BioVector® KUP5 Immortalized Mouse Kupffer Cell Line / KUP5 小鼠肝巨噬（库普弗）永生化细胞系

一 产品基本信息与细胞生物学背景

• 细胞名称：KUP5。

• 物种与组织来源：小鼠（Mus musculus），源自 C57BL/6 成年小鼠的健康肝脏组织（富集纯化的肝脏定居巨噬细胞/库普弗细胞）。

• 细胞系建立背景：KUP5 细胞系由日本科学团队通过将原代分离的 C57BL/6 小鼠肝巨噬细胞进行混合原代培养，随后利用携带人源 c-myc 癌基因以及新霉素抗性基因（Neomycin resistance gene）的复制缺陷型慢病毒/逆转录病毒载体转导建立的。传统的原代库普弗细胞（Kupffer Cells, KCs）体外分离产量极低、不均一且无法传代（迅速进入自发衰老死亡），KUP5 细胞系的成功克隆和鉴定，彻底解决了免疫学和肝脏病学界长期缺乏稳定、均一、可无限增殖的库普弗模式细胞的痛点。

• 核心表型与巨噬细胞特征：

  • 标志物高表达：经免疫细胞化学和流式细胞术验证，KUP5 细胞强阳性表达小鼠定居型巨噬细胞的特征性表面标志物 F4/80 和 Mac-1 (CD11b)，完美保留了原代库普弗细胞的免疫系谱特征。

  • 高度活跃的吞噬功能（Phagocytosis）：具有极强的吞噬本能，体外实验中对聚苯乙烯微球（Polystyrene Microbeads）、细胞碎片、异物以及细菌包囊表现出显著的內吞与清理活性。

  • 强烈的炎性响应：对脂多糖（LPS/Endotoxin）极其敏感。在极低浓度 LPS 刺激下，能迅速激活其 NF-κB 通路，并丰度释放肿瘤坏死因子（TNF-alpha）、白介素-6（IL-6）等核心促炎细胞因子。

• 生物安全级别：1级（BSL-1）。经检测无传染性病毒颗粒自发释放。

二 核心科研价值与转化医学应用

KUP5 作为肝脏专职抗原递呈与免疫监视细胞的标杆体外模型，在多个前沿交叉研究领域应用极其广泛：

1. 非酒精性脂肪性肝炎（NASH/MASH）与肝纤维化机制研究：在代谢相关脂肪性肝病中，库普弗细胞的异常激活是驱动静息态肝星状细胞（HSCs）向肌成纤维细胞转变、进而引发肝纤维化（Liver Fibrosis）的核心开关。KUP5 常与小鼠原代肝细胞、小鼠肝星状细胞（如 JS1 系列）构建多细胞共培养体系，用于深度解构“脂毒性-巨噬细胞活化-星状细胞纤维化”的分子网络。

2. 内毒素血症、肝损伤与 NLRP3 炎性小体活化机制：KUP5 被广泛应用于研究外源性毒素（如重金属镉、微塑料、酒精代谢物）介导的肝毒性反应。它是探讨活性氧（ROS）激活 NF-κB 进而触发 NLRP3 炎性小体级联释放 IL-1β 的经典靶板。

3. 纳微米药物载体（Nanoparticles）的肝脏清除与靶向评价：绝大多数静脉注射的纳米药物制剂、核酸脂质纳米粒（LNPs）都会在肝血窦内被库普弗细胞作为异物疯狂拦截并吞噬（即肝脏首过效应/免疫清除）。KUP5 是体外定量评估纳米载体“免疫逃逸”能力、筛选抗巨噬细胞拦截涂层、开发肝脏主动靶向给药系统（DDS）必不可少的吞噬屏障模型。

三 实验室细胞复苏、贴壁常规培养、传代与保存标准步骤

KUP5 细胞体积相对较小，对贴壁器皿表面无特殊包被要求，但在复苏和传代阶段，其对液体温度的变化非常敏感。在 37°C 下该细胞的附着极快，因此在洗涤和离心阶段需要使用冷培养基操作以防止发生非特异性抱团。

