BioVector? MPC-5 條件永生化小鼠腎足細(xì)胞株——產(chǎn)品技術(shù)說(shuō)明書(shū)

1 產(chǎn)品基本信息與鑒定譜系產(chǎn)品名稱：BioVector MPC-5 條件永生化小鼠腎足細(xì)胞株常用別名：MPC5，Mouse Podocyte Clone-5國(guó)際數(shù)據(jù)庫(kù)登錄號(hào)：Cellosaurus登錄號(hào)：CVCL_AS87，Cytion貨號(hào)：305481生物學(xué)來(lái)源：小鼠 Mus musculus品系背景：源自 CBA/Ca x C57BL/10 Tg(H2Kb-tsA58) Immortomouse 永生鼠 遺傳背景組織器官：腎臟 / 腎小球足細(xì)胞 Glomerular Podocyte生長(zhǎng)特性：貼壁型上皮樣單層，細(xì)胞形態(tài)隨分化狀態(tài)發(fā)生劇烈改變核心科研價(jià)值：體外研究腎小球?yàn)V過(guò)屏障損傷、裂隙隔膜重塑、糖尿病腎病病理機(jī)制及足細(xì)胞骨架重排的國(guó)際公認(rèn)標(biāo)準(zhǔn)細(xì)胞模型。

2 條件永生化調(diào)控機(jī)制與標(biāo)志物特征MPC-5 細(xì)胞株內(nèi)嵌一套高度精準(zhǔn)的溫度敏感型條件永生化系統(tǒng)，使研究者能自由調(diào)控細(xì)胞在增殖擴(kuò)展與終端分化兩種狀態(tài)間切換：

核心轉(zhuǎn)化基因：基因組整合了溫度敏感型猿猴病毒40大T抗原 tsA58 SV40LT，其啟動(dòng)子受干擾素-gamma（IFN-gamma）誘導(dǎo)的 mouse H2Kb 啟動(dòng)子調(diào)控。允許期/增殖期（Permissive Phase）：在 33 ℃ 并添加 IFN-gamma 的條件下栽培。此時(shí) SV40 大 T 抗原結(jié)構(gòu)穩(wěn)定并高效表達(dá)，強(qiáng)行驅(qū)動(dòng)細(xì)胞進(jìn)入持續(xù)的有絲分裂和快速擴(kuò)增階段。細(xì)胞鏡下多呈成纖維狀、三角形或鋪路石狀。非允許期/分化期（Non-Permissive Phase）：切換至 37 ℃ 且不含 IFN-gamma 的條件下栽培。在此環(huán)境下，tsA58 突變體蛋白發(fā)生自發(fā)性構(gòu)象改變并迅速降解，細(xì)胞完全停止分裂，向成熟足細(xì)胞表型分化。細(xì)胞體積急劇增大，并自發(fā)延伸出粗大的主突起以及成排的、指狀的足突。核心標(biāo)志物定性：基線標(biāo)志物：在增殖和分化階段均穩(wěn)定表達(dá) Wilms 瘤抑癌蛋白 WT1。成熟度分化指標(biāo)：當(dāng)完全切斷 IFN-gamma 并移至 37 ℃ 培養(yǎng) 10-14 天后，細(xì)胞內(nèi)會(huì)特異性高表達(dá)足細(xì)胞成熟標(biāo)志物——突觸素 Synaptopodin、Nephrin 和 Podocin。

3 專用培養(yǎng)基配方規(guī)范

由于 MPC-5 的雙軌生長(zhǎng)特性，必須根據(jù)實(shí)驗(yàn)?zāi)康呐渲脙商壮煞纸厝徊煌耐耆囵B(yǎng)基：

A 允許期增殖完全培養(yǎng)基（用于 33 ℃ 細(xì)胞日常傳代與擴(kuò)增）基礎(chǔ)培養(yǎng)基：高糖 DMEM（含 4.5 g/L 葡萄糖、4 mM L-谷氨酰胺，不含丙酮酸鈉）。血清添加：10% 優(yōu)質(zhì)胎牛血清 FBS。關(guān)鍵永生化因子：重組小鼠干擾素-gamma（IFN-gamma，終濃度：10 到 50 Units/mL；若缺失此成分，SV40LT 表達(dá)將大幅下調(diào)導(dǎo)致細(xì)胞提前終止分裂）。雙抗：1% 青霉素-鏈霉素溶液（終濃度：100 U/mL 青霉素，100 ug/mL 鏈霉素）。

