BioVector? hTERT-RPE1 人視網(wǎng)膜色素上皮細(xì)胞株

BioVector? hTERT-RPE1 Human Retinal Pigment Epithelial 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? hTERT-RPE1 人視網(wǎng)膜色素上皮細(xì)胞

• 系統(tǒng)學(xué) Accession Cellosaurus CVCL 4388

• 保藏機(jī)構(gòu)貨號(hào) ATCC CRL 4000

• 物種來(lái)源 人類 Homo sapiens

• 組織與疾病背景 該細(xì)胞株源自一名女性捐獻(xiàn)者的正常眼底視網(wǎng)膜色素上皮（RPE）組織。原代細(xì)胞通過轉(zhuǎn)染編碼人端粒酶逆轉(zhuǎn)錄酶的質(zhì)粒（pGRN145）實(shí)現(xiàn)無(wú)限增殖化。

• 遺傳與表型特征

  • 核型特征 表現(xiàn)為近二倍體核型，主要包含 46條染色體，其中一條 X染色體末端帶有衍生自 10號(hào)染色體的易位片段。盡管經(jīng)過端粒酶改造，該細(xì)胞仍保持了極高的基因組穩(wěn)定性。

  • 標(biāo)志物與細(xì)胞特性 保持了正常的接觸抑制特性。在匯合度達(dá)到100%且血清饑餓誘導(dǎo)后，該細(xì)胞能高效地在其表面自主生長(zhǎng)出單根初級(jí)纖毛（Primary Cilia）。

• 生物安全級(jí)別 1級(jí)。

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)典型且規(guī)則的上皮樣、多角形或鋪路石狀細(xì)胞結(jié)構(gòu)。在低密度時(shí)細(xì)胞呈細(xì)長(zhǎng)形伸展，完全融合后排列緊密。

• 生長(zhǎng)模式 貼壁生長(zhǎng)。

• 倍增時(shí)間 增殖極其活躍，倍增時(shí)間大約為 20到25小時(shí)。

• 標(biāo)準(zhǔn)完全培養(yǎng)基配方

  • 基礎(chǔ)培養(yǎng)基 BioVector? DMEM/F-12 1比1 培養(yǎng)基。

  • 維持添加

    • 10% BioVector? 優(yōu)質(zhì)胎牛血清。

    • 0.01 mg/mL 潮霉素B（Hygromycin B）。注意：潮霉素B用于維持端粒酶轉(zhuǎn)基因的篩選壓力，但在常規(guī)實(shí)驗(yàn)或功能驗(yàn)證傳代中可選擇不加。

• 物理培養(yǎng)參數(shù) 37攝氏度恒溫、5% 二氧化碳、空氣飽和濕度。

三 細(xì)胞傳代與復(fù)蘇標(biāo)準(zhǔn)操作步驟

1. 常規(guī)傳代操作

   • 當(dāng)細(xì)胞密度達(dá)到 80% 到 90% 匯合度時(shí)需要進(jìn)行傳代。切勿讓細(xì)胞過度生長(zhǎng)或重疊，否則會(huì)影響其后續(xù)的纖毛誘導(dǎo)分化能力。

   • 吸除舊培養(yǎng)基，用無(wú)菌 PBS 輕輕洗滌。

   • 加入適量 BioVector? 0.25% Trypsin 消化液，在 37攝氏度下孵育 2到4分鐘。

   • 顯微鏡下觀察到細(xì)胞開始變圓并脫落后，加入等體積含血清的完全培養(yǎng)基終止消化。

   • 輕輕吹打產(chǎn)生單細(xì)胞懸液，按 1比4 至 1比8 的傳代比例注入新的器皿中。

2. 凍存細(xì)胞復(fù)蘇

   • 從液氮中取出冷凍管，立即投入 37攝氏度 BioVector? 水浴鍋中快速搖動(dòng)使其融化，控制在 1到2分鐘內(nèi)。

   • 將解凍的細(xì)胞懸液移至含 5 mL 預(yù)熱培養(yǎng)基的離心管中，常規(guī)速度離心 5分鐘以沉淀細(xì)胞。

   • 棄去上清，加入新鮮 BioVector? 完全培養(yǎng)基重懸，接種到培養(yǎng)瓶?jī)?nèi)正常培養(yǎng)。

四 核心科研應(yīng)用方向

1. 細(xì)胞生物學(xué)與初級(jí)纖毛（Primary Cilia）研究：BioVector? hTERT-RPE1 是全球公認(rèn)最經(jīng)典的初級(jí)纖毛生物學(xué)研究模型。通過撤去血清（血清饑餓法）培養(yǎng) 24到48小時(shí)，可誘導(dǎo)細(xì)胞表面同步化長(zhǎng)出纖毛，用于篩選調(diào)控纖毛發(fā)生、纖毛內(nèi)轉(zhuǎn)運(yùn)（IFT）以及纖毛?。–iliopathies）的靶向基因。

2. 細(xì)胞周期調(diào)控與有絲分裂質(zhì)控：由于其具備接近正常的二倍體基因組且不含有抑癌基因（如 p53, pRb）的病毒癌基因失活，它被廣泛用于有絲分裂紡錘體組裝檢查點(diǎn)（SAC）、中心體復(fù)制以及 DNA損傷修復(fù)的精準(zhǔn)機(jī)制解析。

3. 視網(wǎng)膜及上皮屏障功能模擬：用于體外研究視網(wǎng)膜色素上皮細(xì)胞的極性發(fā)育、胞吞作用（如視桿細(xì)胞外節(jié)段的吞噬模擬）以及上皮-間充質(zhì)轉(zhuǎn)化（EMT）病理過程。

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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