BioVector® Hep3B 2.1-7-GFP Polyclonal Stable Cell Line / Hep3B 2.1-7-GFP 稳转细胞系

一 产品基本信息与遗传学背景

• 细胞名称：Hep3B 2.1-7-GFP 绿色荧光标记稳转细胞系。

• 物种来源：人源（Human），来源于一名 8 岁黑人男性的肝细胞癌（Hepatocellular Carcinoma, HCC）组织切块。

• 母本细胞背景（Hep3B 2.1-7）：

  • 病毒整合状态：Hep3B 是国际公认的乙型肝炎病毒（HBV）基因组整合阳性的细胞模型。其染色体中含有整合的 HBV DNA 片段，能够持续分泌乙型肝炎表面抗原（HBsAg），但不产生完整的传染性病毒颗粒。同时，该细胞系呈 p53 基因缺陷/缺失状态（p53-deficient），且不含丙型肝炎病毒（HCV）。

  • 标志物表达：保留了分化良好的实质肝细胞功能，能够高效分泌人血清白蛋白（Albumin）、alpha-胎儿蛋白（AFP）、载脂蛋白（Apolipoprotein A-II）以及补体 C3 等关键肝源性蛋白。此外，该细胞对缺氧环境极其敏感，是研究低氧诱导因子（HIF）及红细胞生成素（EPO）表达的经典靶细胞。

• GFP 稳转特性：利用带有强启动子（如 CMV 或 EF1a）的慢病毒（Lentivirus）表达载体转导母本 Hep3B 2.1-7 细胞，经过抗生素（如 Puromycin）长期定向筛选获得。GFP（绿色荧光蛋白）在细胞质内呈结构性、高水平稳定表达。

• 生长特性：贴壁生长（Adherent），主要呈现上皮细胞样（Epithelial-like）、多角形排列或铺路石状集落生长形态。

• 生物安全级别：2级（BSL-2）。由于该细胞含有整合的 HBV 基因组，活细胞操作必须在二级生物安全柜内进行。

二 核心科研价值与转化医学应用

Hep3B 2.1-7-GFP 细胞将原位肝癌的生物学特征与可视化示踪技术完美结合，具备极高的实验便利性：

1. 肿瘤活体体内示踪与多维成像（In Vivo Track & Bioimaging）：

   在构建小鼠同种/异种皮下移植瘤、原位肝癌（Orthotopic Liver Cancer）模型时，无需借助繁琐的活体染色。通过小鼠体内小动物荧光成像系统（IVIS），可实时、无创、动态地监测肝癌细胞在体内的定殖增殖速度、三维空间肿瘤生长体积以及晚期肿瘤向肺、腹膜等远端器官的微小转移灶。

2. 三维细胞培养与肿瘤生物膜动态观察（3D Culture & Biofilm Microenvironment）：

   在体外开展微流控芯片培养、多细胞球体（Spheroids）器官样三维培养时，借助共聚焦显微镜（CLSM）可通过单色绿色通道清晰分辨肝癌细胞的形态边界、极性组装以及在空间基质中的浸润动力学。

3. 抗肿瘤药物高通量筛选（HTS & Cytotoxicity assays）：

   通过微孔板荧光读数仪（Fluorescence Plate Reader），可直接通过检测全孔绿色荧光总强度（RFU）来定量推算活细胞相对密度，极大简化了传统 MTT/CCK-8 等破坏性显色步骤，适合用于抗肝癌小分子药物、靶向抗体、CAR-T 细胞毒杀动力学的实时在线筛选。

三 实验室细胞复苏、扩增传代与冷冻保存标准步骤

1. 完全培养基配置

Hep3B 2.1-7-GFP 细胞对营养基质要求较常规，但添加足量血清对维持其强健的贴壁形态至关重要：

• 基础培养基：MEM 培养基（推荐） 或 高糖 DMEM（含 NEAA 非必需氨基酸）。

• 完全添加剂成分：

  • 10% 优质胎牛血清（FBS）。

  • 1% 灭菌双抗（Penicillin-Streptomycin）。

  • 1% 丙酮酸钠（Sodium Pyruvate，可选，促进细胞能量代谢）。

  • 维持抗生素（可选）：为了在长期传代中杜绝 GFP 的自发丢失，可在常规扩增维持液中添加最终工作浓度为 0.5 - 1.5 ug/mL 的标准嘌呤霉素（Puromycin）；但在开展常规体外药敏毒性测试及动物接种前，建议提前 1-2 代撤除维持抗生素。

2. 细胞复苏步骤

1. 将完全培养基放入 37 摄氏度水浴中预热。

2. 从液氮罐中取出 Hep3B 2.1-7-GFP 冻存管，立即投入 37 摄氏度恒温水浴箱中，轻微晃动。

3. 在 1 分钟内令其急速融化（至仅剩极小冰芯时捞出）。迅速用 75% 酒精擦拭外部。

4. 在生物安全柜内，用移液管将细胞悬液吸出，置于含有 5 mL 预热完全培养基的 15 mL 离心管中，轻柔颠倒混匀。

5. 以 200 - 300 x g 离心 3 分钟，小心吸除含有 DMSO 的上清液。

6. 加入 5 mL 新鲜完全培养基，用移液枪轻柔吹打重悬细胞。接种于 T25 培养瓶中，置于 37 摄氏度、5% CO2 孵箱中培养。次日通过倒置荧光显微镜观察细胞贴壁状态及绿色荧光表达效率。

