MGG152 BioVector®患者来源人类胶质瘤神经干细胞株

BioVector® MGG152 患者来源人类胶质瘤神经干细胞株——产品技术说明书

1 产品基本信息与遗传背景产品名称：BioVector® MGG152 患者来源人类胶质瘤神经干细胞株常用别名：MGG152，MGG152 Tumorsphere Line，MGH-152 胶质瘤干细胞生物学来源：人类 Homo sapiens组织器官：大脑 / 脑部恶性神经胶质瘤（源自临床患者手术切除标本原代分化）生长特性：悬浮型神经肿瘤球（Tumorsphere / Neurosphere）生长核心突变特征：天然携带 IDH1 杂合热点突变（IDH1 R132H），属于典型的 G-CIMP（胶质瘤 CpG 岛甲基化表型）底盘。同时常伴有 O-6-甲基鸟嘌呤-DNA 甲基转移酶（MGMT）启动子区域的高度甲基化。核心科研价值：MGG152 是全球极少数能够保持天然 IDH1 突变表型的患者来源原代肿瘤干细胞模型。它是研究 IDH 突变型恶性胶质瘤进展、癌细胞代谢重塑、高浓度癌代谢物 2-羟基戊二酸（2-HG）致病机制、NAD+ 代谢通路缺陷、以及评价新型小分子 IDH1 抑制剂或烷化剂（如替莫唑胺 TMZ）耐药机理的国际公认金标准模型。

2 细胞生物学特征与核心标志物

MGG152 作为无血清神经干细胞培养体系维持的肿瘤球模型，展现出极强的干性特征：

致瘤与干性机制：细胞在体外不依赖于血清，完全依靠表皮生长因子（EGF）和碱性成纤维细胞生长因子（bFGF）的刺激维持未分化状态。其细胞内新形态酶活性（Neomorphic enzymatic activity）会将 alpha-酮戊二酸（alpha-KG）源源不断地转化为 2-羟基戊二酸（2-HG）。细胞形态观察：在无血清生长培养基中，细胞会自发聚集，在数天内折光度良好、边缘清晰的圆形或桑葚状悬浮“神经肿瘤球”（Neurosphere）。直接贴壁栽培或加入血清会导致其发生不可逆的分化和干性丧失。特异性标志物：细胞稳定高表达神经干细胞标志物——Nestin、Sox2、Musashi-1 以及胶质前体标志物 Olig2。此外，免疫生化检测可见突变型 IDH1 R132H 蛋白的强阳性表达。

3 专用无血清神经干细胞完全培养基配方规范

红线警告：MGG152 为原代干细胞株，在日常常规扩增和维持阶段，绝对禁止添加任何成分的动物或人类血清（如胎牛血清 FBS），血清中的未知因子会强行诱导其分化并彻底丧失 IDH1 突变表型。

标准无血清扩增完全培养基配方基础培养基：DMEM/F12 1:1 复合培养基（或者 Neurobasal 培养基）。核心干性补剂：1% 或 2% B-27 Supplement（50X，无维生素 A 型，即 B-27 Supplement, minus vitamin A）。细胞生长因子（必须在使用前新鲜加入）：重组人表皮生长因子（Recombinant Human EGF）：终浓度 20 ng/mL。重组人碱性成纤维细胞生长因子（Recombinant Human bFGF）：终浓度 20 ng/mL。肝素补剂（可选）：Heparin 溶液，终浓度 2 到 5 ug/mL（用于稳定生长因子活性并促进肿瘤球聚拢）。双抗：1% 青霉素-链霉素溶液（终浓度：100 U/mL 青霉素，100 ug/mL 链霉素）。

细胞冻存保护液（无血清配方）无血清标准配方：90% 上述无血清完全生长培养基 + 10% 细胞级二甲基亚砜 DMSO。商业级配方：直接采用优质的无血清细胞冻存液（如 CryoStor CS10 级别）。

4 物理环境控制参数孵育温度：37.0 ℃（温控误差需小于正负 0.3 ℃）气相环境：5% 二氧化碳（CO2），加湿平衡常压无菌空气（部分实验室采用 2% 到 5% 低氧低初生态环境，有助于更好的维持突变干性）相对湿度：大于 95% 恒湿物理外貌特征：悬浮于培养液中大小不一的细胞团块。当肿瘤球直径达到 150 到 200 微米，或者球体核心开始出现发黑发暗倾向时，必须立即进行机械或酶学解离传代。

