BioVector? IPTI002-A 人誘導多能干細胞株

BioVector? IPTI002-A Human Induced Pluripotent Stem Cell Line

第一部分 中文說明

一 產品基本信息與遺傳學背景

• 細胞名稱：BioVector? IPTI002-A 人誘導多能干細胞

• 系統(tǒng)學 Accession：Cellosaurus CVCL_C5X8

• 物種來源：人類 (Homo sapiens)

• 性別來源：女性 (Female)

• 組織與重編程背景：該細胞株建立于一名健康女性捐獻者的外周血單核細胞（PBMC）。通過使用非整合型的非對稱仙臺病毒（Sendai Virus）載體系統(tǒng)，導入標準的 Yamanaka 四因子（OCT4, SOX2, KLF4, c-MYC）進行重編程創(chuàng)制。由于重編程載體在隨后的傳代過程中被完全清除，該細胞株表現(xiàn)為無外源基因殘留的非整合特性。

• 多能性分子特征：

  • 核型特征：經嚴格鑒定表現(xiàn)為正常的女性二倍體核型（46, XX），在體外長期傳代過程中能夠保持極高的染色體核型穩(wěn)定性。

  • 多能性標志物：高水平表達干細胞核心轉錄因子 OCT4、NANOG、SOX2，且其細胞表面強陽性表達階段特異性胚胎抗原 SSEA-4 以及腫瘤排斥抗原 TRA-1-60、TRA-1-81。

  • 分化潛能：具備標準的三胚層（內胚層、中胚層、外胚層）全能分化潛能，在體內異種移植實驗中可高效形成包含多組織成分的畸胎瘤（Teratoma）。

• 生物安全級別：1級（BSL-1）。

二 細胞形態(tài)學與特殊培養(yǎng)環(huán)境

• 形態(tài)學特征：在優(yōu)質的基質膠（如 Matrigel）或人源重組玻璃體結合蛋白（Vitronectin）包被的基底上呈經典的貼壁克隆集落（Colony）狀生長。集落邊界清晰、極為緊密。單顆干細胞體積較小，核質比極高，且克隆內部細胞排列緊密。

• 生長模式：貼壁集落生長。

• 標準完全培養(yǎng)基配方：

  • 基礎與維持體系：BioVector? 無血清、無飼養(yǎng)層多能干細胞完全培養(yǎng)基（如經典穩(wěn)定的 mTeSR Plus 或 Essential 8 增強型無血清制劑）。

  • 基質包被：培養(yǎng)前基質表面必須經過 BioVector? 1比100稀釋的基質膠（Matrigel）或高純度 Vitronectin 溶液在 37攝氏度下包被 1 小時。

• 物理培養(yǎng)參數(shù)：37攝氏度恒溫、5% 二氧化碳、空氣飽和濕度（或選用 5% 氧氣、5% 二氧化碳的三氣低氧生理環(huán)境培養(yǎng)以維持其最高干性狀態(tài)）。

三 細胞傳代與復蘇標準操作步驟

1. 常規(guī)傳代操作（建議采用溫和的常規(guī)團塊傳代法法）：

   • 當多能干細胞克隆集落覆蓋培養(yǎng)皿面積達到 75% 到 85%，且部分集落中心開始出現(xiàn)過密堆積時需要進行傳代。切勿讓克隆過度生長或相互融合，否則會導致細胞發(fā)生自發(fā)性分化。

   • 吸除舊培養(yǎng)基，用無菌的無鈣鎂離子 1x PBS 洗滌 1 次。

   • 加入適量溫和的 BioVector? 0.5 mM EDTA 傳代分離液（如 ReLeSR 或常規(guī)無酶分散液），在 37攝氏度下孵育 2 到 4 分鐘。

   • 顯微鏡下觀察到集落邊緣開始輕微回縮、變亮后，立即吸除分離液。加入新鮮完全培養(yǎng)基，用移液槍極其輕柔地吹吸 1 2 次，將克隆片層剝離并碎裂成大小適中的細胞團塊（Clumps）。切勿將其消化吹打成單細胞，否則會引起大規(guī)模細胞凋亡。

   • 推薦常規(guī)傳代比例為 1比4 至 1比8，每 4 到 6 天傳代一次。

2. 特殊單細胞傳代/接種策略（若實驗需要單細胞鋪板）：

   • 若需要進行高通量篩選或轉染而必須打散成單細胞時，須使用 BioVector? Accutase 酶消化液，且在接種后的前 24 小時內，完全培養(yǎng)基中必須添加最終濃度為 10 μM 的 Rock 抑制劑（Y-27632），以特異性阻斷單細胞懸浮引發(fā)的失巢凋亡（Anoikis）。

