中国组织工程研究
中國組織工程研究
중국조직공정연구
Journal of Clinical Rehabilitative Tissue Engineering Research
2014年
37期
5947-5954
,共8页
吴洁莹%陆琰%陈劲松%朱璐%甘文婷
吳潔瑩%陸琰%陳勁鬆%硃璐%甘文婷
오길형%륙염%진경송%주로%감문정
干细胞%脐带脐血干细胞%脐血%脐带间充质干细胞%体外扩增%免疫表型%成骨分化%成脂分化%冷冻保存%复苏
榦細胞%臍帶臍血榦細胞%臍血%臍帶間充質榦細胞%體外擴增%免疫錶型%成骨分化%成脂分化%冷凍保存%複囌
간세포%제대제혈간세포%제혈%제대간충질간세포%체외확증%면역표형%성골분화%성지분화%냉동보존%복소
fetal blood%plasma%umbilical cord%mesenchymal stem cells%freezing
背景：采用胎牛血清扩增培养及冷冻保存的间充质干细胞，在临床应用中存在一定的风险。目的：探讨以脐血血浆替代胎牛血清体外分离培养、扩增及冷冻保存脐带间充质干细胞的可行性。方法：选择符合广州脐血库供者合格性筛选标准的脐血，收集脐血干细胞制备过程中去除的血浆，经病原学及微生物检测合格，多份混合用于细胞培养。采用酶消化法从健康足月分娩新生儿的脐带组织分离获得脐带间充质干细胞，分为两组，分别以DMEM/F12为基础培养基，添加胎牛血清或混合脐血血浆，经培养扩增后，采用流式细胞仪检测细胞免疫表型，并进行间充质干细胞成骨、成脂分化鉴定。在含有10%二甲基亚砜的DMEM/F12培养基中，分别添加体积分数为20%的胎牛血清或混合脐血血浆作为冷冻保存液，对扩增至第3代的细胞冷冻保存至6个月以上，观察复苏后细胞的活率、贴壁情况、增殖、免疫表型及向成骨细胞分化的能力。结果与结论：两种培养体系下的脐带间充质干细胞均呈现典型的梭形漩涡状生长，间充质干细胞免疫表型的表达谱系基本无差异，均具有成骨、成脂分化的能力，但脐血血浆培养条件下细胞的增殖速度显著高于胎牛血清。冻存复苏后的细胞可正常贴壁，且具有向成骨细胞分化的能力，采用脐血血浆冻存的细胞具有更高的贴壁效率及扩增能力。上述结果表明，脐血血浆可以替代胎牛血清用于脐带间充质干细胞的扩增培养及冷冻保存，并维持了间充质干细胞的基本生物学特性，是大量扩增间充质干细胞用于临床治疗较为安全的选择。
揹景：採用胎牛血清擴增培養及冷凍保存的間充質榦細胞，在臨床應用中存在一定的風險。目的：探討以臍血血漿替代胎牛血清體外分離培養、擴增及冷凍保存臍帶間充質榦細胞的可行性。方法：選擇符閤廣州臍血庫供者閤格性篩選標準的臍血，收集臍血榦細胞製備過程中去除的血漿，經病原學及微生物檢測閤格，多份混閤用于細胞培養。採用酶消化法從健康足月分娩新生兒的臍帶組織分離穫得臍帶間充質榦細胞，分為兩組，分彆以DMEM/F12為基礎培養基，添加胎牛血清或混閤臍血血漿，經培養擴增後，採用流式細胞儀檢測細胞免疫錶型，併進行間充質榦細胞成骨、成脂分化鑒定。在含有10%二甲基亞砜的DMEM/F12培養基中，分彆添加體積分數為20%的胎牛血清或混閤臍血血漿作為冷凍保存液，對擴增至第3代的細胞冷凍保存至6箇月以上，觀察複囌後細胞的活率、貼壁情況、增殖、免疫錶型及嚮成骨細胞分化的能力。結果與結論：兩種培養體繫下的臍帶間充質榦細胞均呈現典型的梭形漩渦狀生長，間充質榦細胞免疫錶型的錶達譜繫基本無差異，均具有成骨、成脂分化的能力，但臍血血漿培養條件下細胞的增殖速度顯著高于胎牛血清。凍存複囌後的細胞可正常貼壁，且具有嚮成骨細胞分化的能力，採用臍血血漿凍存的細胞具有更高的貼壁效率及擴增能力。上述結果錶明，臍血血漿可以替代胎牛血清用于臍帶間充質榦細胞的擴增培養及冷凍保存，併維持瞭間充質榦細胞的基本生物學特性，是大量擴增間充質榦細胞用于臨床治療較為安全的選擇。
배경：채용태우혈청확증배양급냉동보존적간충질간세포，재림상응용중존재일정적풍험。목적：탐토이제혈혈장체대태우혈청체외분리배양、확증급냉동보존제대간충질간세포적가행성。방법：선택부합엄주제혈고공자합격성사선표준적제혈，수집제혈간세포제비과정중거제적혈장，경병원학급미생물검측합격，다빈혼합용우세포배양。채용매소화법종건강족월분면신생인적제대조직분리획득제대간충질간세포，분위량조，분별이DMEM/F12위기출배양기，첨가태우혈청혹혼합제혈혈장，경배양확증후，채용류식세포의검측세포면역표형，병진행간충질간세포성골、성지분화감정。재함유10%이갑기아풍적DMEM/F12배양기중，분별첨가체적분수위20%적태우혈청혹혼합제혈혈장작위냉동보존액，대확증지제3대적세포냉동보존지6개월이상，관찰복소후세포적활솔、첩벽정황、증식、면역표형급향성골세포분화적능력。결과여결론：량충배양체계하적제대간충질간세포균정현전형적사형선와상생장，간충질간세포면역표형적표체보계기본무차이，균구유성골、성지분화적능력，단제혈혈장배양조건하세포적증식속도현저고우태우혈청。동존복소후적세포가정상첩벽，차구유향성골세포분화적능력，채용제혈혈장동존적세포구유경고적첩벽효솔급확증능력。상술결과표명，제혈혈장가이체대태우혈청용우제대간충질간세포적확증배양급냉동보존，병유지료간충질간세포적기본생물학특성，시대량확증간충질간세포용우림상치료교위안전적선택。
