A supercritical CO2 injection system for the production of polymer/mammalian cell composites

We have recently described a technique whereby supercritical CO2 (scCO2) was used to process mammalian cells into polymeric tissue engineering scaffolds. However, the time-dependent survival of the cells in scCO2 places significant restrictions on polymer processing times and pressures, which are both important factors in the final characteristics of the foamed scaffold. Here, we report an extension to that work that facilitates adequate plasticization time followed by the introduction of cells into the already plasticized polymer via a high-pressure CO2 injection port. Live murine C2C12 cells were shown to not only survive this injection process, via LIVE/DEAD staining, but also to retain their ability to undergo osteogenic differentiation after induction with bone morphogenetic protein-2 (BMP-2) as indicated by an alkaline phosphatase activity. Cells were injected into plasticized poly(DL-lactic acid) (PDLLA) under scCO2 conditions and were visualised in the resultant 3D porous foams by both SEM and micro-CT. After subsequent culture of the cell loaded scaffolds, the cells were shown to retain both metabolic and enzyme activity. This work represents an important development in the production of polymer/cell composite materials for biotechnological applications using a single scCO2 processing step.