BioVector? SW48 KRAS (G12D/+) 人類同基因型結(jié)直腸癌細(xì)胞株——產(chǎn)品技術(shù)說明書 (Product Datasheet)

1. 產(chǎn)品基本信息與鑒定譜系 (Product & Identification General Information)

• 產(chǎn)品名稱：BioVector? SW48 KRAS (G12D/+) 人類同基因型結(jié)直腸癌細(xì)胞株

• 國際數(shù)據(jù)庫登錄號：Horizon Discovery (HD 103-011) / Cellosaurus登錄號：CVCL_LC92

• 生物學(xué)來源：人類（Homo sapiens）

• 組織來源：大腸/結(jié)腸

• 疾病背景：結(jié)直腸腺癌（Dukes' C 期，IV 級）

• 性別：女性

• 供體年齡：83歲

• 生長特性：貼壁型上皮細(xì)胞（體外傾向于形成緊密的鋪路石狀克隆團(tuán)塊）

2. 基因組架構(gòu)與突變特征 (Genetic Architecture & Mutation Profile)

• 同基因修飾策略：利用高精度基因編輯技術(shù)，在天然野生型（WT）SW48 細(xì)胞的內(nèi)源性 KRAS 位點的一個等位基因上精確引入了激活型點突變。

• 目標(biāo)基因型：雜合子基因敲入（KRAS G12D/+）

• 突變分子細(xì)節(jié)：編碼區(qū)：c.35G>A（第2外顯子，第12密碼子） $\rightarrow$ 蛋白質(zhì)水平：p.Gly12Asp（甘氨酸被天冬氨酸取代）

• 背景突變（承襲自親本株）：

  • 純合型 ACVR2A (p.Lys437Argfs*5)

  • 純合型 TGFBR2 (p.Lys128Serfs*35)

  • 雜合型 CTNNB1 (p.Ser33Tyr)

  • 維持野生型（WT）狀態(tài)的基因：APC、TP53、PTEN、BRAF。

• 微衛(wèi)星狀態(tài)：高突變/微衛(wèi)星不穩(wěn)定（MSI-H）

3. 常規(guī)培養(yǎng)基配方規(guī)范 (Routine Culturing Medium & Formulations)

為確保該株細(xì)胞的基因型與表型穩(wěn)態(tài)，防止細(xì)胞自發(fā)成團(tuán)脫落，必須嚴(yán)格執(zhí)行以下優(yōu)化的培養(yǎng)基配置：

標(biāo)準(zhǔn)完全生長培養(yǎng)基 (Standard Complete Growth Medium)

• 基礎(chǔ)培養(yǎng)基：McCoy's 5A 改進(jìn)型培養(yǎng)基（含 L-谷氨酰胺及高糖）。

  • 經(jīng)質(zhì)控驗證的替代培養(yǎng)基：含 4 mM L-谷氨酰胺的高糖 DMEM 培養(yǎng)基。

• 血清添加：10% 優(yōu)質(zhì)胎牛血清（FBS）（日常增殖無需進(jìn)行熱滅活）。

• 抗生素混劑：1% 標(biāo)準(zhǔn)青霉素-鏈霉素雙抗溶液（終濃度：100 units/mL 青霉素，100 $\mu$g/mL 鏈霉素）。

細(xì)胞凍存保護(hù)液 (Cryopreservation Medium)

• 標(biāo)準(zhǔn)配方：90% 完全生長培養(yǎng)基（McCoy's 5A + 10% FBS） + 10% 細(xì)胞級二甲基亞砜（DMSO）。

• 高回收率替代配方：45% 基礎(chǔ) McCoy's 5A 培養(yǎng)基 + 45% 優(yōu)質(zhì)胎牛血清 + 10% DMSO。

4. 物理環(huán)境控制參數(shù) (Controlled Culture Environmental Conditions)

• 孵育溫度：37.0 ℃（恒溫，嚴(yán)禁劇烈波動）。

• 氣相環(huán)境：5% 二氧化碳 ($CO_2$)，平衡常壓無菌空氣。

• 相對濕度：$\gt 95\%$（高濕環(huán)境，嚴(yán)防培養(yǎng)基微量蒸餾濃縮）。

• 特別注意：雖然原代 ATCC 親本野生型 SW48 細(xì)胞具有在無 $CO_2$ 環(huán)境下使用 Leibovitz's L-15 培養(yǎng)基生長的全空氣適應(yīng)能力，但本款基因編輯純化后的同基因 variant 突變株，必須在 5% $CO_2$ 的 McCoy's 5A 緩沖體系下栽培，以維持其最優(yōu)的代謝與增殖速率。

