BioVector? Hep-56.1D Murine Hepatocellular Carcinoma Cell Line / Hep-56.1D 小鼠肝細(xì)胞癌細(xì)胞株

一 產(chǎn)品基本信息與細(xì)胞生物學(xué)背景

• 細(xì)胞名稱：Hep-56.1D（亦常寫作 Hep56.1D 或 Hep 56.1D）。

• 物種與品系來源：小鼠（Mus musculus），源自經(jīng)典的C57BL/6（黑六）近交系小鼠。

• 組織源起與病理背景：

  Hep-56.1D 是一株源自 C57BL/6 小鼠原發(fā)性肝細(xì)胞癌（Hepatocellular Carcinoma, HCC）組織塊的惡性腫瘤細(xì)胞系。在生物醫(yī)學(xué)研究中，它是模擬人類肝癌發(fā)生、發(fā)展以及探索宿主免疫系統(tǒng)與腫瘤微環(huán)境相互作用最常用的同系（Syngeneic）小鼠腫瘤模型底盤之一。

• 核心表型與細(xì)胞生物學(xué)特征：

  • 形態(tài)學(xué)表現(xiàn)：貼壁生長。在倒置顯微鏡下，Hep-56.1D 細(xì)胞表現(xiàn)為極其典型的上皮樣（Epithelial-like）形態(tài)。細(xì)胞多呈多角形，胞質(zhì)致密，中央常含有明顯的單核或雙核。在細(xì)胞生長匯合、密集成片時(shí)，它們會(huì)緊密排列成類似鋪路石（Cobblestone-like）的單層圖案。

  • 同系移植（Syngeneic Grafting）優(yōu)勢(shì)：由于該細(xì)胞系完全來源于 C57BL/6 小鼠背景，當(dāng)將其重新接種（皮下注射或原位肝臟接種）回免疫健全的 C57BL/6 模式小鼠體內(nèi)時(shí)，宿主不會(huì)發(fā)生針對(duì)外源組織的免疫排斥反應(yīng)。這一特征使其在腫瘤免疫治療研究中的應(yīng)用價(jià)值遠(yuǎn)超常規(guī)的免疫缺陷人源異種移植（CDX）模型。

• 生物安全級(jí)別：1級(jí)（BSL-1）。

二 核心科研價(jià)值與腫瘤免疫醫(yī)學(xué)轉(zhuǎn)化應(yīng)用

Hep-56.1D 細(xì)胞株在現(xiàn)代癌癥生物學(xué)、免疫檢查點(diǎn)阻斷及藥物開發(fā)中扮演著核心角色：

1. 構(gòu)建免疫健全的小鼠原位/皮下肝癌模型：

   通過將 Hep-56.1D 細(xì)胞直接注射入 C57BL/6 小鼠的皮下或直接接種于肝左葉（原位移植，Orthotopic transplantation），可誘導(dǎo)出高成瘤率的肝癌病灶。由于宿主小鼠擁有完全健康的 T 細(xì)胞、B 細(xì)胞、NK 細(xì)胞以及巨噬細(xì)胞系統(tǒng)，該模型能夠完美重現(xiàn)實(shí)體瘤內(nèi)部復(fù)雜的免疫微環(huán)境（Tumor Microenvironment, TME）。

2. 抗腫瘤免疫檢查點(diǎn)藥物（如 Anti-PD-1/PD-L1, Anti-CTLA-4）體外與體內(nèi)藥效評(píng)估：

   用于測(cè)試各類新型單克隆抗體、溶瘤病毒、雙特異性抗體或小分子免疫調(diào)節(jié)劑在健全免疫背景下的抗癌靶向效能。它是評(píng)估人類免疫療法走向臨床前的關(guān)鍵臨床前試驗(yàn)（Pre-clinical trials）臺(tái)表。

3. 肝癌轉(zhuǎn)移機(jī)制與血管生成（Angiogenesis）探索：

   用于解構(gòu)肝癌細(xì)胞如何通過分泌血管內(nèi)皮生長因子（VEGF）誘導(dǎo)周圍微血管形成，以及如何逃逸宿主免疫監(jiān)視并向肺部、淋巴結(jié)發(fā)生遠(yuǎn)處轉(zhuǎn)移（Metastasis）的分子病理機(jī)制。