1. 专用培养基与核心成分配置

为了维持 KUP5 细胞长期的旺盛分裂能力与巨噬表型，其完全培养基配方比普通巨噬细胞更为特殊：

• 基础培养基：高糖 DMEM 培养基。

• 特殊完全培养基配方（关键控制点）：

  • 高糖 DMEM 基础培养基

  • 加 10% 优质胎牛血清（FBS）

  • 加 10 μg/mL 人胰岛素（Human Insulin）（维持其肝源性代谢因子的耐受与增殖促进）

  • 加 250 μM 单硫代甘油（Monothioglycerol）（强效还原剂，保护巨噬细胞免受氧化应激损伤，维持正常分裂，可用等效 beta-巯基乙醇替代但单硫代甘油最佳）

  • 加 1% 青霉素-链霉素双抗。

• 细胞解离液：推荐使用温和的解离液（如 Accutase 或 0.25% Trypsin-EDTA）。

• 生长常数：37 摄氏度，5% 二氧化碳，倍增时间约为 19 - 24 小时（增殖较为迅速）。

2. 冷冻细胞复苏步骤

1. 核心避坑指南：准备 1 支包含 9 mL 冷完全培养基（4 摄氏度左右）的 15 mL 离心管，置于冰上备用。由于库普弗细胞在 37°C 预热介质中会瞬间粘附在离心管塑料壁上，导致离心洗涤时细胞严重损失，因此洗涤去除 DMSO 的过程必须使用冷培养基并在低温/冰上短暂进行。

2. 从液氮罐中取出 KUP5 冻存管，立刻投入 37 摄氏度恒温水浴箱中快速摇晃解冻，确保在 1 分钟内完全融化。

3. 用 75% 酒精消毒管外壁，移入无菌生物安全柜。

4. 用移液枪吸取全量融化的菌悬液，极其缓慢地逐滴滴入上述准备好的 9 mL 冷培养基离心管中，轻柔颠倒一次。

5. 以 300 g（约 1000 - 1200 rpm）室温或 4 度离心 5 分钟，小心抽干含有 DMSO 的上清液。

6. 加入 1 mL 预热至 37 摄氏度的完全培养基，使用 P1000 移液枪轻轻重悬沉淀。(注意：由于巨噬细胞沉淀极小且易聚集成团，切勿使用大号血清移液管粗暴吹打，应使用枪头轻柔化开)。

7. 接种至干净未包被的 T25 培养瓶中，补足 4 - 5 mL 预热完全培养基，混匀后置于 37 摄氏度孵箱中培养。

8. 孵育 4 - 6 小时或过夜后，待细胞完全贴壁，全量更换一次新鲜的、预热完全培养基以清除极微量残留的死细胞。

3. 日常贴壁常规传代操作

• 传代时机：KUP5 细胞呈多角形、多伪足的经典巨噬细胞形态。当细胞汇合度达到 80% - 90% 时必须传代。由于其倍增极快，若任其长满至 100% 极度过密，KUP5 会由静息态发生非特异性自发极化（M1/M2 状态漂移），导致背景炎症因子基础释放量飙升，严重污染后续实验。

• 操作流程：

  1. 吸除旧培养基，使用无菌的、不含钙镁离子的 PBS 缓冲液轻轻漂洗细胞表面 2 次，彻底洗净血清残余。

  2. 加入适量 0.25% 胰酶或 Accutase（T25 瓶加入 1 - 2 mL），全面覆盖细胞层。置于 37 摄氏度孵箱中消化 3 - 5 分钟。

  3. 每隔 2 分钟在显微镜下动态观察。巨噬细胞贴壁较为牢固，消化时可配合用手掌轻敲培养瓶侧壁。当发现伪足收回、胞体变圆并大面积发生成片脱落位移时，立即加入 2 倍体积的含血清完全培养基终止消化。

