関 実

Technologies for efficiently expanding Chinese hamster ovary (CHO) cells, the primary host cells for antibody production, are of growing industrial importance. Various processes for the use of microcarriers in CHO suspension cultures have been developed, but there have been very few studies on cell-adhesive microcarriers that are similar in size to cells. In this study, we proposed a new approach to suspension cultures of CHO cells using cell-sized condensed and crosslinked gelatin microparticles (GMPs) as carriers. Unlike commercially available carriers with sizes typically greater than 100 μm, each cell can adhere to the surface of multiple particles and form loose clusters with voids. We prepared GMPs of different average diameters (27 and 48 μm) and investigated their effects on cell adhesion and cluster formation. In particular, small GMPs promoted cell proliferation and increased IgG4 production by the antibody-producing CHO cell line. The data obtained in this study suggest that cell-sized particles, rather than larger ones, enhance cell proliferation and function, providing useful insights for improving suspension-culture-based cell expansion and cell-based biologics production for a wide range of applications.

Spheroid formation assisted by microengineered chambers is a versatile approach for morphology-controlled three-dimensional (3D) cell cultivation with physiological relevance to human tissues. However, the limitation in diffusion-based oxygen/nutrient transport has been a critical issue for the densely packed cells in spheroids, preventing maximization of cellular functions and thus limiting their biomedical applications. Here, we have developed a multiscale microfluidic system for the perfusion culture of spheroids, in which porous microchambers, connected with microfluidic channels, were engineered. A newly developed process of centrifugation-assisted replica molding and salt-leaching enabled the formation of single micrometer-sized pores on the chamber surface and in the substrate. The porous configuration generates a vertical flow to directly supply the medium to the spheroids, while avoiding the formation of stagnant flow regions. We created seamlessly integrated, all PDMS/silicone-based microfluidic devices with an array of microchambers. Spheroids of human liver cells (HepG2 cells) were formed and cultured under vertical-flow perfusion, and the proliferation ability and liver cell-specific functions were compared with those of cells cultured in non-porous chambers with a horizontal flow. The presented system realizes both size-controlled formation of spheroids and direct medium supply, making it suitable as a precision cell culture platform for drug development, disease modelling, and regenerative medicine.

Numerous attempts have been made to organize isolated primary hepatocytes into functional three-dimensional (3D) constructs, but technologies to introduce extracellular matrix (ECM) components into such assemblies have not been fully developed. Here we report a new approach to forming hepatocyte-based 3D tissues using fibrillized collagen microparticles (F-CMPs) as intercellular binders. We created thick tissues with a thickness of w200 mm simply by mixing FCMPs with isolated primary rat hepatocytes and culturing them in cell culture inserts. Owing to the incorporated FCMPs, the circular morphology of the formed tissues was stabilized, which was strong enough to be manually manipulated and retrieved from the chamber of the insert. We confirmed that the F-CMPs dramatically improved the cell viability and hepatocyte-specific functions such as albumin production and urea synthesis in the formed tissues. The presented approach provides a versatile strategy for hepatocyte-based tissue engineering, and will have a significant impact on biomedical applications and pharmaceutical research. (c) 2021, The Society for Biotechnology, Japan. All rights reserved.

Numerous attempts have been made to develop efficient systems to purify trace amounts of circulating tumor cells (CTCs) from blood samples. However, current technologies are limited by complexities in device fabrication, system design, and process operability. Here we describe a facile, scalable, and highly efficient approach to physically capturing CTCs using a rationally designed microfluidic isolator with an array of microslit channels. The wide but thin microslit channels with a depth of several micrometers selectively capture CTCs, which are larger and less deformable than other blood cells, while allowing other blood cells to just flow through. We investigated in detail the effects of the microchannel geometry and operating parameters on the capture efficiency and selectivity of several types of cultured tumor cells spiked in blood samples as the CTC model. Additionally, in situ post-capture staining of the captured cells was demonstrated to investigate the system's applicability to clinical cancer diagnosis. The presented approach is simple in operation but significantly effective in capturing specific cells and hence it may have great potential in implementating cell physics-based CTC isolation techniques for cancer liquid biopsy.