KCl-MOH1 BioVector® 人类髓系白血病细胞株

BioVector® KCl-MOH1 人类髓系白血病细胞株——产品技术说明书

1 产品基本信息与起源背景产品名称：BioVector® KCl-MOH1 人类髓系白血病细胞株常用别名：KCl-MOH1，KCL-MOH-1生物学来源：人类 Homo sapiens组织器官：血液 / 骨髓（源自人类慢性髓系白血病 CML 急变期患者）生长特性：悬浮型生长，常呈单个细胞或松散的微小细胞团块形核心科研价值：该细胞株是研究慢性髓系白血病急性变机制、BCR-ABL 酪氨酸激酶信号通路转导、造血干细胞定向分化障碍以及筛选新型靶向抗白血病药物（如二代、三代 BCR-ABL 抑制剂）的重要体外模型。

2 细胞生物学特征与核心标志物KCl-MOH1 细胞株作为经典的悬浮系白血病底盘，表现出高度特异性的分子特征：

核型与基因特征：细胞基因组中携带代表性的费城染色体 Philadelphia chromosome，即 t(9;22)(q34;q11) 染色体易位，导致 BCR-ABL 融合基因的高度表达以及下游激酶通路的持续激活。细胞形态观察：在倒置显微镜下，细胞呈现典型的原粒细胞或原始单核细胞样特征，胞体呈圆形或类圆形，大小相对均一。由于是完全悬浮生长，细胞不会附着于瓶壁，随着密度增加会自发形成松散的小多细胞聚集体。表面标志物谱系：稳定表达髓系相关分化抗原，如 CD13、CD33 以及早期造血标志物 CD34 呈现不同程度的阳性表达。

3 专用培养基配方规范

悬浮细胞对培养基的营养消耗和酸碱度变化极为敏感。为了维持 KCl-MOH1 细胞的高活力和对数期分裂速度，必须严格遵循以下完全生长培养基配方：

标准完全生长培养基配方基础培养基：高糖 RPMI-1640 培养基（含 2.0 g/L 碳酸氢钠、4.0 mM L-谷氨酰胺，不含丙酮酸钠）。血清添加：10% 至 15% 优质胎牛血清 FBS（建议首次复苏以及细胞活力低时使用 15% 胎牛血清，进入常规传代周期后可稳定在 10%）。双抗补剂：1% 青霉素-链霉素溶液（终浓度：100 U/mL 青霉素，100 ug/mL 链霉素）。

细胞冻存保护液标准配方：90% 完全生长培养基（RPMI-1640 + 15% FBS） + 10% 细胞级二甲基亚砜 DMSO。高回收率配方：45% 基础 RPMI-1640 + 45% 优质胎牛血清 + 10% DMSO。

4 物理环境控制参数孵育温度：37.0 ℃（恒温设置，温控误差需小于正负 0.5 ℃）气相环境：5% 二氧化碳（CO2），加湿平衡常压无菌空气相对湿度：大于 95% 恒湿环境物理生长状态：细胞完全独立或成团悬浮于培养基中，随着营养消耗，培养基颜色会迅速由红变黄。

5 悬浮传代操作规范与红线传代临界控制点：悬浮细胞不计算汇合度，而是严格通过细胞计数器监测细胞密度。常规维持密度必须控制在 2.0 x 10^5 cells/mL 到 1.0 x 10^6 cells/mL 之间。当细胞密度达到 1.0 x 10^6 cells/mL 时必须立即传代。切勿让细胞密度超过 2.0 x 10^6 cells/mL，否则会导致培养基严重酸化、细胞迅速爆发式绝死并释放大量有毒碎片。建议传代稀释比例：1:2 到 1:4（常规每 2-3 天进行一次半量换液或分瓶传代）。

标准传代步骤（半量分瓶法/全量离心法）：方法 A：半量分瓶法（适用于细胞状态良好、无大量碎片时）1 静置培养瓶，用移液枪直接吸出 50% 体积的饱含细胞的旧悬液并弃去。2 补充等体积的预热新鲜完全生长培养基，轻柔吹匀，分装回原瓶或新瓶中，放回 37 ℃ 孵箱。

方法 B：标准离心换液法（适用于日常传代或细胞碎片较多时）1 将培养瓶中的全部细胞悬液收集至无菌 15 mL 离心管中。2 在 200 x g 至 250 x g（低转速，约 800-1000 rpm）下低速离心 5 分钟。悬浮细胞较为脆弱，切忌使用高转速离心造成细胞机械碎裂。3 抽干并弃去陈旧的上清液，注意切勿触动底部的暗白色细胞沉淀。4 加入新鲜的预热完全生长培养基，用移液枪轻弹管底使细胞块完全散开，重新调整细胞计数密度至 3.0 x 10^5 cells/mL，接种回培养瓶中，放入 37 ℃ 孵箱。

