Chester Rosie, Das Anupam A K, Medlock Jevan, Nees Dieter, Allsup David J, Madden Leigh A, Paunov Vesselin N
Department of Chemistry and Biochemistry, University of Hull, Cottingham Road, Hull, HU67RX, U.K.
Joanneum Research FmbH, Leonhardstrasse 59, 8010 Graz, Austria.
ACS Appl Bio Mater. 2020 Feb 17;3(2):789-800. doi: 10.1021/acsabm.9b00770. Epub 2020 Jan 17.
We report a cell recognition chromatography approach for blood cancer cell separation from healthy peripheral blood mononuclear cells (PBMCs) based on size-matched functionalized particle imprints. Negative imprints were prepared from layers of 15 μm polymeric microbeads closely matching the size of cultured human leukemic cells (HL60). We replicated these imprints on a large scale with UV curable polyurethane resin using nanoimprinting lithography. The imprints were functionalized with branched polyethylene imine (bPEI) and passivated by Poloxamer 407 to promote a weak attraction toward cells. When a matching cell fits into an imprint cavity, its contact area with the imprint is maximized, which amplifies the attraction and the binding selectivity. We tested these imprints specificity for depleting myeloblasts from a mixture with healthy human PBMCs in a cell recognition chromatography setup hosting the imprint. The mixture of fixed HL60/PBMCs ratio was circulated over the imprint and at each step the selectivity toward HL60 was assessed by flow cytometry. The role of the imprint length, flow rate, channel depth, and the bPEI coating concentration were examined. The results show that HL60 cells, closely matching the imprint cavities, get trapped on the imprint, while the smaller PBMCs are carried away by the drag force of the flow. Lower flow rates, longer imprints, and interim channel depth favor HL60 specific retention. The bPEI concentration higher than 1 wt % on the imprint made it less selective toward the HL60 because of indiscriminate attraction with all cells. Particle imprint based cell recognition chromatography was able to achieve selective myeloblast depletion from initial 11.7% HL60 (88.3% PBMC) to less than 1.3% HL60 for 3 h of circulation. The cell recognition chromatography with size-matched microbead imprints can be employed as an efficient cell separation technique and potentially lead to alternative therapies for myeloblasts removal from peripheral blood of patients with acute myeloid leukemia.
我们报道了一种基于尺寸匹配的功能化颗粒印记从健康外周血单核细胞（PBMC）中分离血癌细胞的细胞识别色谱方法。阴性印记由与培养的人白血病细胞（HL60）大小紧密匹配的15μm聚合物微珠层制备而成。我们使用纳米压印光刻技术，用紫外光固化聚氨酯树脂大规模复制这些印记。印记用支化聚乙烯亚胺（bPEI）功能化，并通过泊洛沙姆407钝化，以促进对细胞的弱吸引力。当匹配的细胞适合印记腔时，其与印记的接触面积最大化，从而增强吸引力和结合选择性。我们在容纳印记的细胞识别色谱装置中测试了这些印记从与健康人PBMC的混合物中耗尽成髓细胞的特异性。固定HL60/PBMC比例的混合物在印记上循环，并且在每个步骤通过流式细胞术评估对HL60的选择性。研究了印记长度、流速、通道深度和bPEI涂层浓度的作用。结果表明，与印记腔紧密匹配的HL60细胞被困在印记上，而较小的PBMC被流动的拖曳力带走。较低的流速、较长的印记和中间通道深度有利于HL60的特异性保留。印记上bPEI浓度高于1 wt%时，由于对所有细胞的无差别吸引，使其对HL60的选择性降低。基于颗粒印记的细胞识别色谱能够在3小时的循环中实现从初始11.7%的HL60（88.3%的PBMC）选择性耗尽成髓细胞至低于1.3%的HL60。具有尺寸匹配微珠印记的细胞识别色谱可作为一种有效的细胞分离技术，并有可能为从急性髓性白血病患者外周血中去除成髓细胞带来替代疗法。
