MPC-5 BioVector®条件永生化小鼠肾足细胞株

BioVector® MPC-5 条件永生化小鼠肾足细胞株——产品技术说明书

1 产品基本信息与鉴定谱系产品名称：BioVector MPC-5 条件永生化小鼠肾足细胞株常用别名：MPC5，Mouse Podocyte Clone-5国际数据库登录号：Cellosaurus登录号：CVCL_AS87，Cytion货号：305481生物学来源：小鼠 Mus musculus品系背景：源自 CBA/Ca x C57BL/10 Tg(H2Kb-tsA58) Immortomouse 永生鼠 遗传背景组织器官：肾脏 / 肾小球足细胞 Glomerular Podocyte生长特性：贴壁型上皮样单层，细胞形态随分化状态发生剧烈改变核心科研价值：体外研究肾小球滤过屏障损伤、裂隙隔膜重塑、糖尿病肾病病理机制及足细胞骨架重排的国际公认标准细胞模型。

2 条件永生化调控机制与标志物特征MPC-5 细胞株内嵌一套高度精准的温度敏感型条件永生化系统，使研究者能自由调控细胞在增殖扩展与终端分化两种状态间切换：

核心转化基因：基因组整合了温度敏感型猿猴病毒40大T抗原 tsA58 SV40LT，其启动子受干扰素-gamma（IFN-gamma）诱导的 mouse H2Kb 启动子调控。允许期/增殖期（Permissive Phase）：在 33 ℃ 并添加 IFN-gamma 的条件下栽培。此时 SV40 大 T 抗原结构稳定并高效表达，强行驱动细胞进入持续的有丝分裂和快速扩增阶段。细胞镜下多呈成纤维状、三角形或铺路石状。非允许期/分化期（Non-Permissive Phase）：切换至 37 ℃ 且不含 IFN-gamma 的条件下栽培。在此环境下，tsA58 突变体蛋白发生自发性构象改变并迅速降解，细胞完全停止分裂，向成熟足细胞表型分化。细胞体积急剧增大，并自发延伸出粗大的主突起以及成排的、指状的足突。核心标志物定性：基线标志物：在增殖和分化阶段均稳定表达 Wilms 瘤抑癌蛋白 WT1。成熟度分化指标：当完全切断 IFN-gamma 并移至 37 ℃ 培养 10-14 天后，细胞内会特异性高表达足细胞成熟标志物——突触素 Synaptopodin、Nephrin 和 Podocin。

3 专用培养基配方规范

由于 MPC-5 的双轨生长特性，必须根据实验目的配置两套成分截然不同的完全培养基：

A 允许期增殖完全培养基（用于 33 ℃ 细胞日常传代与扩增）基础培养基：高糖 DMEM（含 4.5 g/L 葡萄糖、4 mM L-谷氨酰胺，不含丙酮酸钠）。血清添加：10% 优质胎牛血清 FBS。关键永生化因子：重组小鼠干扰素-gamma（IFN-gamma，终浓度：10 到 50 Units/mL；若缺失此成分，SV40LT 表达将大幅下调导致细胞提前终止分裂）。双抗：1% 青霉素-链霉素溶液（终浓度：100 U/mL 青霉素，100 ug/mL 链霉素）。

B 非允许期分化完全培养基（用于 37 ℃ 诱导成熟足细胞，禁止传代）基础培养基：高糖 DMEM。血清添加：10% 优质胎牛血清 FBS。核心剔除成分：完全不添加任何干扰素-gamma（IFN-gamma，即 0 U/mL）。双抗：1% 青霉素-链霉素溶液。

C 细胞冻存保护液标准配方：90% 允许期完全生长培养基 + 10% 细胞级二甲基亚砜 DMSO。高回收率配方：45% 基础 DMEM + 45% 优质胎牛血清 + 10% DMSO。

