BioVector® RBL15 人视网膜母细胞瘤细胞株

BioVector® RBL15 Human Retinoblastoma Cell Line

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：BioVector® RBL15

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• 保藏机构货号：DSMZ ACC 943

• 物种来源：人类 (Homo sapiens)

• 性别与年龄：女性，11个月大婴儿

• 组织与疾病背景：该细胞株建立于1986年，源自一名11个月大女婴患者的原发性双侧视网膜母细胞瘤（Primary bilateral retinoblastoma）组织，在进行眼球摘除术（Enucleation）后分离构建。

• 抑癌基因 RB1 突变与启动子状态特征：

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  • 作为经典的遗传性/双侧视网膜母细胞瘤体外模型，该细胞系两条抑癌基因 RB1（Retinoblastoma 1） 等位基因均携带明确的病理性非 sense 突变。

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  • 具体双位点突变：等位基因 1 发生位于外显子 10 的 R320* 突变（chr13:48367512C>T, hg38）；等位基因 2 发生位于外显子 23 的 L797* 突变（chr13:48465269T>G, hg38），导致 RB1 蛋白功能完全丧失。

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  • 甲基化特征：经甲基化敏感性 PCR 验证，其 RB1 启动子区（CpG106）处于 0% 的未甲基化状态，而 RB1 内含子 2 的差异甲基化区（DMR CpG85）呈现 100% 的完全甲基化状态。

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• 生物安全级别与病毒状态：1级（BSL-1）。经 PCR 多重筛查，EBV、HBV、HCV、HIV-1/2、HPV、HTLV-1/2 均为阴性，小鼠白血病病毒（MLV）呈现阳性。

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二 细胞形态学与培养环境

• 形态学特征：展现典型的神经外胚层恶性肿瘤生长特性。在刚完成复苏解冻时通常呈单个散在的单细胞状态，随着培养时间的延长，细胞会自发聚集生长，最终形成直径可达数毫米（several mm）、表面具有明显结节状结构（Nodular structures）的巨大球状或类器官样悬浮聚合体。

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• 生长模式：完全悬浮生长（高度自聚集成球体）。

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• 群体倍增/传代时间：大约 5 至 7 天。

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• 标准特殊完全培养基配方：

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  • 85% BioVector® 高糖 DMEM 培养基（含 4.5 g/L 葡萄糖）

  • 15% 优质热灭活胎牛血清（h.i. FBS）

  • 10微克/毫升（10 µg/ml）人类胰岛素（Human insulin）

  • 2 mM L-谷氨酰胺（L-Glutamine）

  • 1 mM 丙酮酸钠（Sodium pyruvate）

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

• 物理培养参数：37摄氏度恒温、10% 二氧化碳（CO2）、空气饱和湿度。注：必须使用 10% 密度的高浓度二氧化碳孵育系统。

三 细胞传代与复苏标准操作步骤

1. 复苏早期极度敏感特性与适应期管理：

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   • 初次接种限制：从冻存管初次复苏时，务必将全部细胞一并接种入 6孔培养板的一个单一孔中，并加入大约 4 毫升 含有全组分添加的完全培养基。

   • 首期 1-3 周不传代原则：在复苏后的前 1 到 3 周内，严禁进行分裂传代（Do not split）。此时细胞处于缓慢聚集成球的适应期，应维持原孔培养，每周仅需一次或两次通过吸除旧基并补充 50% 的新鲜完全培养基 来执行半换液补液。

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2. 常规日常传代执行：

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   • 聚合体物理敏感度警告：RBL15 形成的类器官球状聚合体对普通小口径移液器的物理剪切力极其敏感，极易由于过度剧烈吹打而遭到机械性破坏（Sensitive to destruction by pipetting）。因此，在任何传代、补液或转移操作中，必须强制使用广口移液器（如常规无菌血清移液管、宽口/宽头枪头，或人工使用无菌剪刀剪去尖端的吸头）。

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   • 传代策略：当细胞顺利进入稳定扩增阶段后（此时 6 孔板单孔内的成熟聚合体球中所蕴含的活细胞总量可高达 $6\times 10^6$ 个），可以开始常规传代。通常每隔 5 到 7 天（约一周）按 1比2 至 1比3 的比例分裂入新器皿中。

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四 核心科研应用方向

1. RB1 双等位基因非sense突变（R320* / L797*）的眼科遗传学病理研究：BioVector® RBL15 是国际上用于探索儿童视网膜母细胞瘤发生、染色体 13q 缺陷以及 RB1 恶性失活突变网络机制的标准内源性疾病模型。

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2. 肿瘤多细胞类器官球体（Spheroids/Aggregates）三维形貌与缺氧微环境研究：由于其能自发形成数毫米大且带结节的球体，非常适用于作为非人工支架依赖性的眼科视网膜肿瘤 3D 模型，用于分析实体瘤核心区的养分梯度、坏死演变及缺氧诱导因子（HIF）调控轴。

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3. 靶向药物穿透力评价与高效抗肿瘤小分子高通量筛选：该细胞株是评估新型靶向化疗药物或表观遗传学调节剂是否能成功穿透致密肿瘤结节、杀死内部未分化癌细胞的优良体外阻抗评价屏障模型。

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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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