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

1. 專用培養(yǎng)基配置與生長環(huán)境

• 基礎(chǔ)培養(yǎng)基：高糖 DMEM（含 4.5 g/L 葡萄糖、L-谷氨酰胺及丙酮酸鈉）。

• 完全培養(yǎng)基配方：

  • 高糖 DMEM 基礎(chǔ)培養(yǎng)基

  • 加 10% 優(yōu)質(zhì)胎牛血清（FBS）

  • 加 1% 青霉素-鏈霉素雙抗溶液。

• 細(xì)胞解離液：0.25% Trypsin - 0.02% EDTA 消化液。

• 培養(yǎng)物理常數(shù)：標(biāo)準(zhǔn) 37 攝氏度，含 5% 二氧化碳（$CO_2$） 的恒溫高濕度飽和無菌孵箱。

2. 冷凍細(xì)胞復(fù)蘇步驟

1. 提前在生物安全柜中準(zhǔn)備好干凈的 T25 培養(yǎng)瓶，注入 5 - 6 mL 預(yù)熱至 37 ℃ 的完全培養(yǎng)基。

2. 從液氮罐或 -80 ℃ 冰箱中取出 Hep-56.1D 凍存管，立刻全量投入 37 ℃ 恒溫水浴箱中，快速用力搖晃以期在 1 分鐘內(nèi)令管內(nèi)冰塊完全融化（切忌慢速融化，否則冰晶重結(jié)晶會(huì)嚴(yán)重刺破細(xì)胞膜）。

3. 噴灑 75% 酒精消毒外壁，移入生物安全柜。

4. 吸取細(xì)胞懸液，緩慢滴加至盛有 4 - 5 mL 預(yù)熱完全培養(yǎng)基的 15 mL 離心管中，輕柔顛倒一次以稀釋 DMSO。

5. 以 1000 rpm（約 200 g）室溫溫和離心 5 分鐘，小心抽干含有 DMSO 的上清液。

6. 加入 1 mL 新鮮完全培養(yǎng)基，使用 P1000 移液槍輕柔吹打重懸細(xì)胞沉淀。

7. 將懸液接種至準(zhǔn)備好的 T25 瓶中，輕柔“十字形”搖勻。

8. 擰松瓶蓋（或使用透氣膜蓋），放入 37 ℃、5% $CO_2$ 孵箱中培養(yǎng)。

9. 24 小時(shí)后觀察貼壁匯合度，全量更換一次新鮮培養(yǎng)基，洗去殘留的死細(xì)胞碎屑。

3. 日常貼壁常規(guī)傳代操作

• 傳代時(shí)機(jī)：當(dāng)上皮樣鋪路石狀的細(xì)胞密集成片，匯合度（Confluency）達(dá)到 80% - 90% 時(shí)必須傳代。Hep-56.1D 屬于惡性腫瘤細(xì)胞，生長速度較快，切忌使其過密（100% 接觸抑制），否則會(huì)導(dǎo)致細(xì)胞局部堆疊、狀態(tài)老化。傳代頻率通常為每 2 - 3 天一次。

• 操作流程：

  1. 吸除舊培養(yǎng)基，使用無菌、無鈣鎂離子的 PBS 緩沖液輕輕漂洗細(xì)胞表面 1 次，徹底洗凈殘余血清（血清中的蛋白質(zhì)會(huì)強(qiáng)力抑制胰酶活性）。

  2. 加入適量 0.25% Trypsin-EDTA 消化液（T25 瓶常規(guī)加入 1 mL），使其均勻覆蓋細(xì)胞層，隨后放入 37 ℃ 孵箱中溫和消化。

  3. 鏡下動(dòng)態(tài)觀察：通常在 37 ℃ 下消化 1 - 3 分鐘。當(dāng)在倒置顯微鏡下觀察到多角形細(xì)胞連接變松、胞體收縮變圓、且輕敲培養(yǎng)瓶一側(cè)時(shí)有成片細(xì)胞自發(fā)向下滑落，說明消化達(dá)標(biāo)。

  4. 立刻向瓶?jī)?nèi)倒入 2 倍體積的含血清完全培養(yǎng)基，利用血清終止胰酶的剪切作用。

  5. 用移液槍反復(fù)輕柔沖洗瓶壁，將細(xì)胞完全洗脫下來，打散調(diào)理成均勻的單細(xì)胞懸液。

  6. 將懸液收集至離心管中，1000 rpm 離心 5 分鐘，棄去酶解上清。

  7. 加入新鮮完全培養(yǎng)基重懸沉淀，按照 1:3 至 1:6 的常規(guī)稀釋比例接種至新的培養(yǎng)瓶中，補(bǔ)充完全培養(yǎng)基，放回孵箱中繼續(xù)擴(kuò)增。

