BioVector? MGG152 患者來(lái)源人類(lèi)膠質(zhì)瘤神經(jīng)干細(xì)胞株——產(chǎn)品技術(shù)說(shuō)明書(shū)

1 產(chǎn)品基本信息與遺傳背景產(chǎn)品名稱：BioVector? MGG152 患者來(lái)源人類(lèi)膠質(zhì)瘤神經(jīng)干細(xì)胞株常用別名：MGG152，MGG152 Tumorsphere Line，MGH-152 膠質(zhì)瘤干細(xì)胞生物學(xué)來(lái)源：人類(lèi) Homo sapiens組織器官：大腦 / 腦部惡性神經(jīng)膠質(zhì)瘤（源自臨床患者手術(shù)切除標(biāo)本原代分化）生長(zhǎng)特性：懸浮型神經(jīng)腫瘤球（Tumorsphere / Neurosphere）生長(zhǎng)核心突變特征：天然攜帶 IDH1 雜合熱點(diǎn)突變（IDH1 R132H），屬于典型的 G-CIMP（膠質(zhì)瘤 CpG 島甲基化表型）底盤(pán)。同時(shí)常伴有 O-6-甲基鳥(niǎo)嘌呤-DNA 甲基轉(zhuǎn)移酶（MGMT）啟動(dòng)子區(qū)域的高度甲基化。核心科研價(jià)值：MGG152 是全球極少數(shù)能夠保持天然 IDH1 突變表型的患者來(lái)源原代腫瘤干細(xì)胞模型。它是研究 IDH 突變型惡性膠質(zhì)瘤進(jìn)展、癌細(xì)胞代謝重塑、高濃度癌代謝物 2-羥基戊二酸（2-HG）致病機(jī)制、NAD+ 代謝通路缺陷、以及評(píng)價(jià)新型小分子 IDH1 抑制劑或烷化劑（如替莫唑胺 TMZ）耐藥機(jī)理的國(guó)際公認(rèn)金標(biāo)準(zhǔn)模型。

2 細(xì)胞生物學(xué)特征與核心標(biāo)志物

MGG152 作為無(wú)血清神經(jīng)干細(xì)胞培養(yǎng)體系維持的腫瘤球模型，展現(xiàn)出極強(qiáng)的干性特征：

致瘤與干性機(jī)制：細(xì)胞在體外不依賴于血清，完全依靠表皮生長(zhǎng)因子（EGF）和堿性成纖維細(xì)胞生長(zhǎng)因子（bFGF）的刺激維持未分化狀態(tài)。其細(xì)胞內(nèi)新形態(tài)酶活性（Neomorphic enzymatic activity）會(huì)將 alpha-酮戊二酸（alpha-KG）源源不斷地轉(zhuǎn)化為 2-羥基戊二酸（2-HG）。細(xì)胞形態(tài)觀察：在無(wú)血清生長(zhǎng)培養(yǎng)基中，細(xì)胞會(huì)自發(fā)聚集，在數(shù)天內(nèi)折光度良好、邊緣清晰的圓形或桑葚狀懸浮“神經(jīng)腫瘤球”（Neurosphere）。直接貼壁栽培或加入血清會(huì)導(dǎo)致其發(fā)生不可逆的分化和干性喪失。特異性標(biāo)志物：細(xì)胞穩(wěn)定高表達(dá)神經(jīng)干細(xì)胞標(biāo)志物——Nestin、Sox2、Musashi-1 以及膠質(zhì)前體標(biāo)志物 Olig2。此外，免疫生化檢測(cè)可見(jiàn)突變型 IDH1 R132H 蛋白的強(qiáng)陽(yáng)性表達(dá)。

3 專用無(wú)血清神經(jīng)干細(xì)胞完全培養(yǎng)基配方規(guī)范

紅線警告：MGG152 為原代干細(xì)胞株，在日常常規(guī)擴(kuò)增和維持階段，絕對(duì)禁止添加任何成分的動(dòng)物或人類(lèi)血清（如胎牛血清 FBS），血清中的未知因子會(huì)強(qiáng)行誘導(dǎo)其分化并徹底喪失 IDH1 突變表型。

