BioVector® Mero-48a 人胸膜双相型恶性间皮瘤细胞技术指南

BioVector® Mero-48a Human Pleural Biphasic Malignant Mesothelioma Cell Line Technical Guide

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：Mero-48a 人胸膜双相型恶性间皮瘤细胞

• 系统学 Accession：Cellosaurus CVCL_2593

• 保藏机构货号：ECACC 09100104

• 物种来源：人类 (Homo sapiens)

• 组织与疾病背景：该细胞株由 Dr Marjan A.Versnel（伊拉斯姆斯医学中心免疫学系）构建并保藏。其衍生自一名男性患者胸膜腔内的尸检肿瘤组织，临床与病理分型为明确的胸膜双相型恶性间皮瘤（Pleural Biphasic Mesothelioma），该类疾病往往与环境中的特定致癌物（如石棉）接触史高度相关。

• 遗传与分子特征：

  • 核型特征：呈现高非整倍体变异，体外传代培养物中的染色体数目稳定介于 71–75 之间。

  • 基因组与信号通路变异：携带间皮瘤中经典的染色体异常与基因功能缺陷，常伴有 Hippo 信号通路相关基因的突变特征。

  • 特异性标志物：阳性表达上皮膜抗原（Epithelial Membrane Antigen, EMA）。

• 生物安全级别：2级（Hazard Group 2 / BSL-2）。鉴于其人类恶性肿瘤来源，所有无菌细胞操作均必须在二级生物安全柜中进行。

二、 细胞形态学与培养环境

• 形态学特征：由于其源自双相型（混合型）间皮瘤，细胞在显微镜下展现出特异性的混合型形态，即上皮样细胞与梭形（肉瘤样）细胞混杂生长。

• 生长模式：贴壁生长（Adherent）。

• 倍增时间（Doubling Time）：大约 24 小时，细胞增殖速度较快。

• 标准完全培养基配方：

  • 基础培养基：Ham's F10 培养基。

  • 常规维持添加：15% 优质胎牛血清（FCS/FBS）+ 2 mM 谷氨酰胺（Glutamine）+ 1% 双抗（青霉素-链霉素）。

• 物理培养参数：37°C 恒温、5% 二氧化碳（$CO_2$）、饱和空气湿度培养箱。

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 70%–80% 时）：

   • 吸除上清旧培养基，用无菌 PBS 轻轻洗涤细胞层 1–2 次。

   • 加入适量 0.05% Trypsin 或 Trypsin-EDTA 消化液，置于 37°C 孵育 2–3 分钟。

   • 显微镜下观察到大部分细胞开始变圆并脱落，立即加入含血清的完全培养基终止消化反应。

   • 用移液枪温和吹打细胞以使其完全脱落并打散，移入离心管中，以 1000 RPM (约 200g) 离心 5 分钟。

   • 弃去上清，加入新鲜完全培养基重悬，推荐按 1:4 至 1:10 的比例进行传代接种（或按 2–4×$10^4\text{ cells/cm}^2$ 的接种密度种植至新培养瓶中）。

2. 冻存细胞复苏：

   • 将冷冻管从液氮或超低温冰箱中取出，立即投入 37°C 水浴中快速摇动使其融化（确保在 1–2 分钟内完全融解）。

   • 将解冻的细胞悬液移至含 5 mL 预热完全培养基的离心管中，1000 RPM 离心 5 分钟。

   • 弃去含保护剂（DMSO）的上清液，用新鲜完全培养基重悬细胞并接种于 T25 培养瓶中，置于 37°C 培养箱中静态培养。

四、 核心科研应用方向

1. 恶性间皮瘤发病机制研究：作为经典的双相型间皮瘤细胞株，用于研究上皮样向间质样/肉瘤样转化的分子机制，以及 Hippo 通路障碍在间皮瘤发生中的作用。

2. 新型诊断方法与生物标志物开发：利用其高表达上皮膜抗原（EMA）的分子特征，用于开发和评估针对临床间皮瘤的新型多克隆/单克隆抗体、免疫组化检测靶点或分子诊断技术。

3. 肿瘤耐药性与药物高通量筛选：针对恶性胸膜间皮瘤临床化疗响应差的特点，用于新型靶向药物、谷氨酰胺转化酶（GST）抑制剂、解毒酶抑制剂以及基于 CRISPR/Cas9 系统的全基因组依赖性靶点筛选。