5 肿瘤球解离传代操作规范传代控制点：当 70% 以上的肿瘤球体积达到临界大小（直径大于 150 微米）时启动传代。由于球体内部缺乏血管，过大的肿瘤球会导致中心区细胞缺氧坏死并释放大量有毒酸性物质。解离分瓶比例：1:2 到 1:3（一般 4-7 天传代一次，取决于球体的生长速率）。

标准温和解离传代步骤：1 将包含所有悬浮肿瘤球的培养液吸出，收集至无菌 15 mL 离心管中。2 在 200 x g 下低速离心 5 分钟，轻轻吸去上清液。3 向细胞沉淀中加入 1.0 mL 预热的温和干性细胞专用解离液（如 Accutase 或 TrypLE Express）。切勿使用高浓度的 0.25% 传统粗制胰酶，否则会导致干细胞表面生长因子受体被降解，从而引发不可逆的细胞绝死。4 将离心管置于 37 ℃ 孵箱中静置消化 3 到 5 分钟。期间每隔 1.5 分钟，用移液枪轻柔吹打管底 3-5 次，加速球体机械剥离。5 当在显微镜下观察到大球基本消退，转化为均匀的单细胞或 2-3 个细胞的微小微团时，立即加入 3 到 4 倍体积的冷基础 DMEM/F12 培养基稀释终止消化。6 200 x g 离心 5 分钟，彻底弃去上清液。7 加入足量含有新鲜加入的 EGF、bFGF 和 B-27 的完全无血清生长培养基，轻柔弹散重悬，接种回超低黏附培养瓶（Ultra-Low Attachment Flask）或悬浮培养皿中，放入 37 ℃ 孵箱。

6 复苏与冻存后恢复流线1 提前准备超低黏附 T25 培养瓶，注入 5.0 mL 已经补齐 B-27、EGF、bFGF 的无血清完全培养基，置于 37 ℃、5% CO2 孵箱中进行 20 分钟的物理平衡。2 从液氮罐中取出冷冻的 MGG152 冻存管，立刻全量投入 37 ℃ 恒温水浴箱中快速水平摇晃。3 在 60 秒内使其完全融化。由于无血清体系中的干细胞极其娇嫩，冰晶复融过程必须控制在 1 分钟以内。4 用 75% 酒精消毒管壁后移入超净台，用大口径移液枪吸出复苏的胞悬液，极其缓慢地逐滴注入盛有 4.0 mL 预热基础 DMEM/F12 培养基的 15 mL 离心管中。5 在 180 x g 到 200 x g 的极低转速下离心 5 分钟，彻底吸干含有毒 DMSO 的上清液。6 加入 1.5 mL Fresh 无血清完全培养基轻弹悬起细胞，随后全量接种到已预热平衡的超低黏附 T25 瓶中，置于 37 ℃ 孵箱栽培。7 复苏后前 48 小时细胞多呈现单个悬浮或 2-3 个紧挨生长，通常到第 3-4 天会正式聚拢形成规则的初代肿瘤球。

7 超低温长期保存与质控规范生物安全级别：BSL-2。因直接来源于人类神经系统临床活检组织，必须在二级生物安全柜内进行无菌技术操作，严防气溶胶污染。长期封存温控：必须永久存放于液氮蒸汽或液相（-150 ℃ 至 -196 ℃）中。绝对禁止在 -80 ℃ 普通机械冰箱内保存。致癌代谢物 2-HG 周期性抽检：为了确保该原代干细胞株未发生长期传代导致的表型自发分化或突变丢失，每传代 5-10 代后，应收集 1 x 10^6 个细胞的培养上清液，利用液相色谱-质谱联用仪（LC-MS/MS）或酶学比色法定量测定 2-羟基戊二酸（2-HG）的细胞内/外累积浓度。若发现 2-HG 释放量发生断崖式下跌，则证实细胞干性及 IDH1 突变特征已丧失，需立刻废弃并更换更低代次的冻存管。

