BioVector® SW48 KRAS (G12D/+) 人类同基因型结直肠癌细胞株——产品技术说明书 (Product Datasheet)

1. 产品基本信息与鉴定谱系 (Product & Identification General Information)

• 产品名称：BioVector® SW48 KRAS (G12D/+) 人类同基因型结直肠癌细胞株

• 国际数据库登录号：Horizon Discovery (HD 103-011) / Cellosaurus登录号：CVCL_LC92

• 生物学来源：人类（Homo sapiens）

• 组织来源：大肠/结肠

• 疾病背景：结直肠腺癌（Dukes' C 期，IV 级）

• 性别：女性

• 供体年龄：83岁

• 生长特性：贴壁型上皮细胞（体外倾向于形成紧密的铺路石状克隆团块）

2. 基因组架构与突变特征 (Genetic Architecture & Mutation Profile)

• 同基因修饰策略：利用高精度基因编辑技术，在天然野生型（WT）SW48 细胞的内源性 KRAS 位点的一个等位基因上精确引入了激活型点突变。

• 目标基因型：杂合子基因敲入（KRAS G12D/+）

• 突变分子细节：编码区：c.35G>A（第2外显子，第12密码子） $\rightarrow$ 蛋白质水平：p.Gly12Asp（甘氨酸被天冬氨酸取代）

• 背景突变（承袭自亲本株）：

  • 纯合型 ACVR2A (p.Lys437Argfs*5)

  • 纯合型 TGFBR2 (p.Lys128Serfs*35)

  • 杂合型 CTNNB1 (p.Ser33Tyr)

  • 维持野生型（WT）状态的基因：APC、TP53、PTEN、BRAF。

• 微卫星状态：高突变/微卫星不稳定（MSI-H）

3. 常规培养基配方规范 (Routine Culturing Medium & Formulations)

为确保该株细胞的基因型与表型稳态，防止细胞自发成团脱落，必须严格执行以下优化的培养基配置：

标准完全生长培养基 (Standard Complete Growth Medium)

• 基础培养基：McCoy's 5A 改进型培养基（含 L-谷氨酰胺及高糖）。

  • 经质控验证的替代培养基：含 4 mM L-谷氨酰胺的高糖 DMEM 培养基。

• 血清添加：10% 优质胎牛血清（FBS）（日常增殖无需进行热灭活）。

• 抗生素混剂：1% 标准青霉素-链霉素双抗溶液（终浓度：100 units/mL 青霉素，100 $\mu$g/mL 链霉素）。

细胞冻存保护液 (Cryopreservation Medium)

• 标准配方：90% 完全生长培养基（McCoy's 5A + 10% FBS） + 10% 细胞级二甲基亚砜（DMSO）。

• 高回收率替代配方：45% 基础 McCoy's 5A 培养基 + 45% 优质胎牛血清 + 10% DMSO。

4. 物理环境控制参数 (Controlled Culture Environmental Conditions)

• 孵育温度：37.0 ℃（恒温，严禁剧烈波动）。

• 气相环境：5% 二氧化碳 ($CO_2$)，平衡常压无菌空气。

• 相对湿度：$\gt 95\%$（高湿环境，严防培养基微量蒸馏浓缩）。

• 特别注意：虽然原代 ATCC 亲本野生型 SW48 细胞具有在无 $CO_2$ 环境下使用 Leibovitz's L-15 培养基生长的全空气适应能力，但本款基因编辑纯化后的同基因 variant 突变株，必须在 5% $CO_2$ 的 McCoy's 5A 缓冲体系下栽培，以维持其最优的代谢与增殖速率。

5. 贴壁传代操作红线 (Subculturing Protocol & Metrics)

• 标准传代汇合度：70% 到 80% 汇合度。

• 核心操作红线：严禁让细胞长满至 100% 汇合度。 SW48 具有极强的密集重叠生长倾向。一旦长满，细胞会迅速向上堆积形成复杂的三维重叠块，此时胰酶极难渗透，极易导致消化不均、细胞大规模成团损伤以及复苏后活力断崖式下跌。