B 非允許期分化完全培養(yǎng)基（用于 37 ℃ 誘導(dǎo)成熟足細(xì)胞，禁止傳代）基礎(chǔ)培養(yǎng)基：高糖 DMEM。血清添加：10% 優(yōu)質(zhì)胎牛血清 FBS。核心剔除成分：完全不添加任何干擾素-gamma（IFN-gamma，即 0 U/mL）。雙抗：1% 青霉素-鏈霉素溶液。

C 細(xì)胞凍存保護(hù)液標(biāo)準(zhǔn)配方：90% 允許期完全生長(zhǎng)培養(yǎng)基 + 10% 細(xì)胞級(jí)二甲基亞砜 DMSO。高回收率配方：45% 基礎(chǔ) DMEM + 45% 優(yōu)質(zhì)胎牛血清 + 10% DMSO。

4 雙軌環(huán)境控制參數(shù)

栽培 MPC-5 細(xì)胞時(shí)，必須嚴(yán)格在兩臺(tái)設(shè)置不同的獨(dú)立避光孵箱中進(jìn)行物理切換：

允許期增殖階段（日常傳代/擴(kuò)增）：孵箱設(shè)定溫度：33.0 ℃干擾素-gamma（IFN-gamma）：強(qiáng)陽(yáng)性添加（10-50 U/mL）二氧化碳濃度：5%，加濕平衡常壓空氣相對(duì)濕度：大于 95%細(xì)胞命運(yùn)走向：細(xì)胞對(duì)數(shù)分裂，維持未分化狀態(tài)

非允許期分化階段（功能學(xué)研究/損傷模型）：孵箱設(shè)定溫度：37.0 ℃干擾素-gamma（IFN-gamma）：絕對(duì)缺失（0 U/mL）二氧化碳濃度：5%，加濕平衡常壓空氣相對(duì)濕度：大于 95%細(xì)胞命運(yùn)走向：徹底退出細(xì)胞周期，長(zhǎng)出交織足突，需 10-14 天

5 允許期貼壁傳代操作規(guī)范傳代臨界匯合度：日常在 33 ℃ 栽培時(shí)，必須在細(xì)胞單層達(dá)到 75% - 85% 匯合度時(shí)進(jìn)行傳代。切勿讓細(xì)胞在 33 ℃ 下 100% 匯合過(guò)密，否則會(huì)導(dǎo)致嚴(yán)重的接觸抑制并誘發(fā)自發(fā)性衰亡。建議傳代稀釋比例：1:3 到 1:5（一般每 2-3 天傳代一次）。

標(biāo)準(zhǔn)消化分散步驟：1 完全吸除舊培養(yǎng)基。2 使用無(wú)菌、不含鈣鎂離子的 PBS 緩沖液輕柔漂洗細(xì)胞單層 1 次。3 加入適量預(yù)熱的 0.25% Trypsin - 0.02% EDTA 消化液（T25 瓶加入 1.0 mL）。4 將培養(yǎng)瓶置于 33 ℃ 孵箱中（或室溫下）靜置消化 1 到 2 分鐘。MPC-5 對(duì)胰酶極為敏感，極易消化過(guò)度。全程需在倒置顯微鏡下密切監(jiān)控。5 一旦發(fā)現(xiàn)單層細(xì)胞回縮變圓，立即加入等體積的含血清允許期完全培養(yǎng)基終止消化。6 輕柔吹打貼壁面，分散為單細(xì)胞懸液。在 300 x g 下離心 3 到 5 分鐘。7 棄去上清液，加入新鮮的含 IFN-gamma 的允許期完全培養(yǎng)基重懸彈散細(xì)胞塊，轉(zhuǎn)接至新器皿中，放回 33 ℃ 孵箱繼續(xù)擴(kuò)增。

6 復(fù)蘇與凍存后恢復(fù)流線1 提前在無(wú)菌 T25 培養(yǎng)瓶中注入 5.0 mL 含 IFN-gamma 的增殖完全培養(yǎng)基，置于 33 ℃、5% CO2 孵箱中進(jìn)行氣相平衡。2 從液氮罐中取出冷凍的 MPC-5 凍存管，立刻全量投入 37 ℃ 恒溫水浴箱中快速搖晃。3 在 40 至 60 秒內(nèi)融化完畢，直至管內(nèi)只剩一絲小冰芯。4 用 75% 酒精消毒管壁后移入超凈臺(tái)，將融化后的胞懸液逐滴緩慢注入盛有 5.0 mL 預(yù)熱增殖完全培養(yǎng)基的 15 mL 離心管中。5 300 x g 離心 3 到 5 分鐘，徹底吸干含有毒 DMSO 的上清液。6 加入 1.5 mL 新鮮增殖完全培養(yǎng)基輕彈懸起細(xì)胞，隨后全量接種到已預(yù)熱平衡的 T25 瓶中，置于 33 ℃ 孵箱栽培。7 24 小時(shí)后，必須進(jìn)行一次全量換液以清除殘余的未貼壁細(xì)胞碎片。