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 完全生长培养基（建议使用 15% FBS 比例），置于 37 ℃、5% CO2 孵箱中进行气相与 pH 平衡。2 从液氮罐中取出冷冻的 KCl-MOH1 冻存管，立刻全量投入 37 ℃ 恒温水浴箱中快速水平摇晃。3 必须在 60 至 90 秒内使其完全融化，直至管内只剩最后一丝微小冰芯。4 用 75% 酒精消毒管壁后移入超净台，用移液枪吸出融化后的胞悬液，缓慢逐滴注入盛有 4.0 mL 预热完全培养基的 15 mL 离心管中，轻柔混匀。5 在 200 x g 下离心 5 分钟，彻底吸干含有毒 DMSO 的上清液。6 加入 2.0 mL 新鲜完全培养基轻弹悬起细胞，随后全量接种到已完成预热平衡的 T25 瓶中。悬浮细胞在复苏初期需要较高的局部细胞密度来释放生长因子。7 置于 37 ℃ 孵箱栽培。接种 24 小时后，对细胞进行计数。若发现死细胞碎片较多，必须再次通过 200 x g 低速离心清除碎片并更换全新培养基。

7 超低温长期保存与质控规范生物安全级别：BSL-1 或 BSL-2（取决于各地区对人类来源血液细胞的规范）。由于细胞源自人类临床样本，日常必须严格执行标准无菌防护屏障和医疗废弃物高压灭菌规范。长期封存温控：冻存管必须永久存放于液氮蒸汽或液相（-150 ℃ 至 -196 ℃）中。严禁在机械式 -80 ℃ 普通冰箱中存放超过 1 个月，温度波动会导致悬浮细胞复苏率出现灾难性断崖式下跌。支原体污染定期筛查：由于悬浮细胞对环境压力敏感度高，在开展大规模药物敏感性（IC50）检测前，应定期抽取细胞上清液进行 PCR 支原体检测。确保细胞在无支原体污染、活力大于 90% 的对数生长状态下进行科研实验，以保证实验数据的严谨性。

BioVector® KCl-MOH1 Human Myeloid Leukemia Cell Line Product Datasheet

1 Product and Identification General InformationProduct Name: BioVector® KCl-MOH1 Human Myeloid Leukemia Cell LineSynonyms: KCl-MOH1, KCL-MOH-1Organism Source: Human Homo sapiensTissue/Organ Site: Blood / Bone Marrow (Derived from a patient with chronic myeloid leukemia CML in blastic crisis)Growth Properties: Suspension growth, occurs as single cells or loose micro-clustersCore Research Significance: This cell line serves as a key in vitro model to investigate the mechanisms of chronic myeloid leukemia blast crisis, BCR-ABL tyrosine kinase signal transduction pathways, myeloid differentiation blockade, and to screen novel targeted anti-leukemic agents (such as second- and third-generation BCR-ABL inhibitors).

2 Cellular Properties and Biomarker ProfilesThe KCl-MOH1 cell line functions as a classic suspension myeloid chassis, demonstrating highly specific molecular hallmarks:

Karyotypic and Genetic Profiles: The genome carries the characteristic Philadelphia chromosome, resulting from a t(9;22)(q34;q11) reciprocal translocation. This rearrangement drives the strong expression of the constitutive active BCR-ABL fusion protein and its downstream kinase cascades.Microscopic Morphology: Under an inverted microscope, cells present typical myeloblastoid or monoblastoid features, exhibiting round or spherical profiles with relatively uniform dimensions. As a purely suspension culture, cells do not attach to the substrate and will spontaneously form loose, multi-cellular micro-aggregates as density increases.Surface Marker Antigen Spectrum: Exhibits stable expression of myeloid-associated differentiation antigens, including positive expressions of CD13, CD33, and variable intensities of the early hematopoietic stem cell marker CD34.

3 Dedicated Culturing Medium and Formulations

Suspension cells are exceptionally vulnerable to nutrient depletion and shifting pH dynamics. To guarantee peak viability and maintain log-phase division velocity, the complete growth medium must be formulated precisely as follows:

Standard Complete Growth Medium FormulationBasal Medium Base: High-Glucose RPMI-1640 Medium (containing 2.0 g/L sodium bicarbonate, 4.0 mM L-glutamine, without sodium pyruvate).Serum Supplement: 10% to 15% premium Fetal Bovine Serum FBS (15% FBS is recommended immediately post-thaw or during low-vitality periods; it can be stabilized at 10% during standard routine passaging).Antibiotic Supplement: 1% Penicillin-Streptomycin Solution (Final concentration: 100 U/mL Penicillin, 100 ug/mL Streptomycin).

