BioVector® Hep-56.1D Murine Hepatocellular Carcinoma Cell Line / Hep-56.1D 小鼠肝细胞癌细胞株

一 产品基本信息与细胞生物学背景

• 细胞名称：Hep-56.1D（亦常写作 Hep56.1D 或 Hep 56.1D）。

• 物种与品系来源：小鼠（Mus musculus），源自经典的C57BL/6（黑六）近交系小鼠。

• 组织源起与病理背景：

  Hep-56.1D 是一株源自 C57BL/6 小鼠原发性肝细胞癌（Hepatocellular Carcinoma, HCC）组织块的恶性肿瘤细胞系。在生物医学研究中，它是模拟人类肝癌发生、发展以及探索宿主免疫系统与肿瘤微环境相互作用最常用的同系（Syngeneic）小鼠肿瘤模型底盘之一。

• 核心表型与细胞生物学特征：

  • 形态学表现：贴壁生长。在倒置显微镜下，Hep-56.1D 细胞表现为极其典型的上皮样（Epithelial-like）形态。细胞多呈多角形，胞质致密，中央常含有明显的单核或双核。在细胞生长汇合、密集成片时，它们会紧密排列成类似铺路石（Cobblestone-like）的单层图案。

  • 同系移植（Syngeneic Grafting）优势：由于该细胞系完全来源于 C57BL/6 小鼠背景，当将其重新接种（皮下注射或原位肝脏接种）回免疫健全的 C57BL/6 模式小鼠体内时，宿主不会发生针对外源组织的免疫排斥反应。这一特征使其在肿瘤免疫治疗研究中的应用价值远超常规的免疫缺陷人源异种移植（CDX）模型。

• 生物安全级别：1级（BSL-1）。

二 核心科研价值与肿瘤免疫医学转化应用

Hep-56.1D 细胞株在现代癌症生物学、免疫检查点阻断及药物开发中扮演着核心角色：

1. 构建免疫健全的小鼠原位/皮下肝癌模型：

   通过将 Hep-56.1D 细胞直接注射入 C57BL/6 小鼠的皮下或直接接种于肝左叶（原位移植，Orthotopic transplantation），可诱导出高成瘤率的肝癌病灶。由于宿主小鼠拥有完全健康的 T 细胞、B 细胞、NK 细胞以及巨噬细胞系统，该模型能够完美重现实体瘤内部复杂的免疫微环境（Tumor Microenvironment, TME）。

2. 抗肿瘤免疫检查点药物（如 Anti-PD-1/PD-L1, Anti-CTLA-4）体外与体内药效评估：

   用于测试各类新型单克隆抗体、溶瘤病毒、双特异性抗体或小分子免疫调节剂在健全免疫背景下的抗癌靶向效能。它是评估人类免疫疗法走向临床前的关键临床前试验（Pre-clinical trials）台表。

3. 肝癌转移机制与血管生成（Angiogenesis）探索：

   用于解构肝癌细胞如何通过分泌血管内皮生长因子（VEGF）诱导周围微血管形成，以及如何逃逸宿主免疫监视并向肺部、淋巴结发生远处转移（Metastasis）的分子病理机制。

三 实验室细胞复苏、贴壁培养、常规传代与保存标准步骤

1. 专用培养基配置与生长环境

• 基础培养基：高糖 DMEM（含 4.5 g/L 葡萄糖、L-谷氨酰胺及丙酮酸钠）。

• 完全培养基配方：

  • 高糖 DMEM 基础培养基

  • 加 10% 优质胎牛血清（FBS）

  • 加 1% 青霉素-链霉素双抗溶液。

• 细胞解离液：0.25% Trypsin - 0.02% EDTA 消化液。

• 培养物理常数：标准 37 摄氏度，含 5% 二氧化碳（$CO_2$） 的恒温高湿度饱和无菌孵箱。

2. 冷冻细胞复苏步骤

1. 提前在生物安全柜中准备好干净的 T25 培养瓶，注入 5 - 6 mL 预热至 37 ℃ 的完全培养基。

2. 从液氮罐或 -80 ℃ 冰箱中取出 Hep-56.1D 冻存管，立刻全量投入 37 ℃ 恒温水浴箱中，快速用力摇晃以期在 1 分钟内令管内冰块完全融化（切忌慢速融化，否则冰晶重结晶会严重刺破细胞膜）。