• 建议传代稀释比例：1:3 到 1:5（依接种密度动态调节）。

• 全量换液频率：每周 2 至 3 次。

标准传代消化步骤：

1. 完全吸除培养瓶中的陈旧培养基。

2. 使用不含钙、镁离子的无菌 PBS 缓冲液轻柔洗涤细胞单层 1 次，以彻底清除残余血清（血清中的抗胰蛋白酶会强烈抑制消化液活性）。

3. 视培养器皿面积加入适量预热的 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. 将培养瓶置于 37 ℃ 孵箱中消化 3 到 5 分钟。全程通过倒置显微镜观察。

5. 操作禁忌：在等待细胞脱落期间，切勿大力拍打或剧烈摇晃培养瓶，否则极易诱发 SW48 自发性拉丝结块。一旦镜下观察到大部分细胞回缩变圆、单层出现缝隙、轻 tilt 瓶身见细胞呈流沙状整体下滑，立即加入等体积的含血清完全生长培养基终止消化。

6. 用移液枪顺着贴壁面轻柔吹打，将细胞团块彻底分散为均一的单细胞悬液。

7. 将悬液收集至离心管中，在 300 × g（约 1000 rpm）下低速离心 5 分钟。

8. 抽干弃去上清液，加入新鲜的完全生长培养基重悬弹散细胞块，按传代比例分装至新的培养器皿中。

6. 复苏与冻存后恢复流线 (Thawing & Post-Cryo Recovery Workflow)

1. 提前在无菌 T25 培养瓶中注入 5.0 mL 完全 McCoy's 5A 培养基，置于 37 ℃、5% $CO_2$ 孵箱中平衡 20 分钟以稳定 pH 值。

2. 从液氮储存罐中小心取出 SW48 KRAS (G12D/+) 冻存管。

3. 立刻将冻存管完全浸入 37 ℃ 恒温水浴箱中。 保持管帽 O 型圈处于水面以上，剧烈水平摇动。必须在 60 至 90 秒内实现全量融化（快融原则，防止胞内冰晶二次重结晶刺破细胞膜）。

4. 用 75% 酒精喷洒管壁消杀，移入超净台内。

5. 用移液枪吸出融化后的浓稠胞悬液，缓慢滴加到盛有 4.0 mL 预热完全培养基的 15 mL 离心管中，轻柔混匀。

6. 300 × g 离心 5 分钟，使活细胞沉降成块。

7. 彻底吸除上清液，以完全清除具有细胞毒性的微量 DMSO 残留。

8. 加入 1.5 mL 新鲜完全培养基，轻弹管底重悬细胞，随后全量接种到已完成气相平衡的 T25 瓶中。

9. 置于 37 ℃ 孵箱静置培养。接种 24 小时后，必须进行一次全量换液，以彻底清除死细胞碎片及未贴壁的细胞废渣。

7. 生物安全、长期保存与质控标准 (Biosafety, Storage & Quality Control)

• 生物安全等级 (BSL)：BSL-2（2级生物安全柜操作）。本细胞株源自人类组织且经过基因编辑载体改造，操作过程必须严格遵守实验室防护规范。

• 长期封存参数：冻存管必须永久存放于 液氮的液相或气相超低温环境（-196 ℃）中。严禁在机械式 -80 ℃ 普通普通冰箱中长期存放超过 1-2 个月，否则会导致细胞活性呈指数级衰减，并可能导致编辑成功的 mutant 突变等位基因发生自发性表观遗传学沉默。

• 基因型稳定性复核：建议每传代 10 到 15 代，提取细胞全基因组，针对 KRAS Exon 2 进行 PCR 扩增并提交 Sanger 测序。唯有在密码子 12（c.35 位点）清晰测出 G 和 A 的等高杂合双峰（比值接近 1:1），方可判定该品系未发生突变位点丢失。

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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