Rapid Engineering of Three-Dimensional, Multicellular Tissues With Polymeric Scaffolds

Engineered tissues could be grown in weeks or days instead of months.

A process has been developed for the rapid tissue engineering of multicellular- tissue-equivalent assemblies by the controlled enzymatic degradation of polymeric beads in a low-fluid- shear bioreactor. In this process, the porous polymeric beads serve as temporary scaffolds to support the assemblies of cells in a tissuelike 3D configuration during the critical initial growth phases of attachment of anchorage-dependent cells, aggregation of the cells, and formation of a 3D extracellular matrix. Once the cells are assembled into a 3D array and enmeshed in a structural supportive 3D extracellular matrix (ECM), the polymeric scaffolds can be degraded in the low-fluid-shear environment of the NASA-designed bioreactor. The natural 3D tissuelike assembly, devoid of any artificial support structure, is maintained in the low-shear bioreactor environment by the newly formed natural cellular/ECM. The elimination of the artificial scaffold allows normal tissue structure and function.

The advantages afforded by the enzymatic- digestion method, relative to the prior method, arise in connection with much greater speed of digestion. The biodegradable polymers commonly used heretofore as scaffolding materials have been poly(lactic acid), poly(glycolic acid), and copolymers of lactic and glycolic acids. The time needed for complete degradation of scaffolding made from these polymers typically ranges from 10 to 52 weeks, the exact time depending on the chemical composition of the polymer. Such long degradation times are problematic, especially when 3D tissue assemblies without artificial materials are needed in much shorter times (for example, for growth of autologous tissue to be implanted to replace damaged or diseased tissue). In contrast, the enzymatic-degradation method enables complete digestion of polymeric scaffolding within days.

For a tissue-engineering process that incorporates this enzymatic-digestion process, one must select a scaffolding material amenable to enzymatic degradation. For an experiment in which such a process was demonstrated, dextranbased beads were selected as the scaffolding and dispase (a neutral protease) was selected as an enzyme that could digest the beads without damaging cell membranes or disrupting the 3D tissuelike infrastructure. The beads were initially incubated with rat fibroblasts for four days on a rotary shaker, then the fibroblast-coated beads (see upper part of figure) were inoculated into a nutrient fluid in a horizontal-axis rotatingvessel (HARV) bioreactor, which provided a low-shear flow environment. After one day of incubation in the HARV, human epithelial cells were inoculated and cultured for three days to allow the formation of a natural structural infrastrucure comprising fibroblast-epithelial cell layers and a prominent ECM. Next, dispase was introduced into the culture medium to digest the beads and incubation was continued for another week. Microscopic examination of spheroids showed (see lower part of figure) that the controlled enzymatic degradation of an artificial matrix in the low shear environment of the NASAdesigned bioreactor could rapidly produce 3D tissuelike spheroids free of any artificial infrastructure.

This work was done by Steve R. Gonda of Johnson Space Center, Jacqueline Jordan of Universities Space Research Association, and Denise N. Fraga of Enterprise Advisory Service, Inc. For further information, contact the Johnson Innovative Partnerships Office at (281) 483-3809.