BioVector® CW1474 人食管腺癌细胞

BioVector® CW1474 Human Esophageal Adenocarcinoma 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：CW1474 人食管腺癌细胞

• 系统学 Accession：Cellosaurus CVCL_D4Z7

• 保藏机构货号：ATCC CRL-3529

• 物种来源：人类 (Homo sapiens)

• 组织与疾病背景：该细胞株衍生自一名 86 岁高龄高加索（白种人）男性患者的食管原位腺癌（Esophageal Adenocarcinoma）。该细胞系最初是通过建立患者来源的异种移植模型（PDX），随后进一步进行体外连续传代培养从而实现细胞系永生化。

• 遗传与分子特征：

  • 基因组学：经全外显子组测序（WES）验证。

  • 关键基因突变：携带肿瘤抑制基因 TP53 的纯合缺失突变（具体变异位点为 p.Gly279_Thr284del 纯合缺失）。

  • 特异性表达谱：该细胞属于 lincPRKD 基因和 lincRTL 基因双阴性（Null/阴性表达） 的特殊食管腺癌细胞模型。

• 生物安全级别：2级（BSL-2）。

二、 细胞形态学与培养环境

• 形态学特征：展现典型的上皮样特征。

• 生长模式：贴壁生长（Adherent）。

• 倍增时间（Doubling Time）：大约 41 小时，增殖速度相对温和。

• 标准完全培养基配方（高精密要求）：

  • 基础培养基为 L-WRN 条件培养基（内含 Wnt-3A、R-spondin 和 Noggin 生长因子）。

  • 完全培养基添加剂（每 245 mL L-WRN 条件培养基中需添加）：

    • SB202190 抑制剂（最终浓度 10 μM）

    • A83-01 抑制剂（最终浓度 200 nM）

    • 胃泌素（Gastrin，最终浓度 10 nM）

    • 成纤维细胞生长因子-10（FGF-10，最终浓度 200 ng/mL）

  • 保存注意：配制好的完全培养基需严格避光保存，在 2–8°C 下稳定性通常为 14 天。

• 物理培养参数：37°C 恒温、5% 二氧化碳（$CO_2$）、空气 95% 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（当细胞汇合度达到 50%–80% 时）：

   • 吸除旧培养基，用无菌 PBS 轻轻洗涤。

   • 加入适量消化液（如 Trypsin-EDTA 溶液），置于 37°C 孵育促进细胞分散。

   • 特殊注意事项：为防止细胞产生不可逆的聚集（Clumping），在等待细胞脱落期间严禁拍打或剧烈摇晃培养瓶。

   • 细胞完全脱落后，加入完全培养基（不含 Rock 抑制剂）终止消化，温和吹打。

   • 以 150–400 ×g 离心 8–12 分钟以去除残留的消化剂。

   • 推荐的传代分瓶比例为 1:3，建议维持细胞种植密度在 4.0×$10^4$ 至 7.0×$10^4\text{ cells/cm}^2$ 之间。每周定期更换培养基 2–3 次。

2. 冻存细胞复苏：

   • 从液氮中取出冷冻管，立即投入 37°C 水浴中快速摇动使其融化（控制在 2 分钟内）。注意：该细胞长期保藏必须存放于液氮中，否则会导致细胞失去活力。

   • 将解冻的细胞悬液移至含 9.0 mL 预热培养基的离心管中，以 125 ×g 离心 5–7 分钟。

   • 弃去上清，用完全培养基重悬。为避免细胞在复苏初期因培养基过碱而受损，建议在接种细胞前，将含有培养基的培养瓶提前置于 37°C、5% $CO_2$ 培养箱中孵育至少 15 分钟，使其 pH 值平衡至正常范围。

四、 核心科研应用方向

1. 食管腺癌分子分型与靶向治疗：作为 HNF4A 调控网络及转化生长因子 β（TGF-beta）通路研究的重要细胞模型，用于评估针对远端食管恶性肿瘤的通路靶向耐药与脆弱性机制。

2. 非编码 RNA 功能筛选控制研究：由于其天然缺失 lincRTL 和 lincPRKD 的双阴性遗传背景，CW1474 常被用作理想的阴性对照细胞株，用于确证反义寡核苷酸（ASO）或 Gapmer 针对食管腺癌长链非编码 RNA 靶向敲降时的特异性与脱靶效应。

3. 新型生物仿生纳米载药系统开发：利用其食管腺癌特异性的细胞膜组分，通过技术包被纳米颗粒，用于肿瘤同源靶向性（Homotypic tumor targeting）以及体外药物递送微系统的研发。

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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