BioVector? KUP5 Immortalized Mouse Kupffer Cell Line / KUP5 小鼠肝巨噬（庫普弗）永生化細(xì)胞系

一 產(chǎn)品基本信息與細(xì)胞生物學(xué)背景

• 細(xì)胞名稱：KUP5。

• 物種與組織來源：小鼠（Mus musculus），源自 C57BL/6 成年小鼠的健康肝臟組織（富集純化的肝臟定居巨噬細(xì)胞/庫普弗細(xì)胞）。

• 細(xì)胞系建立背景：KUP5 細(xì)胞系由日本科學(xué)團(tuán)隊(duì)通過將原代分離的 C57BL/6 小鼠肝巨噬細(xì)胞進(jìn)行混合原代培養(yǎng)，隨后利用攜帶人源 c-myc 癌基因以及新霉素抗性基因（Neomycin resistance gene）的復(fù)制缺陷型慢病毒/逆轉(zhuǎn)錄病毒載體轉(zhuǎn)導(dǎo)建立的。傳統(tǒng)的原代庫普弗細(xì)胞（Kupffer Cells, KCs）體外分離產(chǎn)量極低、不均一且無法傳代（迅速進(jìn)入自發(fā)衰老死亡），KUP5 細(xì)胞系的成功克隆和鑒定，徹底解決了免疫學(xué)和肝臟病學(xué)界長期缺乏穩(wěn)定、均一、可無限增殖的庫普弗模式細(xì)胞的痛點(diǎn)。

• 核心表型與巨噬細(xì)胞特征：

  • 標(biāo)志物高表達(dá)：經(jīng)免疫細(xì)胞化學(xué)和流式細(xì)胞術(shù)驗(yàn)證，KUP5 細(xì)胞強(qiáng)陽性表達(dá)小鼠定居型巨噬細(xì)胞的特征性表面標(biāo)志物 F4/80 和 Mac-1 (CD11b)，完美保留了原代庫普弗細(xì)胞的免疫系譜特征。

  • 高度活躍的吞噬功能（Phagocytosis）：具有極強(qiáng)的吞噬本能，體外實(shí)驗(yàn)中對聚苯乙烯微球（Polystyrene Microbeads）、細(xì)胞碎片、異物以及細(xì)菌包囊表現(xiàn)出顯著的內(nèi)吞與清理活性。

  • 強(qiáng)烈的炎性響應(yīng)：對脂多糖（LPS/Endotoxin）極其敏感。在極低濃度 LPS 刺激下，能迅速激活其 NF-κB 通路，并豐度釋放腫瘤壞死因子（TNF-alpha）、白介素-6（IL-6）等核心促炎細(xì)胞因子。

• 生物安全級別：1級（BSL-1）。經(jīng)檢測無傳染性病毒顆粒自發(fā)釋放。

二 核心科研價值與轉(zhuǎn)化醫(yī)學(xué)應(yīng)用

KUP5 作為肝臟專職抗原遞呈與免疫監(jiān)視細(xì)胞的標(biāo)桿體外模型，在多個前沿交叉研究領(lǐng)域應(yīng)用極其廣泛：

1. 非酒精性脂肪性肝炎（NASH/MASH）與肝纖維化機(jī)制研究：在代謝相關(guān)脂肪性肝病中，庫普弗細(xì)胞的異常激活是驅(qū)動靜息態(tài)肝星狀細(xì)胞（HSCs）向肌成纖維細(xì)胞轉(zhuǎn)變、進(jìn)而引發(fā)肝纖維化（Liver Fibrosis）的核心開關(guān)。KUP5 常與小鼠原代肝細(xì)胞、小鼠肝星狀細(xì)胞（如 JS1 系列）構(gòu)建多細(xì)胞共培養(yǎng)體系，用于深度解構(gòu)“脂毒性-巨噬細(xì)胞活化-星狀細(xì)胞纖維化”的分子網(wǎng)絡(luò)。

2. 內(nèi)毒素血癥、肝損傷與 NLRP3 炎性小體活化機(jī)制：KUP5 被廣泛應(yīng)用于研究外源性毒素（如重金屬鎘、微塑料、酒精代謝物）介導(dǎo)的肝毒性反應(yīng)。它是探討活性氧（ROS）激活 NF-κB 進(jìn)而觸發(fā) NLRP3 炎性小體級聯(lián)釋放 IL-1β 的經(jīng)典靶板。

