BioVector? CW1474 人食管腺癌細(xì)胞

BioVector? CW1474 Human Esophageal Adenocarcinoma Cell Line

第一部分：中文說明

一、 產(chǎn)品基本信息與遺傳學(xué)背景

• 細(xì)胞名稱：CW1474 人食管腺癌細(xì)胞

• 系統(tǒng)學(xué) Accession：Cellosaurus CVCL_D4Z7

• 保藏機(jī)構(gòu)貨號(hào)：ATCC CRL-3529

• 物種來源：人類 (Homo sapiens)

• 組織與疾病背景：該細(xì)胞株衍生自一名 86 歲高齡高加索（白種人）男性患者的食管原位腺癌（Esophageal Adenocarcinoma）。該細(xì)胞系最初是通過建立患者來源的異種移植模型（PDX），隨后進(jìn)一步進(jìn)行體外連續(xù)傳代培養(yǎng)從而實(shí)現(xiàn)細(xì)胞系永生化。

• 遺傳與分子特征：

  • 基因組學(xué)：經(jīng)全外顯子組測(cè)序（WES）驗(yàn)證。

  • 關(guān)鍵基因突變：攜帶腫瘤抑制基因 TP53 的純合缺失突變（具體變異位點(diǎn)為 p.Gly279_Thr284del 純合缺失）。

  • 特異性表達(dá)譜：該細(xì)胞屬于 lincPRKD 基因和 lincRTL 基因雙陰性（Null/陰性表達(dá)） 的特殊食管腺癌細(xì)胞模型。

• 生物安全級(jí)別：2級(jí)（BSL-2）。

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)典型的上皮樣特征。

• 生長(zhǎng)模式：貼壁生長(zhǎng)（Adherent）。

• 倍增時(shí)間（Doubling Time）：大約 41 小時(shí)，增殖速度相對(duì)溫和。

• 標(biāo)準(zhǔn)完全培養(yǎng)基配方（高精密要求）：

  • 基礎(chǔ)培養(yǎng)基為 L-WRN 條件培養(yǎng)基（內(nèi)含 Wnt-3A、R-spondin 和 Noggin 生長(zhǎng)因子）。

  • 完全培養(yǎng)基添加劑（每 245 mL L-WRN 條件培養(yǎng)基中需添加）：

    • SB202190 抑制劑（最終濃度 10 μM）

    • A83-01 抑制劑（最終濃度 200 nM）

    • 胃泌素（Gastrin，最終濃度 10 nM）

    • 成纖維細(xì)胞生長(zhǎng)因子-10（FGF-10，最終濃度 200 ng/mL）

  • 保存注意：配制好的完全培養(yǎng)基需嚴(yán)格避光保存，在 2–8°C 下穩(wěn)定性通常為 14 天。

• 物理培養(yǎng)參數(shù)：37°C 恒溫、5% 二氧化碳（$CO_2$）、空氣 95% 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)胞匯合度達(dá)到 50%–80% 時(shí)）：

   • 吸除舊培養(yǎng)基，用無(wú)菌 PBS 輕輕洗滌。

   • 加入適量消化液（如 Trypsin-EDTA 溶液），置于 37°C 孵育促進(jìn)細(xì)胞分散。

   • 特殊注意事項(xiàng)：為防止細(xì)胞產(chǎn)生不可逆的聚集（Clumping），在等待細(xì)胞脫落期間嚴(yán)禁拍打或劇烈搖晃培養(yǎng)瓶。

   • 細(xì)胞完全脫落后，加入完全培養(yǎng)基（不含 Rock 抑制劑）終止消化，溫和吹打。

   • 以 150–400 ×g 離心 8–12 分鐘以去除殘留的消化劑。

   • 推薦的傳代分瓶比例為 1:3，建議維持細(xì)胞種植密度在 4.0×$10^4$ 至 7.0×$10^4\text{ cells/cm}^2$ 之間。每周定期更換培養(yǎng)基 2–3 次。

2. 凍存細(xì)胞復(fù)蘇：

   • 從液氮中取出冷凍管，立即投入 37°C 水浴中快速搖動(dòng)使其融化（控制在 2 分鐘內(nèi)）。注意：該細(xì)胞長(zhǎng)期保藏必須存放于液氮中，否則會(huì)導(dǎo)致細(xì)胞失去活力。

   • 將解凍的細(xì)胞懸液移至含 9.0 mL 預(yù)熱培養(yǎng)基的離心管中，以 125 ×g 離心 5–7 分鐘。

   • 棄去上清，用完全培養(yǎng)基重懸。為避免細(xì)胞在復(fù)蘇初期因培養(yǎng)基過堿而受損，建議在接種細(xì)胞前，將含有培養(yǎng)基的培養(yǎng)瓶提前置于 37°C、5% $CO_2$ 培養(yǎng)箱中孵育至少 15 分鐘，使其 pH 值平衡至正常范圍。

四、 核心科研應(yīng)用方向

1. 食管腺癌分子分型與靶向治療：作為 HNF4A 調(diào)控網(wǎng)絡(luò)及轉(zhuǎn)化生長(zhǎng)因子 β（TGF-beta）通路研究的重要細(xì)胞模型，用于評(píng)估針對(duì)遠(yuǎn)端食管惡性腫瘤的通路靶向耐藥與脆弱性機(jī)制。

