A previous report on an end-over-end mixed batch adsorption process using large beads did not perform well and this was attributed to poor suspension of the beads, a lack of contact of cells with the beads and the possibility of mechanical disruption of the cells on the bead surface. In contrast to these earlier reports we have shown that the use of large beads in a ��roller bottle�� format has proved to be a very effective method of cell capture. We AbMole Oleandrin attribute this to the ease with which the large number of high density beads move through SVF without mechanical restriction, the repeated sedimentation and resupension cycles that they go through and the very high affinity of the interaction when both beads and cell surfaces are populated with antibody. This implies 2 levels of interaction during capture; the antibody on the beads interacting with cell surface antigen in addition to the antibody on the cells binding the Ig specific ligand on the beads. Initial experiments were with Protein A-coated large beads: this is a recombinant microbial protein that binds immunoglobulin at neutral pH; elution can only be achieved at pH,3. Protein A binding affinity can vary between antibody species and sub-class, however previous studies have demonstrated that Protein A binds murine IgG with high affinity therefore it was not surprising that the murine IgG anti-rat CD90 bound to these Protein A coated beads. Efficient cell capture was still obtained when beads were loaded with an incredibly small quantity of antibody. CD90 + cell depletion was reported using flow cytometry, as the essential technique at the analytical stage to quantify cell capture on a population scale supported by fluorescent microscopy and qRTPCR which identified CD90 transcript specifically associated with the beads. Collectively this cross referenced and validated this technique for isolation of CD90 + cells from heterogeneous SVF. Although isolation of cells with Protein A beads was demonstrated to be of high efficiency an effective cellular release method was still required and so an alternative cell capture bead was explored for this purpose. This bead was modified with a covalently bound mixed-mode ligand coating based on an aromatic acid moiety; these ligands bind and release immunoglobins in a pH dependant manner over a narrow range and have been used in preparative chromatographic processes. Otherwise, the overall structure and density of this mixed mode ligand bead was identical to the Protein A counterpart. This investigation successfully demonstrated loading of FITC-conjugated antibody onto mixed mode ligand beads at pH5-6, whilst raising the pH to 8.4 instantly released the antibody and subsequent bound cells. Pre-incubating ligand beads with an excess of polyclonal IgG prior to release significantly increased release efficiency. This suggested that saturating the ligand binding sites on the beads with non-specific IgG reduced the possibility of multiple interactions with the cellspecific antibody leading to optimal release kinetics. This study therefore presents the initial steps in the validation of a new, minimally invasive stem cell harvesting system. Future research will focus on confirming and quantifying cell viability and phenotype maintenance in response to subjection to this novel pH mediated sorting strategy. A new approach to isolating highly purified populations of cells from primary complex mammalian tissues has been experimentally evidenced and validated.