BioVector? Hep3B 2.1-7-GFP Polyclonal Stable Cell Line / Hep3B 2.1-7-GFP 穩(wěn)轉(zhuǎn)細(xì)胞系

一 產(chǎn)品基本信息與遺傳學(xué)背景

• 細(xì)胞名稱：Hep3B 2.1-7-GFP 綠色熒光標(biāo)記穩(wěn)轉(zhuǎn)細(xì)胞系。

• 物種來源：人源（Human），來源于一名 8 歲黑人男性的肝細(xì)胞癌（Hepatocellular Carcinoma, HCC）組織切塊。

• 母本細(xì)胞背景（Hep3B 2.1-7）：

  • 病毒整合狀態(tài)：Hep3B 是國際公認(rèn)的乙型肝炎病毒（HBV）基因組整合陽性的細(xì)胞模型。其染色體中含有整合的 HBV DNA 片段，能夠持續(xù)分泌乙型肝炎表面抗原（HBsAg），但不產(chǎn)生完整的傳染性病毒顆粒。同時，該細(xì)胞系呈 p53 基因缺陷/缺失狀態(tài)（p53-deficient），且不含丙型肝炎病毒（HCV）。

  • 標(biāo)志物表達(dá)：保留了分化良好的實質(zhì)肝細(xì)胞功能，能夠高效分泌人血清白蛋白（Albumin）、alpha-胎兒蛋白（AFP）、載脂蛋白（Apolipoprotein A-II）以及補體 C3 等關(guān)鍵肝源性蛋白。此外，該細(xì)胞對缺氧環(huán)境極其敏感，是研究低氧誘導(dǎo)因子（HIF）及紅細(xì)胞生成素（EPO）表達(dá)的經(jīng)典靶細(xì)胞。

• GFP 穩(wěn)轉(zhuǎn)特性：利用帶有強啟動子（如 CMV 或 EF1a）的慢病毒（Lentivirus）表達(dá)載體轉(zhuǎn)導(dǎo)母本 Hep3B 2.1-7 細(xì)胞，經(jīng)過抗生素（如 Puromycin）長期定向篩選獲得。GFP（綠色熒光蛋白）在細(xì)胞質(zhì)內(nèi)呈結(jié)構(gòu)性、高水平穩(wěn)定表達(dá)。

• 生長特性：貼壁生長（Adherent），主要呈現(xiàn)上皮細(xì)胞樣（Epithelial-like）、多角形排列或鋪路石狀集落生長形態(tài)。

• 生物安全級別：2級（BSL-2）。由于該細(xì)胞含有整合的 HBV 基因組，活細(xì)胞操作必須在二級生物安全柜內(nèi)進(jìn)行。

二 核心科研價值與轉(zhuǎn)化醫(yī)學(xué)應(yīng)用

Hep3B 2.1-7-GFP 細(xì)胞將原位肝癌的生物學(xué)特征與可視化示蹤技術(shù)完美結(jié)合，具備極高的實驗便利性：

1. 腫瘤活體體內(nèi)示蹤與多維成像（In Vivo Track & Bioimaging）：

   在構(gòu)建小鼠同種/異種皮下移植瘤、原位肝癌（Orthotopic Liver Cancer）模型時，無需借助繁瑣的活體染色。通過小鼠體內(nèi)小動物熒光成像系統(tǒng)（IVIS），可實時、無創(chuàng)、動態(tài)地監(jiān)測肝癌細(xì)胞在體內(nèi)的定殖增殖速度、三維空間腫瘤生長體積以及晚期腫瘤向肺、腹膜等遠(yuǎn)端器官的微小轉(zhuǎn)移灶。

2. 三維細(xì)胞培養(yǎng)與腫瘤生物膜動態(tài)觀察（3D Culture & Biofilm Microenvironment）：

   在體外開展微流控芯片培養(yǎng)、多細(xì)胞球體（Spheroids）器官樣三維培養(yǎng)時，借助共聚焦顯微鏡（CLSM）可通過單色綠色通道清晰分辨肝癌細(xì)胞的形態(tài)邊界、極性組裝以及在空間基質(zhì)中的浸潤動力學(xué)。

3. 抗腫瘤藥物高通量篩選（HTS & Cytotoxicity assays）：

   通過微孔板熒光讀數(shù)儀（Fluorescence Plate Reader），可直接通過檢測全孔綠色熒光總強度（RFU）來定量推算活細(xì)胞相對密度，極大簡化了傳統(tǒng) MTT/CCK-8 等破壞性顯色步驟，適合用于抗肝癌小分子藥物、靶向抗體、CAR-T 細(xì)胞毒殺動力學(xué)的實時在線篩選。