  4. 轻轻吹打瓶壁，将脱落的细胞全部收集至 15 mL 离心管中，300 g 离心 5 分钟。

  5. 弃上清，用预热的完全培养基重悬，打散成单细胞悬液。按照 1 比 4 至 1 比 6 的稀释比例接种入新培养器皿中。

  6. 通常每 2 - 3 天传代一次。若发现局部有未完全消化的抱团细胞，传代时可适当减少接种密度。

4. 细胞长期保存标准

• 冻存液配方：70% 基础高糖 DMEM 培养基 加 20% 优质胎牛血清（FBS） 加 10% 分析级二甲基亚砜（DMSO）（或直接使用市售免程序降温的专用高内皮型无血清细胞冻存液）。

• 冷冻规范：

  1. 必须收集处于连续传代期间、处于对数生长最旺盛期（汇合度约 80%）、未受过任何 LPS 或炎性因子刺激的健康状态 KUP5 细胞。

  2. 经消化、离心后，用配制好的冷冻液悬浮并调整密度至 每毫升 1,500,000 到 2,000,000 个细胞。

  3. 分装至无菌冻存管中，立即放入标准程序降温盒（如 Mr. Frosty），将其置于 零下 80 摄氏度超低温冰箱内过夜完成每分钟稳定降温 1 摄氏度的梯度降温过程。

  4. 次日，必须以极快速度将冻存管投递至液氮罐（零下 196 摄氏度）的气相或液相中锁死长期保存。绝对禁止在 零下 80 度冰箱中存放超过 2 周，以防止 myc 永生化表型在亚稳态低温下由于冰晶重组而发生隐性衰退。

Part 2 English Section

I General Information and Cell Biological Background

• Cell Line Name: KUP5 (Standardly referenced as KUP5 clone).

• Organism and Tissue Extraction Origin: Mus musculus (mouse); highly purified resident liver macrophages (Kupffer cells) isolated from the hepatic tissue of an adult adult C57BL/6 strain donor.

• Cell Line Establishment Background:The KUP5 cell line was successfully generated by a Japanese investigative cohort through a modified mixed primary culture of adult C57BL/6 mouse liver cells. The enriched primary Kupffer matrices were subsequently transduced with a replication-deficient retroviral vector carrying the human c-myc oncogene partnered with a neomycin resistance cassette (G418 selection framework). Because wild-type primary Kupffer cells (KCs) isolate with low cell yield, suffer from heavy subset heterogeneity, and fail to expand in vitro (entering rapid mitotic senescence), the clone validation of KUP5 offered an indispensable, infinitely expanding human/murine analog within immunology and hepatology.

• Core Morphological Phenotype and Macrophage Expression Patterns:

  • Lineage Expression Matrix: Confirmed via immunocytochemistry and flow cytometric sorting, KUP5 cells display robust, stable positive expression of definitive murine resident macrophage markers, including F4/80 and Mac-1 (CD11b).

  • Phagocytic Competence: Retains an active baseline endocytic profile, manifesting intense engulfment dynamics toward foreign particles, dead cell frameworks, and fluorescent-labeled polystyrene microbeads.

  • Hyper-Sensitive Inflammatory Response: Highly responsive to Lipopolysaccharide (LPS/Endotoxin) molecular challenges. Exposure to trace scales of LPS drives immediate NF-κB nuclear translocation, sparking a massive transcriptional output of pro-inflammatory cytokines such as Tumor Necrosis Factor-alpha (TNF-alpha) and Interleukin-6 (IL-6).

• Biosafety Matrix: Classified under Biosafety Level 1 (BSL-1) containment boundaries. Bioassays demonstrate zero shedding of infectious replication-competent retroviral particles.