BioVector® MGG152 Patient-Derived Human Glioma Stem Cell Line Product Datasheet

1 Product and Identification General InformationProduct Name: BioVector® MGG152 Patient-Derived Human Glioma Stem Cell LineSynonyms: MGG152, MGG152 Tumorsphere Line, MGH-152 Glioma Stem CellsOrganism Source: Human Homo sapiensTissue/Organ Site: Brain / Malignant Glioma (Derived via primary separation of clinical post-operative surgical biopsy specimens)Growth Properties: Suspension Tumorsphere / Neurosphere GrowthCore Genetic Signatures: Carries a endogenous heterozygous hot-spot IDH1 mutation (IDH1 R132H), representing a classic G-CIMP (Glioma CpG Island Methylator Phenotype) framework. It is typically accompanied by hypermethylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promoter region.Core Research Significance: MGG152 stands as one of the very few globally accessible patient-derived primary glioma models that stably preserve the native IDH1 mutant genotype in vitro. It is an internationally recognized gold standard for studying IDH-mutant malignant glioma progression, metabolic rewiring, toxic oncometabolite D-2-hydroxyglutarate (2-HG) oncogenesis, metabolic dependencies related to NAD+ biosynthesis pathways, and for testing novel small-molecule IDH1 inhibitors or alkylating chemotherapies (e.g., Temozolomide TMZ).

2 Cellular Properties and Biomarker ProfilesMaintained under a rigid serum-free neural stem cell culture pipeline, MGG152 manifests powerful stemness and self-renewal attributes:

Stemness & Oncogenic Mechanisms: The culture isolates entirely bypass animal serum dependencies, relying on targeted Epidermal Growth Factor (EGF) and basic Fibroblast Growth Factor (bFGF) signals to sustain an undifferentiated profile. The intracellular neomorphic enzymatic function actively channels alpha-ketoglutarate (alpha-KG) into excessive volumes of D-2-hydroxyglutarate (2-HG).Microscopic Morphology: When propagated in standard serum-free medium, cells cluster spontaneously, establishing highly refractive, sharply bordered, spherical or morula-like floating "neurospheres" within a few days. Exposing the cells to standard adherent plasticware or adding serum leads to irreversible differentiation and loss of mutant status.Specific Antigenic Profiles: Stably hyper-expresses neural stem cell hallmarks including Nestin, Sox2, Musashi-1, alongside the glial progenitor driver Olig2. Immunochemical testing confirms persistent, robust expressions of the mutated IDH1 R132H protein.

3 Dedicated Serum-Free Neural Stem Cell Medium Formulations

CRITICAL WARNING: MGG152 is a highly sensitive primary stem cell model. During all routine propagation and expansion phases, the addition of any animal or human serum (e.g., Fetal Bovine Serum FBS) is strictly prohibited. Serum factors will enforce terminal differentiation and permanently eliminate the IDH1 mutant phenotype.

Standard Serum-Free Complete Growth FormulationBasal Medium Base: DMEM/F12 1:1 Composite Medium (or Neurobasal Medium).Core Stemness Supplement: 1% or 2% B-27 Supplement (50X, formulated minus Vitamin A to prevent differentiation; B-27 Supplement, minus vitamin A).Recombinant Growth Factors (Must be added fresh before use):Recombinant Human EGF: Final concentration at 20 ng/mL.Recombinant Human bFGF: Final concentration at 20 ng/mL.Heparin Co-Factor (Optional): Heparin solution, final concentration at 2 to 5 ug/mL (stabilizes growth factor kinetics and supports tight tumorsphere formation).Antibiotic Supplement: 1% Penicillin-Streptomycin Solution (Final concentration: 100 U/mL Penicillin, 100 ug/mL Streptomycin).

Cryopreservation Freezing Medium Formulation (Serum-Free)Standard Serum-Free Freezing Formula: 90% Complete Serum-Free Growth Medium + 10% Analytical Grade DMSO (Dimethyl Sulfoxide).Commercial Matrix: Direct application of high-quality serum-free cryopreservation media (such as CryoStor CS10).

4 Controlled Culture Environmental ConditionsIncubator Temperature: 37.0 ℃ (constant temperature, variations must be strictly locked within plus or minus 0.3 ℃)Gas Composition (CO2): 5% Carbon Dioxide balanced with humidified atmospheric air (Some laboratories adopt a hypoxic workspace of 2% to 5% O2, which aids in preserving primary stemness kinetics).Relative Humidity: Greater than 95% constant humidityPhysical Manifestation: Occurs as free-floating multicellular aggregates varying in size. When tumorsphere diameters reach 150 to 200 micrometers, or when the central core manifests dark/black necrotic traits, immediate enzymatic or mechanical dissociation passaging must be executed.