三 實驗室細(xì)胞復(fù)蘇、擴增傳代與冷凍保存標(biāo)準(zhǔn)步驟

1. 完全培養(yǎng)基配置

Hep3B 2.1-7-GFP 細(xì)胞對營養(yǎng)基質(zhì)要求較常規(guī)，但添加足量血清對維持其強健的貼壁形態(tài)至關(guān)重要：

• 基礎(chǔ)培養(yǎng)基：MEM 培養(yǎng)基（推薦） 或 高糖 DMEM（含 NEAA 非必需氨基酸）。

• 完全添加劑成分：

  • 10% 優(yōu)質(zhì)胎牛血清（FBS）。

  • 1% 滅菌雙抗（Penicillin-Streptomycin）。

  • 1% 丙酮酸鈉（Sodium Pyruvate，可選，促進(jìn)細(xì)胞能量代謝）。

  • 維持抗生素（可選）：為了在長期傳代中杜絕 GFP 的自發(fā)丟失，可在常規(guī)擴增維持液中添加最終工作濃度為 0.5 - 1.5 ug/mL 的標(biāo)準(zhǔn)嘌呤霉素（Puromycin）；但在開展常規(guī)體外藥敏毒性測試及動物接種前，建議提前 1-2 代撤除維持抗生素。

2. 細(xì)胞復(fù)蘇步驟

1. 將完全培養(yǎng)基放入 37 攝氏度水浴中預(yù)熱。

2. 從液氮罐中取出 Hep3B 2.1-7-GFP 凍存管，立即投入 37 攝氏度恒溫水浴箱中，輕微晃動。

3. 在 1 分鐘內(nèi)令其急速融化（至僅剩極小冰芯時撈出）。迅速用 75% 酒精擦拭外部。

4. 在生物安全柜內(nèi)，用移液管將細(xì)胞懸液吸出，置于含有 5 mL 預(yù)熱完全培養(yǎng)基的 15 mL 離心管中，輕柔顛倒混勻。

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

6. 加入 5 mL 新鮮完全培養(yǎng)基，用移液槍輕柔吹打重懸細(xì)胞。接種于 T25 培養(yǎng)瓶中，置于 37 攝氏度、5% CO2 孵箱中培養(yǎng)。次日通過倒置熒光顯微鏡觀察細(xì)胞貼壁狀態(tài)及綠色熒光表達(dá)效率。

3. 細(xì)胞傳代與消化

• 傳代時機：該細(xì)胞生長具有明顯的島狀/集落密集特征。當(dāng)細(xì)胞總?cè)诤隙冗_(dá)到約 80% - 85% 時必須執(zhí)行傳代。切勿讓細(xì)胞完全長滿或重疊生長，否則接觸抑制會導(dǎo)致細(xì)胞分化或熒光表達(dá)減弱。

• 傳代步驟：

  1. 吸除舊培養(yǎng)基，用無菌 PBS 輕輕洗滌細(xì)胞表面 1-2 次，以徹底清除殘留的血清（血清會嚴(yán)重抑制胰酶活性）。

  2. 加入適量 0.25% Trypsin-EDTA 消化液（T25 瓶常規(guī)加 1 mL），確保覆蓋細(xì)胞。

  3. 置于 37 攝氏度 孵箱中消化 2 至 4 分鐘。在顯微鏡下連續(xù)觀察，由于該細(xì)胞貼壁較牢固，當(dāng)觀察到大部分上皮樣細(xì)胞收縮變圓、細(xì)胞間隙增大時，可輕敲瓶壁配合，待細(xì)胞脫落后立即加入 2 倍體積的含血清完全培養(yǎng)基終止消化。

  4. 用移液槍輕柔吹打瓶壁，使細(xì)胞完全分散為單細(xì)胞懸液。收集至離心管中，300 x g 離心 3 分鐘，棄上清。

  5. 按照 1:3 至 1:5 的傳代比例接種到新的培養(yǎng)瓶中。通常 3-4 天即可再次長滿。

4. 細(xì)胞冷凍保存

• 凍存液配方：90% 完全培養(yǎng)基（或純 FBS） + 10% 優(yōu)質(zhì) DMSO。

• 凍存操作：收集處于對數(shù)生長活躍期且熒光強度飽滿的健康細(xì)胞，離心棄上清。調(diào)整細(xì)胞密度至 $1 \times 10^6$ 至 $3 \times 10^6$ cells/vial。加入凍存液重懸分裝后，立即投入標(biāo)準(zhǔn)程序降溫盒（梯度降溫盒，1 攝氏度/min），置于 -80 攝氏度過夜，次日必須轉(zhuǎn)移至 -196 攝氏度液氮中長期冷凍保存。

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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