FIBROBLAST AGGREGATION BY SUSPENSION WITH CONJUGATES OF POLY(ETHYLENEGLYCOL) AND RGD

Cell aggregates may be useful components of artificial organs and mamm alian cell bioreactors, but many cells do not naturally aggregate. In a previous report, we described a method for promoting neural cell agg regation by addition of water-soluble conjugates of cell adhesion pept ides, containing the th ree amino acid sequence Arg-Gly-Asp (RGD), and poly(ethylene glycol) (PEG). Here, we examined the mechanism of conju gate-induced aggregation using fibroblasts and a variety PEG-peptide c onjugates. Aggregation was monitored during rotation culture of fibrob lasts in the presence of unconjugated GRGDY and PEG; monofunctional (P EG-GRGDY) and bifunctional (GRGDY-PEG-GRGDY) conjugates; and bifunctio nal conjugates produced with a similar, but non-cell-binding, peptide (GRGEY-PEG-GRGEY). GRGDY-PEG-GRGDY conjugates induced rapid and pronou nced fibroblast aggregation that was dose-dependent: at the highest co ncentration tested (5 mg/mL GRGDY-PEG-GRGDY), cell aggregates were pro duced more quickly (similar to 1 h) and were significantly larger at 2 4 h (mean radius similar to 66 mu m) than at slightly lower concentrat ions (1.7 and 3.3 mg/m L). Aggregation with GRGDY-PEG-GRGDY was comple tely inhibited by dissolved GRGDY (1.7 mg/ml). Neither unmodified GRGD Y, unmodified PEG, PEG-GRGDY, nor GRGEY-PEG-GRGEY conjugates led to si gnificant aggregation. The extent of aggregation depended on PEG molec ular weight: conjugates with 3400 M(w) PEG produced aggregates with si gnificantly larger mean radius than conjugates with 20,000 M(w) PEG. W hen 1 N-8A fibroblasts, genetically engineered to produce recombinant nerve growth factor(NGF), were aggregated with GRGDY-PEG-GRGDY, aggreg ated cells produced more NGF per cell than nonaggregated cells. Aggreg ation of cells may lead to improved cell function, such as the increas e in NGF production observed here, which could be useful in large-scal e cell culture and construction of artificial organs or tissue transpl ants for tissue engineering. (C) 1996 John Wiley & Sons, Inc.