標(biāo)準(zhǔn)無(wú)血清擴(kuò)增完全培養(yǎng)基配方基礎(chǔ)培養(yǎng)基：DMEM/F12 1:1 復(fù)合培養(yǎng)基（或者 Neurobasal 培養(yǎng)基）。核心干性補(bǔ)劑：1% 或 2% B-27 Supplement（50X，無(wú)維生素 A 型，即 B-27 Supplement, minus vitamin A）。細(xì)胞生長(zhǎng)因子（必須在使用前新鮮加入）：重組人表皮生長(zhǎng)因子（Recombinant Human EGF）：終濃度 20 ng/mL。重組人堿性成纖維細(xì)胞生長(zhǎng)因子（Recombinant Human bFGF）：終濃度 20 ng/mL。肝素補(bǔ)劑（可選）：Heparin 溶液，終濃度 2 到 5 ug/mL（用于穩(wěn)定生長(zhǎng)因子活性并促進(jìn)腫瘤球聚攏）。雙抗：1% 青霉素-鏈霉素溶液（終濃度：100 U/mL 青霉素，100 ug/mL 鏈霉素）。

細(xì)胞凍存保護(hù)液（無(wú)血清配方）無(wú)血清標(biāo)準(zhǔn)配方：90% 上述無(wú)血清完全生長(zhǎng)培養(yǎng)基 + 10% 細(xì)胞級(jí)二甲基亞砜 DMSO。商業(yè)級(jí)配方：直接采用優(yōu)質(zhì)的無(wú)血清細(xì)胞凍存液（如 CryoStor CS10 級(jí)別）。

4 物理環(huán)境控制參數(shù)孵育溫度：37.0 ℃（溫控誤差需小于正負(fù) 0.3 ℃）氣相環(huán)境：5% 二氧化碳（CO2），加濕平衡常壓無(wú)菌空氣（部分實(shí)驗(yàn)室采用 2% 到 5% 低氧低初生態(tài)環(huán)境，有助于更好的維持突變干性）相對(duì)濕度：大于 95% 恒濕物理外貌特征：懸浮于培養(yǎng)液中大小不一的細(xì)胞團(tuán)塊。當(dāng)腫瘤球直徑達(dá)到 150 到 200 微米，或者球體核心開(kāi)始出現(xiàn)發(fā)黑發(fā)暗傾向時(shí)，必須立即進(jìn)行機(jī)械或酶學(xué)解離傳代。

5 腫瘤球解離傳代操作規(guī)范傳代控制點(diǎn)：當(dāng) 70% 以上的腫瘤球體積達(dá)到臨界大?。ㄖ睆酱笥?150 微米）時(shí)啟動(dòng)傳代。由于球體內(nèi)部缺乏血管，過(guò)大的腫瘤球會(huì)導(dǎo)致中心區(qū)細(xì)胞缺氧壞死并釋放大量有毒酸性物質(zhì)。解離分瓶比例：1:2 到 1:3（一般 4-7 天傳代一次，取決于球體的生長(zhǎng)速率）。

標(biāo)準(zhǔn)溫和解離傳代步驟：1 將包含所有懸浮腫瘤球的培養(yǎng)液吸出，收集至無(wú)菌 15 mL 離心管中。2 在 200 x g 下低速離心 5 分鐘，輕輕吸去上清液。3 向細(xì)胞沉淀中加入 1.0 mL 預(yù)熱的溫和干性細(xì)胞專用解離液（如 Accutase 或 TrypLE Express）。切勿使用高濃度的 0.25% 傳統(tǒng)粗制胰酶，否則會(huì)導(dǎo)致干細(xì)胞表面生長(zhǎng)因子受體被降解，從而引發(fā)不可逆的細(xì)胞絕死。4 將離心管置于 37 ℃ 孵箱中靜置消化 3 到 5 分鐘。期間每隔 1.5 分鐘，用移液槍輕柔吹打管底 3-5 次，加速球體機(jī)械剝離。5 當(dāng)在顯微鏡下觀察到大球基本消退，轉(zhuǎn)化為均勻的單細(xì)胞或 2-3 個(gè)細(xì)胞的微小微團(tuán)時(shí)，立即加入 3 到 4 倍體積的冷基礎(chǔ) DMEM/F12 培養(yǎng)基稀釋終止消化。6 200 x g 離心 5 分鐘，徹底棄去上清液。7 加入足量含有新鮮加入的 EGF、bFGF 和 B-27 的完全無(wú)血清生長(zhǎng)培養(yǎng)基，輕柔彈散重懸，接種回超低黏附培養(yǎng)瓶（Ultra-Low Attachment Flask）或懸浮培養(yǎng)皿中，放入 37 ℃ 孵箱。