3. 納微米藥物載體（Nanoparticles）的肝臟清除與靶向評價：絕大多數(shù)靜脈注射的納米藥物制劑、核酸脂質(zhì)納米粒（LNPs）都會在肝血竇內(nèi)被庫普弗細(xì)胞作為異物瘋狂攔截并吞噬（即肝臟首過效應(yīng)/免疫清除）。KUP5 是體外定量評估納米載體“免疫逃逸”能力、篩選抗巨噬細(xì)胞攔截涂層、開發(fā)肝臟主動靶向給藥系統(tǒng)（DDS）必不可少的吞噬屏障模型。

三 實(shí)驗(yàn)室細(xì)胞復(fù)蘇、貼壁常規(guī)培養(yǎng)、傳代與保存標(biāo)準(zhǔn)步驟

KUP5 細(xì)胞體積相對較小，對貼壁器皿表面無特殊包被要求，但在復(fù)蘇和傳代階段，其對液體溫度的變化非常敏感。在 37°C 下該細(xì)胞的附著極快，因此在洗滌和離心階段需要使用冷培養(yǎng)基操作以防止發(fā)生非特異性抱團(tuán)。

1. 專用培養(yǎng)基與核心成分配置

為了維持 KUP5 細(xì)胞長期的旺盛分裂能力與巨噬表型，其完全培養(yǎng)基配方比普通巨噬細(xì)胞更為特殊：

• 基礎(chǔ)培養(yǎng)基：高糖 DMEM 培養(yǎng)基。

• 特殊完全培養(yǎng)基配方（關(guān)鍵控制點(diǎn)）：

  • 高糖 DMEM 基礎(chǔ)培養(yǎng)基

  • 加 10% 優(yōu)質(zhì)胎牛血清（FBS）

  • 加 10 μg/mL 人胰島素（Human Insulin）（維持其肝源性代謝因子的耐受與增殖促進(jìn)）

  • 加 250 μM 單硫代甘油（Monothioglycerol）（強(qiáng)效還原劑，保護(hù)巨噬細(xì)胞免受氧化應(yīng)激損傷，維持正常分裂，可用等效 beta-巰基乙醇替代但單硫代甘油最佳）

  • 加 1% 青霉素-鏈霉素雙抗。

• 細(xì)胞解離液：推薦使用溫和的解離液（如 Accutase 或 0.25% Trypsin-EDTA）。

• 生長常數(shù)：37 攝氏度，5% 二氧化碳，倍增時間約為 19 - 24 小時（增殖較為迅速）。

2. 冷凍細(xì)胞復(fù)蘇步驟

1. 核心避坑指南：準(zhǔn)備 1 支包含 9 mL 冷完全培養(yǎng)基（4 攝氏度左右）的 15 mL 離心管，置于冰上備用。由于庫普弗細(xì)胞在 37°C 預(yù)熱介質(zhì)中會瞬間粘附在離心管塑料壁上，導(dǎo)致離心洗滌時細(xì)胞嚴(yán)重?fù)p失，因此洗滌去除 DMSO 的過程必須使用冷培養(yǎng)基并在低溫/冰上短暫進(jìn)行。

2. 從液氮罐中取出 KUP5 凍存管，立刻投入 37 攝氏度恒溫水浴箱中快速搖晃解凍，確保在 1 分鐘內(nèi)完全融化。

3. 用 75% 酒精消毒管外壁，移入無菌生物安全柜。

4. 用移液槍吸取全量融化的菌懸液，極其緩慢地逐滴滴入上述準(zhǔn)備好的 9 mL 冷培養(yǎng)基離心管中，輕柔顛倒一次。

5. 以 300 g（約 1000 - 1200 rpm）室溫或 4 度離心 5 分鐘，小心抽干含有 DMSO 的上清液。

6. 加入 1 mL 預(yù)熱至 37 攝氏度的完全培養(yǎng)基，使用 P1000 移液槍輕輕重懸沉淀。(注意：由于巨噬細(xì)胞沉淀極小且易聚集成團(tuán)，切勿使用大號血清移液管粗暴吹打，應(yīng)使用槍頭輕柔化開)。

