應(yīng)用指南 - 使用CTS Rotea逆流離心系統(tǒng)洗脫殘留物

在用于細(xì)胞治療前，細(xì)胞可能要經(jīng)過解凍、篩選、擴(kuò)增、基因改造、分化和/或冷凍保存這些步驟。每個(gè)步驟可能需要不同的培養(yǎng)基或緩沖液，其中可能含有對(duì)細(xì)胞有害的物質(zhì)。Gibco?CTS?Rotea?逆流離心系統(tǒng)可有效去除殘留物質(zhì)。通過流化床泵入洗滌緩沖液，以替換用于上樣細(xì)胞的培養(yǎng)基，可去除95%以上的殘留物質(zhì)且使細(xì)胞損失最小。在此，我們描述了如何使用CTSRotea系統(tǒng)來有效去除T細(xì)胞培養(yǎng)基中的各種殘留物質(zhì)。洗滌T細(xì)胞去除雜質(zhì)能使細(xì)胞更安全且更有利于下游應(yīng)用。CTSRotea系統(tǒng)的細(xì)胞洗滌和濃???CTS免疫細(xì)胞SR?CTSDPBS預(yù)充系統(tǒng)細(xì)胞上樣預(yù)充系統(tǒng)細(xì)胞上樣Theworldleaderinservingsciencegibco材料和方法首先使用CTSRotea系統(tǒng)和Gibco?CTS?Rotea?一次性管路套件，從leukopakGibco?CTS?Dynabeads?CD3/CD28磁珠在以下T細(xì)胞完全培養(yǎng)基中分離和擴(kuò)增T細(xì)胞：添加了Gibco?CTS?免疫細(xì)胞血清替代物（SR）、Gibco?L-谷氨酰?CTS?DPBS或使用加入2%人圖1.用于洗滌和濃縮T細(xì)胞的CTSRotea一次性g力102034A至B560–7J至K89J至KK至J889個(gè)細(xì)胞9個(gè)細(xì)胞DPBS9個(gè)細(xì)胞DPBS9個(gè)細(xì)胞可去除培養(yǎng)基中99%以上的殘留葡萄糖。用120mL相同洗滌緩ResidualglucoseWashvolume(mL):DPBS+2%HSAWashvolume(mL)Washvolume(mL)20304050607095120Glucoseremoved25.08%58.96%81.12%93.81%97.45%99.19%99.79%99.98%100.00%*ResidualIL-2Washvolume(mL):DPBS+2%HSAWashvolume(mL)Washvolume(mL)20304050607095120145IL-2removed16.72%13.68%63.54%78.89%82.61%93.73%97.25%97.62%93.11%98.78%ResidualDMSOWashvolume(mL):DPBSWashvolume(mL):DPBSWashvolume(mL)20Washvolume(mL)20304050607095120DMSOremoved14.64%49.19%62.18%80.12%89.62%93.85%95.97%97.91%100.00%*Washvolume(mL)20304050607095120145HSAremoved28.01%44.49%76.65%86.64%83.89%89.94%95.62%96.35%94.11%94.02%T細(xì)胞起始量8ABC908070605040302001024.31%Washvolume(mL)Removedglucose*Noglucose1024.31%Washvolume(mL)Removedglucose*Noglucosedetectedinoutput.0255075100125150Washvolume(mL):DPBS+22060.61%2060.61%3078.89%4088.72%5095.05%6098.07%7099.57%9599.60%12099.89%145100.00%*908070605040302005x10Tcells0255075100125150WashvolumeWashvolume(mL):DPBS+2Washvolume(mL)Removedglucose25.08%2058.96%3081.12%4093.81%5097.45%6099.19%7099.79%9599.98%100.00%*Noglucosedetectedinoutput.*908070605040302005x10Tcells0255075100125150Washvolume(mL):DPBS+2Washvolume(mL)Removedglucose33.68%2081.19%3094.94%4098.28%5099.18%6099.35%7099.49%9599.53%99.58%99.67%24599.99%CF比率的影響心力g設(shè)定為1,200×g、2,000×g或2,500×g，以此ABCExpanded(1,200xg,25mL/min)908070605040302002550751001251509080706050403020090807060504030200Compressed(2,500xg,25mL/min)Standard(2,000xg,25mL/min)255075100125150255075100125150000Washvolume(mL)Removedglucose1010.09%Washvolume(mL)Removedglucose1010.09%2061.14%3081.25%4094.15%5097.68%6099.20%7099.59%95100.00%*oglucosedetectedinoutput.Washvolume(mL)Removedglucose25.08%2058.96%3081.12%4093.81%5097.45%6099.19%99.79%9599.98%100.00%*Noglucosedetectedinoutput.Washvolume(mL)Removedglucose13.48%2055.21%3076.09%4090.44%5096.74%6098.54%99.28%9599.69%99.77%99.81%1x10TcellswashedontheCTSRoteasystem~45minutes~45minutes01x10Tcellswashedmanually0~135minutesIL-2removalontheCTSRoteasystem84.62%29.76%85.47%61.64%91.79%85.95%89.83%94.27%92.25%98.74%99.23%97.74%97.19%98.36%96.20%圖5.使用CTSRotea系統(tǒng)和使用臺(tái)式離心機(jī)手動(dòng)洗滌的IL-2去除效率的比較。0圖6.洗滌和濃縮過程后的平均T細(xì)胞活性和 0mLwash10mLwash20mLwash30mLwash 40mLwash50mLwash60mLwash70mLwash圖7.CTSRotea系統(tǒng)更換培養(yǎng)基的可視化。CTSRotea系統(tǒng)可以有效去除細(xì)胞培養(yǎng)基中的殘留物質(zhì)，并最大