6 復(fù)蘇與凍存后恢復(fù)流線1 提前準(zhǔn)備超低黏附 T25 培養(yǎng)瓶，注入 5.0 mL 已經(jīng)補(bǔ)齊 B-27、EGF、bFGF 的無(wú)血清完全培養(yǎng)基，置于 37 ℃、5% CO2 孵箱中進(jìn)行 20 分鐘的物理平衡。2 從液氮罐中取出冷凍的 MGG152 凍存管，立刻全量投入 37 ℃ 恒溫水浴箱中快速水平搖晃。3 在 60 秒內(nèi)使其完全融化。由于無(wú)血清體系中的干細(xì)胞極其嬌嫩，冰晶復(fù)融過(guò)程必須控制在 1 分鐘以內(nèi)。4 用 75% 酒精消毒管壁后移入超凈臺(tái)，用大口徑移液槍吸出復(fù)蘇的胞懸液，極其緩慢地逐滴注入盛有 4.0 mL 預(yù)熱基礎(chǔ) DMEM/F12 培養(yǎng)基的 15 mL 離心管中。5 在 180 x g 到 200 x g 的極低轉(zhuǎn)速下離心 5 分鐘，徹底吸干含有毒 DMSO 的上清液。6 加入 1.5 mL Fresh 無(wú)血清完全培養(yǎng)基輕彈懸起細(xì)胞，隨后全量接種到已預(yù)熱平衡的超低黏附 T25 瓶中，置于 37 ℃ 孵箱栽培。7 復(fù)蘇后前 48 小時(shí)細(xì)胞多呈現(xiàn)單個(gè)懸浮或 2-3 個(gè)緊挨生長(zhǎng)，通常到第 3-4 天會(huì)正式聚攏形成規(guī)則的初代腫瘤球。

7 超低溫長(zhǎng)期保存與質(zhì)控規(guī)范生物安全級(jí)別：BSL-2。因直接來(lái)源于人類(lèi)神經(jīng)系統(tǒng)臨床活檢組織，必須在二級(jí)生物安全柜內(nèi)進(jìn)行無(wú)菌技術(shù)操作，嚴(yán)防氣溶膠污染。長(zhǎng)期封存溫控：必須永久存放于液氮蒸汽或液相（-150 ℃ 至 -196 ℃）中。絕對(duì)禁止在 -80 ℃ 普通機(jī)械冰箱內(nèi)保存。致癌代謝物 2-HG 周期性抽檢：為了確保該原代干細(xì)胞株未發(fā)生長(zhǎng)期傳代導(dǎo)致的表型自發(fā)分化或突變丟失，每傳代 5-10 代后，應(yīng)收集 1 x 10^6 個(gè)細(xì)胞的培養(yǎng)上清液，利用液相色譜-質(zhì)譜聯(lián)用儀（LC-MS/MS）或酶學(xué)比色法定量測(cè)定 2-羥基戊二酸（2-HG）的細(xì)胞內(nèi)/外累積濃度。若發(fā)現(xiàn) 2-HG 釋放量發(fā)生斷崖式下跌，則證實(shí)細(xì)胞干性及 IDH1 突變特征已喪失，需立刻廢棄并更換更低代次的凍存管。

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質(zhì)粒載體菌株細(xì)胞蛋白抗體基因保藏中心

電話:400-800-2947

工作QQ/微信同號(hào):1843439339

網(wǎng)址http://www.nedfriskphoto.com