With the recent progress in cell-based regenerative therapies, the challenge of isolating functional cells for cell therapy applications has gained significance. Traditional chromatography-based purification techniques cannot be extended to cell separations due to limitations like high shear stress environment for cells, requirement of special columns, long processing time. There is an urgent need to develop an alternative cell purification technique that will guarantee optimum recovery, cell viability, scalability, purity and fast processing times.<br/>In this work, we designed a phage display technique to identify affinity peptides that target and bind to extracellular proteins expressed on dermal fibroblasts and endothelial cells (HUVECs) with high affinity and specificity. Three parallel phage bio-panning rounds were conducted separately against both cell types. Appropriate subtraction steps and washes were employed in the screen to eliminate the possibility of selecting non-specifically binding phage. Immunofluorescence assays were carried out to determine the binding strength and specificity of the selected phage clones to the two cell types. Preliminary data indicated the requirement of more biopanning rounds to achieve more selective ligands. Therefore, five additional rounds of biopanning were performed. This led to a significant improvement in the selectivity of phage clones toward the two cell types. Two strongly performing candidates B8 and F12 (out of 80 tested) showed high specific binding to HUVECs and fibroblasts respectively and were further evaluated for cell separation. Flow cytometric experiments confirmed the high affinity and specificity of the two lead peptides. To evaluate the efficacy of these peptides for cell separation applications, the peptides were immobilized on Dynabeads®. Significantly, F12 and B8 functionalized dynabeads® were successful in isolating pure samples of fibroblast and endothelial cells respectively from a complex mixture. This work serves as a proof-of-concept for the purification of specific cell types using strategic phage biopanning approaches, and can be extended for the effective downstream processing of more therapeutically relevant cell types.

Developing affinity peptide-based cell separation using phage display

2019 Spring National Meeting

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