BioVector? RBL15 人視網(wǎng)膜母細(xì)胞瘤細(xì)胞株

BioVector? RBL15 Human Retinoblastoma Cell Line

第一部分 中文說(shuō)明

一 產(chǎn)品基本信息與遺傳學(xué)背景

• 細(xì)胞名稱：BioVector? RBL15

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• 保藏機(jī)構(gòu)貨號(hào)：DSMZ ACC 943

• 物種來(lái)源：人類 (Homo sapiens)

• 性別與年齡：女性，11個(gè)月大嬰兒

• 組織與疾病背景：該細(xì)胞株建立于1986年，源自一名11個(gè)月大女嬰患者的原發(fā)性雙側(cè)視網(wǎng)膜母細(xì)胞瘤（Primary bilateral retinoblastoma）組織，在進(jìn)行眼球摘除術(shù)（Enucleation）后分離構(gòu)建。

• 抑癌基因 RB1 突變與啟動(dòng)子狀態(tài)特征：

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  • 作為經(jīng)典的遺傳性/雙側(cè)視網(wǎng)膜母細(xì)胞瘤體外模型，該細(xì)胞系兩條抑癌基因 RB1（Retinoblastoma 1） 等位基因均攜帶明確的病理性非 sense 突變。

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  • 具體雙位點(diǎn)突變：等位基因 1 發(fā)生位于外顯子 10 的 R320* 突變（chr13:48367512C>T, hg38）；等位基因 2 發(fā)生位于外顯子 23 的 L797* 突變（chr13:48465269T>G, hg38），導(dǎo)致 RB1 蛋白功能完全喪失。

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  • 甲基化特征：經(jīng)甲基化敏感性 PCR 驗(yàn)證，其 RB1 啟動(dòng)子區(qū)（CpG106）處于 0% 的未甲基化狀態(tài)，而 RB1 內(nèi)含子 2 的差異甲基化區(qū)（DMR CpG85）呈現(xiàn) 100% 的完全甲基化狀態(tài)。

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• 生物安全級(jí)別與病毒狀態(tài)：1級(jí)（BSL-1）。經(jīng) PCR 多重篩查，EBV、HBV、HCV、HIV-1/2、HPV、HTLV-1/2 均為陰性，小鼠白血病病毒（MLV）呈現(xiàn)陽(yáng)性。

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二 細(xì)胞形態(tài)學(xué)與培養(yǎng)環(huán)境

• 形態(tài)學(xué)特征：展現(xiàn)典型的神經(jīng)外胚層惡性腫瘤生長(zhǎng)特性。在剛完成復(fù)蘇解凍時(shí)通常呈單個(gè)散在的單細(xì)胞狀態(tài)，隨著培養(yǎng)時(shí)間的延長(zhǎng)，細(xì)胞會(huì)自發(fā)聚集生長(zhǎng)，最終形成直徑可達(dá)數(shù)毫米（several mm）、表面具有明顯結(jié)節(jié)狀結(jié)構(gòu)（Nodular structures）的巨大球狀或類器官樣懸浮聚合體。

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• 生長(zhǎng)模式：完全懸浮生長(zhǎng)（高度自聚集成球體）。

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• 群體倍增/傳代時(shí)間：大約 5 至 7 天。

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• 標(biāo)準(zhǔn)特殊完全培養(yǎng)基配方：

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  • 85% BioVector? 高糖 DMEM 培養(yǎng)基（含 4.5 g/L 葡萄糖）

  • 15% 優(yōu)質(zhì)熱滅活胎牛血清（h.i. FBS）

  • 10微克/毫升（10 μg/ml）人類胰島素（Human insulin）

  • 2 mM L-谷氨酰胺（L-Glutamine）

  • 1 mM 丙酮酸鈉（Sodium pyruvate）

  • 50 μM 2-麥角乙醇（beta-mercaptoethanol）

• 物理培養(yǎng)參數(shù)：37攝氏度恒溫、10% 二氧化碳（CO2）、空氣飽和濕度。注：必須使用 10% 密度的高濃度二氧化碳孵育系統(tǒng)。