4 双轨环境控制参数

栽培 MPC-5 细胞时，必须严格在两台设置不同的独立避光孵箱中进行物理切换：

允许期增殖阶段（日常传代/扩增）：孵箱设定温度：33.0 ℃干扰素-gamma（IFN-gamma）：强阳性添加（10-50 U/mL）二氧化碳浓度：5%，加湿平衡常压空气相对湿度：大于 95%细胞命运走向：细胞对数分裂，维持未分化状态

非允许期分化阶段（功能学研究/损伤模型）：孵箱设定温度：37.0 ℃干扰素-gamma（IFN-gamma）：绝对缺失（0 U/mL）二氧化碳浓度：5%，加湿平衡常压空气相对湿度：大于 95%细胞命运走向：彻底退出细胞周期，长出交织足突，需 10-14 天

5 允许期贴壁传代操作规范传代临界汇合度：日常在 33 ℃ 栽培时，必须在细胞单层达到 75% - 85% 汇合度时进行传代。切勿让细胞在 33 ℃ 下 100% 汇合过密，否则会导致严重的接触抑制并诱发自发性衰亡。建议传代稀释比例：1:3 到 1:5（一般每 2-3 天传代一次）。

标准消化分散步骤：1 完全吸除旧培养基。2 使用无菌、不含钙镁离子的 PBS 缓冲液轻柔漂洗细胞单层 1 次。3 加入适量预热的 0.25% Trypsin - 0.02% EDTA 消化液（T25 瓶加入 1.0 mL）。4 将培养瓶置于 33 ℃ 孵箱中（或室温下）静置消化 1 到 2 分钟。MPC-5 对胰酶极为敏感，极易消化过度。全程需在倒置显微镜下密切监控。5 一旦发现单层细胞回缩变圆，立即加入等体积的含血清允许期完全培养基终止消化。6 轻柔吹打贴壁面，分散为单细胞悬液。在 300 x g 下离心 3 到 5 分钟。7 弃去上清液，加入新鲜的含 IFN-gamma 的允许期完全培养基重悬弹散细胞块，转接至新器皿中，放回 33 ℃ 孵箱继续扩增。

6 复苏与冻存后恢复流线1 提前在无菌 T25 培养瓶中注入 5.0 mL 含 IFN-gamma 的增殖完全培养基，置于 33 ℃、5% CO2 孵箱中进行气相平衡。2 从液氮罐中取出冷冻的 MPC-5 冻存管，立刻全量投入 37 ℃ 恒温水浴箱中快速摇晃。3 在 40 至 60 秒内融化完毕，直至管内只剩一丝小冰芯。4 用 75% 酒精消毒管壁后移入超净台，将融化后的胞悬液逐滴缓慢注入盛有 5.0 mL 预热增殖完全培养基的 15 mL 离心管中。5 300 x g 离心 3 到 5 分钟，彻底吸干含有毒 DMSO 的上清液。6 加入 1.5 mL 新鲜增殖完全培养基轻弹悬起细胞，随后全量接种到已预热平衡的 T25 瓶中，置于 33 ℃ 孵箱栽培。7 24 小时后，必须进行一次全量换液以清除残余的未贴壁细胞碎片。

7 超低温长期保存与质控规范生物安全级别：BSL-2。因细胞携带 SV40 病毒基因片段，必须在二级生物安全柜内进行无菌技术操作。长期封存温控：必须永久存放于液氮蒸汽或液相（-150 ℃ 至 -196 ℃）中。严禁在 -80 ℃ 普通冰箱中长期存放超过 1 个月，温度过高会导致其条件永生化温敏开关失灵，导致细胞复苏后无法增殖或丧失分化能力。分化能力定期复核：在大规模实验前，应抽取一管传代细胞切换至 37 ℃ 无 IFN-gamma 培养基中连续培养 10 天。若镜下细胞完全停止分裂、体积显著扩张并形成蛛网状相互交织的丝状突起，且免疫荧光检测 Synaptopodin 呈强阳性，方可确证细胞品系未发生表型漂移。

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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