3. 细胞传代与消化

• 传代时机：该细胞生长具有明显的岛状/集落密集特征。当细胞总融合度达到约 80% - 85% 时必须执行传代。切勿让细胞完全长满或重叠生长，否则接触抑制会导致细胞分化或荧光表达减弱。

• 传代步骤：

  1. 吸除旧培养基，用无菌 PBS 轻轻洗涤细胞表面 1-2 次，以彻底清除残留的血清（血清会严重抑制胰酶活性）。

  2. 加入适量 0.25% Trypsin-EDTA 消化液（T25 瓶常规加 1 mL），确保覆盖细胞。

  3. 置于 37 摄氏度 孵箱中消化 2 至 4 分钟。在显微镜下连续观察，由于该细胞贴壁较牢固，当观察到大部分上皮样细胞收缩变圆、细胞间隙增大时，可轻敲瓶壁配合，待细胞脱落后立即加入 2 倍体积的含血清完全培养基终止消化。

  4. 用移液枪轻柔吹打瓶壁，使细胞完全分散为单细胞悬液。收集至离心管中，300 x g 离心 3 分钟，弃上清。

  5. 按照 1:3 至 1:5 的传代比例接种到新的培养瓶中。通常 3-4 天即可再次长满。

4. 细胞冷冻保存

• 冻存液配方：90% 完全培养基（或纯 FBS） + 10% 优质 DMSO。

• 冻存操作：收集处于对数生长活跃期且荧光强度饱满的健康细胞，离心弃上清。调整细胞密度至 $1 \times 10^6$ 至 $3 \times 10^6$ cells/vial。加入冻存液重悬分装后，立即投入标准程序降温盒（梯度降温盒，1 摄氏度/min），置于 -80 摄氏度过夜，次日必须转移至 -196 摄氏度液氮中长期冷冻保存。

Part 2 English Section

I General Information and Genetic Architecture

• Cell Line Name: Hep3B 2.1-7-GFP Polyclonal Green Fluorescent Tagged Stable Cell Line.

• Species Origin: Human. Originally isolated from the liver biopsy fragments of an 8-year-old Black male diagnosed with Hepatocellular Carcinoma (HCC).

• Parental Cell Architecture (Hep3B 2.1-7):

  • Viral Integration Architecture: Hep3B represents a premier globally accepted reference paradigm for Hepatitis B Virus (HBV) genome-integrated malignant lineages. Its chromosome anchors integrated HBV DNA segments, driving continuous expression and secretion of Hepatitis B surface antigen (HBsAg) without facilitating the assembly of intact, infectious viral particles. Additionally, this line is characterized by a p53-deficient status (deleted/non-functional p53 gene clusters) and tests strictly negative for Hepatitis C Virus (HCV).

  • Phenotypic Differentiation Markers: It preserves prominent native parenchymal enterocyte functional pathways, demonstrating robust output of human serum Albumin, alpha-fetoprotein (AFP), Apolipoprotein A-II, and Complement component C3. Moreover, its intense cellular reaction to oxygen depletion patterns makes it a gold standard platform to monitor hypoxia-inducible factor (HIF) networks and erythropoietin (EPO) induction kinetics.

• GFP Transgenic Integration Profiles: Established via continuous transduction of the parental Hep3B 2.1-7 line using high-titer lentiviral vectors harboring a constitutive promoter matrix (e.g., CMV or EF1a). Stable integrated expression yields structural, permanent, high-intensity green fluorescent protein (GFP) accumulation throughout the target cytoplasm, unlocked via directed antibiotic selection channels (e.g., Puromycin selection pressures).

• Growth Topology: Adherent growth matrix. Exhibits classical differentiated epithelial-like, polygonal, clustering layouts that pack together into paving-stone colony patterns.

• Biosafety Matrix: Classified under Biosafety Level 2 (BSL-2) containment envelopes. Due to its historical profile bearing integrated active HBV DNA segments, all continuous handling loops must be strictly deployed inside certified Class II Biosafety Cabinets.

II Strategic Research Value and Translational Fields

The Hep3B 2.1-7-GFP line elegantly couples native clinical liver carcinoma traits with visual tracking utilities, yielding streamlined operational profiles for complex workflows:

1. In Vivo Oncogenic Longitudinal Tracking & Deep Bioimaging:

   During the modeling of subcutaneous mouse xenografts or orthotopic liver carcinoma tracking cascades, this line eliminates the need for messy ex-vivo tissue dye processing. Utilizing specialized in vivo imaging architectures (IVIS channels), investigators can dynamically capture tumor engraftment timelines, chart 3D volumetric growth velocities, and map micro-metastatic homing into distal pulmonary or peritoneal niches in real-time.