4. 動(dòng)物實(shí)驗(yàn)前成瘤細(xì)胞準(zhǔn)備（體內(nèi)接種質(zhì)控核心）

如果要將 Hep-56.1D 用于 C57BL/6 小鼠體內(nèi)（In vivo）接種構(gòu)建肝癌模型，必須嚴(yán)格遵循以下質(zhì)控規(guī)范以保證高成瘤率與實(shí)驗(yàn)可重復(fù)性：

1. 細(xì)胞狀態(tài)限制：必須選擇處于對(duì)數(shù)生長期（Log phase）最旺盛階段、匯合度約 75% - 80% 且未發(fā)生任何變異退化的健康細(xì)胞。細(xì)胞傳代代數(shù)（Passage number）建議控制在復(fù)蘇后的 10 代以內(nèi)，過高代數(shù)的癌癥細(xì)胞其體內(nèi)成瘤率和對(duì)免疫藥物的敏感性會(huì)發(fā)生自發(fā)飄移。

2. 純化與解離：消化時(shí)要確保細(xì)胞被充分打散成高純度的單細(xì)胞，嚴(yán)禁帶有多細(xì)胞聚集團(tuán)塊。

3. 洗滌與懸浮緩沖液：利用完全培養(yǎng)基終止消化離心后，必須用無菌基底 DMEM 培養(yǎng)基或無菌無重金屬生理鹽水（PBS）連續(xù)離心洗滌細(xì)胞 2 - 3 次，以徹底清除任何外源牛血清蛋白（FBS 原料殘留在小鼠體內(nèi)會(huì)引發(fā)強(qiáng)烈的非特異性異種異源免疫反應(yīng)，干擾真實(shí)的腫瘤免疫藥物評(píng)價(jià)）。

4. 接種密度與冷鏈控制：

   • 最終接種時(shí)，用無血清 DMEM 或 PBS（必要時(shí)可混入 1:1 體積的 Matrigel 基底膜基質(zhì)以促進(jìn)皮下成瘤）重懸細(xì)胞，調(diào)整密度至每 100 $\mu$L 含有 $1 \times 10^6$ 到 $5 \times 10^6$ 個(gè)細(xì)胞。

   • 全流程必須置于冰水混合物（0 - 4 ℃）中冷鏈保存，并在離心解離后的 1 小時(shí)內(nèi)完成全部小鼠的皮下或原位注射，以防細(xì)胞因在常溫下失巢擱置過久發(fā)生缺氧失活或沉降聚集。

5. 細(xì)胞長期保存標(biāo)準(zhǔn)

• 凍存液配方：90% 新鮮完全培養(yǎng)基（或 80% 基礎(chǔ) DMEM + 10% FBS）加 10% 優(yōu)質(zhì)分析級(jí)二甲基亞砜（DMSO）。亦可使用高效的商用無血清細(xì)胞凍存液。

• 冷凍降溫規(guī)范：

  1. 收集形態(tài)標(biāo)桿健康的對(duì)數(shù)生長期 Hep-56.1D 細(xì)胞，離心棄上清。

  2. 用配制好的冷凍液懸浮，調(diào)整細(xì)胞終密度至 每毫升 1,000,000 到 3,000,000 個(gè)細(xì)胞。

  3. 分裝入無菌凍存管，立刻移入標(biāo)準(zhǔn)程序降溫盒（如 Mr. Frosty）。

  4. 將降溫盒投入 -80 ℃ 超低溫冰箱中過夜梯度降溫（確保達(dá)到 $-1\text{ }^\circ\text{C/min}$ 的標(biāo)稱降溫速率）。

  5. 次日，迅速將凍存管轉(zhuǎn)移入液氮罐（-196 ℃）中鎖死長期保存。嚴(yán)禁在 -80 ℃ 冰箱中無限期存放，以防微小的溫度震蕩導(dǎo)致細(xì)胞內(nèi)部冰晶融化破壞質(zhì)膜結(jié)構(gòu)，引發(fā)后續(xù)復(fù)蘇時(shí)存活率的雪崩式下跌。

Part 2 English Section

I General Information and Cell Biological Background

• Cell Line Name: Hep-56.1D (also alternative standard nomenclature variations include Hep56.1D or Hep 56.1D).

• Organism and Inbred Strain Origin: Mouse (Mus musculus); explicitly derived from the foundational C57BL/6 (Black 6) inbred mammalian background.

• Tissue Extract and Pathological Framework:

  Hep-56.1D represents a solid malignant lineage isolated from a primary Hepatocellular Carcinoma (HCC) lesion induced in a C57BL/6 mouse specimen. Within cancer research, it serves as a highly reliable syngeneic murine liver cancer matrix leveraged to evaluate oncogenesis, tumor progression, and host immuno-oncology networks.