三 細(xì)胞傳代與復(fù)蘇標(biāo)準(zhǔn)操作步驟

1. 復(fù)蘇早期極度敏感特性與適應(yīng)期管理：

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   • 初次接種限制：從凍存管初次復(fù)蘇時(shí)，務(wù)必將全部細(xì)胞一并接種入 6孔培養(yǎng)板的一個(gè)單一孔中，并加入大約 4 毫升 含有全組分添加的完全培養(yǎng)基。

   • 首期 1-3 周不傳代原則：在復(fù)蘇后的前 1 到 3 周內(nèi)，嚴(yán)禁進(jìn)行分裂傳代（Do not split）。此時(shí)細(xì)胞處于緩慢聚集成球的適應(yīng)期，應(yīng)維持原孔培養(yǎng)，每周僅需一次或兩次通過(guò)吸除舊基并補(bǔ)充 50% 的新鮮完全培養(yǎng)基 來(lái)執(zhí)行半換液補(bǔ)液。

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2. 常規(guī)日常傳代執(zhí)行：

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   • 聚合體物理敏感度警告：RBL15 形成的類器官球狀聚合體對(duì)普通小口徑移液器的物理剪切力極其敏感，極易由于過(guò)度劇烈吹打而遭到機(jī)械性破壞（Sensitive to destruction by pipetting）。因此，在任何傳代、補(bǔ)液或轉(zhuǎn)移操作中，必須強(qiáng)制使用廣口移液器（如常規(guī)無(wú)菌血清移液管、寬口/寬頭槍頭，或人工使用無(wú)菌剪刀剪去尖端的吸頭）。

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   • 傳代策略：當(dāng)細(xì)胞順利進(jìn)入穩(wěn)定擴(kuò)增階段后（此時(shí) 6 孔板單孔內(nèi)的成熟聚合體球中所蘊(yùn)含的活細(xì)胞總量可高達(dá) $6\times 10^6$ 個(gè)），可以開(kāi)始常規(guī)傳代。通常每隔 5 到 7 天（約一周）按 1比2 至 1比3 的比例分裂入新器皿中。

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四 核心科研應(yīng)用方向

1. RB1 雙等位基因非sense突變（R320* / L797*）的眼科遺傳學(xué)病理研究：BioVector? RBL15 是國(guó)際上用于探索兒童視網(wǎng)膜母細(xì)胞瘤發(fā)生、染色體 13q 缺陷以及 RB1 惡性失活突變網(wǎng)絡(luò)機(jī)制的標(biāo)準(zhǔn)內(nèi)源性疾病模型。

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2. 腫瘤多細(xì)胞類器官球體（Spheroids/Aggregates）三維形貌與缺氧微環(huán)境研究：由于其能自發(fā)形成數(shù)毫米大且?guī)ЫY(jié)節(jié)的球體，非常適用于作為非人工支架依賴性的眼科視網(wǎng)膜腫瘤 3D 模型，用于分析實(shí)體瘤核心區(qū)的養(yǎng)分梯度、壞死演變及缺氧誘導(dǎo)因子（HIF）調(diào)控軸。

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3. 靶向藥物穿透力評(píng)價(jià)與高效抗腫瘤小分子高通量篩選：該細(xì)胞株是評(píng)估新型靶向化療藥物或表觀遺傳學(xué)調(diào)節(jié)劑是否能成功穿透致密腫瘤結(jié)節(jié)、殺死內(nèi)部未分化癌細(xì)胞的優(yōu)良體外阻抗評(píng)價(jià)屏障模型。

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PART 2 ENGLISH SECTION

I General Information and Genetic Background

• Cell Line Name: BioVector? RBL15

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• Repository Catalog Number: DSMZ ACC 943

• Species Origin: Human (Homo sapiens)

• Sex and Age of Donor: Female, 11-month-old infant

• Tissue and Disease Background: Established in 1986 from the primary bilateral retinoblastoma mass of an 11-month-old female infant, isolated successfully following therapeutic eye enucleation.

• RB1 Tumour Suppressor Mutation & Methylation Blueprint:

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  • An authentic reference system for bilateral/hereditary retinoblastoma modeling, carrying severe pathognomonic biallelic nonsense variations within the RB1 (Retinoblastoma 1) locus.

