Thermoresponsive block copolymer brush for temperature-modulated hepatocyte separation
Thermoresponsive block copolymer brush for temperature-modulated hepatocyte separation
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用于温度调节肝细胞分离的热响应嵌段共聚物刷
DOI：
10.1039/d2tb01384c
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发表时间：
2022
影响因子：
7
通讯作者：
Hideko Kanazawa
中科院分区：
文献类型：
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作者：
Kenichi Nagase;Naoto Kojima;Mitsuaki Goto;Toshihiro Akaike;Hideko Kanazawa
肝组织工程可能是治疗肝病的有效途径；然而，其实际应用需要不涉及细胞表面修饰并保持细胞活性的肝细胞分离技术。在这项研究中，我们使用热响应性聚合物和对肝细胞具有高亲和力的聚合物开发了肝细胞分离材料。通过两步原子转移自由基聚合反应制备了聚(N-对乙烯基苄基-O-β-D-吡喃半乳糖基-(1→4)-D-葡萄糖酰胺)(PVLA)和聚(N-异丙基丙烯酰胺)(PNIPAAm)嵌段共聚物[PVLA-b-PNIPAAm]。在制备的 PVLA-b-PNIPAAm 刷上，HepG2 细胞（模型肝细胞）在 37 °C 时粘附，在 20 °C 时分离，这归因于 PVLA 和 HepG2 之间的温度调节亲和力。来自永生化人肝星状细胞系 (TWNT-1) 的细胞不粘附到共聚物刷上，而 RAW264.7 细胞（小鼠巨噬细胞；模型库普弗细胞）粘附到共聚物刷上，无论温度如何。利用共聚物刷上细胞粘附特性的差异，成功地证明了温度调节细胞分离。将 HepG2、RAW264.7 和 TWNT-1 细胞的混合物在 37°C 下接种到共聚物刷上以进行粘附。通过将温度降低至 20 °C，选择性回收粘附的 HepG2 细胞，纯度约为 85%，且活性正常。此外，诱导多能干（iPS）细胞来源的肝细胞在37℃时粘附在PVLA-b-PNIPAAm刷上，并在20℃时从共聚物刷上脱离，而未分化的iPS细胞不粘附，表明制备的PVLA-b-PNIPAAm刷可用于分离肝细胞分化和未分化细胞。这些结果表明，新开发的PVLA-b-PNIPAAm刷可以通过温度调节将肝细胞与污染物细胞分离，而不影响细胞活性或改变细胞表面。因此，共聚物刷有望成为使用肝细胞进行细胞治疗和组织工程的有用分离工具。
Hepatic tissue engineering may be an effective approach for the treatment of liver disease; however, its practical application requires hepatic cell separation technologies that do not involve cell surface modification and maintain cell activity. In this study, we developed hepatocyte cell separation materials using a thermoresponsive polymer and a polymer with high affinity to hepatocytes. A block copolymer of poly(N-p-vinylbenzyl-O-β-D-galactopyranosyl-(1→4)-D-gluconamide) (PVLA) and poly(N-isopropylacrylamide) (PNIPAAm) [PVLA-b-PNIPAAm] was prepared through two steps of atom transfer radical polymerization. On the prepared PVLA-b-PNIPAAm brush, HepG2 cells (model hepatocytes) adhered at 37 °C and detached at 20 °C, attributed to the temperature-modulated affinity between PVLA and HepG2. Cells from the immortalized human hepatic stellate cell line (TWNT-1) did not adhere to the copolymer brush, and RAW264.7 cells (mouse macrophage; model Kupffer cells) adhered to the copolymer brush, regardless of temperature. Using the difference in cell adhesion properties on the copolymer brush, temperature-modulated cell separation was successfully demonstrated. A mixture of HepG2, RAW264.7, and TWNT-1 cells was seeded on the copolymer brush at 37 °C for adherence. By reducing the temperature to 20 °C, adhered HepG2 cells were selectively recovered with a purity of approximately 85% and normal activity. In addition, induced pluripotent stem (iPS) cell-derived hepatocytes adhered on the PVLA-b-PNIPAAm brush at 37 °C and detached from the copolymer brush at 20 °C, whereas the undifferentiated iPS cells did not adhere, indicating that the prepared PVLA-b-PNIPAAm brush could be utilized to separate hepatocyte differentiated and undifferentiated cells. These results indicated that the newly developed PVLA-b-PNIPAAm brush can separate hepatic cells from contaminant cells by temperature modulation, without affecting cell activity or modifying the cell surface. Thus, the copolymer brush is expected to be a useful separation tool for cell therapy and tissue engineering using hepatocytes.