3. 喷洒 75% 酒精消毒外壁，移入生物安全柜。

4. 吸取细胞悬液，缓慢滴加至盛有 4 - 5 mL 预热完全培养基的 15 mL 离心管中，轻柔颠倒一次以稀释 DMSO。

5. 以 1000 rpm（约 200 g）室温温和离心 5 分钟，小心抽干含有 DMSO 的上清液。

6. 加入 1 mL 新鲜完全培养基，使用 P1000 移液枪轻柔吹打重悬细胞沉淀。

7. 将悬液接种至准备好的 T25 瓶中，轻柔“十字形”摇匀。

8. 拧松瓶盖（或使用透气膜盖），放入 37 ℃、5% $CO_2$ 孵箱中培养。

9. 24 小时后观察贴壁汇合度，全量更换一次新鲜培养基，洗去残留的死细胞碎屑。

3. 日常贴壁常规传代操作

• 传代时机：当上皮样铺路石状的细胞密集成片，汇合度（Confluency）达到 80% - 90% 时必须传代。Hep-56.1D 属于恶性肿瘤细胞，生长速度较快，切忌使其过密（100% 接触抑制），否则会导致细胞局部堆叠、状态老化。传代频率通常为每 2 - 3 天一次。

• 操作流程：

  1. 吸除旧培养基，使用无菌、无钙镁离子的 PBS 缓冲液轻轻漂洗细胞表面 1 次，彻底洗净残余血清（血清中的蛋白质会强力抑制胰酶活性）。

  2. 加入适量 0.25% Trypsin-EDTA 消化液（T25 瓶常规加入 1 mL），使其均匀覆盖细胞层，随后放入 37 ℃ 孵箱中温和消化。

  3. 镜下动态观察：通常在 37 ℃ 下消化 1 - 3 分钟。当在倒置显微镜下观察到多角形细胞连接变松、胞体收缩变圆、且轻敲培养瓶一侧时有成片细胞自发向下滑落，说明消化达标。

  4. 立刻向瓶内倒入 2 倍体积的含血清完全培养基，利用血清终止胰酶的剪切作用。

  5. 用移液枪反复轻柔冲洗瓶壁，将细胞完全洗脱下来，打散调理成均匀的单细胞悬液。

  6. 将悬液收集至离心管中，1000 rpm 离心 5 分钟，弃去酶解上清。

  7. 加入新鲜完全培养基重悬沉淀，按照 1:3 至 1:6 的常规稀释比例接种至新的培养瓶中，补充完全培养基，放回孵箱中继续扩增。

4. 动物实验前成瘤细胞准备（体内接种质控核心）

如果要将 Hep-56.1D 用于 C57BL/6 小鼠体内（In vivo）接种构建肝癌模型，必须严格遵循以下质控规范以保证高成瘤率与实验可重复性：

1. 细胞状态限制：必须选择处于对数生长期（Log phase）最旺盛阶段、汇合度约 75% - 80% 且未发生任何变异退化的健康细胞。细胞传代代数（Passage number）建议控制在复苏后的 10 代以内，过高代数的癌症细胞其体内成瘤率和对免疫药物的敏感性会发生自发飘移。