5 Tumorsphere Dissociation and Subculturing Passaging ProtocolOptimal Passaging Control: Initiate passaging promptly when over 70% of the active tumorspheres surpass a diameter threshold of 150 micrometers. Lacking vascular pathways, oversized spheres experience central core hypoxia, triggering cellular necrosis and the release of toxic acidic matrices into the medium.Subcultivation Splitting Ratio: 1:2 to 1:3 (typically passaged every 4 to 7 days depending on aggregate expansion speed).

Step-by-Step Gentle Dissociation Method:1 Aspirate the entirety of the floating sphere suspension and collect it into a sterile 15 mL conical tube.2 Centrifuge at 200 x g (low speed) for 5 minutes, then carefully decant and discard the supernatant.3 Add 1.0 mL of pre-warmed gentle cell dissociation fluid (such as Accutase or TrypLE Express). Never use standard coarse 0.25% Trypsin-EDTA, as it will digest cell-surface growth factor receptors, leading to irreversible loss of stem cell viability.4 Incubate the conical tube at 37 ℃ for 3 to 5 minutes. Every 1.5 minutes, use a pipette tip to gently break up the pellet 3-5 times to accelerate physical dissociation.5 Once microscopic inspection reveals that the large spheres have dissolved into a uniform single-cell suspension or small micro-clusters of 2-3 cells, instantly add 3 to 4 volumes of cold basal DMEM/F12 to neutralize the enzyme.6 Centrifuge at 200 x g for 5 minutes and discard the supernatant completely.7 Resuspend the cell pellet in an appropriate volume of fresh serum-free complete medium (pre-fortified with fresh B-27, EGF, and bFGF), and distribute the solution into Ultra-Low Attachment Flasks or suspension plates. Return to the 37 ℃ incubator.

6 Thawing and Post-Cryo Recovery Workflow1 Pre-warm 5.0 mL of serum-free complete medium (fully supplemented with B-27, EGF, and bFGF) in a sterile Ultra-Low Attachment T25 flask, and equilibrate inside the 37 ℃, 5% CO2 incubator for 20 minutes to stabilize the gas phase.2 Retrieve the MGG152 cryovial from liquid nitrogen storage.3 Plunge the vial instantly into a 37 ℃ water bath and rapidly agitate horizontally. Complete the thawing sequence within 60 seconds. Because serum-free primary lines are delicate, rapid warming is vital to bypass toxic recrystallization damage.4 Sanitize the vial exterior with 75% ethanol before moving it into the biosafety cabinet.5 Transfer the thawed cell matrix slowly and dropwise into a 15 mL sterile conical tube containing 4.0 mL of pre-warmed basal DMEM/F12 medium.6 Centrifuge at an ultra-low speed of 180 x g to 200 x g for 5 minutes. Decant the supernatant completely to clear toxic DMSO remnants.7 Add 1.5 mL of fresh complete serum-free growth fluid, gently tap the tube wall to free the pellet, and seed the entire suspension into the pre-stabilized ultra-low attachment T25 flask.8 Incubate at 37 ℃, 5% CO2. For the initial 48 hours, cells will float mostly as single entries or small duos; they will aggregate into typical spherical clusters by day 3 to 4.

7 Biosafety, Storage and Quality Control StandardsBiosafety Level: BSL-2 (Class II Biosafety Cabinet Operations). Directly isolated from human neural tumor clinical tissue; standard clinical barrier protections and biohazard medical waste autoclaving rules must be followed rigorously.Long-Term Storage Parameters: Cryovials must be stored permanently within the vapor or liquid phase of liquid nitrogen (-150 ℃ to -196 ℃). Storage in mechanical -80 ℃ freezers is completely prohibited.Oncometabolite 2-HG Quantitation QC: To verify that long-term passaging has not triggered spontaneous differentiation or mutant silencing, harvest 1 x 10^6 cells along with their spent culture fluid every 5-10 passages. Quantify the absolute concentrations of accumulated D-2-hydroxyglutarate (2-HG) utilizing Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) or enzymatic colorimetric assays. If 2-HG profiles reveal a precipitous drop, the line has drifted; discard the culture immediately and thaw a lower-passage master vial.

BioVector NTCC质粒载体菌株细胞蛋白抗体基因保藏中心

电话:400-800-2947

工作QQ/微信同号:1843439339

网址http://www.biovector.net