BioVector® hTERT-RPE1 人视网膜色素上皮细胞株

BioVector® hTERT-RPE1 Human Retinal Pigment Epithelial 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® hTERT-RPE1 人视网膜色素上皮细胞

• 系统学 Accession Cellosaurus CVCL 4388

• 保藏机构货号 ATCC CRL 4000

• 物种来源 人类 Homo sapiens

• 组织与疾病背景 该细胞株源自一名女性捐献者的正常眼底视网膜色素上皮（RPE）组织。原代细胞通过转染编码人端粒酶逆转录酶的质粒（pGRN145）实现无限增殖化。

• 遗传与表型特征

  • 核型特征 表现为近二倍体核型，主要包含 46条染色体，其中一条 X染色体末端带有衍生自 10号染色体的易位片段。尽管经过端粒酶改造，该细胞仍保持了极高的基因组稳定性。

  • 标志物与细胞特性 保持了正常的接触抑制特性。在汇合度达到100%且血清饥饿诱导后，该细胞能高效地在其表面自主生长出单根初级纤毛（Primary Cilia）。

• 生物安全级别 1级。

二 细胞形态学与培养环境

• 形态学特征 展现典型且规则的上皮样、多角形或铺路石状细胞结构。在低密度时细胞呈细长形伸展，完全融合后排列紧密。

• 生长模式 贴壁生长。

• 倍增时间 增殖极其活跃，倍增时间大约为 20到25小时。

• 标准完全培养基配方

  • 基础培养基 BioVector® DMEM/F-12 1比1 培养基。

  • 维持添加

    • 10% BioVector® 优质胎牛血清。

    • 0.01 mg/mL 潮霉素B（Hygromycin B）。注意：潮霉素B用于维持端粒酶转基因的筛选压力，但在常规实验或功能验证传代中可选择不加。

• 物理培养参数 37摄氏度恒温、5% 二氧化碳、空气饱和湿度。

三 细胞传代与复苏标准操作步骤

1. 常规传代操作

   • 当细胞密度达到 80% 到 90% 汇合度时需要进行传代。切勿让细胞过度生长或重叠，否则会影响其后续的纤毛诱导分化能力。

   • 吸除旧培养基，用无菌 PBS 轻轻洗涤。

   • 加入适量 BioVector® 0.25% Trypsin 消化液，在 37摄氏度下孵育 2到4分钟。

   • 显微镜下观察到细胞开始变圆并脱落后，加入等体积含血清的完全培养基终止消化。

   • 轻轻吹打产生单细胞悬液，按 1比4 至 1比8 的传代比例注入新的器皿中。

2. 冻存细胞复苏

   • 从液氮中取出冷冻管，立即投入 37摄氏度 BioVector® 水浴锅中快速摇动使其融化，控制在 1到2分钟内。

   • 将解冻的细胞悬液移至含 5 mL 预热培养基的离心管中，常规速度离心 5分钟以沉淀细胞。

   • 弃去上清，加入新鲜 BioVector® 完全培养基重悬，接种到培养瓶内正常培养。

四 核心科研应用方向

1. 细胞生物学与初级纤毛（Primary Cilia）研究：BioVector® hTERT-RPE1 是全球公认最经典的初级纤毛生物学研究模型。通过撤去血清（血清饥饿法）培养 24到48小时，可诱导细胞表面同步化长出纤毛，用于筛选调控纤毛发生、纤毛内转运（IFT）以及纤毛病（Ciliopathies）的靶向基因。

2. 细胞周期调控与有丝分裂质控：由于其具备接近正常的二倍体基因组且不含有抑癌基因（如 p53, pRb）的病毒癌基因失活，它被广泛用于有丝分裂纺锤体组装检查点（SAC）、中心体复制以及 DNA损伤修复的精准机制解析。

3. 视网膜及上皮屏障功能模拟：用于体外研究视网膜色素上皮细胞的极性发育、胞吞作用（如视杆细胞外节段的吞噬模拟）以及上皮-间充质转化（EMT）病理过程。

PART 2 ENGLISH SECTION

I General Information and Genetic Background

• Cell Line Name BioVector® hTERT-RPE1

• Cellosaurus Accession CVCL 4388

• Repository Catalog Number ATCC CRL 4000

• Species Origin Human Homo sapiens

• Tissue and Disease Background Derived from normal retinal pigment epithelial tissue of a female donor. The primary cells were immortalized via stable transfection with the pGRN145 plasmid encoding human telomerase reverse transcriptase.