2. 纯化与解离：消化时要确保细胞被充分打散成高纯度的单细胞，严禁带有多细胞聚集团块。

3. 洗涤与悬浮缓冲液：利用完全培养基终止消化离心后，必须用无菌基底 DMEM 培养基或无菌无重金属生理盐水（PBS）连续离心洗涤细胞 2 - 3 次，以彻底清除任何外源牛血清蛋白（FBS 原料残留在小鼠体内会引发强烈的非特异性异种异源免疫反应，干扰真实的肿瘤免疫药物评价）。

4. 接种密度与冷链控制：

   • 最终接种时，用无血清 DMEM 或 PBS（必要时可混入 1:1 体积的 Matrigel 基底膜基质以促进皮下成瘤）重悬细胞，调整密度至每 100 $\mu$L 含有 $1 \times 10^6$ 到 $5 \times 10^6$ 个细胞。

   • 全流程必须置于冰水混合物（0 - 4 ℃）中冷链保存，并在离心解离后的 1 小时内完成全部小鼠的皮下或原位注射，以防细胞因在常温下失巢搁置过久发生缺氧失活或沉降聚集。

5. 细胞长期保存标准

• 冻存液配方：90% 新鲜完全培养基（或 80% 基础 DMEM + 10% FBS）加 10% 优质分析级二甲基亚砜（DMSO）。亦可使用高效的商用无血清细胞冻存液。

• 冷冻降温规范：

  1. 收集形态标杆健康的对数生长期 Hep-56.1D 细胞，离心弃上清。

  2. 用配制好的冷冻液悬浮，调整细胞终密度至 每毫升 1,000,000 到 3,000,000 个细胞。

  3. 分装入无菌冻存管，立刻移入标准程序降温盒（如 Mr. Frosty）。

  4. 将降温盒投入 -80 ℃ 超低温冰箱中过夜梯度降温（确保达到 $-1\text{ }^\circ\text{C/min}$ 的标称降温速率）。

  5. 次日，迅速将冻存管转移入液氮罐（-196 ℃）中锁死长期保存。严禁在 -80 ℃ 冰箱中无限期存放，以防微小的温度震荡导致细胞内部冰晶融化破坏质膜结构，引发后续复苏时存活率的雪崩式下跌。

Part 2 English Section

I General Information and Cell Biological Background

• Cell Line Name: Hep-56.1D (also alternative standard nomenclature variations include Hep56.1D or Hep 56.1D).

• Organism and Inbred Strain Origin: Mouse (Mus musculus); explicitly derived from the foundational C57BL/6 (Black 6) inbred mammalian background.

• Tissue Extract and Pathological Framework:

  Hep-56.1D represents a solid malignant lineage isolated from a primary Hepatocellular Carcinoma (HCC) lesion induced in a C57BL/6 mouse specimen. Within cancer research, it serves as a highly reliable syngeneic murine liver cancer matrix leveraged to evaluate oncogenesis, tumor progression, and host immuno-oncology networks.

• Core Morphological Phenotype and Identity Characterization:

  • Morphological Structure: Adherent growth matrix. Under phase-contrast inverted profiling, Hep-56.1D exhibits a classical epithelial-like topology. Individual cellular frames present as distinctly polygonal units featuring compact cytoplasm and centralized single or prominent prominent binucleated nuclei. Upon reaching full confluency across the surface plane, they organize into a dense, interlocking "cobblestone-like" configuration.

  • Syngeneic Engraftment Execution Matrix: Because this cancerous cell line is derived from an un-mutated C57BL/6 background, delivering these cells back into immunocompetent C57BL/6 recipient mice avoids any foreign histocompatibility antigen rejection. This critical property enables investigators to study solid tumor dynamics in the presence of an intact host immune system, a feature unavailable in traditional human xenograft (CDX) models hosted in immunodeficient mice.

• Biosafety Threshold: Rated at Biosafety Level 1 (BSL-1).

