Development of Novel Cell Separation System Using Poly(n- Isopropylacrylamide)-graft-polypropylene Non-woven Membrane with Antibody

Many types of cell-separation methods have been studied and developed. However, these methods are expensive, cannot be operated continuously on a large scale, and are troublesome. The purpose of our study is to develop a novel cell separation method that can capture or enrich a specific cell type continuously and in high yield and that can be operated easily and quickly in clinical settings. We have designed a novel cell-separation system that uses a poly(N-isopropylacrylamide)-graftpolypropylene (PNIPAAm-g-PP) membrane containing an adsorbed monoclonal antibody that binds specifically to the surface of the target cells. Non-woven poly(propylene) (PP) membrane has many advantages, such as large specific surface area, chemical and physical stability, good resistance to biodegradation, and low cost. Poly(N-isopropylacrylamide) (PNIPAAm) has its unique thermal properties: PNIPAAm undergoes a reversible thermoresponsive phase transition in an aqueous solution at approximately 32°C (the lower critical solution temperature, LCST). PNIPAAm is insoluble (in a hydrophobic state) in water above the LCST and reversibly soluble (in a hydrophilic state) below the LCST. We previously prepared a PNIPAAm-g-PP membrane by plasma-induced graft polymerization and found that the permeability of water through this membrane could be controlled by changing the temperature around 32°C. We hypothesized that a PNIPAAm-g-PP membrane adsorbed monoclonal antibody (mAb) would selectively capture a specific cell type having a certain receptor on its surface above the LCST and released the captured cells below the LCST as shown schematically in Figure 1. We previously reported that the PNIPAAm-g-PP membrane containing antibody had the separation ability from a mixture of cells, which had adherent property. In this study, we investigated the ability of these PNIPAAm-g-PP membranes to selectively separate a specific cell type from a mixture of non-adherent cell types. In addition, the mechanism of separating cells was elucidated. Selective adhesion of CD34-positive, human bone marrow acute myelogenous leukemia cells (KG-1a) to PNIPAAm-g-PP membranes containing adsorbed anti-human CD34 mAb was examined. Next, the detachment of attaching cells from the PNIPAAm-g-PP membrane by changing the temperature was evaluated. The ability of the PNIPAAm-g-PP membranes containing anti-human CD34 mAb to selectively separate cells from a 1:1 suspension of KG-1a cells and CD-34 negative, human T lymphoma cells (Jurkat) was confirmed. Finally, the mechanism of this cell separation was discussed focusing on the functions of the mouse IgG1, which was used as a model of mAb, and the grafted PNIPAAm chains.