应用指南 - 使用CTS Rotea逆流离心系统洗脱残留物

在用于细胞治疗前，细胞可能要经过解冻、筛选、扩增、基因改造、分化和/或冷冻保存这些步骤。每个步骤可能需要不同的培养基或缓冲液，其中可能含有对细胞有害的物质。Gibco™CTS™Rotea™逆流离心系统可有效去除残留物质。通过流化床泵入洗涤缓冲液，以替换用于上样细胞的培养基，可去除95%以上的残留物质且使细胞损失最小。在此，我们描述了如何使用CTSRotea系统来有效去除T细胞培养基中的各种残留物质。洗涤T细胞去除杂质能使细胞更安全且更有利于下游应用。CTSRotea系统的细胞洗涤和浓•••CTS免疫细胞SR•CTSDPBS预充系统细胞上样预充系统细胞上样Theworldleaderinservingsciencegibco材料和方法首先使用CTSRotea系统和Gibco™CTS™Rotea™一次性管路套件，从leukopakGibco™CTS™Dynabeads™CD3/CD28磁珠在以下T细胞完全培养基中分离和扩增T细胞：添加了Gibco™CTS™免疫细胞血清替代物（SR）、Gibco™L-谷氨酰™CTS™DPBS或使用加入2%人图1.用于洗涤和浓缩T细胞的CTSRotea一次性g力102034A至B560–7J至K89J至KK至J889个细胞9个细胞DPBS9个细胞DPBS9个细胞可去除培养基中99%以上的残留葡萄糖。用120mL相同洗涤缓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细胞起始量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比率的影响心力g设定为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%图5.使用CTSRotea系统和使用台式离心机手动洗涤的IL-2去除效率的比较。0图6.洗涤和浓缩过程后的平均T细胞活性和 0mLwash10mLwash20mLwash30mLwash 40mLwash50mLwash60mLwash70mLwash图7.CTSRotea系统更换培养基的可视化。CTSRotea系统可以有效去除细胞培养基中的残留物质，并最大