• Core Morphological Phenotype and Identity Characterization:

  • Morphological Structure: Adherent growth matrix. Under phase-contrast inverted profiling, Hep-56.1D exhibits a classical epithelial-like topology. Individual cellular frames present as distinctly polygonal units featuring compact cytoplasm and centralized single or prominent prominent binucleated nuclei. Upon reaching full confluency across the surface plane, they organize into a dense, interlocking "cobblestone-like" configuration.

  • Syngeneic Engraftment Execution Matrix: Because this cancerous cell line is derived from an un-mutated C57BL/6 background, delivering these cells back into immunocompetent C57BL/6 recipient mice avoids any foreign histocompatibility antigen rejection. This critical property enables investigators to study solid tumor dynamics in the presence of an intact host immune system, a feature unavailable in traditional human xenograft (CDX) models hosted in immunodeficient mice.

• Biosafety Threshold: Rated at Biosafety Level 1 (BSL-1).

II Strategic Research Value and Immuno-Oncology Applications

The Hep-56.1D platform functions as a critical preclinical engine for exploring tumor-host interplay and accelerating drug screening cascades:

1. Assembling Immunocompetent Syngeneic Orthotopic or Subcutaneous HCC Models:

   By injecting Hep-56.1D cells into either the subcutaneous flank or directly within the left hepatic lobe (orthotopic transplantation) of healthy C57BL/6 hosts, investigators can rapidly induce solid tumor pathology. Because the host animal retains fully operational T-cell, B-cell, NK-cell, and specialized myeloid macrophage systems, the resulting lesions accurately recreate the complex Tumor Microenvironment (TME) observed in clinical scenarios.

2. Preclinical Validation of Immune Checkpoint Inhibitors (e.g., Anti-PD-1/PD-L1, Anti-CTLA-4 Drugs):

   The line is widely standardly implemented to screen the therapeutic efficacy of novel checkpoint monoclonal antibodies, oncolytic viral therapies, bispecific antibody platforms, or small-molecule immunomodulators. It serves as a necessary preclinical proving ground before bringing novel immunology drug configurations into human clinical testing.

3. Deconstructing Metastasis Cascades and Angiogenesis Profiles:

   Leveraged to study how liver cancer targets exploit endogenous vascular networks by secreting vascular endothelial growth factors (VEGF), signaling neo-angiogenesis, bypassing host immune detection loops, and establishing distant secondary metastatic lesions inside pulmonary or lymph systems.

III Laboratory Thawing, Cultivation, Maintenance, and In Vivo Inoculation Protocols

1. Basal Media Formulation and Atmospheric Environment Variables

• Basal Medium Base: High-Glucose DMEM matrix (outfitted with 4.5 g/L D-Glucose, L-Glutamine, and Sodium Pyruvate).

• Complete Growth Matrix Formulation:

  • High-Glucose DMEM basal matrix

  • Supplemented with 10% premium Fetal Bovine Serum (FBS)

  • Fortified with 1% standard Penicillin-Streptomycin dual antibiotic cocktail.

• Cell Dissociation Solution: Standard 0.25% Trypsin - 0.02% EDTA mix.

• Physical Environmental Settings: Calibrate the incubator strictly to 37 °C, packed with 5% Carbon Dioxide ($CO_2$) under continuous humified saturation conditions.

3. Cryovial Thawing and Monolayer Recovery Protocol

1. Pre-warm a sterile T25 culture flask filled with 5 - 6 mL of complete growth medium to 37 °C inside the biosafety workstation.

2. Retrieve the Hep-56.1D cryovial from storage and submerge it instantly inside a 37 °C water bath. Agitate the vial continuously to melt the internal matrix within 60 seconds. Never allow slow warming; rapid melting prevents ice recrystallization from puncturing cellular membranes.

3. Mist the exterior shell with 75% ethanol before transfer into the biosafety clean room.

4. Draw up the liquid and transfer it slowly into a 15 mL conical tube containing 4 - 5 mL of pre-warmed complete growth medium to dilute the toxic DMSO footprint.

5. Centrifuge the suspension at 1000 rpm (~200 g) at room temperature for 5 minutes, then aspirate the chemical-laden supernatant.

6. Administer 1 mL of fresh complete growth medium onto the pellet and resuspend very gently using a P1000 micro-pipette.

7. Dispense the cells evenly into the prepared T25 flask, mix gently in a cross pattern, and place into the 37 °C, 5% $CO_2$ incubator.

