CAJ | 학술논문

目的：研究脐带来源间充质干细胞（UC-MSCs）的扩增工艺及质量检验方法，提出质量控制标准，为临床研究提供合格的干细胞产品。方法新鲜脐带去羊膜及血管，剪碎，组织块放入无菌培养皿，在培养箱进行脐带间充质干细胞培养、扩增。细胞培养至 P3代，按照《干细胞制剂质量控制及临床前研究指导原则》（征求意见稿）进行干细胞质量的全面检定。检定内容包括无菌检测，支原体检测，人源特定病毒及猪源病毒检测，内毒素检测，细胞形态、细胞数及细胞活率检测，染色体核型分析，干细胞免疫表型检测，STR 图谱分析，免疫抑制活性检测及分化能力检测。结果按照标准化流程对18根脐带进行了 UC-MSCs 的分离、纯化及培养，获得一致性较好的干细胞。通过对干细胞进行质量检定，很好地控制了干细胞的质量。干细胞活性在冻存前≥90%，冻存复苏后≥80%；该工艺生产的脐带MSC 产品无细菌、人源特定病毒及猪细小病毒、支原体等微生物污染，内毒素检测阴性；干细胞免疫表型检测 CD90、CD105呈阳性，阳性率不低于95%；CD31、CD34、HLA-DR呈阴性，阳性率不高于2%；染色体核型分析为46XX 或46XY，无缺失及插入突变；STR 图谱分析证实干细胞为单一人来源；UC-MSCs 具有成脂及成骨分化潜能；UC-MSCs能抑制异源淋巴细胞增殖。结论按本工艺及标准制备的 UC-MSCs 符合《干细胞制剂质量控制及临床前研究指导原则》的质量控制标准，为同类干细胞制备及检定过程标准化提供了实验依据。
목적：연구제대래원간충질간세포（UC-MSCs）적확증공예급질량검험방법，제출질량공제표준，위림상연구제공합격적간세포산품。방법신선제대거양막급혈관，전쇄，조직괴방입무균배양명，재배양상진행제대간충질간세포배양、확증。세포배양지 P3대，안조《간세포제제질량공제급림상전연구지도원칙》（정구의견고）진행간세포질량적전면검정。검정내용포괄무균검측，지원체검측，인원특정병독급저원병독검측，내독소검측，세포형태、세포수급세포활솔검측，염색체핵형분석，간세포면역표형검측，STR 도보분석，면역억제활성검측급분화능력검측。결과안조표준화류정대18근제대진행료 UC-MSCs 적분리、순화급배양，획득일치성교호적간세포。통과대간세포진행질량검정，흔호지공제료간세포적질량。간세포활성재동존전≥90%，동존복소후≥80%；해공예생산적제대MSC 산품무세균、인원특정병독급저세소병독、지원체등미생물오염，내독소검측음성；간세포면역표형검측 CD90、CD105정양성，양성솔불저우95%；CD31、CD34、HLA-DR정음성，양성솔불고우2%；염색체핵형분석위46XX 혹46XY，무결실급삽입돌변；STR 도보분석증실간세포위단일인래원；UC-MSCs 구유성지급성골분화잠능；UC-MSCs능억제이원림파세포증식。결론안본공예급표준제비적 UC-MSCs 부합《간세포제제질량공제급림상전연구지도원칙》적질량공제표준，위동류간세포제비급검정과정표준화제공료실험의거。
Objective To establish a practical quality control standard of the product of umbilical cord-derived mesenchymal stem cells (UC-MSCs) for clinical research. Methods Fresh umbilical cord from donors was obtained. After amnion and blood vessel was removed, the Wharton Jelly was minced into 1-2 mm3 fragments and then suspended in an animal serum-free MSC growth medium and incubated in a humidified atmosphere with 5% CO2 at 37 ℃. Cells at passage 3 were used for experiments. According to guideline of quality control and preclinical research of stem cells, UC-MSCs were performed overall examination. The examined contents contained sterility detection, mycoplasma detection, human-derived and swine-derived virus screening, endotoxin detection, cell morphology observation, cell number and cell viability assay, chromatosome karyotype analysis, immunophenotype assay, short tandem repeat profiling, immuno-supress activity and differentiation assay. Results UC-MSCs from donors were prepared according to the standard operation procedure for cell isolation, purification and culture. Through overall quality arbitration, the quality of UC-MSCs could be controlled. The cell viability was more than or equal to 90 percent before preservation and more than or equal to 80 percent after preservation. UC-MSCs was sterile, mycoplasma-free, endotoxin-free and non-specific human- and swine-derived virus. The UC-MSCs were positive for CD90 and CD105, whereas negative for CD34, CD45, and HLA-DR. Chromatosome karyotype was in the form of 46XX or 46XY, no deletion and insertion mutation. STR profiling verified that UC-MSCs showed characteristic human STR profiles and no cross contamination. UC-MSCs possessed multi-differentiation potential and suppressed heterogenous lymphocyte proliferation. Conclusion We have established the quality-control standard and supplied experimental data for preparation and examination procedure of umbilical cord-derived mesenchymal stem cells.