5. 貼壁傳代操作紅線 (Subculturing Protocol & Metrics)

• 標(biāo)準(zhǔn)傳代匯合度：70% 到 80% 匯合度。

• 核心操作紅線：嚴(yán)禁讓細(xì)胞長滿至 100% 匯合度。 SW48 具有極強(qiáng)的密集重疊生長傾向。一旦長滿，細(xì)胞會迅速向上堆積形成復(fù)雜的三維重疊塊，此時胰酶極難滲透，極易導(dǎo)致消化不均、細(xì)胞大規(guī)模成團(tuán)損傷以及復(fù)蘇后活力斷崖式下跌。

• 建議傳代稀釋比例：1:3 到 1:5（依接種密度動態(tài)調(diào)節(jié)）。

• 全量換液頻率：每周 2 至 3 次。

標(biāo)準(zhǔn)傳代消化步驟：

1. 完全吸除培養(yǎng)瓶中的陳舊培養(yǎng)基。

2. 使用不含鈣、鎂離子的無菌 PBS 緩沖液輕柔洗滌細(xì)胞單層 1 次，以徹底清除殘余血清（血清中的抗胰蛋白酶會強(qiáng)烈抑制消化液活性）。

3. 視培養(yǎng)器皿面積加入適量預(yù)熱的 0.25% Trypsin - 0.02% EDTA 消化液（T25 瓶通常加入 1.0 mL；T75 瓶加入 2.0–3.0 mL）。

4. 將培養(yǎng)瓶置于 37 ℃ 孵箱中消化 3 到 5 分鐘。全程通過倒置顯微鏡觀察。

5. 操作禁忌：在等待細(xì)胞脫落期間，切勿大力拍打或劇烈搖晃培養(yǎng)瓶，否則極易誘發(fā) SW48 自發(fā)性拉絲結(jié)塊。一旦鏡下觀察到大部分細(xì)胞回縮變圓、單層出現(xiàn)縫隙、輕 tilt 瓶身見細(xì)胞呈流沙狀整體下滑，立即加入等體積的含血清完全生長培養(yǎng)基終止消化。

6. 用移液槍順著貼壁面輕柔吹打，將細(xì)胞團(tuán)塊徹底分散為均一的單細(xì)胞懸液。

7. 將懸液收集至離心管中，在 300 × g（約 1000 rpm）下低速離心 5 分鐘。

8. 抽干棄去上清液，加入新鮮的完全生長培養(yǎng)基重懸彈散細(xì)胞塊，按傳代比例分裝至新的培養(yǎng)器皿中。

6. 復(fù)蘇與凍存后恢復(fù)流線 (Thawing & Post-Cryo Recovery Workflow)

1. 提前在無菌 T25 培養(yǎng)瓶中注入 5.0 mL 完全 McCoy's 5A 培養(yǎng)基，置于 37 ℃、5% $CO_2$ 孵箱中平衡 20 分鐘以穩(wěn)定 pH 值。

2. 從液氮儲存罐中小心取出 SW48 KRAS (G12D/+) 凍存管。

3. 立刻將凍存管完全浸入 37 ℃ 恒溫水浴箱中。 保持管帽 O 型圈處于水面以上，劇烈水平搖動。必須在 60 至 90 秒內(nèi)實現(xiàn)全量融化（快融原則，防止胞內(nèi)冰晶二次重結(jié)晶刺破細(xì)胞膜）。

4. 用 75% 酒精噴灑管壁消殺，移入超凈臺內(nèi)。

5. 用移液槍吸出融化后的濃稠胞懸液，緩慢滴加到盛有 4.0 mL 預(yù)熱完全培養(yǎng)基的 15 mL 離心管中，輕柔混勻。

6. 300 × g 離心 5 分鐘，使活細(xì)胞沉降成塊。

7. 徹底吸除上清液，以完全清除具有細(xì)胞毒性的微量 DMSO 殘留。

8. 加入 1.5 mL 新鮮完全培養(yǎng)基，輕彈管底重懸細(xì)胞，隨后全量接種到已完成氣相平衡的 T25 瓶中。

9. 置于 37 ℃ 孵箱靜置培養(yǎng)。接種 24 小時后，必須進(jìn)行一次全量換液，以徹底清除死細(xì)胞碎片及未貼壁的細(xì)胞廢渣。

7. 生物安全、長期保存與質(zhì)控標(biāo)準(zhǔn) (Biosafety, Storage & Quality Control)