2. 3D Biomimetic Culturing & Microfluidic Microenvironment Inspection:

   When integrated into organ-on-a-chip architectures, microfluidic platforms, or multi-cellular spheroid assemblies, confocal laser scanning microscopy (CLSM) easily resolves cellular boundary kinetics, target cell polarity layout shifts, and matrix invasion dynamics through clean single-channel green fluorescence sorting.

3. High-Throughput Cytotoxicity & Drug Discovery Platforms (HTS Matrices):

   Utilizing basic fluorescence plate reading setups, automated pipelines measure total green relative fluorescence units (RFU) per well to deduce relative viable cell density scores. This bypasses tedious destructive metabolic endpoints (such as routine MTT/CCK-8 processing), permitting automated, real-time tracking of targeted anti-cancer drug chemical screening, monoclonal antibody binding kinetics, and CAR-T cell-mediated cytolytic performance.

III Thawing, Proliferation, Passaging, and Cryopreservation Routines

1. Formulating the Complete Growth Medium

While Hep3B 2.1-7-GFP is biologically resilient, adequate serum conditioning is recommended to preserve sharp epithelial matrix anchorage structures:

• Basal Medium: Minimum Essential Medium (MEM - Highly Recommended) or high-glucose DMEM fortified with Non-Essential Amino Acids (NEAA).

• Complete Media Supplements:

  • 10% Premium Fetal Bovine Serum (FBS).

  • 1% Penicillin-Streptomycin cocktail.

  • 1% Sodium Pyruvate (Optional metabolic booster).

  • Maintenance Selective Pressure (Optional): To guarantee absolute mitigation against spontaneous transgene drift or silencing during long-term passaging across months, supplement standard proliferation maintenance media with 0.5 - 1.5 ug/mL analytic-grade Puromycin. Ensure selection drugs are entirely omitted 1 to 2 passages prior to initiating standard in vitro cytotoxicity screening assays or in vivo animal inoculation loops.

2. Cryovial Thawing Routine

1. Pre-warm the formulated complete growth medium within a 37 degree Celsius water bath.

2. Extract the Hep3B 2.1-7-GFP cryovial from liquid nitrogen storage and instantly plunge it into the 37 degree Celsius water bath with continuous gentle agitation.

3. Achieve complete liquefaction rapidly within 1 minute (extract when a minute ice core remains). Swab the exterior thoroughly with 70% ethanol.

4. Inside a biosafety enclosure, pipette the cell slurry directly into a 15 mL conical tube filled with 5 mL of pre-warmed complete growth medium. Mix by gentle inversion to cushion osmotic pressure variations.

5. Centrifuge the suspension at 200 - 300 x g for 3 minutes, then cleanly aspirate the DMSO-laden supernatant.

6. Replenish with 5 mL of fresh complete medium, gently resuspend the pellet, transfer into the target culture flask, and incubate at 37 degree Celsius under a humidified 5% CO2 atmosphere. Confirm target cell morphology and green fluorescence output indices using an inverted fluorescent microscope the following day.

3. Subculturing and Cell Passaging Guide

• Confluency Threshold: This line displays distinct focal island-like cell cluster growth dynamics. Subculturing must be performed precisely when the monolayer hits 80%–85% total confluency. Do not allow cells to reach absolute 100% saturation or form multi-layered stacks, as contact inhibition will degrade the baseline phenotype and damp transgene expression.

• Step-by-Step Harvesting:

  1. Aspirate spent medium and rinse the sheet 1–2 times with sterile, calcium/magnesium-free PBS to remove residual serum (residual serum proteins rapidly inhibit trypsin performance).

  2. Add an appropriate volume of 0.25% Trypsin-EDTA Solution (approx. 1 mL for standard T25 flasks) to fully submerge the cell layer.

  3. Incubate at 37 degree Celsius for 2 to 4 minutes. Monitor closely under an inverted microscope; since this line adheres firmly to plastic substrates, gentle mechanical tapping against the vessel wall can assist detachment. As soon as the epithelial-like cells round up and release, instantly introduce a double volume of complete serum-supplemented medium to quench enzymatic degradation.

  4. Gently pipette the vessel walls to yield a single-cell suspension. Spin down at 300 x g for 3 minutes and discard the supernatant.

  5. Re-plate into fresh culture vessels utilizing a standard split ratio ranging from 1:3 to 1:5. Monolayers typically achieve optimal density within 72–96 hours.

4. Cryopreservation Protocol

• Freezing Medium Matrix: 90% Complete Growth Medium (or pure premium FBS) supplemented with 10% analytical-grade DMSO.

• Freezing Routine: Harvest healthy, log-phase cells showing optimal viability profiles and crisp fluorescent outputs. Centrifuge, discard the supernatant fluid, and re-suspend the cell mass to log a final target density of $1 \times 10^6$ to $3 \times 10^6$ viable cells/vial. Aliquot into sterile cryovials and transfer immediately to a standardized isopropyl alcohol controlled-rate freezing box. Store at -80 degree Celsius overnight, and shift the vials into liquid nitrogen (-196 degree Celsius) the following day for indefinite preservation.

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