On-demand killing of adherent cells on photo-acid-generating culture substrates.

As a powerful tool of cell screening and cell purification, we developed a novel method to kill adherent cells as cultured on a substrate by micro-projection of incoherent visible light. To kill the cells by the mild light irradiated by electrically controllable micro-projection systems currently available, we introduced the assist of the photo-responsive culture substrates functionalized with a photo-acid-generating polymer. In clear contrast to the existing laser-based methods requiring point scanning, areal micro-projection of blue light with the wavelength 436 nm killed many CHO-K1 cells at a time in the irradiated area on the substrate. The effect of the photo-generated acid was so confined that selective killing of targeted cells was achieved without critical damage to the neighboring cells. Further, we demonstrated the photo-selective killing of the adherent cells after preliminarily patterning through the photo-induced removal of cell adhesion-inhibiting polymer.

Effective cell separation utilizing poly(N-isopropylacrylamide)-grafted polypropylene membrane containing adsorbed antibody.

We previously developed a cell separation method using a poly(N-isopropylacrylamide)-grafted polypropylene (PNIPAAm-g-PP) membrane containing an adsorbed monoclonal antibody (mAb). The purpose of this study is to elucidate the cell separation mechanism in detail and to design an optimal method. As the grafting yield of PNIPAAm increased, the level of the adsorption of IgG(1) and cell adhesion to the membrane decreased. After BSA was adsorbed to a PNIPAAm-g-PP membrane at 6 degrees C, where PNIPAAm was hydrophilic, a small amount of IgG(1) was adsorbed to the membrane at 37 degrees C, where PNIPAAm was hydrophobic. The desorption of the adsorbed IgG(1) was not enhanced even though temperature was reduced to 10 degrees C, where PNIPAAm was hydrophilic. These results indicate that the antibody adsorbed to the intact PP surface of the membrane predominantly contributes to the capture of target cells through the antigen-antibody reaction and that a thermoresponsive transition of PNIPAAm contributes to the detachment of the captured cells. The total number of cells recovered from a PNIPAAm-g-PP membrane containing the adsorbed mAb decreased as the grafting yield increased. A PNIPAAm-g-PP membrane with a 1.7% grafting yield containing adsorbed anti-human CD34 mAb enriched CD34-positive KG-1a cells to 85% from a 1:1 cell suspension of KG-1a cells and CD34-negative Jurkat cells.

Poly(N-isopropylacrylamide)-graft-polypropylene membranes containing adsorbed antibody for cell separation.

We developed a novel selective cell-separation method based on using a poly(N-isopropylacrylamide)-graft-polypropylene (PNIPAAm-g-PP) membrane containing adsorbed monoclonal antibody specific to the target cell. This membrane was prepared by plasma-induced polymerization and soaking in an antibody solution at 37 degrees C. Poly(N-isopropylacrylamide) has a thermoresponsive phase transition: at 32 degrees C water-insoluble (hydrophobic) and water-soluble (hydrophilic) states interconvert. Adsorption of antibody onto PNIPAAm-g-PP membrane at 37 degrees C and its desorption at 4 degrees C was verified by fluorescence-microscopy of the PNIPAAm-g-PP membrane after soaking it in fluorescein-conjugated goat anti-mouse IgG in phosphate-buffered saline. PNIPAAm-g-PP membranes containing adsorbed anti-mouse CD80 monoclonal antibody preferentially captured mouse-CD80 transfected cells at 37 degrees C compared with membranes lacking antibody or containing anti-mouse CD86 monoclonal antibody. Detachment of captured cells from PNIPAAm-g-PP membranes was facilitated by washing at 4 degrees C because of the thermoresponsive phase transition of PNIPAAm. With this method, mouse CD80- or mouse CD86-transfected cells were enriched from a 1:1 cell suspension to 72% or 66%, simply and with high yield.

Surface modification of poly(L: -lactic acid) affects initial cell attachment, cell morphology, and cell growth.

The object of this study was to develop a highly porous scaffold to be used in regeneration of blood vessels, nerves, and other hollow tissues with small openings. Using the phase-inversion method and a mixture of water and methanol as a coagulating agent, we prepared highly porous flat membranes from poly(L: -lactic acid) (PLLA) with numerous pores both on the surface and in the interior of the membranes. Chinese hamster ovary (CHO) cells were cultured on the membranes to evaluate initial cell adhesion, cell proliferation, and cell morphology. Adhesion of CHO cells to PLLA was poor: the cells adhered at approximately half the rate observed with a tissue culture polystyrene dish (TCPS). In contrast, adhesion of cells to PLLA treated with a low-temperature oxygen plasma was good; the adhesion rate was the same as that on TCPS. The rate of cell proliferation on the treated membranes was no different from that on the nontreated membranes, but cell morphologies were quite different. The cells on the nontreated membranes were small and round and proliferated separately from one another. In contrast, the cells on the plasma-treated membranes proliferated in close contact with other cells, spreading out extensively in sheet-like formations. Since the plasma treatment not only accelerated cell adhesion but also enabled cells to proliferate in the form of sheets resembling biological tissue, we believe that oxygen-plasma treatment is extremely effective for modifying surfaces of materials used for tissue regeneration.

Critical roles of Pten in B cell homeostasis and immunoglobulin class switch recombination.

Pten is a tumor suppressor gene mutated in human cancers. We used the Cre-loxP system to generate a B cell-specific mutation of Pten in mice (bPten(flox/flox)mice). bPten(flox/flox) mice showed elevated numbers of B1a cells and increased serum autoantibodies. Among B2 cells in bPten(flox/flox) spleens, numbers of marginal zone B (MZB) cells were significantly increased while those of follicular B (FOB) cells were correspondingly decreased. Pten-deficient B cells hyperproliferated, were resistant to apoptotic stimuli, and showed enhanced migration. The survival kinase PKB/Akt was highly activated in Pten-deficient splenic B cells. In addition, immunoglobulin class switch recombination was defective and induction of activation-induced cytidine deaminase (AID) was impaired. Thus, Pten plays a role in developmental fate determination of B cells and is an indispensable regulator of B cell homeostasis.