3. 凍存細胞復蘇：

   • 從液氮中取出冷凍管，立即投入 37攝氏度 BioVector? 水浴鍋中高頻搖動使其快速融化，控制在 1到2 分鐘內。

   • 將解凍的細胞懸液移至含 5 mL 預熱干細胞完全培養(yǎng)基的管中，常規(guī)速度低速離心 4 分鐘（約 100 g）以沉淀團塊。

   • 棄去含有 DMSO 的上清，加入含有 10 μM Y-27632 的新鮮干細胞完全培養(yǎng)基重懸細胞團，接種于提前包被好基質膠的培養(yǎng)皿中。次日務必更換為不含 Y-27632 的常規(guī)完全培養(yǎng)基。

四 核心科研應用方向

1. 人體器官類器官（Organoids）分化與發(fā)育生物學模型：BioVector? IPTI002-A 作為源自健康女性個體的無外源殘留經典誘導多能干細胞系，廣泛用作體外定向分化的高質量起始材料。通過多步法小分子組合誘導，可高效定向分化為腦類器官（Brain organoids）、心肌類器官（Cardiac organoids）及高純度的定形內胚層細胞，用于解析人類胚胎早期器官發(fā)育的基因調控網絡。

2. 新型小分子/抗體藥物體外發(fā)育毒性與致畸性評價：在制藥工業(yè)的臨床前安全評價中，該細胞常被用于搭建體外人胚胎干細胞試驗（EST）替代模型。通過監(jiān)測該株在藥物干預下三胚層自發(fā)分化標志物（如內胚層 AFP、中胚層 Brachyury、外胚層 Nestin）的轉錄變異，評估待測候選化合物的生殖和發(fā)育毒性風險。

3. 基于患者特異性的疾病模擬與基因編輯平臺：利用 CRISPR/Cas9 基因編輯技術在該細胞株中引入特定的致病突變，或利用其作為正常對照組（Control line）與特定遺傳病患者來源的 iPSCs 進行并行研究，用于揭示神經退行性疾病、代謝性心肌病等復雜疾病的分子病理，開展高通量的先導化合物藥效篩選。

PART 2 ENGLISH SECTION

I General Information and Genetic Background

• Cell Line Name: BioVector? IPTI002-A

• Synonyms: IPTI002-A, IPTIi002-A

• Cellosaurus Accession: CVCL_C5X8

• Species Origin: Human (Homo sapiens)

• Sex of Donor: Female

• Tissue and Reprogramming History: Established from peripheral blood mononuclear cells (PBMCs) isolated from a healthy female donor. Reprogramming was executed utilizing a non-integrating, replication-incompetent Sendai Virus delivery system driving the standard Yamanaka pluripotency repertoire (OCT4, SOX2, KLF4, and c-MYC). The viral vectors are completely cleared out during downstream passaging, ensuring a transgene-free and footprint-free pluripotent genomic status.

• Pluripotency and Karyotypic Traits:

  • Karyotype: Validated to harbor a stable, normal female diploid chromosome configuration (46, XX), demonstrating excellent genomic architecture retention across prolonged in vitro expansion cycles.

  • Core Markers: Robustly expresses essential stemness transcription factor networks including OCT4, NANOG, and SOX2, while displaying heavy apical presentation of stage-specific embryonic antigen SSEA-4 and tumor rejection antigens TRA-1-60 and TRA-1-81.

  • Lineage Competence: Possesses standard tri-lineage differentiation potential. Spontaneously differentiates into recognizable functional cell configurations representing the ectoderm, mesoderm, and endoderm layers, and reliably organizes multi-tissue teratomas during in vivo immunodeficient mouse transplantation validations.

• Biosafety Level: BSL-1.

II Morphological Attributes and Cultivation Media

• Morphology: Proliferates as classic adherent pluripotent colonies when cultured on validated ECM substrates such as Matrigel or recombinant human Vitronectin. Colonies display highly defined, sharp boundaries and compact cellular clustering. Individual cells within the colony maintain small diameters, high nuclear-to-cytoplasmic volume ratios, and dense intercellular contact arrangements.

• Growth Mode: Adherent colony growth.

