Dedifferentiated Chondrocyte Culture using Alginate Microbead Prepared from Microfluidic Technique

Abstract

In this study, alginate microbeads were fabricated using a microfluidic system and these microbeads were used as a culture matrix of dedifferentiated chondrocytes. Chondrocytes mixed in the alginate solution were naturally encapsulated within the microbeads during the processes of creating tiny drops of microbeads(similar to 300 mu m). The cell numbers ranged from 10 to 15 cells for each microbead were observed after 3 weeks of culture. Although the cell number remained similar with time, cell viability was found substantially reduced from 59 +/- 18% at 1 week to 40 +/- 14% at 3 weeks. To have chondrocytes dedifferentiated, a series of passages were carried out in monolayer culture for up to P5. These passaged cells(P3 and P5) were encapsulated in the microbeads and then subjected to the administration Of parathyroid hormone related peptide(PTHrP, 0.3 mu g/mL) twice a week for the effect of redifferentiating potential. Without the passage(PO), the results of RT-PCR showed that cells could maintain their phenotype, highly expressing two major extracellular matrix(ECM) molecules, type II collagen and aggrecan. As the number of passage grows, however the mRNA level of type I collagen greatly increased but that of type II collagen significantly faded out. When the PTHrP-treated chondrocytes were tested for the cartilage-specific markers, they exhibited that gene expression of type II collagen was pronounced as compared to the control but the signal from aggrecan was very weak. The present work suggested that alginate microbeads uniformly fabricated from a microfluidic chip can be very useful for chondrocyte encapsulation and that a certain level of redifferentiation of the dedifferentiated chondrocytes may be feasible in three-dimensional(3D) alginate matrix culture.