BACKGROUND:Fetal bovine serum based media used for expanding and cryopreserving human mesenchymal stem cells raise safety concerns in the clinical setting. OBJECTIVE:To investigate the feasibility of human umbilical cord blood plasma as a replacement for fetal bovine serum in culture and cryopreservation of human mesenchymal stem cells derived from umbilical cord. METHODS:Umbilical cord blood units were suitable for this research if they fulfil ed the donor selection criteria of the Guangzhou Cord Blood Bank strictly. Cord blood plasma was ready to use after col ected from the plasma reduction during the suitable cord blood units processing and pooling. Umbilical cord mesenchymal stem cells were harvested from the umbilical cord tissue of health ful-term newborns after delivery by enzyme digestion and were cultured in the presence of Dulbecco’s modified Eagle’s medium/Ham’s nutrient mixture F-12 containing either fetal bovine serum or pooled cord blood plasma. Morphology, proliferation, immunophenotype detected by flow cytometry and differentiation toward adipogenic and osteogenic lineages were utilized for investigating the effect of media on umbilical cord mesenchymal stem cells after 3-5 passages. Then cells were cryopreserved in media containing 10%dimethyl sulfoxide, 20%fetal bovine serum or 20%pooled cord blood plasma for at least 6 months. Viability, adhesion, proliferation, immunophenotype and osteogenic differentiation of the cells were assessed after thawing. RESULTS AND CONCLUSION:The morphology (spindle-shaped and plastic-adherent), phenotype and differentiation potential (osteogenic and adipogenic) were almost indistinguishable between cells cultured in fetal bovine serum or cord blood plasma medium, while cells grown in cord blood plasma medium demonstrated significantly higher proliferation rates than those in medium containing fetal bovine serum. After thawing, the cells maintained their adherence to the culture surface and differentiation potential to osteoblasts, but cells from cord blood plasma cryopreservation medium showed significantly better plastic attachment and produced greater cellnumbers than fetal bovine serum for the first three post-thaw passages. The results demonstrate that cord blood plasma can sever as an effective substitute to fetal bovine serum for growth, maintenance and differentiation of umbilical cord mesenchymal stem cells, and thus it wil be a safe choice for clinical-scale production of human mesenchymal stem cells.