• 生物安全等級 (BSL)：BSL-2（2級生物安全柜操作）。本細(xì)胞株源自人類組織且經(jīng)過基因編輯載體改造，操作過程必須嚴(yán)格遵守實驗室防護(hù)規(guī)范。

• 長期封存參數(shù)：凍存管必須永久存放于 液氮的液相或氣相超低溫環(huán)境（-196 ℃）中。嚴(yán)禁在機(jī)械式 -80 ℃ 普通普通冰箱中長期存放超過 1-2 個月，否則會導(dǎo)致細(xì)胞活性呈指數(shù)級衰減，并可能導(dǎo)致編輯成功的 mutant 突變等位基因發(fā)生自發(fā)性表觀遺傳學(xué)沉默。

• 基因型穩(wěn)定性復(fù)核：建議每傳代 10 到 15 代，提取細(xì)胞全基因組，針對 KRAS Exon 2 進(jìn)行 PCR 擴(kuò)增并提交 Sanger 測序。唯有在密碼子 12（c.35 位點）清晰測出 G 和 A 的等高雜合雙峰（比值接近 1:1），方可判定該品系未發(fā)生突變位點丟失。

BioVector? SW48 KRAS (G12D/+) Human Isogenic Colorectal Cancer Cell Line Product Datasheet

1. Product & Identification General Information

• Product Name: BioVector? SW48 KRAS (G12D/+) Human Isogenic Colorectal Cancer Cell Line

• Catalog/Reference Cross-References: Horizon Discovery (HD 103-011) / Cellosaurus Accession: CVCL_LC92

• Organism Source: Human (Homo sapiens)

• Tissue/Organ Site: Large Intestine / Colon

• Disease Background: Colorectal Adenocarcinoma (Dukes' C, Grade IV)

• Biological Sex: Female

• Donor Age: 83 years old

• Growth Properties: Adherent Epithelial Monolayer (forms compact cobblestone-like colonial clusters)

2. Genetic Architecture & Mutation Profile

• Isogenic Modification Strategy: Precise single-nucleotide point mutation knocked into one endogenous allele of the KRAS locus within a parental wild-type SW48 context.

• Target Genotype: Heterozygous knock-in (KRAS G12D/+)

• Target Mutation Molecular Details: Coding Sequence: c.35G>A (Exon 2, Codon 12) $\rightarrow$ Protein Level: p.Gly12Asp (Glycine replaced by Aspartic Acid)

• Background Mutations (Inherited from Parental Line):

  • Homozygous ACVR2A (p.Lys437Argfs*5)

  • Homozygous TGFBR2 (p.Lys128Serfs*35)

  • Heterozygous CTNNB1 (p.Ser33Tyr)

  • Wild-Type status maintained for APC, TP53, PTEN, and BRAF.

• Microsatellite Status: Hypermutated / Microsatellite Unstable (MSI-H)

3. Routine Culturing Medium & Formulations

To preserve phenotypic stability and prevent cellular detachment, it is critical to implement the optimized culture medium configuration below.

Standard Complete Growth Medium Formulation

• Basal Medium Base: McCoy's 5A Modified Medium (with L-glutamine and high glucose).

  • Alternative validated medium: High-Glucose DMEM containing 4 mM L-glutamine.

• Serum Supplement: 10% Premium Fetal Bovine Serum (FBS) (heat inactivation is optional but generally unnecessary for routine propagation).

• Antibiotic Cocktail: 1% Penicillin-Streptomycin Solution (Final concentration: 100 units/mL Penicillin, 100 $\mu$g/mL Streptomycin).

Cryopreservation (Freezing) Medium Formulation

• Standard Freezing Mixture: 90% Complete Growth Medium (McCoy's 5A + 10% FBS) + 10% Analytical Grade Dimethyl Sulfoxide (DMSO).

• Alternative high-recovery formulation: 45% Basal McCoy's 5A medium + 45% Premium FBS + 10% DMSO.

4. Controlled Culture Environmental Conditions

• Incubation Temperature: 37.0 °C (constant, stable thermal window).