7. 接種至干凈未包被的 T25 培養(yǎng)瓶中，補(bǔ)足 4 - 5 mL 預(yù)熱完全培養(yǎng)基，混勻后置于 37 攝氏度孵箱中培養(yǎng)。

8. 孵育 4 - 6 小時或過夜后，待細(xì)胞完全貼壁，全量更換一次新鮮的、預(yù)熱完全培養(yǎng)基以清除極微量殘留的死細(xì)胞。

3. 日常貼壁常規(guī)傳代操作

• 傳代時機(jī)：KUP5 細(xì)胞呈多角形、多偽足的經(jīng)典巨噬細(xì)胞形態(tài)。當(dāng)細(xì)胞匯合度達(dá)到 80% - 90% 時必須傳代。由于其倍增極快，若任其長滿至 100% 極度過密，KUP5 會由靜息態(tài)發(fā)生非特異性自發(fā)極化（M1/M2 狀態(tài)漂移），導(dǎo)致背景炎癥因子基礎(chǔ)釋放量飆升，嚴(yán)重污染后續(xù)實(shí)驗(yàn)。

• 操作流程：

  1. 吸除舊培養(yǎng)基，使用無菌的、不含鈣鎂離子的 PBS 緩沖液輕輕漂洗細(xì)胞表面 2 次，徹底洗凈血清殘余。

  2. 加入適量 0.25% 胰酶或 Accutase（T25 瓶加入 1 - 2 mL），全面覆蓋細(xì)胞層。置于 37 攝氏度孵箱中消化 3 - 5 分鐘。

  3. 每隔 2 分鐘在顯微鏡下動態(tài)觀察。巨噬細(xì)胞貼壁較為牢固，消化時可配合用手掌輕敲培養(yǎng)瓶側(cè)壁。當(dāng)發(fā)現(xiàn)偽足收回、胞體變圓并大面積發(fā)生成片脫落位移時，立即加入 2 倍體積的含血清完全培養(yǎng)基終止消化。

  4. 輕輕吹打瓶壁，將脫落的細(xì)胞全部收集至 15 mL 離心管中，300 g 離心 5 分鐘。

  5. 棄上清，用預(yù)熱的完全培養(yǎng)基重懸，打散成單細(xì)胞懸液。按照 1 比 4 至 1 比 6 的稀釋比例接種入新培養(yǎng)器皿中。

  6. 通常每 2 - 3 天傳代一次。若發(fā)現(xiàn)局部有未完全消化的抱團(tuán)細(xì)胞，傳代時可適當(dāng)減少接種密度。

4. 細(xì)胞長期保存標(biāo)準(zhǔn)

• 凍存液配方：70% 基礎(chǔ)高糖 DMEM 培養(yǎng)基 加 20% 優(yōu)質(zhì)胎牛血清（FBS） 加 10% 分析級二甲基亞砜（DMSO）（或直接使用市售免程序降溫的專用高內(nèi)皮型無血清細(xì)胞凍存液）。

• 冷凍規(guī)范：

  1. 必須收集處于連續(xù)傳代期間、處于對數(shù)生長最旺盛期（匯合度約 80%）、未受過任何 LPS 或炎性因子刺激的健康狀態(tài) KUP5 細(xì)胞。

  2. 經(jīng)消化、離心后，用配制好的冷凍液懸浮并調(diào)整密度至 每毫升 1,500,000 到 2,000,000 個細(xì)胞。

  3. 分裝至無菌凍存管中，立即放入標(biāo)準(zhǔn)程序降溫盒（如 Mr. Frosty），將其置于 零下 80 攝氏度超低溫冰箱內(nèi)過夜完成每分鐘穩(wěn)定降溫 1 攝氏度的梯度降溫過程。

  4. 次日，必須以極快速度將凍存管投遞至液氮罐（零下 196 攝氏度）的氣相或液相中鎖死長期保存。絕對禁止在 零下 80 度冰箱中存放超過 2 周，以防止 myc 永生化表型在亞穩(wěn)態(tài)低溫下由于冰晶重組而發(fā)生隱性衰退。

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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