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  • Biallelic Disruption Profile: Verified via Sanger sequencing showing allele 1 carries an exon 10 R320* truncation (chr13:48367512C>T, hg38), while allele 2 harbors an exon 23 L797* nonsense alteration (chr13:48465269T>G, hg38), culminating in complete loss of functional RB1 protein assembly.

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  • Epigenetic Profile: Methylation-sensitive PCR tracks the RB1 promoter (CpG106) at a 0% unmethylated profile, while the differentially methylated region CpG85 inside RB1 intron 2 is 100% methylated.

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• Biosafety Level & Viral Profile: BSL-1.Tested negative via targeted PCR lines for EBV, HBV, HCV, HIV-1/2, HPV, and HTLV-1/2 genomes; verified positive for Murine Leukemia Virus (MLV) sequences.

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II Morphological Attributes and Cultivation Media

• Morphology: Exhibits typical features of malignant ocular neuroectodermal clusters.Immediately following thaw procedures, cultures present predominantly as isolated singular units.Over time, cells spontaneously condense to grow as massive multicellular spheroids extending up to several millimeters in diameter, showing unique nodular structures.

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• Growth Mode: Strict suspension growth (expanding as macro-aggregates and heavy cluster spheres).

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• Population Doubling / Interval Sequence: Average cycle spans 5 to 7 days.

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• Standard Specialized Complete Growth Medium Formulation:

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  • 85% BioVector? High-Glucose DMEM medium (supplemented with 4.5 g/L glucose)

  • 15% premium BioVector? Heat-Inactivated Fetal Bovine Serum (h.i. FBS)

  • 10 μg/ml recombinant Human Insulin

  • 2 mM L-Glutamine

  • 1 mM Sodium Pyruvate

  • 50 μM 2-Mercaptoethanol

• Physical Incubation Parameters: Regulated at 37 degrees Celsius under a custom humidified atmosphere containing 10% Carbon Dioxide (CO2). Note: Standard 5% CO2 environments are inadequate for this line.

III Subculturing and Thawing Protocols

1. Critical Seeding Phase and Delayed Post-Thaw Recovery Dynamics:

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   • Initialization Parameters: Upon thawing a cryovial, seed out the entire cell content into a single well of a standard 6-well culture plate using approximately 4 mL of fresh fully-supplemented complete medium.

   • First 1 to 3 Weeks No-Split Restriction: Crucially, do not split or pass the culture during the first 1 to 3 weeks of adaptation.Instead, focus on maintaining the primary aggregate niche by executing a 50% medium replacement protocol once or twice per week.

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2. Routine Continuous Passaging Schedule:

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   • Shear-Force Sensitivity Warning: The floating multicellular macro-aggregates generated by RBL15 are highly sensitive to mechanical destruction caused by aggressive pipetting. Consequently, investigators must exclusively utilize wide-opening pipetting systems (such as serological glass pipettes, wide-bore tips, or sterile manually-cut plastic tips) to handle the cultures safely.

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   • Subculturing Routine: Once the suspension establishes strong expansion kinetics (a split-ready single well in a 6-well plate may contain up to $6\times 10^6$ active cells), split the cluster suspension at ratios of 1:2 to 1:3 once per week.

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IV Strategic Research Applications

1. Deciphering RB1 Biallelic Nonsense Mutations (R320* / L797*) in Ocular Oncology: BioVector? RBL15 represents an endogenous reference matrix to evaluate hereditary infant bilateral retinoblastoma biology and map downstream cell-cycle deregulation caused by specific exon truncations.

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2. Modeling 3D Spheroid Microenvironments and Intratumoral Hypoxia: The spontaneous generation of nodular macro-spheroids provides a reliable scaffold-free 3D model for tracing spatial nutrient depletion, necrotic core formation, and hypoxia-inducible factor (HIF) downstream activation loops in solid eye tumors.

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3. Evaluating Drug Intratumoral Penetration and Small-Molecule Screening: This cell line is an ideal platform for high-throughput screening to determine whether candidate chemotherapeutic entities or epigenetic small molecules can successfully penetrate dense, multi-millimeter cell aggregates to eliminate underlying tumor-initiating populations.

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