BioVector? KCl-MOH1 人類(lèi)髓系白血病細(xì)胞株——產(chǎn)品技術(shù)說(shuō)明書(shū)

1 產(chǎn)品基本信息與起源背景產(chǎn)品名稱(chēng)：BioVector? KCl-MOH1 人類(lèi)髓系白血病細(xì)胞株常用別名：KCl-MOH1，KCL-MOH-1生物學(xué)來(lái)源：人類(lèi) Homo sapiens組織器官：血液 / 骨髓（源自人類(lèi)慢性髓系白血病 CML 急變期患者）生長(zhǎng)特性：懸浮型生長(zhǎng)，常呈單個(gè)細(xì)胞或松散的微小細(xì)胞團(tuán)塊形核心科研價(jià)值：該細(xì)胞株是研究慢性髓系白血病急性變機(jī)制、BCR-ABL 酪氨酸激酶信號(hào)通路轉(zhuǎn)導(dǎo)、造血干細(xì)胞定向分化障礙以及篩選新型靶向抗白血病藥物（如二代、三代 BCR-ABL 抑制劑）的重要體外模型。

2 細(xì)胞生物學(xué)特征與核心標(biāo)志物KCl-MOH1 細(xì)胞株作為經(jīng)典的懸浮系白血病底盤(pán)，表現(xiàn)出高度特異性的分子特征：

核型與基因特征：細(xì)胞基因組中攜帶代表性的費(fèi)城染色體 Philadelphia chromosome，即 t(9;22)(q34;q11) 染色體易位，導(dǎo)致 BCR-ABL 融合基因的高度表達(dá)以及下游激酶通路的持續(xù)激活。細(xì)胞形態(tài)觀察：在倒置顯微鏡下，細(xì)胞呈現(xiàn)典型的原粒細(xì)胞或原始單核細(xì)胞樣特征，胞體呈圓形或類(lèi)圓形，大小相對(duì)均一。由于是完全懸浮生長(zhǎng)，細(xì)胞不會(huì)附著于瓶壁，隨著密度增加會(huì)自發(fā)形成松散的小多細(xì)胞聚集體。表面標(biāo)志物譜系：穩(wěn)定表達(dá)髓系相關(guān)分化抗原，如 CD13、CD33 以及早期造血標(biāo)志物 CD34 呈現(xiàn)不同程度的陽(yáng)性表達(dá)。

3 專(zhuān)用培養(yǎng)基配方規(guī)范

懸浮細(xì)胞對(duì)培養(yǎng)基的營(yíng)養(yǎng)消耗和酸堿度變化極為敏感。為了維持 KCl-MOH1 細(xì)胞的高活力和對(duì)數(shù)期分裂速度，必須嚴(yán)格遵循以下完全生長(zhǎng)培養(yǎng)基配方：

標(biāo)準(zhǔn)完全生長(zhǎng)培養(yǎng)基配方基礎(chǔ)培養(yǎng)基：高糖 RPMI-1640 培養(yǎng)基（含 2.0 g/L 碳酸氫鈉、4.0 mM L-谷氨酰胺，不含丙酮酸鈉）。血清添加：10% 至 15% 優(yōu)質(zhì)胎牛血清 FBS（建議首次復(fù)蘇以及細(xì)胞活力低時(shí)使用 15% 胎牛血清，進(jìn)入常規(guī)傳代周期后可穩(wěn)定在 10%）。雙抗補(bǔ)劑：1% 青霉素-鏈霉素溶液（終濃度：100 U/mL 青霉素，100 ug/mL 鏈霉素）。

細(xì)胞凍存保護(hù)液標(biāo)準(zhǔn)配方：90% 完全生長(zhǎng)培養(yǎng)基（RPMI-1640 + 15% FBS） + 10% 細(xì)胞級(jí)二甲基亞砜 DMSO。高回收率配方：45% 基礎(chǔ) RPMI-1640 + 45% 優(yōu)質(zhì)胎牛血清 + 10% DMSO。

4 物理環(huán)境控制參數(shù)孵育溫度：37.0 ℃（恒溫設(shè)置，溫控誤差需小于正負(fù) 0.5 ℃）氣相環(huán)境：5% 二氧化碳（CO2），加濕平衡常壓無(wú)菌空氣相對(duì)濕度：大于 95% 恒濕環(huán)境物理生長(zhǎng)狀態(tài)：細(xì)胞完全獨(dú)立或成團(tuán)懸浮于培養(yǎng)基中，隨著營(yíng)養(yǎng)消耗，培養(yǎng)基顏色會(huì)迅速由紅變黃。

5 懸浮傳代操作規(guī)范與紅線(xiàn)傳代臨界控制點(diǎn)：懸浮細(xì)胞不計(jì)算匯合度，而是嚴(yán)格通過(guò)細(xì)胞計(jì)數(shù)器監(jiān)測(cè)細(xì)胞密度。常規(guī)維持密度必須控制在 2.0 x 10^5 cells/mL 到 1.0 x 10^6 cells/mL 之間。當(dāng)細(xì)胞密度達(dá)到 1.0 x 10^6 cells/mL 時(shí)必須立即傳代。切勿讓細(xì)胞密度超過(guò) 2.0 x 10^6 cells/mL，否則會(huì)導(dǎo)致培養(yǎng)基嚴(yán)重酸化、細(xì)胞迅速爆發(fā)式絕死并釋放大量有毒碎片。建議傳代稀釋比例：1:2 到 1:4（常規(guī)每 2-3 天進(jìn)行一次半量換液或分瓶傳代）。