7 超低溫長(zhǎng)期保存與質(zhì)控規(guī)范生物安全級(jí)別：BSL-2。因細(xì)胞攜帶 SV40 病毒基因片段，必須在二級(jí)生物安全柜內(nèi)進(jìn)行無(wú)菌技術(shù)操作。長(zhǎng)期封存溫控：必須永久存放于液氮蒸汽或液相（-150 ℃ 至 -196 ℃）中。嚴(yán)禁在 -80 ℃ 普通冰箱中長(zhǎng)期存放超過(guò) 1 個(gè)月，溫度過(guò)高會(huì)導(dǎo)致其條件永生化溫敏開(kāi)關(guān)失靈，導(dǎo)致細(xì)胞復(fù)蘇后無(wú)法增殖或喪失分化能力。分化能力定期復(fù)核：在大規(guī)模實(shí)驗(yàn)前，應(yīng)抽取一管傳代細(xì)胞切換至 37 ℃ 無(wú) IFN-gamma 培養(yǎng)基中連續(xù)培養(yǎng) 10 天。若鏡下細(xì)胞完全停止分裂、體積顯著擴(kuò)張并形成蛛網(wǎng)狀相互交織的絲狀突起，且免疫熒光檢測(cè) Synaptopodin 呈強(qiáng)陽(yáng)性，方可確證細(xì)胞品系未發(fā)生表型漂移。

BioVector MPC-5 Conditionally Immortalized Mouse Podocyte Cell Line Product Datasheet

1 Product and Identification General InformationProduct Name: BioVector MPC-5 Conditionally Immortalized Mouse Podocyte Cell LineSynonyms: MPC5, Mouse Podocyte Clone-5Cellosaurus Accession: CVCL_AS87, Cytion Cat No 305481Organism Source: Mouse Mus musculusBreed/Subspecies Background: Derived from the CBA/Ca x C57BL/10 Tg(H2Kb-tsA58) Immortomouse backgroundTissue/Organ Site: Kidney / Glomerular PodocyteGrowth Properties: Adherent Epithelial-like Monolayer, undergoes morphological transitions depending on differentiation statesCore Research Significance: Serving as the international gold standard cell model to investigate glomerular filtration barrier dynamics, slit diaphragm remodeling, diabetic nephropathy injury pathways, and podocyte foot process cytoskeletal rearrangements in vitro.

2 Conditional Immortalization Mechanism and Marker ProfileThe MPC-5 cell line is engineered via a conditional, temperature-sensitive transformation system, permitting precise experimental control between proliferation and mature differentiation states:

Genetic Transformant: Integrated with the temperature-sensitive Simian Virus 40 Large T Antigen tsA58 SV40LT under the control of an interferon-gamma (IFN-gamma) inducible mouse Major Histocompatibility Complex H2Kb promoter.Permissive Proliferative Phase: Cultured at 33 ℃ with IFN-gamma. The SV40 large T antigen is active and stable, driving cell division and rapid expansion. Morphologically, cells appear cobblestone, fusiform, or triangular.Non-Permissive Differentiated Phase: Cultured at 37 ℃ without IFN-gamma. The SV40 large T antigen undergoes rapid protein degradation, resulting in complete growth arrest. Cells switch to terminal differentiation, expanding significantly in cell body size and sprouting long, branched primary and finger-like secondary foot processes.Biomarker Expression Matrix:Baseline Proliferation Markers: Wilms tumor suppressor protein WT1 is consistently expressed across both stages.Mature Podocyte Differentiation Endpoints: Synaptopodin, Nephrin, and Podocin are strongly upregulated and assembled in the cytoplasm specifically when shifted to the differentiated phase (37 ℃ without IFN-gamma).

3 Dedicated Culturing Medium and Formulations

The MPC-5 baseline formulation requires strict micro-environment supplements to prevent premature differentiation or cell death during routine propagation.

A Permissive Expansion Medium (For Active Cell Proliferation at 33 ℃)Basal Medium Base: High-Glucose DMEM (Dulbecco Modified Eagle Medium containing 4.5 g/L glucose and 4 mM L-glutamine).Serum Supplement: 10% premium Fetal Bovine Serum FBS.Permissive Growth Factor: Recombinant Mouse Interferon-gamma (IFN-gamma, Final concentration: 10 to 50 Units/mL; crucial for maintaining SV40LT expression).Antibiotics: 1% Penicillin-Streptomycin Solution (100 U/mL Penicillin, 100 ug/mL Streptomycin).