2. 非編碼 RNA 功能篩選控制研究：由于其天然缺失 lincRTL 和 lincPRKD 的雙陰性遺傳背景，CW1474 常被用作理想的陰性對(duì)照細(xì)胞株，用于確證反義寡核苷酸（ASO）或 Gapmer 針對(duì)食管腺癌長(zhǎng)鏈非編碼 RNA 靶向敲降時(shí)的特異性與脫靶效應(yīng)。

3. 新型生物仿生納米載藥系統(tǒng)開發(fā)：利用其食管腺癌特異性的細(xì)胞膜組分，通過技術(shù)包被納米顆粒，用于腫瘤同源靶向性（Homotypic tumor targeting）以及體外藥物遞送微系統(tǒng)的研發(fā)。

PART 2: ENGLISH SECTION

I. General Information and Genetic Background

• Cell Line Name: CW1474

• Cellosaurus Accession: CVCL_D4Z7

• Repository Catalog Number: ATCC CRL-3529

• Species Origin: Human (Homo sapiens)

• Tissue & Disease Background: Derived from an in situ esophageal adenocarcinoma (EAC) isolated from an 86-year-old Caucasian male patient. This cancer cell line was established via a patient-derived xenograft (PDX) model, followed by continuous in vitro passaging.

• Genomic & Molecular Profile:

  • Genomics: Fully verified via Whole Exome Sequencing (WES).

  • Tumor Suppressor Mutation: Carries a distinctive homozygous deletion variant in the TP53 gene (specifically p.Gly279_Thr284del; Zygosity=Homozygous).

  • LncRNA Profile: Characterized as a lincPRKD and lincRTL null-expression model, making it a unique tool in esophageal oncology genomics.

• Biosafety Level: BSL-2.

II. Morphological Attributes and Cultivation Media

• Morphology: Epithelial-like phenotype.

• Growth Mode: Adherent monolayer.

• Population Doubling Time: Approximately 41 hours, showing moderate growth kinetics.

• Standard Complete Growth Medium Formulation (Highly Specific):

  • Basal Medium: Requires L-WRN Conditioned Medium (rich in Wnt-3A, R-spondin, and Noggin).

  • Essential Additives (Supplements required per 245 mL of L-WRN Conditioned Medium):

    • SB202190 inhibitor (Final concentration: 10 μM)

    • A83-01 inhibitor (Final concentration: 200 nM)

    • Gastrin (Final concentration: 10 nM)

    • Fibroblast Growth Factor-10 (FGF-10, Final concentration: 200 ng/mL)

  • Storage Protocol: The completely assembled formulation must be strictly protected from light and remains stable at 2 to 8°C for up to 14 days.

• Physical Incubation Thresholds: Regulated tightly at 37°C under an atmospheric layer of 5% Carbon Dioxide ($CO_2$) and 95% air humidity.

III. Subculturing and Thawing Protocols

1. Routine Passaging Schedule (At 50%–80% Confluence):

   • Aspirate the spent culture medium and gently rinse the cell monolayer with sterile PBS.

   • Dispense an appropriate volume of dissociation agent (such as Trypsin-EDTA) and incubate at 37°C to facilitate cellular dispersal.

   • Critical Handling Caution: To eliminate cellular aggregation or clumping, do not hit, agitate, or shake the flask while waiting for detachment.

   • Stop the reaction by adding 6.0 to 8.0 mL of complete growth medium (without Rock inhibitor) and collect via gentle pipetting.

   • Centrifuge the suspension at 150 to 400 ×g for 8 to 12 minutes to remove any remaining enzyme residues.

   • Seed into fresh vessels at a recommended split ratio of 1:3, keeping the concentration strictly between 4.0×$10^4$ and 7.0×$10^4\text{ cells/cm}^2$. Renew medium 2 to 3 times per week.

2. Cryovial Thawing and Recovery:

   • Retrieve the cryovial from liquid nitrogen storage and submerge it into a 37°C water bath with rapid agitation (within 2 minutes). Note: Storage at stable liquid nitrogen temperatures is mandatory to preserve viability.

   • Transfer the matrix into a tube containing 9.0 mL of pre-warmed complete growth medium and centrifuge at approximately 125 ×g for 5 to 7 minutes.

   • Decant the supernatant and resuspend the pellet. To avoid excessive alkalinity during recovery, pre-incubate the culture vessel containing fresh complete growth medium inside the 5% $CO_2$ incubator for at least 15 minutes before adding cells to stabilize the normal pH window.

IV. Strategic Research Applications

1. EAC Molecular Subtyping & Targeted Therapies: Serves as a primary clinical model for deciphering HNF4A-driven lineage traits and evaluating transforming growth factor-beta (TGF-β) pathway-targeted vulnerabilities in distal esophagus carcinomas.

2. LncRNA Target Knockdown Specificity Controls: Owing to its unique lincRTL-null and lincPRKD-null baseline genotype, CW1474 is widely used as an ideal negative control line to assess the specificity and potential off-target effects of antisense gapmer designs in esophageal adenocarcinoma research.

3. Biomimetic Nanoparticle Drug Delivery Platforms: Utilized in advanced bio-engineering fields where its distinct cancer cell membranes are harvested and wrapped around functional nanoparticles to evaluate homotypic tumor targeting, cellular uptake kinetics, and drug platform stabilities.

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