BioVector? AML12 小鼠正常肝細胞系說明書

BioVector? AML12 Mouse Normal Hepatocyte Cell Line Manual

第一部分 中文說明

一 產(chǎn)品基本信息與遺傳學背景

• 細胞名稱：AML12（全稱 Alpha Mouse Liver 12）小鼠正常肝細胞系。

• 物種來源：小鼠（Mouse, Mus musculus），由 CD-1 正常品系（MT42轉基因小鼠品系）的3個月齡雄性小鼠正常肝臟組織中分離出的肝細胞建立。

• 核心遺傳學與分子標志：

  • hTGF-alpha 轉基因背景：該細胞系是在小鼠金屬硫蛋白-I（MT-I）啟動子控制下，穩(wěn)定整合并高表達人源轉化生長因子 alpha（hTGF-alpha）轉基因的小鼠肝細胞模型。由于高表達 hTGF-alpha，賦予了其在體外長期增殖和傳代的能力。

  • 非致瘤性與高分化特性：雖然具備長久傳代能力，但 AML12 細胞在免疫缺陷小鼠體內不具備致瘤性（不形成腫瘤）。在電子顯微鏡下，該細胞保留了極其典型的高分化正常肝細胞超微結構，如豐富的過氧化物酶體（Peroxisomes）和膽小管樣結構（Bile canalicular-like structures）。

  • 功能性蛋白表達：該細胞能夠持續(xù)表達和分泌高水平的正常肝特異性功能指標，包括小鼠血清白蛋白（Albumin）、alpha 1-抗胰蛋白酶（alpha 1 antitrypsin）以及轉鐵蛋白（Transferrin）；同時表達間隙連接蛋白 connexins 26 和 32，并僅含有乳酸脫氫酶（LDH）的同工酶5。

• 生長特性：貼壁生長（Adherent），呈現(xiàn)典型的上皮樣（Epithelial-like）多角形形態(tài)。

• 生物安全級別：1級（BSL-1）。已通過嚴格的支原體、細菌及真菌檢測。

二 核心科研價值與轉化醫(yī)學應用

AML12 是國際上公認最常用于模擬“正常肝細胞”的體外標準化非癌變模型之一：

1. 肝臟毒理學與藥物代謝（Toxicology & Drug Metabolism）：廣泛用于化學毒物、重金屬、藥物誘導性肝損傷（DILI）的體外高通量毒性篩選，是評價外源物質對正常肝細胞代謝干擾的經(jīng)典對照組。

2. 非酒精性脂肪性肝病與代謝紊亂（NAFLD/NASH）：常通過在培養(yǎng)基中添加高濃度游離脂肪酸（如油酸 OA 或棕櫚酸 PA）誘導其脂質過載，構建高度逼真的脂肪肝、脂毒性及線粒體/內質網(wǎng)應激模型。

3. 理想的轉染宿主（Transfection Host）：細胞具有優(yōu)良的核酸或病毒載體轉染受體特性，適用于基因敲低、過表達等功能基因組學研究。

三 實驗室細胞復蘇、擴增傳代與冷凍保存標準步驟

1. 完全培養(yǎng)基配置（Complete Growth Medium）

由于其特殊的轉基因背景，AML12 需要嚴格配比的激素及微量元素支持以維持其正常的肝功能和穩(wěn)態(tài)：

• 基礎培養(yǎng)基：DMEM/F12（1:1混合） 基礎培養(yǎng)基。

• 必須添加劑成分（按最終完全培養(yǎng)基濃度計）：

  • 10% 優(yōu)質胎牛血清（FBS）

  • 0.005 mg/mL (5 microgram/mL) 胰島素 (Insulin)

  • 0.005 mg/mL (5 microgram/mL) 轉鐵蛋白 (Transferrin)

  • 5 ng/mL 硒 (Selenium)

  • 注：以上三者通常可直接使用市售的 1% ITS 添加劑混合物代替。

  • 40 ng/mL 醋酸地塞米松 (Dexamethasone)。（地塞米松極不穩(wěn)定，建議在每次使用前現(xiàn)用現(xiàn)加）。

  • 1% 雙抗（Penicillin-Streptomycin）。

2. 細胞復蘇（Thawing Protocol）

1. 將完全培養(yǎng)基置于 37攝氏度 水浴中預熱。

2. 從液氮中取出 AML12 凍存管，立即投入 37攝氏度 水浴箱中急速搖晃。在 1分鐘 左右令其完全融化。

3. 消毒凍存管外部，移入生物安全柜內，將細胞懸液吸出并置于含有 8-9 mL 預熱完全培養(yǎng)基的 15 mL 離心管中，輕柔混勻以降低 DMSO 的滲透壓沖擊。

4. 以 200-300 x g 離心 3-5 分鐘，小心棄去含有 DMSO 的上清液。

5. 加入 新鮮完全培養(yǎng)基 重懸細胞，接種于培養(yǎng)瓶中，置于 37攝氏度、5% CO2 孵箱中培養(yǎng)。

• 特異性注意事項：AML12 在復蘇后的前幾天里，可能會有部分活細胞在培養(yǎng)基中保持游離懸浮狀態(tài)（這在正常肝細胞系中屬于正常現(xiàn)象）。在換液時，建議通過輕微離心回收這些懸浮的活細胞并重新放回原培養(yǎng)瓶中，不要直接丟棄。

3. 細胞傳代（Passaging / Subculture）

• 傳代時機：當細胞密度達到約 80% 融合度時必須執(zhí)行傳代。若細胞長至過密（超過 80%），AML12 胞質內會自發(fā)出現(xiàn)大量的空泡（Vacuoles），這會影響其生長狀態(tài)。

• 傳代步驟：

  1. 吸除舊培養(yǎng)基，使用無菌 PBS（不含鈣鎂離子）輕輕洗滌細胞表面 1-2 次，徹底洗去殘留的血清（血清含有胰酶抑制劑）。

  2. 加入適量 0.25% Trypsin-EDTA 消化液 或 Accutase，確保覆蓋細胞層。

  3. 置于 37攝氏度 孵箱或室溫下消化 2-5 分鐘。在倒置顯微鏡下觀察到大部分細胞變圓并開始自瓶壁脫落。

  4. 切勿劇烈拍打瓶身以防細胞成團。立即加入 2-3 倍體積的含血清完全培養(yǎng)基終止消化。

  5. 用移液管輕輕吹打使細胞完全分散，300 x g 離心 3 分鐘棄上清。按照 1:4 至 1:6 的傳代比例接種到新的培養(yǎng)容器中。

4. 細胞冷凍保存（Cryopreservation）

• 凍存液配方：55% 基礎培養(yǎng)基 + 40% 優(yōu)質胎牛血清（FBS） + 5% DMSO；或者直接使用 95% FBS + 5% DMSO。

• 凍存操作：冷凍保存必須選擇處于對數(shù)生長旺盛期的細胞。消化計數(shù)后，將細胞密度調整至 1,000,000 cells/vial 或更高。分裝后移入標準程序降溫盒，置于 -80攝氏度 過夜，次日必須轉移至 液氮（-196攝氏度） 中長期保存。

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.

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