II Strategic Research Value and Translational Fields

KUP5 stands as a standard in vitro chassis representing specialized hepatic resident innate immunity, operating extensively across several core disease models:

1. Modeling Metabolic Dysfunction-Associated Steatohepatitis (MASH/NASH) & Fibrosis Cascades:During chronic metabolic lipid accumulation, the activation trajectory of Kupffer cells serves as the primary paracrine switch that forces quiescent Hepatic Stellate Cells (HSCs) to transdifferentiate into collagen-secreting myofibroblasts. KUP5 cells are standardly integrated into multi-cellular co-culture webs pairing them with murine hepatocytes and stellate lineages (e.g., the JS1 system) to map the "lipotoxicity-macrophage polarization-stellate activation" pathway.

2. Endotoxemia, Acute Liver Injury & NLRP3 Inflammasome Kinetics:The line functions as a definitive screening asset to analyze liver damage driven by environmental xenobiotics, heavy metals (e.g., low-dose Cadmium exposure), and alcohol metabolites. It acts as an essential platform to track how Reactive Oxygen Species (ROS) activate the downstream NF-κB matrix to assembly the NLRP3 inflammasome complex, controlling the release of bio-active IL-1β.

3. Quantifying Hepatic Clearance of Nano-Drug Delivery Systems (LNPs):The structural default of circulating macromolecular therapeutic carriers, including Lipid Nanoparticles (LNPs) and nucleic acid vectors, is immediate entrapment by sinusoidal Kupffer guards via the hepatic first-pass clearing effect. KUP5 cells are used to evaluate the biomimetic "immune-evasive" properties of advanced formulations, optimize anti-phagocytic surface cloaking matrices, and baseline liver-directed targeted Drug Delivery Systems (DDS).

III Laboratory Thawing, Cultivation, Passaging, and Cryopreservation Protocols

KUP5 macrophages proliferate rapidly on conventional, non-coated tissue culture-treated plasticware. However, they display extreme thermal attachment sensitivity. Because they stick rapidly to standard plasticware at 37°C, the chemical clearing and washing steps during thawing must utilize chilled matrices to prevent loss from premature cell adhesion.

1. Growth Medium & Critical Nutrient Formulation

To sustain the long-term c-myc driven vegetative expansion without losing resident macrophage identities, the complete growth formulation requires specific protective adaptors:

• Basal Medium: High-glucose DMEM medium.

• Complete Growth Matrix Formulation (Critical Operation Control):

  • High-glucose DMEM basal medium

  • Enriched with 10% high-grade Fetal Bovine Serum (FBS)

  • Supplemented with 10 μg/mL analytical Human Insulin (to optimize hepatic metabolic stability and drive division)

  • Fortified with 250 μM Monothioglycerol (an essential anti-oxidant reducing agent that shields cells from oxidative stress during rapid splitting; can be substituted with beta-mercaptoethanol if empirically required, though monothioglycerol yields maximum longevity benchmarks)

  • Infused with 1% standard Penicillin-Streptomycin dual antibiotics.

• Cell Dissociation Enzyme: Standard Accutase or 0.25% Trypsin-EDTA solution.

• Kinetic Constants: Cultivate at 37 degrees Celsius inside a humidified atmosphere charged with 5% Carbon Dioxide. Population doubling time averages a crisp 19 to 24 hours.

2. Cryovial Thawing and Recovery Sequence

1. Critical Pre-thaw Operational Mandate: Aliquot 9 mL of ice-cold complete growth medium (approximately 4 degrees Celsius) into a sterile 15 mL conical tube and anchor it inside an ice bucket. Because KUP5 cells adhere instantly to container boundaries when exposed to warm solutions, utilizing chilled growth media protects against losing significant cell volumes during the DMSO removal centifugation step.

2. Extract the KUP5 cryovial from liquid nitrogen storage and submerge it immediately within a 37 degrees Celsius constant-temperature water bath. Shake rapidly and continuously to secure absolute liquefaction within 60 seconds.

3. Sterilize the external shell with 75% ethanol before transferring it into the sterile Class II Biosafety Cabinet.

4. Using a pipettor, extract the thawed slurry and deliver it extremely slowly, dropwise into the 9 mL of ice-cold medium in the conical tube, inverting gently once to equalize.