標(biāo)準(zhǔn)傳代步驟（半量分瓶法/全量離心法）：方法 A：半量分瓶法（適用于細(xì)胞狀態(tài)良好、無(wú)大量碎片時(shí)）1 靜置培養(yǎng)瓶，用移液槍直接吸出 50% 體積的飽含細(xì)胞的舊懸液并棄去。2 補(bǔ)充等體積的預(yù)熱新鮮完全生長(zhǎng)培養(yǎng)基，輕柔吹勻，分裝回原瓶或新瓶中，放回 37 ℃ 孵箱。

方法 B：標(biāo)準(zhǔn)離心換液法（適用于日常傳代或細(xì)胞碎片較多時(shí)）1 將培養(yǎng)瓶中的全部細(xì)胞懸液收集至無(wú)菌 15 mL 離心管中。2 在 200 x g 至 250 x g（低轉(zhuǎn)速，約 800-1000 rpm）下低速離心 5 分鐘。懸浮細(xì)胞較為脆弱，切忌使用高轉(zhuǎn)速離心造成細(xì)胞機(jī)械碎裂。3 抽干并棄去陳舊的上清液，注意切勿觸動(dòng)底部的暗白色細(xì)胞沉淀。4 加入新鮮的預(yù)熱完全生長(zhǎng)培養(yǎng)基，用移液槍輕彈管底使細(xì)胞塊完全散開(kāi)，重新調(diào)整細(xì)胞計(jì)數(shù)密度至 3.0 x 10^5 cells/mL，接種回培養(yǎng)瓶中，放入 37 ℃ 孵箱。

6 復(fù)蘇與凍存后恢復(fù)流線(xiàn)1 提前在無(wú)菌 T25 培養(yǎng)瓶中注入 5.0 mL 完全生長(zhǎng)培養(yǎng)基（建議使用 15% FBS 比例），置于 37 ℃、5% CO2 孵箱中進(jìn)行氣相與 pH 平衡。2 從液氮罐中取出冷凍的 KCl-MOH1 凍存管，立刻全量投入 37 ℃ 恒溫水浴箱中快速水平搖晃。3 必須在 60 至 90 秒內(nèi)使其完全融化，直至管內(nèi)只剩最后一絲微小冰芯。4 用 75% 酒精消毒管壁后移入超凈臺(tái)，用移液槍吸出融化后的胞懸液，緩慢逐滴注入盛有 4.0 mL 預(yù)熱完全培養(yǎng)基的 15 mL 離心管中，輕柔混勻。5 在 200 x g 下離心 5 分鐘，徹底吸干含有毒 DMSO 的上清液。6 加入 2.0 mL 新鮮完全培養(yǎng)基輕彈懸起細(xì)胞，隨后全量接種到已完成預(yù)熱平衡的 T25 瓶中。懸浮細(xì)胞在復(fù)蘇初期需要較高的局部細(xì)胞密度來(lái)釋放生長(zhǎng)因子。7 置于 37 ℃ 孵箱栽培。接種 24 小時(shí)后，對(duì)細(xì)胞進(jìn)行計(jì)數(shù)。若發(fā)現(xiàn)死細(xì)胞碎片較多，必須再次通過(guò) 200 x g 低速離心清除碎片并更換全新培養(yǎng)基。

7 超低溫長(zhǎng)期保存與質(zhì)控規(guī)范生物安全級(jí)別：BSL-1 或 BSL-2（取決于各地區(qū)對(duì)人類(lèi)來(lái)源血液細(xì)胞的規(guī)范）。由于細(xì)胞源自人類(lèi)臨床樣本，日常必須嚴(yán)格執(zhí)行標(biāo)準(zhǔn)無(wú)菌防護(hù)屏障和醫(yī)療廢棄物高壓滅菌規(guī)范。長(zhǎng)期封存溫控：凍存管必須永久存放于液氮蒸汽或液相（-150 ℃ 至 -196 ℃）中。嚴(yán)禁在機(jī)械式 -80 ℃ 普通冰箱中存放超過(guò) 1 個(gè)月，溫度波動(dòng)會(huì)導(dǎo)致懸浮細(xì)胞復(fù)蘇率出現(xiàn)災(zāi)難性斷崖式下跌。支原體污染定期篩查：由于懸浮細(xì)胞對(duì)環(huán)境壓力敏感度高，在開(kāi)展大規(guī)模藥物敏感性（IC50）檢測(cè)前，應(yīng)定期抽取細(xì)胞上清液進(jìn)行 PCR 支原體檢測(cè)。確保細(xì)胞在無(wú)支原體污染、活力大于 90% 的對(duì)數(shù)生長(zhǎng)狀態(tài)下進(jìn)行科研實(shí)驗(yàn)，以保證實(shí)驗(yàn)數(shù)據(jù)的嚴(yán)謹(jǐn)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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