• Atmospheric Gas Composition: 5% Carbon Dioxide ($CO_2$) balanced with humidified atmospheric air.

• Relative Humidity: $\gt 95\%$ (to limit micro-evaporation within culture vessels).

• Atmospheric Note: Unlike the original ATCC parental line which can adapt to Leibovitz's L-15 medium in 100% atmospheric air (0% $CO_2$), the engineered isogenic variant undergoes optimal metabolic performance in McCoy's 5A under a strict 5% $CO_2$ buffer system.

5. Subculturing (Passaging) Protocol & Metrics

• Optimal Passaging Confluency: 70% to 80% confluency.

• Critical Operational Threshold: Do not allow the monolayer to achieve 100% confluency. SW48 cells form tight, multi-layered aggregates if overcrowded. Once clustered into three-dimensional heaps, they become highly resistant to enzymatic dissociation and exhibit reduced post-passage viability.

• Subcultivation Splitting Ratio: 1:3 to 1:5 (depending on the baseline seeding density).

• Medium Renewal Frequency: 2 to 3 times per week.

Step-by-Step Dissociation Method:

1. Aspirate the spent growth medium completely from the vessel.

2. Rinse the cell monolayer gently with sterile, Calcium/Magnesium-free Phosphate-Buffered Saline (PBS) once to remove trace serum (which contains trypsin inhibitors).

3. Apply a minimal volume of pre-warmed 0.25% Trypsin - 0.02% EDTA Solution (typically 1.0 mL for a T25 flask; 2.0–3.0 mL for a T75 flask).

4. Incubate the vessel at 37 °C for 3 to 5 minutes. Monitor under an inverted microscope.

5. Handling Precaution: Do not hit or shake the flask while waiting for cells to loosen, as mechanical agitation can promote severe cell clumping. Once the cells round up and shift upon gentle tilting, add an equal volume of serum-containing complete growth medium to immediately deserialize enzymatic activity.

6. Gently pipette the suspension against the vessel wall to break down multi-cellular sheets into a uniform single-cell suspension.

7. Transfer the suspension to a conical tube and centrifuge at 300 × g (~1000 rpm) for 5 minutes.

8. Discard the supernatant, gently resuspend the cell pellet in fresh, pre-warmed complete growth medium, and distribute the target aliquots into new culture vessels.

6. Thawing & Post-Cryo Recovery Workflow

1. Pre-warm 5.0 mL of complete McCoy's 5A growth medium in a sterile T25 flask and balance it in the 37 °C, 5% $CO_2$ incubator for 20 minutes to stabilize the pH.

2. Retrieve the SW48 KRAS (G12D/+) cryovial from liquid nitrogen storage.

3. Plunge the vial instantly into a 37 °C water bath. Rapidly shake horizontally, ensuring the O-ring cap remains above the water line. Achieve complete thawing within 60 to 90 seconds.

4. Disinfect the vial exterior with 75% ethanol before wiping it dry and transferring it to 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 medium.

6. Centrifuge at 300 × g for 5 minutes to form a clean cell pellet.

7. Aspirate the supernatant completely to remove toxic DMSO residue.

8. Add 1.5 mL of fresh complete medium, gently tap the bottom of the tube to loosen the pellet, and seed the entire biomass into the pre-stabilized T25 flask.

9. Incubate at 37 °C, 5% $CO_2$. Perform a complete medium exchange 24 hours post-thaw to eliminate any non-adherent dead cell debris.

7. Biosafety, Storage & Quality Control Standards

• Biosafety Level (BSL): BSL-2. This cell line contains genomic editing vectors and is of human origin; appropriate personal protective equipment (PPE) and containment protocols must be strictly followed.

• Long-Term Storage Parameters: Cryovials must be submerged permanently within the liquid nitrogen vapor/liquid phase (-196 °C). Short- or long-term storage in mechanical -80 °C freezers beyond 1-2 months is prohibited, as it leads to an exponential drop in post-thaw viability and may induce spontaneous phenotypic drifting of the mutant allele.

• Genotypic Stability Quality Control: It is recommended to perform Sanger sequencing across KRAS Exon 2 every 10 to 15 passages. A stable heterozygous knock-in profile is confirmed when overlapping G and A peaks are maintained at an approximate 1:1 signal ratio at codon 12 (c.35 position).

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