5. Sediment the cells via centrifugation at 300 g (approximately 1000 - 1200 rpm) for 5 minutes at room temperature (or 4°C), then carefully aspirate the DMSO-laden supernatant.

6. Dispense 1 mL of pre-warmed (37 degrees Celsius) complete growth medium onto the pellet and resuspend gently using a P1000 micro-pipette. Avoid using large serological pipettes for aggressive up-and-down mixing, as macrophage pellets can be dense and prone to structural clumping if handled roughly.

7. Transfer the entire suspension into a pristine, non-coated T25 culture flask, supplement with an additional 4 - 5 mL of pre-warmed complete growth medium, and incubate under standard atmospheric constants.

8. Allow cells to attach securely for 4 - 6 hours or overnight. Once attachment is confirmed, perform a complete medium change using pre-warmed complete medium to clear non-adherent fragments and cellular debris.

3. Adherent Passaging Mechanics and Maintenance

• Confluency Control Window: KUP5 cells present typical multi-pseudopodial, polymorphic macrophage traits. Subculturing routines must be initiated when monolayers hit an optimal 80% - 90% confluency range. Allowing cultures to reach absolute 100% saturation or overcrowding forces cells into contact-dependent non-specific polarization (spontaneous M1/M2 drifting), skewing baseline cytokine measurements and invalidating subsequent immunotoxicological assays.

• Passaging Execution Steps:

  1. Aspirate the spent growth matrix and carefully rinse the cell sheet 2 times using sterile, calcium/magnesium-free PBS to remove any residual serum proteins that could deactivate the dissociation enzyme.

  2. Administer a suitable volume of 0.25% Trypsin-EDTA or Accutase enzyme (typically 1 - 2 mL for a T25 format), slide the fluid to cover the monolayer completely, and place inside the 37 degrees Celsius incubator for 3 - 5 minutes.

  3. Monitor cell detachment kinetics under an inverted microscope. Macrophages maintain strong attachment parameters; investigators can firmly tap the flask sidewall with their palm to accelerate detachment. Once cells retract their pseudopodia, round up, and slide freely, immediately add 2 volumes of serum-fortified complete growth medium to arrest enzymatic activity.

  4. Gently pipette the suspension against the flask interior surfaces to clear remaining clusters, collect the fluid into a conical tube, and centrifuge at 300 g for 5 minutes.

  5. Discard the supernatant, resuspend the cell pellet in fresh, pre-warmed complete growth medium, and inoculate new flasks using optimal split ratios ranging from 1:4 to 1:6.

  6. Execute subculturing every 2 - 3 days. If localized clusters remain post-seeding, adjust the initial inoculation densities downward.

4. Long-Term Cryopreservation Standards

• Cryoprotectant Preservation Matrix: 70% basal high-glucose DMEM medium combined with 20% premium Fetal Bovine Serum (FBS) and supplemented with 10% analytical-grade Dimethyl Sulfoxide (DMSO) (or validated high-end commercial serum-free preservation media).

• Freezing Protocol Validation:

  1. Exclusively harvest healthy, log-phase KUP5 cultures showing an optimal confluency of approximately 80% that have never been exposed to LPS or inflammatory stimulants.

  2. Following enzymatic detachment and sediment centrifugation, resuspend the cells inside the formulated cold cryoprotectant matrix to achieve a target range of 1,500,000 to 2,000,000 cells per milliliter.

  3. Aliquot into sterile cryovials, insert them immediately into a standard rate-controlled freezing container (e.g., Mr. Frosty), and place into a minus 80 degrees Celsius freezer overnight to ensure a steady gradient cooling rate of 1 degree Celsius per minute.

  4. The following day, swiftly transfer the cryovials into liquid nitrogen storage tanks (minus 196 degrees Celsius) for long-term preservation. Do not store vials inside a minus 80 degrees Celsius freezer for more than 2 weeks; extended storage at this sub-optimal temperature will lead to cryogenic matrix degradation, compromising the c-myc immortalized phenotype and lowering post-thaw recovery rates.

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