8. Perform a complete medium replacement 24 hours post-thaw to clear residual dead cell debris.

3. Routine Adherent Subculturing and Passaging Routines

• Confluency Assessment Control: Subculturing mechanics must be executed when the interlocking epithelial sheets reach 80% - 90% confluency. Hep-56.1D cells are aggressive cancer models; allowing the monolayer to achieve 100% saturation triggers crowding and aging phenotypes. Expect a standard passaging schedule every 2 - 3 days.

• Passaging Execution Steps:

  1. Aspirate the spent growth fluid and wash the monolayer once with sterile, calcium/magnesium-free PBS to remove any residual serum proteins that could inactivate the trypsin.

  2. Administer an appropriate thin layer of 0.25% Trypsin-EDTA solution (typically 1 mL for a T25 format) and incubate at 37 °C.

  3. Microscopic Tracking: Maintain continuous visual monitoring under the microscope. Trypsinization typically completes within 1 - 3 minutes at 37 °C. The moment cells lose their polygonal junctions, round up, and slide off the surface upon gentle tapping, the process is complete.

  4. Immediately add 2 volumes of serum-fortified complete growth medium to stop the enzymatic cleavage.

  5. Gently pipette the suspension against the flask wall to break up clusters, resulting in a homogenous single-cell suspension.

  6. Spin the cells down at 1000 rpm for 5 minutes, discard the trypsin-laden fluid, and resuspend in fresh complete growth medium.

  7. Seed the cells into new flasks utilizing standard split ratios of 1:3 to 1:6, top off with complete medium, and return to the incubator.

4. Critical Pre-Inoculation Preparation for Animal Modeling (In Vivo Quality Metrics)

When preparing Hep-56.1D cells for syngeneic transplantation into C57BL/6 mice, strict adherence to these quality parameters is required to ensure consistent tumor take and reliable, reproducible experimental outcomes:

1. Passage Threshold and Longevity Control: Exclusively harvest cells from highly viable, log-phase cultures displaying approximately 75% - 80% confluency. To prevent genetic drift and altered drug responsiveness, ensure cells have been passaged fewer than 10 times post-thaw before mouse injection.

2. Homogenous Dissociation: Ensure complete enzymatic dissociation during passaging to yield a high-purity single-cell suspension. The injection of multi-cellular cell aggregate clumps will cause inconsistent tumor morphology or vascular embolisms during in vivo injection.

3. Serum Clearance Washing Sequence: Following trypsin deactivation, the cells must be washed 2 - 3 times via sequential centrifugation using serum-free basal DMEM or sterile saline (PBS). Residual bovine serum proteins introduced into C57BL/6 recipients will trigger non-specific xenogeneic immune responses, obscuring the evaluation of candidate immunotherapy drugs.

4. Cold-Chain Maintenance and Density Control:

   • Resuspend the final serum-free cell pellet in basal DMEM or PBS (a 1:1 mixture with Matrigel can be utilized for challenging subcutaneous setups) to a final injection concentration of $1 \times 10^6$ to $5 \times 10^6$ cells per 100 $\mu$L.

   • Keep the cell suspension on ice (0 - 4 °C) throughout the procedure. Execute all subcutaneous or orthotopic animal micro-injections within 1 hour post-harvest to prevent cell death from prolonged detachment or cell precipitation within the syringe barrel.

5. Long-Term Cryopreservation Parameters

• Cryoprotectant Preservation Formula: 90% fresh complete growth medium (or 80% basal DMEM + 10% premium FBS) packed with 10% analytical-grade Dimethyl Sulfoxide (DMSO).

• Controlled Gradient Freezing Protocol:

  1. Harvest healthy, log-phase Hep-56.1D monolayers showing robust epithelial integrity. Centrifuge and isolate the pellet.

  2. Resuspend the cells in the chilled cryoprotectant matrix to achieve a target cell density of 1,000,000 to 3,000,000 cells per milliliter.

  3. Transfer the solution into sterile cryovials and place them immediately into a standard controlled-rate cooling container (e.g., Mr. Frosty).

  4. Deposit the cooling container inside a -80 °C ultra-low freezer overnight to execute a steady cooling rate of -1 °C/minute.

  5. Within 24 hours, quickly transfer the vials into liquid nitrogen storage tanks (-196 °C) for long-term preservation. Do not store vials indefinitely inside a -80 °C freezer; minor temperature variations can compromise membrane integrity and lead to poor post-thaw viability.

BioVector NTCC質(zhì)粒載體菌株細(xì)胞蛋白抗體基因保藏中心

電話:400-800-2947

工作QQ/微信同號(hào):1843439339

網(wǎng)址http://www.nedfriskphoto.com