yī)學應用

普通的膠質(zhì)瘤細胞系（如 U87、U251 等）缺乏真實的內(nèi)源性 IDH 突變。TS603 細胞能夠在長期的體外傳代中，極其罕見地同時穩(wěn)定維持內(nèi)源性 IDH1 突變與 1p/19q 雙缺失表型：

1. 膠質(zhì)瘤表觀遺傳學（G-CIMP 現(xiàn)象）研究：用于揭示胞內(nèi)高濃度 D-2HG 蓄積如何特異性抑制 DNA 去甲基化酶和組蛋白去甲基化酶，進而啟動全基因組 CpG 島惡性超甲基化（G-CIMP 陽性表型）并阻斷神經(jīng)膠質(zhì)細胞正常分化的精細機制。

2. 突變型 IDH1 靶向小分子藥物篩選：作為測試突變型 IDH1 專屬抑制劑（如 AGI-5198、Vorasidenib）及表觀遺傳修復藥物（如地西他濱 / DAC）效能的標準陽性對照模型，用以觀察 D-2HG 滴度下降、表觀標記重塑以及誘導星形膠質(zhì)細胞分化（GFAP 表達上調(diào)）的動力學曲線。

3. 合成致死與代謝靶點挖掘：利用 TS603 的特殊代謝缺陷，在大規(guī)模文庫中篩查能夠與 IDH 突變產(chǎn)生協(xié)同殺傷效應的代謝阻斷劑（如谷胱甘肽 GSH 代謝抑制劑、細胞周期阻斷劑 Zotiraciclib 等）。

三 實驗室細胞復蘇、懸浮球培養(yǎng)傳代與冷凍保存標準步驟

1. 專用無血清神經(jīng)球培養(yǎng)基配置（NBE 培養(yǎng)基）

為了維持 TS603 細胞的干細胞樣腫瘤球空間構(gòu)象并防止其發(fā)生自發(fā)性分化，必須使用完全無血清的定向增殖體系：

• 標準完全培養(yǎng)基配方：

  • Neurobasal 基礎培養(yǎng)基（或 50% DMEM/F12 混合基礎培養(yǎng)基）

  • 補充添加：1% B27 或 N2 細胞生長添加劑、20 ng/mL 重組人表皮生長因子（EGF）、20 ng/mL 重組人堿性成纖維細胞生長因子（bFGF）、2 micrograms/mL 肝素鈉（Heparin Sodium），以及 1% 滅菌雙抗（青霉素-鏈霉素）。

• 注意：在維持生長期間切勿加入任何常規(guī)動物血清（如 FBS），血清因子會誘導 TS603 發(fā)生不可逆的貼壁分化改變并喪失原有的干細胞特性。

2. 細胞復蘇（Thawing Protocol）

1. 從液氮罐中取出 TS603 凍存管，立即完全浸入 37 攝氏度恒溫水浴箱中快速搖晃。

2. 在 1 到 2 分鐘內(nèi)令其急速融化至僅剩微小冰芯。復蘇必須迅速，以減少 DMSO 在融化狀態(tài)下對脆弱干細胞的細胞毒性。

3. 用 70% 酒精對凍存管外部進行消毒后移入生物安全柜，用移液管將細胞懸液轉(zhuǎn)移至 15 mL 干凈離心管中。

4. 吸取 9 mL 預熱的完全無血清 NBE 培養(yǎng)基，以滴加的方式（Dropwise）緩慢加入離心管中，期間輕輕轉(zhuǎn)動離心管以防止?jié)B透壓休克。

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

6. 加入 5 mL 新鮮的完全無血清 NBE 培養(yǎng)基，用移液槍極輕柔地吹打 1-2 次重懸細胞，接種于超低貼壁（Ultra-low attachment）培養(yǎng)瓶或培養(yǎng)皿中，置于 37 攝氏度、5% CO2 孵箱中培養(yǎng)，促進其自發(fā)聚集形成腫瘤球。

3. 腫瘤球消化與傳代（Passaging / Tumorsphere Dissociation）

• 傳代時機：TS603 細胞生長相對緩慢（群體倍增時間通常長達 7 天左右）。當顯微鏡下觀察到大量腫瘤球直徑增大（達到 150 至 200 微米以上），或部分球體中心開始發(fā)黑（提示中心營養(yǎng)匱乏或發(fā)生壞死）時，必須執(zhí)行消化傳代。

• 傳代步驟：

  1. 用移液管將包含所有懸浮腫瘤球的細胞懸液全部收集至 15 mL 離心管中。

  2. 250 x g 離心 3 分鐘使腫瘤球沉淀，輕輕吸除舊培養(yǎng)基。

  3. 向沉淀中加入 1 至 2 mL 預熱的 Accutase（阿庫酶）或 TrypLE Express 消化液，確保覆蓋細胞球。

  4. 置于 37 攝氏度孵箱中消化 5 至 8 分鐘。期間每隔 2-3 分鐘用 p1000 移液器對其進行適度的機械吹打，直至肉眼觀察到大塊的腫瘤球完全分散成單個細胞懸液。

  5. 加入 5 倍體積的完全 NBE 培養(yǎng)基終止消化，300 x g 離心 4 分鐘棄上清。

  6. 進行細胞計數(shù)并評估活率，按照 50,000 至 100,000 活細胞/mL 的密度重新接種至新的超低貼壁培養(yǎng)瓶中。

4. 細胞冷凍保存（Cryopreservation）

• 凍存液配方：90% 新鮮完全無血清 NBE 培養(yǎng)基 + 10% 醫(yī)用級高純度 DMSO，或直接使用市售經(jīng)證實的無血清無蛋白干細胞專屬凍存液。

• 凍存操作：收集處于對數(shù)生長旺盛期且直徑適中的腫瘤球，通過上述步驟消化分散為單細胞懸液。離心棄上清，調(diào)整細胞密度至每支凍存管包含至少 2,000,000 個活細胞。加入凍存液重懸后迅速分裝，立即投入標準程序降溫盒（異丙醇梯度降溫盒，1 攝氏度/分鐘），置于 -80 攝氏度過夜。次日必須迅速轉(zhuǎn)移至 -196 攝氏度液氮罐中進行無限期冷凍長期保存。

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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