BioVector® AML12 小鼠正常肝细胞系说明书

BioVector® AML12 Mouse Normal Hepatocyte Cell Line Manual

第一部分 中文说明

一 产品基本信息与遗传学背景

• 细胞名称：AML12（全称 Alpha Mouse Liver 12）小鼠正常肝细胞系。

• 物种来源：小鼠（Mouse, Mus musculus），由 CD-1 正常品系（MT42转基因小鼠品系）的3个月龄雄性小鼠正常肝脏组织中分离出的肝细胞建立。

• 核心遗传学与分子标志：

  • hTGF-alpha 转基因背景：该细胞系是在小鼠金属硫蛋白-I（MT-I）启动子控制下，稳定整合并高表达人源转化生长因子 alpha（hTGF-alpha）转基因的小鼠肝细胞模型。由于高表达 hTGF-alpha，赋予了其在体外长期增殖和传代的能力。

  • 非致瘤性与高分化特性：虽然具备长久传代能力，但 AML12 细胞在免疫缺陷小鼠体内不具备致瘤性（不形成肿瘤）。在电子显微镜下，该细胞保留了极其典型的高分化正常肝细胞超微结构，如丰富的过氧化物酶体（Peroxisomes）和胆小管样结构（Bile canalicular-like structures）。

  • 功能性蛋白表达：该细胞能够持续表达和分泌高水平的正常肝特异性功能指标，包括小鼠血清白蛋白（Albumin）、alpha 1-抗胰蛋白酶（alpha 1 antitrypsin）以及转铁蛋白（Transferrin）；同时表达间隙连接蛋白 connexins 26 和 32，并仅含有乳酸脱氢酶（LDH）的同工酶5。

• 生长特性：贴壁生长（Adherent），呈现典型的上皮样（Epithelial-like）多角形形态。

• 生物安全级别：1级（BSL-1）。已通过严格的支原体、细菌及真菌检测。

二 核心科研价值与转化医学应用

AML12 是国际上公认最常用于模拟“正常肝细胞”的体外标准化非癌变模型之一：

1. 肝脏毒理学与药物代谢（Toxicology & Drug Metabolism）：广泛用于化学毒物、重金属、药物诱导性肝损伤（DILI）的体外高通量毒性筛选，是评价外源物质对正常肝细胞代谢干扰的经典对照组。

2. 非酒精性脂肪性肝病与代谢紊乱（NAFLD/NASH）：常通过在培养基中添加高浓度游离脂肪酸（如油酸 OA 或棕榈酸 PA）诱导其脂质过载，构建高度逼真的脂肪肝、脂毒性及线粒体/内质网应激模型。

3. 理想的转染宿主（Transfection Host）：细胞具有优良的核酸或病毒载体转染受体特性，适用于基因敲低、过表达等功能基因组学研究。

三 实验室细胞复苏、扩增传代与冷冻保存标准步骤

1. 完全培养基配置（Complete Growth Medium）

由于其特殊的转基因背景，AML12 需要严格配比的激素及微量元素支持以维持其正常的肝功能和稳态：

• 基础培养基：DMEM/F12（1:1混合） 基础培养基。

• 必须添加剂成分（按最终完全培养基浓度计）：

  • 10% 优质胎牛血清（FBS）

  • 0.005 mg/mL (5 microgram/mL) 胰岛素 (Insulin)

  • 0.005 mg/mL (5 microgram/mL) 转铁蛋白 (Transferrin)

  • 5 ng/mL 硒 (Selenium)

  • 注：以上三者通常可直接使用市售的 1% ITS 添加剂混合物代替。

  • 40 ng/mL 醋酸地塞米松 (Dexamethasone)。（地塞米松极不稳定，建议在每次使用前现用现加）。

  • 1% 双抗（Penicillin-Streptomycin）。

2. 细胞复苏（Thawing Protocol）

1. 将完全培养基置于 37摄氏度 水浴中预热。

2. 从液氮中取出 AML12 冻存管，立即投入 37摄氏度 水浴箱中急速摇晃。在 1分钟 左右令其完全融化。

3. 消毒冻存管外部，移入生物安全柜内，将细胞悬液吸出并置于含有 8-9 mL 预热完全培养基的 15 mL 离心管中，轻柔混匀以降低 DMSO 的渗透压冲击。

4. 以 200-300 x g 离心 3-5 分钟，小心弃去含有 DMSO 的上清液。

5. 加入 新鲜完全培养基 重悬细胞，接种于培养瓶中，置于 37摄氏度、5% CO2 孵箱中培养。

• 特异性注意事项：AML12 在复苏后的前几天里，可能会有部分活细胞在培养基中保持游离悬浮状态（这在正常肝细胞系中属于正常现象）。在换液时，建议通过轻微离心回收这些悬浮的活细胞并重新放回原培养瓶中，不要直接丢弃。

