We have designed a novel microcarrier for cell cluster formation in rotary bioreactor culture. Incorporated is a crosslinking chemistry designed for enzyme-free cell recovery using mild cysteine solutions. The 3-dimensional (3-D) morphology of the resulting cell clusters more closely resembles actual tissue than that of 2-D monolayers, providing more realistic in vitro models for research.

Dextran Sephadex beads (GE Healthcare) were coated with crosslinker-free Extracel under reduced pressure and allowed to oxidatively crosslink. The beads were frozen and lyophilized. For validation, synthesis was repeated with the addition of reacting Alexa Fluor 488 maleimide with thiol groups of the Extracel and imaged at 490 nm. Intestine 407 cells were seeded onto 0.5 grams sterilized microbeads at an amount of 4 million cells in 50 ml media in a 50 ml bioreactor vessel (Synthecon). Rotary culture was continued for 15 days, with media being changed every 2 days. On days 2, 4, 8, 10 and 15, the cells and beads were imaged with light and fluorescent microscopy to track the formation and growth of cell clusters. The cultures were treated with 50 mM N-acetyl-L-cysteine (NAcCys) in media for one hour to reductively cleave the disulfide bonds of the coating. Cell clusters were replated on tissue culture plastic and assayed at 24, 48, and 72 hours for viability.

Reaction of the Extracel thiol groups with Alexafluor 488 maleimide resulted in PBS-rinsed beads fluorescing green under 490 nm, verifying successful coating. During the 15 day culture, cluster growth was consistent. LIVE/DEAD assays showed live cells with healthy cluster morphology. After NAcCys treatment, cells peeled off bead surfaces as the coating was degraded. After replating, cells of healthy monolayer morphology were seen expanding outwards from the initially seeded cluster locations. Furthermore, cell viability on the three days after replating was at least 94%.

Extracel-coated microbeads provide a successful microcarrier/bioreactor system for culturing cells into 3-dimensional cell clusters. This system improves on existing systems by adding an enzyme-free cell recovery method that targets the microbead coating instead of the cell adhesion molecules on the cell surface, allowing non-cell-altering recovery for future culture or use in standard 2-D assays. Intestine 407 clusters similar to ours have already been used to investigate bacterial infections. By varying cell types, our approach could be used in scaled down and inexpensive models for cancer and drug delivery research.