B Non-Permissive Differentiation Medium (To Induce Mature Podocytes at 37 ℃)Basal Medium Base: High-Glucose DMEM.Serum Supplement: 10% premium FBS.Key Modification: Exclude Interferon-gamma (IFN-gamma) completely.Antibiotics: 1% Penicillin-Streptomycin Solution.

C Standard Cryopreservation Freezing MediumStandard Freezing Formula: 90% Complete Expansion Medium with 10% FBS + 10% Analytical Grade DMSO (Dimethyl Sulfoxide).Alternative High-Recovery Formula: 45% Basal DMEM + 45% Premium FBS + 10% DMSO.

4 Dual-State Controlled Incubator Conditions

Unlike traditional cell lines, MPC-5 must be moved between separate physical environments depending on your experimental intent:

Permissive Culture Phase (Expansion/Passaging):Incubator Temperature: 33.0 ℃ (strict control)Interferon-gamma (IFN-gamma): Present (10-50 U/mL)Gas Composition (CO2): 5% Carbon Dioxide in humidified airRelative Humidity: Greater than 95%Cellular Fate: Rapid Division / Undifferentiated State

Non-Permissive Phase (Differentiation/Assay):Incubator Temperature: 37.0 ℃ (strict control)Interferon-gamma (IFN-gamma): Absent (0 U/mL)Gas Composition (CO2): 5% Carbon Dioxide in humidified airRelative Humidity: Greater than 95%Cellular Fate: Growth Arrest / Foot Process Arborization, takes 10-14 days

5 Subculturing Passaging Protocol at Permissive Phase (33 ℃)Optimal Passaging Confluency: Perform subculturing strictly when the monolayer reaches 75% to 85% confluency at 33 ℃. Do not let the cells become 100% confluent, as contact inhibition may trigger localized cell senescence.Subcultivation Splitting Ratio: 1:3 to 1:5 (typically passaged every 2 to 3 days).

Step-by-Step Dissociation Workflow:1 Aspirate the spent growth fluid from the vessel.2 Rinse the cell monolayer gently with sterile, Calcium/Magnesium-free PBS once.3 Apply a minimal volume of pre-warmed 0.25% Trypsin - 0.02% EDTA Solution (1.0 mL for a T25 flask).4 Incubate at 33 ℃ (or room temperature) for 1 to 2 minutes. MPC-5 cells detach very quickly under enzymatic treatment. Monitor closely under an inverted microscope.5 As soon as the cells round up, add an equal volume of serum-containing Complete Expansion Medium to neutralize the trypsin.6 Pipette gently to achieve a single-cell suspension. Centrifuge at 300 x g for 3 to 5 minutes.7 Discard the supernatant, gently resuspend the pellet in fresh IFN-gamma-supplemented growth medium, and plate cells into new flasks. Seed at 33 ℃ for continued expansion.

6 Thawing and Post-Cryo Recovery Workflow1 Pre-warm 5.0 mL of complete MPC-5 expansion medium containing IFN-gamma in a sterile T25 flask and equilibrate it inside a 33 ℃, 5% CO2 incubator.2 Retrieve the MPC-5 cryovial from liquid nitrogen and immerse it immediately into a 37 ℃ water bath. Rapidly agitate horizontally for 40 to 60 seconds until only a tiny ice core remains.3 Disinfect the vial exterior with 75% ethanol before transferring it into the biosafety cabinet.4 Transfer the cell suspension dropwise into a 15 mL conical tube containing 5.0 mL of pre-warmed complete expansion medium.5 Centrifuge at 300 x g for 3 to 5 minutes, discard the DMSO-contaminated supernatant, and resuspend the cells in fresh expansion medium.6 Seed into the pre-equilibrated T25 flask and incubate at 33 ℃, 5% CO2. Perform a complete medium change 24 hours post-thaw to eliminate residual dead debris.

7 Storage, Biosafety and Quality ControlBiosafety Level: BSL-2. Contains an SV40 large T antigen genetic component; use standard tissue culture personal protective equipment and containment controls.Long-Term Storage: Vials must be stored in the vapor phase of liquid nitrogen (-150 ℃ to -196 ℃). Storing at -80 ℃ long-term will cause irreversible drops in recovery rates and may damage the temperature-sensitive switch.Differentiation Verification QC: To verify line identity before major signaling assays, shift a sample aliquot to 37 ℃ without IFN-gamma for 10 days. The culture should completely cease proliferation and exhibit long, delicate interlacing cytoplasmic foot processes accompanied by positive immunofluorescence staining for Synaptopodin.

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