• Genomic and Phenotypic Profiles

  • Karyotype Authenticated as a modal diploid line containing 46 chromosomes. It retains excellent genomic stability, with a single stable translocation event involving an addition to the end of one X chromosome derived from chromosome 10.

  • Cellular Traits Maintains classic contact inhibition in culture. Upon hitting absolute confluence followed by serum starvation, cells arrest in G0 phase and assemble a single, functional primary cilium on their apical surfaces.

• Biosafety Level BSL-1.

II Morphological Attributes and Cultivation Media

• Morphology Displays uniform epithelial like, polygonal, and cobblestone arrangements. Cells stretch out at low densities and form dense monolayers upon confluence.

• Growth Mode Adherent monolayer.

• Standard Complete Growth Medium Formulation

  • Basal Medium BioVector® DMEM/F-12 1比1 medium.

  • Routine Maintenance Supplements

    • 10% premium BioVector® Fetal Bovine Serum.

    • 0.01 mg/mL Hygromycin B. Note Hygromycin B is deployed to maintain the exogenous hTERT transgene selection pressure but may be omitted during temporary experimental assays.

• Physical Incubation Thresholds Regulated strictly at 37 degrees Celsius under an atmospheric layer of 5% Carbon Dioxide and saturated air humidity.

III Subculturing and Thawing Protocols

1. Routine Passaging Schedule

   • Initiate subculturing when the monolayer hits 80% to 90% confluence. Avoid letting the culture overgrow past absolute confluence to prevent a decline in its downstream ciliogenesis induction baseline.

   • Aspirate spent medium and rinse the matrix gently with sterile PBS.

   • Dispense BioVector® 0.25% Trypsin solution and incubate at 37 degrees Celsius for 2 to 4 minutes until cells detach.

   • Quench enzymatic activity by introducing an equal volume of serum containing complete growth medium.

   • Pipette gently to yield single cell droplets and seed fresh vessels at a recommended split ratio of 1比4 to 1比8.

2. Cryovial Thawing and Recovery

   • Retrieve the cryovial from storage and submerge it into a 37 degrees Celsius BioVector® water bath with rapid agitation until completely liquefied within 1 to 2 minutes.

   • Dilute the suspension into 5 mL of pre warmed complete broth and spin down for 5 minutes.

   • Decant the DMSO tainted supernatant, resuspend the pellet thoroughly in fresh BioVector® complete medium, and seed into a culture flask.

IV Strategic Research Applications

1. Primary Cilia and Intraflagellar Transport (IFT) Assays: BioVector® hTERT-RPE1 represents the gold standard mammalian model to inspect ciliogenesis mechanics. Serum deprivation for 24 to 48 hours reliably triggers uniform primary cilia assembly, facilitating structural screening for intraflagellar transport components and molecular phenotypes linked to genetic ciliopathies.

2. Cell Cycle Kinetics and Mitotic Checkpoint Interrogations: Because it harbors a stable diploid genome and intact p53/pRb signaling pathways, this line is heavily used to model spindle assembly checkpoints, centrosome duplication anomalies, and DNA damage recovery signaling tracks without the background mutations seen in cancer lines.

3. Retinal Pigment Epithelium Functional Modeling: Utilized as a standard epithelial model to interrogate cell polarity establishment, epithelial to mesenchymal transition (EMT) dynamics, and phagocytosis events mimicking the clearing of photoreceptor outer segments.

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