• Standard Complete Growth Medium Formulation:

  • Basal Maintenance Matrix: BioVector? serum-free, feeder-free pluripotent stem cell complete medium (e.g., standard high-performance mTeSR Plus formulations or Essential 8 enhanced media parameters).

  • Surface Coating: Culture vessels must be pre-coated with BioVector? Matrigel (typically diluted 1:100 in cold basal medium) or purified recombinant Vitronectin solution, incubated at 37 degrees Celsius for 1 hour prior to cell deployment.

• Physical Incubation Thresholds: Regulated strictly at 37 degrees Celsius under an atmospheric layer of 5% Carbon Dioxide and saturated air humidity (or adjusted to a tri-gas physiological hypoxic environment of 5% $O_2$ and 5% $CO_2$ to shield core stemness states).

III Subculturing and Thawing Protocols

1. Routine Passaging Schedule (Clump Passaging Method):

   • Initiate subculturing when the pluripotent colony aggregates cover 75% to 85% of the total vessel surface or when colony centers exhibit dense multi-layer packing. Avoid letting colonies overgrow or fuse completely, which triggers unwanted spontaneous differentiation lines.

   • Aspirate spent media and rinse the matrix once with sterile, calcium/magnesium-free 1x PBS.

   • Administer an appropriate volume of BioVector? 0.5 mM EDTA-based gentle dissociation reagent (such as ReLeSR or standard enzyme-free separation buffers) and incubate at 37 degrees Celsius for 2 to 4 minutes.

   • Once the colony edges show light contraction under light microscopy, instantly draw off the dissociation reagent. Dispense fresh complete maintenance medium and use a pipette to very gently wash and detach the colony sheets 1 to 2 times, breaking them down into small cell clumps. Do not forcefully dissociate the biomass into absolute single cells, as doing so initiates massive apoptosis.

   • Seed fresh matrix-coated vessels at a recommended split ratio of 1比4 to 1比8 every 4 to 6 days.

2. Single-Cell Dissociation Strategy (For Specific Experimental Setup):

   • If single-cell seeding is mandatory for parallel transfections or high-throughput sorting, dissociate the colonies using BioVector? Accutase reagent. Crucially, the fresh complete medium deployed during the preliminary 24-hour post-seeding window must be enriched with a final concentration of 10 μM Rock Inhibitor (Y-27632) to block single-cell anoikis circuits.

3. Cryovial Thawing and Recovery:

   • Retrieve the cryovial from storage and submerge it into a 37 degrees Celsius BioVector? water bath with rapid agitation until completely liquefied within 1 to 2 minutes.

   • Dilute the slurry immediately into a tube holding 5 mL of pre-warmed stem cell complete medium and spin down at a gentle velocity (around 100 g) for 4 minutes to pellet the clump structures.

   • Decant the DMSO-tainted supernatant, resuspend the remaining cell mass in fresh complete BioVector? medium supplemented with 10 μM Y-27632, and plate onto pre-coated matrix dishes. Perform a total medium refresh the following day to eliminate the Rock inhibitor.

IV Strategic Research Applications

1. Human Organoid Morphogenesis and Lineage Trajectory Mapping: BioVector? IPTI002-A serves as an excellent footprint-free normal reference line for directed lineage differentiation workflows. Guided by specific small-molecule temporal cocktails, it yields high-purity definitive endoderm, functional cardiomyocytes, or three-dimensional brain organoids, enabling the tracing of transcriptional networks governing early human organogenesis.

2. In Vitro Developmental Toxicity and Teratogenicity Bioprofiling: Deployed in preclinical drug safety screening pipelines to construct Embryonic Stem Cell Test (EST) alternatives. By tracking how test candidates impact or skew the tri-lineage spontaneous embryoid body differentiation profile (measuring endodermal AFP, mesodermal Brachyury, and ectodermal Nestin markers), toxicology networks can map embryotoxic risks of candidate small molecules or monoclonal formats.

3. Patient-Specific Isogenic Disease Modeling & Genome Engineering Platforms: Serves as a genetically clean wild-type baseline framework for introducing specific familial or sporadic pathogenic single-nucleotide polymorphisms via CRISPR/Cas9 tool suites. It acts as an excellent, non-mutated control line paired against patient-derived iPSCs to unravel molecular pathologies in neurodegenerative conditions or metabolic cardiomyopathies, driving accelerated high-throughput discovery loops for targeted leads.

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