3. 细胞传代（Passaging / Subculture）

• 传代时机：当细胞密度达到约 80% 融合度时必须执行传代。若细胞长至过密（超过 80%），AML12 胞质内会自发出现大量的空泡（Vacuoles），这会影响其生长状态。

• 传代步骤：

  1. 吸除旧培养基，使用无菌 PBS（不含钙镁离子）轻轻洗涤细胞表面 1-2 次，彻底洗去残留的血清（血清含有胰酶抑制剂）。

  2. 加入适量 0.25% Trypsin-EDTA 消化液 或 Accutase，确保覆盖细胞层。

  3. 置于 37摄氏度 孵箱或室温下消化 2-5 分钟。在倒置显微镜下观察到大部分细胞变圆并开始自瓶壁脱落。

  4. 切勿剧烈拍打瓶身以防细胞成团。立即加入 2-3 倍体积的含血清完全培养基终止消化。

  5. 用移液管轻轻吹打使细胞完全分散，300 x g 离心 3 分钟弃上清。按照 1:4 至 1:6 的传代比例接种到新的培养容器中。

4. 细胞冷冻保存（Cryopreservation）

• 冻存液配方：55% 基础培养基 + 40% 优质胎牛血清（FBS） + 5% DMSO；或者直接使用 95% FBS + 5% DMSO。

• 冻存操作：冷冻保存必须选择处于对数生长旺盛期的细胞。消化计数后，将细胞密度调整至 1,000,000 cells/vial 或更高。分装后移入标准程序降温盒，置于 -80摄氏度 过夜，次日必须转移至 液氮（-196摄氏度） 中长期保存。

Part 2 English Section

I General Information and Genetic Architecture

• Cell Line Name: AML12 (Alpha Mouse Liver 12) Normal Mouse Hepatocyte Cell Line.

• Species Origin: Mouse (Mus musculus). Established from differentiated hepatocytes isolated from the normal liver of a 3-month-old male homozygous transgenic mouse of the CD-1 strain (line MT42).

• Core Genetic and Molecular Signatures:

  • hTGF-alpha Transgenic Construct: This cell line was established from a mouse harboring a transgene for human transforming growth factor alpha (hTGF-alpha) under the upstream transcriptional control of the mouse metallothionein-I (MT-I) promoter.Overexpression of hTGF-alpha drives its sustained long-term replication capacity in vitro.

  • Non-Tumorigenic and Well-Differentiated Status: Despite its prolonged subculturing life, AML12 is strictly non-tumorigenic and does not form colonies in soft agar or cause tumors in immunosuppressed mice.Ultrastructural analysis by electron microscopy displays distinctive normal hepatocyte configurations, including prominent peroxisomes and bile canalicular-like microstructures.

  • Functional Hepatic Expressions: AML12 cells maintain the continuous capacity to synthesize high baseline mRNA and protein levels for liver-specific serum elements (including albumin, alpha 1 antitrypsin, and transferrin), gap junction components (connexins 26 and 32), and display strictly isoenzyme 5 of lactate dehydrogenase.

• Growth Topology: Adherent growth mode. Displays classical differentiated epithelial-like cuboidal morphologies.

• Biosafety Matrix: Biosafety Level 1 (BSL-1).Rigorously pre-screened and negative for mycoplasma, bacterial, and fungal cross-contaminations.

II Strategic Research Value and Translational Fields

AML12 represents one of the world's most widely utilized, non-cancerous benchmarking platforms to mimic steady-state mouse liver tissues in vitro:

1. Hepatic Toxicology and Xenobiotic Metabolism: Extensively harnessed for high-throughput in vitro toxicity assays, drug-induced liver injury (DILI) assessment, and mapping chemical detoxification dynamics in normal cells.