Cryopreservation Freezing Medium FormulationStandard Freezing Formula: 90% Complete Growth Medium (RPMI-1640 with 15% FBS) + 10% Analytical Grade DMSO (Dimethyl Sulfoxide).Alternative High-Recovery Formula: 45% Basal RPMI-1640 + 45% Premium FBS + 10% DMSO.

4 Controlled Culture Environmental ConditionsIncubator Temperature: 37.0 ℃ (constant temperature, variations should be strictly restricted within plus or minus 0.5 ℃)Gas Composition (CO2): 5% Carbon Dioxide balanced with humidified atmospheric airRelative Humidity: Greater than 95% relative humidityPhysical Manifestation: Cells float completely detached or aggregated in suspension; the growth fluid turns rapidly from red to orange-yellow as nutrients are actively metabolized.

5 Suspension Subculturing Passaging Protocols and ThresholdsPassaging Critical Density: Suspension lines are managed by monitoring absolute cell count instead of confluency percentages. The operational cell density must be strictly maintained between 2.0 x 10^5 cells/mL and 1.0 x 10^6 cells/mL. Subculturing must be performed immediately when the saturation threshold reaches 1.0 x 10^6 cells/mL. Never allow densities to surpass 2.0 x 10^6 cells/mL, which triggers intense medium acidification, rapid cell autolysis, and heavy accumulation of toxic debris.Subcultivation Splitting Ratio: 1:2 to 1:4 (typically passaged or replenished with fresh fluid every 2 to 3 days).

Step-by-Step Dissociation Methods:Method A: Split-Volume Passaging (For high-viability cultures with minimal background debris)1 Allow the suspension to settle slightly, or pipette gently to homogenize, then directly aspirate 50% of the total volume and discard it.2 Replenish with an equal volume of pre-warmed fresh complete growth medium, mix gently, redistribute into vessels, and return to the 37 ℃ incubator.

Method B: Standard Centrifugation Medium Replacement (For routine passaging or high debris accumulation)1 Collect the entire cell-containing growth fluid into a sterile 15 mL conical tube.2 Centrifuge at 200 x g to 250 x g (low speed, approx 800-1000 rpm) for 5 minutes. Suspension cells are fragile; excessive gravitational force will rupture viable cell membranes.3 Carefully aspirate and discard the spent supernatant without disturbing the off-white cell pellet at the bottom.4 Add pre-warmed fresh complete growth medium, gently tap the tube to completely break up the pellet, adjust the seeding density back to approx 3.0 x 10^5 cells/mL, and transfer into culture vessels.

6 Thawing and Post-Cryo Recovery Workflow1 Pre-warm 5.0 mL of complete growth medium supplemented with 15% FBS in a sterile T25 flask, and equilibrate inside the 37 ℃, 5% CO2 incubator to stabilize gas phase and pH value.2 Retrieve the KCl-MOH1 cryovial from liquid nitrogen storage.3 Plunge the vial instantly into a 37 ℃ water bath and rapidly shake horizontally. Achieve complete thawing within 60 to 90 seconds until only a tiny ice core remains.4 Sanitize the vial exterior with 75% ethanol before transferring it into the biosafety cabinet.5 Transfer the thawed cell solution slowly and dropwise into a 15 mL sterile conical tube containing 4.0 mL of pre-warmed complete growth medium.6 Centrifuge at 200 x g for 5 minutes to form a clean cell pellet.7 Aspirate the supernatant completely to eliminate toxic DMSO residues.8 Add 2.0 mL of fresh complete medium, gently tap the tube to loosen the pellet, and seed the entire suspension into the pre-stabilized T25 flask. Suspension lines require higher local cell densities during the initial post-thaw hours to exchange auto-regulatory growth factors.9 Incubate at 37 ℃, 5% CO2. Perform a cell count and viability assay 24 hours post-thaw; if dead debris is prominent, execute a low-speed centrifugation step to isolate viable cells and replenish with fresh growth fluid.

7 Biosafety, Storage and Quality Control StandardsBiosafety Level: BSL-1 or BSL-2 (subject to national/institutional definitions regarding human blood-derived lines). Standard tissue culture protective equipment and medical waste autoclaving protocols must be strictly followed.Long-Term Storage Parameters: Cryovials must be stored permanently within the vapor or liquid phase of liquid nitrogen (-150 ℃ to -196 ℃). Short- or long-term storage in mechanical -80 ℃ freezers beyond 1 month is prohibited, as it causes irreversible damage to suspension line membranes and drops post-thaw recovery rates.Mycoplasma Cleanness Screening QC: Because suspension lines exhibit high physiological sensitivity to silent contaminants, regular PCR mycoplasma screenings should be performed before major signaling or drug-screening (IC50) campaigns. Ensure that cultures demonstrate a baseline viability greater than 90% in active log-phase to guarantee the reproducible validity of your scientific data.

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