II Strategic Research Value and Immuno-Oncology Applications

The Hep-56.1D platform functions as a critical preclinical engine for exploring tumor-host interplay and accelerating drug screening cascades:

1. Assembling Immunocompetent Syngeneic Orthotopic or Subcutaneous HCC Models:

   By injecting Hep-56.1D cells into either the subcutaneous flank or directly within the left hepatic lobe (orthotopic transplantation) of healthy C57BL/6 hosts, investigators can rapidly induce solid tumor pathology. Because the host animal retains fully operational T-cell, B-cell, NK-cell, and specialized myeloid macrophage systems, the resulting lesions accurately recreate the complex Tumor Microenvironment (TME) observed in clinical scenarios.

2. Preclinical Validation of Immune Checkpoint Inhibitors (e.g., Anti-PD-1/PD-L1, Anti-CTLA-4 Drugs):

   The line is widely standardly implemented to screen the therapeutic efficacy of novel checkpoint monoclonal antibodies, oncolytic viral therapies, bispecific antibody platforms, or small-molecule immunomodulators. It serves as a necessary preclinical proving ground before bringing novel immunology drug configurations into human clinical testing.

3. Deconstructing Metastasis Cascades and Angiogenesis Profiles:

   Leveraged to study how liver cancer targets exploit endogenous vascular networks by secreting vascular endothelial growth factors (VEGF), signaling neo-angiogenesis, bypassing host immune detection loops, and establishing distant secondary metastatic lesions inside pulmonary or lymph systems.

III Laboratory Thawing, Cultivation, Maintenance, and In Vivo Inoculation Protocols

1. Basal Media Formulation and Atmospheric Environment Variables

• Basal Medium Base: High-Glucose DMEM matrix (outfitted with 4.5 g/L D-Glucose, L-Glutamine, and Sodium Pyruvate).

• Complete Growth Matrix Formulation:

  • High-Glucose DMEM basal matrix

  • Supplemented with 10% premium Fetal Bovine Serum (FBS)

  • Fortified with 1% standard Penicillin-Streptomycin dual antibiotic cocktail.

• Cell Dissociation Solution: Standard 0.25% Trypsin - 0.02% EDTA mix.

• Physical Environmental Settings: Calibrate the incubator strictly to 37 °C, packed with 5% Carbon Dioxide ($CO_2$) under continuous humified saturation conditions.

3. Cryovial Thawing and Monolayer Recovery Protocol

1. Pre-warm a sterile T25 culture flask filled with 5 - 6 mL of complete growth medium to 37 °C inside the biosafety workstation.

2. Retrieve the Hep-56.1D cryovial from storage and submerge it instantly inside a 37 °C water bath. Agitate the vial continuously to melt the internal matrix within 60 seconds. Never allow slow warming; rapid melting prevents ice recrystallization from puncturing cellular membranes.

3. Mist the exterior shell with 75% ethanol before transfer into the biosafety clean room.

4. Draw up the liquid and transfer it slowly into a 15 mL conical tube containing 4 - 5 mL of pre-warmed complete growth medium to dilute the toxic DMSO footprint.

5. Centrifuge the suspension at 1000 rpm (~200 g) at room temperature for 5 minutes, then aspirate the chemical-laden supernatant.

6. Administer 1 mL of fresh complete growth medium onto the pellet and resuspend very gently using a P1000 micro-pipette.

7. Dispense the cells evenly into the prepared T25 flask, mix gently in a cross pattern, and place into the 37 °C, 5% $CO_2$ incubator.

8. Perform a complete medium replacement 24 hours post-thaw to clear residual dead cell debris.

3. Routine Adherent Subculturing and Passaging Routines

• Confluency Assessment Control: Subculturing mechanics must be executed when the interlocking epithelial sheets reach 80% - 90% confluency. Hep-56.1D cells are aggressive cancer models; allowing the monolayer to achieve 100% saturation triggers crowding and aging phenotypes. Expect a standard passaging schedule every 2 - 3 days.