2. Nonalcoholic Fatty Liver Disease (NAFLD/NASH) Simulation: Frequently loaded with high-concentration free fatty acids (e.g., oleic acid or palmitic acid) to systematically induce lipid overload, serving as a robust model to dissect steatosis, lipotoxicity, and mitochondrial/MAM homeostatic failure.

3. Optimized Gene Manipulation Host: Serves as an excellent transfection host for plasmids, siRNA, or lentiviral delivery systems targeting functional genomic pathways or epigenetic remodeling.

III Thawing, Proliferation, Passaging, and Cryopreservation Routines

1. Formulating the Specialized Complete Growth Medium

To actively preserve its hepatic functions, AML12 demands explicit supplements added to the basal matrix:

• Basal Medium: A 1:1 mixture of Dulbecco's Modified Eagle's Medium and Ham's F12 Medium (DMEM/F12).

• Mandatory Complete Media Supplements:

  • 10% Premium Fetal Bovine Serum (FBS).

  • 0.005 mg/mL (5 microgram/mL) Insulin.

  • 0.005 mg/mL (5 microgram/mL) Transferrin.

  • 5 ng/mL Selenium.

  • Note: The above three specific components can be easily substituted using a standard commercial 1% ITS Media Supplement.

  • 40 ng/mL Dexamethasone. Crucial: Dexamethasone degrades quickly at ambient parameters; it must be added freshly to the complete medium before use.

  • 1% Penicillin-Streptomycin cocktail.

2. Cryovial Thawing Routine

1. Pre-warm the formulated complete growth medium in a 37 degree Celsius water bath.

2. Retrieve the AML12 cryovial from liquid nitrogen and submerge it instantly into the 37 degree Celsius water bath with continuous gentle agitation.Thaw rapidly within 1 minute.

3. Decontaminate the exterior with 70% ethanol, move to a biosafety cabinet, and transfer the slurry into a 15 mL conical tube filled with 8-9 mL of pre-warmed complete growth medium.

4. Centrifuge at 200-300 x g for 3-5 minutes, aspirate the DMSO-contaminated supernatant, and gently resuspend the pellet with fresh complete medium.

5. Plate into standard culture vessels and incubate at 37 degree Celsius under a humidified 5% CO2 atmosphere.

• Lineage-Specific Note: It is entirely typical for a fraction of viable AML12 cells to remain floating in suspension during the initial days post-thaw.Do not discard them during media changes; spin down these floating cells gently and add them back into the main culture flask.

3. Subculturing and Cell Passaging Guide

• Confluency Threshold: Do not allow cultures to exceed 80% confluency. Once AML12 monolayers become over-confluent, the cells will rapidly generate large intracellular vacuoles, which impairs downstream growth metrics.Passaging should be performed at around 70-80% confluency.

• Step-by-Step Harvesting:

  1. Aspirate the spent culture medium and gently wash the layer 1-2 times with sterile PBS (without calcium and magnesium) to thoroughly remove remaining serum traces containing trypsin inhibitors.

  2. Dispense an adequate volume of 0.25% Trypsin-EDTA or Accutase onto the flask surface.

  3. Incubate at 37 degree Celsius or room temperature for 2-5 minutes until the cells round up and start to detach under microscope observation.

  4. Avoid mechanical agitation such as hitting or shaking the flask to prevent clumping. Immediately add 2-3 volumes of serum-fortified complete medium to neutralize the enzyme.

  5. Disperse the cells gently via pipetting, centrifuge at 300 x g for 3 minutes, and re-seed into new culture vessels at a standard split ratio of 1:4 to 1:6.

4. Cryopreservation Protocol

• Freezing Medium Matrix: 55% Basal Medium + 40% FBS + 5% DMSO, or 95% FBS + 5% DMSO.

• Freezing Routine: Harvest cells strictly during their log-growth phase (~75% confluent).After cell counting, adjust the final cell density to a minimum of 1,000,000 viable cells/vial.Aliquot into cryovials, immediately transfer into an isopropyl alcohol controlled-rate freezing container, and store at -80 degree Celsius overnight.Relocate the tubes into liquid nitrogen vapor phase (-196 degree Celsius) the next day for indefinite long-term preservation.

BioVector NTCC质粒载体菌株细胞蛋白抗体基因保藏中心

电话:400-800-2947

工作QQ/微信同号:1843439339

网址http://www.biovector.net