Department of Applied Chemistry, Graduate School of Engineering, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka 819-0395, Japan.
Division of Biotechnology, Centre for Future Chemistry, Kyushu University, 744 Motooka, Nishi-Ku, Fukuoka 819-0395, Japan.
ACS Biomater Sci Eng. 2024 Jan 8;10(1):628-636. doi: 10.1021/acsbiomaterials.3c01386. Epub 2023 Dec 4.
Droplet-based high-throughput screening systems are an emerging technology that provides a quick test to screen millions of cells with distinctive characteristics. Biopharmaceuticals, specifically therapeutic proteins, are produced by culturing cells that secrete heterologous recombinant proteins with different populations and expression levels; therefore, a technology to discriminate cells that produce more target proteins is needed. Here, we present a droplet-based microfluidic strategy for encapsulating, screening, and selecting target cells with redox-responsive hydrogel beads (HBs). As a proof-of-concept study, we demonstrate the enrichment of hybridoma cells with enhanced capability of antibody secretion using horseradish peroxidase (HRP)-catalyzed hydrogelation of tetra-thiolate poly(ethylene glycol); hybridoma cells were encapsulated in disulfide-bonded HBs. Recombinant protein G or protein M with a C-terminal cysteine residue was installed in the HBs via disulfide bonding to capture antibodies secreted from the cells. HBs were fluorescently stained by adding the protein L-HRP conjugate using a tyramide signal amplification system. HBs were then separated by fluorescence-activated droplet sorting and degraded by reducing the disulfide bonds to recover the target cells. Finally, we succeeded in the selection of hybridoma cells with enhanced antibody secretion, indicating the potential of this system in the therapeutic protein production.
基于液滴的高通量筛选系统是一种新兴技术，它提供了一种快速测试方法，可以对具有独特特征的数百万个细胞进行筛选。生物制药，特别是治疗性蛋白质，是通过培养分泌具有不同群体和表达水平的异源重组蛋白质的细胞来生产的；因此，需要一种能够区分产生更多目标蛋白质的细胞的技术。在这里，我们提出了一种基于液滴的微流控策略，用于封装、筛选和选择具有氧化还原响应水凝胶珠（HBs）的靶细胞。作为概念验证研究，我们展示了使用辣根过氧化物酶（HRP）催化的四巯基聚乙二醇的水凝胶化来富集具有增强抗体分泌能力的杂交瘤细胞；杂交瘤细胞被封装在二硫键结合的 HBs 中。通过二硫键将带有 C 末端半胱氨酸残基的重组蛋白 G 或蛋白 M 安装在 HBs 中，以捕获从细胞中分泌的抗体。通过添加蛋白 L-HRP 缀合物并用辣根过氧化物酶信号放大系统进行荧光染色 HBs。然后通过荧光激活液滴分选分离 HBs，并通过还原二硫键降解 HBs 以回收靶细胞。最后，我们成功地选择了具有增强抗体分泌能力的杂交瘤细胞，表明该系统在治疗性蛋白质生产中的潜力。