PART 2: ENGLISH SECTION

I. General Information and Genetic Background

• Cell Line Name: Mero-48a

• Cellosaurus Accession: CVCL_2593

• Repository Catalog Number: ECACC 09100104

• Species Origin: Human (Homo sapiens)

• Tissue & Disease Background: Established by Dr Marjan A.Versnel at the Department of Immunology, Erasmus MC, this cell line was derived from autopsy tumor material harvested from the pleural cavity of a male patient.The pathological pathogenesis is strictly classified as Pleural Biphasic Malignant Mesothelioma, a lethal malignancy strongly linked to environmental or industrial carcinogen exposure (such as asbestos).

• Genomic & Molecular Profile:

  • Karyotype: Exhibits a highly aneuploid profile with a chromosomal count ranging stably between 71 and 75.

  • Pathway Variations: Harbors distinct genomic signatures and aberrations matching classic malignant mesotheliomas, including mutational profiles associated with the Hippo signaling cascade.

  • Marker Expression: Verified positive for Epithelial Membrane Antigen (EMA) expression.

• Biosafety Level: Hazard Group 2 (BSL-2).Due to its malignant human origin, all cell manipulations must be executed within certified Class II Biosafety Cabinets.

II. Morphological Attributes and Cultivation Media

• Morphology: Displays a characteristic mixed epithelial and spindle-shaped morphology, which directly mirrors the mixed sarcomatoid and epithelioid presentation of biphasic primary tumors.

• Growth Mode: Adherent monolayer.

• Population Doubling Time: Proliferates dynamically with a doubling period of approximately 24 hours.

• Standard Complete Growth Medium Formulation:

  • Basal Medium: Ham's F10 medium.

  • Routine Maintenance Supplements: 15% high-quality Fetal Calf Serum (FCS/FBS) + 2 mM L-Glutamine + 1% Penicillin-Streptomycin.

• Physical Incubation Thresholds: Regulated strictly at 37°C under an atmospheric layer of 5% Carbon Dioxide ($CO_2$) with saturated humidity.

III. Subculturing and Thawing Protocols

1. Routine Passaging Schedule (At 70%–80% Confluence):

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

   • Introduce a sufficient volume of 0.05% Trypsin or Trypsin-EDTA solution and incubate at 37°C for 2–3 minutes.

   • Monitor under an inverted microscope until cells round up and initiate detachment, then immediately neutralize enzymic cleavage with serum-supplemented complete growth medium.

   • Gently pipette to dissociate cell aggregates, transfer into a conical tube, and centrifuge at 1000 RPM (~200g) for 5 minutes.

   • Decant the supernatant, resuspend the pellet in fresh complete growth medium, and split at a ratio of 1:4 to 1:10 (or seed at a target density of 2–4×$10^4\text{ cells/cm}^2$).

2. Cryovial Thawing and Recovery:

   • Retrieve the cryovial from storage and plunge it into a 37°C water bath with continuous agitation until liquefied (within 1–2 minutes).

   • Transfer the thawed cell slurry into a centrifuge tube containing 5 mL of pre-warmed medium and centrifuge at 1000 RPM for 5 minutes.

   • Discard the DMSO-containing supernatant, gently resuspend the cell sediment pellet in fresh complete growth medium, and transfer into a culture flask for static incubation at 37°C with 5% $CO_2$.

IV. Strategic Research Applications

1. Malignant Mesothelioma Pathobiology: Serves as a standard biphasic cell model to evaluate tumor heterogeneity, epithelial-to-mesenchymal transition (EMT) programs, and molecular mechanisms behind Hippo pathway dysregulations.

2. Diagnostic Methods & Biomarker Engineering: Extensively utilized for the identification, verification, and engineering of novel diagnostic methods, panels of antibodies, or histopathological tissue tracking assays (e.g., EMA-based targets).

3. Chemotherapeutic Resistance & High-Throughput Screening: Given the poor clinical response rate of malignant pleural mesothelioma to standard therapies, this line is used for screening novel targeted compounds, glutathione S-transferase (GST) inhibitors, and performing functional genomic screens to uncover drug resistance liabilities.

BioVector NTCC质粒载体菌株细胞蛋白抗体基因保藏中心

电话:400-800-2947

工作QQ/微信同号:1843439339

网址

http://www.biovector.net