BioVector® ImKC 细胞株（小鼠肝脏库普弗细胞） / BioVector® ImKC Cell Line (Immortalized Mouse Kupffer Cells)

一、 背景与来源说明 / Background & Origin

BioVector® ImKC（全称 Immortalized Mouse Kupffer Cells，小鼠永生化库普弗细胞）是一种通过基因工程（通常采用 SV40 Large T 基因或 hTERT 表达载体）转染永生化的小鼠肝脏库普弗细胞株（Mouse Kupffer Cells）。

库普弗细胞（Kupffer Cells）是定居于肝脏血窦（Sinusoid）内的固有巨噬细胞（Tissue-resident Macrophages），构成体内的肝脏免疫屏障。原代小鼠库普弗细胞分离纯化难度高、得率低且体外存活时间极短。BioVector® ImKC 克服了原代细胞难以长期培养与传代的限制，同时保留了典型库普弗细胞的巨噬与免疫功能，是研究肝脏先天免疫、脂质代谢、非酒精性脂肪性肝病（NAFLD/MASH）、肝纤维化及毒理学机制的理想体外细胞模型。

二、 细胞生物学特征 / Biological Characteristics

• 物种与来源 / Taxonomy & Source：小鼠（Mus musculus），肝脏血窦（Liver Sinusoid）

• 生物安全等级 / Biosafety Level：BSL-1

• 生长特性 / Growth Properties：贴壁生长（Adherent），呈巨噬细胞样（Macrophage-like）多角形或不规则延伸形态

• 关键标志物 / Key Immunological Markers：

  • 巨噬/库普弗细胞特异标记：F4/80(+)、CD11b(+)、CD68(+)、CD14(+)、Clec4f(+)

  • 吞噬能力：具备强颗粒吞噬（Phagocytosis）及内吞作用

• 功能特性 / Functional Attributes：

  • 免疫应答：对 LPS（脂多糖）、TNF-$\alpha$ 及其他 PAMPs/DAMPs 刺激产生强致炎因子释放 response（如 IL-6、IL-1$\beta$、TNF-$\alpha$）

  • 细胞间 Crosstalk：可通过分泌 TGF-$\beta1$ 激活肝星状细胞（HSC），诱导肝纤维化信号

• 主要应用 / Key Applications：肝脏固有免疫与炎症反应、非酒精性脂肪肝病（NAFLD/MASH）病理研究、肝纤维化与炎症微环境、药物诱导性肝损伤（DILI）体外评估

三、 培养条件与传代指南 / Culture & Handling Protocols

1. 推荐培养基 / Complete Growth Medium

• 基础培养基：BioVector® High-Glucose DMEM 或 BioVector® RPMI-1640 Medium

• 添加成分：10% 灭活 BioVector® Fetal Bovine Serum (FBS) + 1% 双抗（Penicillin-Streptomycin）

• 辅助添化物（可选）：M-CSF (20 ng/mL，用于保持优良的巨噬细胞分化表型)

• 培养环境：37 摄氏度，5% $\text{CO}_2$，高湿度培养箱

2. 复苏与传代操作 / Thawing & Subculture

1. 复苏：将冻存管快速置于 37 摄氏度水浴中摇晃融化（1–2 分钟）。加入 8–10 mL 预热的 BioVector® Complete Medium，120 x g 离心 5 分钟，弃上清后重悬接种。

2. 消化传代：

   • 细胞汇合度达到 70%–80% 时即可传代。

   • 特殊注意：库普弗细胞贴壁能力极强，仅用 Trypsin 较难脱落。建议使用 0.25% Trypsin-EDTA 孵育 3–5 分钟后，配合 Cell Scraper（细胞刮）轻轻刮下，或使用巨噬细胞专用无酶消化液/冷 PBS 浸泡脱落，避免过长时间胰酶消化损伤细胞表面受体。

   • 按 1:2 至 1:4 比例传代。

3. 冻存保存 / Cryopreservation

• 冻存液配方：90% BioVector® FBS + 10% DMSO 或 70% BioVector® Base Medium + 20% BioVector® FBS + 10% DMSO

• 保存条件：程序降温后长期保存于液氮（-196 摄氏度）。

四、 技术指标简表 / Technical Data Summary

BioVector® ImKC 细胞株（小鼠肝脏库普弗细胞） / BioVector® ImKC Cell Line (Immortalized Mouse Kupffer Cells)

1. Background and Origin

BioVector® ImKC (Immortalized Mouse Kupffer Cells) is an immortalized macrophage cell line derived from primary mouse Kupffer cells via expression of SV40 Large T antigen or hTERT.

Kupffer cells are resident macrophages stationed inside the hepatic sinusoids, serving as a first line of immune defense in the liver. Primary Kupffer cells are difficult to isolate with high purity and rapidly lose viability in vitro. The BioVector® ImKC cell line circumvents these limitations by providing an expandable model that maintains key macrophage/Kupffer markers and functions. It serves as an essential tool for studying liver innate immunity, non-alcoholic steatohepatitis (NASH/MASH), hepatic fibrosis, and drug-induced liver injury (DILI).

2. Biological Characteristics

• Taxonomy & Source: Mus musculus, Liver Sinusoid

• Biosafety Level: BSL-1

• Growth Properties: Adherent, macrophage-like polygonal morphology

• Key Markers:

  • Kupffer / Macrophage Lineage: F4/80(+), CD11b(+), CD68(+), CD14(+), Clec4f(+)

  • Functional Capacity: Active phagocytosis and receptor-mediated endocytosis

• Immune Signaling:

  • Highly responsive to LPS, TNF-$\alpha$, and other danger signals, secreting pro-inflammatory cytokines (IL-6, IL-1$\beta$, TNF-$\alpha$)

  • Capable of activating Hepatic Stellate Cells (HSCs) via paracrine TGF-$\beta1$ release

• Primary Applications: Hepatic innate immunity, NAFLD/MASH disease pathways, liver fibrosis microenvironment, and hepatotoxicity testing

3. Culture and Handling Protocols

Recommended Growth Medium

• Basal Medium: BioVector® High-Glucose DMEM or BioVector® RPMI-1640 Medium

• Supplements: 10% heat-inactivated BioVector® Fetal Bovine Serum (FBS) + 1% Penicillin-Streptomycin

• Culture Conditions: 37 degrees Celsius, 5% $\text{CO}_2$, humidified incubator

Thawing and Subculture Protocol

1. Thawing: Thaw quickly in a 37 degrees Celsius water bath. Add 8–10 mL pre-warmed BioVector® Complete Growth Medium, centrifuge at 120 x g for 5 minutes, resuspend, and seed into culture vessels.

2. Passaging:

   • Subculture at 70–80% confluence.

   • Crucial Tip: Because Kupffer cells adhere strongly, standard trypsinization alone may yield poor detachment. Treat with 0.25% Trypsin-EDTA for 3–5 minutes at 37 degrees Celsius and gently dislodge cells using a sterile cell scraper, or perform non-enzymatic detachment using cold DPBS.

   • Split ratio: 1:2 to 1:4.

Cryopreservation

• Freezing Medium: 90% BioVector® FBS + 10% DMSO or 70% BioVector® Basal Medium + 20% BioVector® FBS + 10% DMSO

• Storage: Liquid nitrogen (-196 degrees Celsius).

4. Technical Data Summary

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