• Passaging Execution Steps:

  1. Aspirate the spent growth fluid and wash the monolayer once with sterile, calcium/magnesium-free PBS to remove any residual serum proteins that could inactivate the trypsin.

  2. Administer an appropriate thin layer of 0.25% Trypsin-EDTA solution (typically 1 mL for a T25 format) and incubate at 37 °C.

  3. Microscopic Tracking: Maintain continuous visual monitoring under the microscope. Trypsinization typically completes within 1 - 3 minutes at 37 °C. The moment cells lose their polygonal junctions, round up, and slide off the surface upon gentle tapping, the process is complete.

  4. Immediately add 2 volumes of serum-fortified complete growth medium to stop the enzymatic cleavage.

  5. Gently pipette the suspension against the flask wall to break up clusters, resulting in a homogenous single-cell suspension.

  6. Spin the cells down at 1000 rpm for 5 minutes, discard the trypsin-laden fluid, and resuspend in fresh complete growth medium.

  7. Seed the cells into new flasks utilizing standard split ratios of 1:3 to 1:6, top off with complete medium, and return to the incubator.

4. Critical Pre-Inoculation Preparation for Animal Modeling (In Vivo Quality Metrics)

When preparing Hep-56.1D cells for syngeneic transplantation into C57BL/6 mice, strict adherence to these quality parameters is required to ensure consistent tumor take and reliable, reproducible experimental outcomes:

1. Passage Threshold and Longevity Control: Exclusively harvest cells from highly viable, log-phase cultures displaying approximately 75% - 80% confluency. To prevent genetic drift and altered drug responsiveness, ensure cells have been passaged fewer than 10 times post-thaw before mouse injection.

2. Homogenous Dissociation: Ensure complete enzymatic dissociation during passaging to yield a high-purity single-cell suspension. The injection of multi-cellular cell aggregate clumps will cause inconsistent tumor morphology or vascular embolisms during in vivo injection.

3. Serum Clearance Washing Sequence: Following trypsin deactivation, the cells must be washed 2 - 3 times via sequential centrifugation using serum-free basal DMEM or sterile saline (PBS). Residual bovine serum proteins introduced into C57BL/6 recipients will trigger non-specific xenogeneic immune responses, obscuring the evaluation of candidate immunotherapy drugs.

4. Cold-Chain Maintenance and Density Control:

   • Resuspend the final serum-free cell pellet in basal DMEM or PBS (a 1:1 mixture with Matrigel can be utilized for challenging subcutaneous setups) to a final injection concentration of $1 \times 10^6$ to $5 \times 10^6$ cells per 100 $\mu$L.

   • Keep the cell suspension on ice (0 - 4 °C) throughout the procedure. Execute all subcutaneous or orthotopic animal micro-injections within 1 hour post-harvest to prevent cell death from prolonged detachment or cell precipitation within the syringe barrel.

5. Long-Term Cryopreservation Parameters

• Cryoprotectant Preservation Formula: 90% fresh complete growth medium (or 80% basal DMEM + 10% premium FBS) packed with 10% analytical-grade Dimethyl Sulfoxide (DMSO).

• Controlled Gradient Freezing Protocol:

  1. Harvest healthy, log-phase Hep-56.1D monolayers showing robust epithelial integrity. Centrifuge and isolate the pellet.

  2. Resuspend the cells in the chilled cryoprotectant matrix to achieve a target cell density of 1,000,000 to 3,000,000 cells per milliliter.

  3. Transfer the solution into sterile cryovials and place them immediately into a standard controlled-rate cooling container (e.g., Mr. Frosty).

  4. Deposit the cooling container inside a -80 °C ultra-low freezer overnight to execute a steady cooling rate of -1 °C/minute.

  5. Within 24 hours, quickly transfer the vials into liquid nitrogen storage tanks (-196 °C) for long-term preservation. Do not store vials indefinitely inside a -80 °C freezer; minor temperature variations can compromise membrane integrity and lead to poor post-thaw viability.

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