Cell aggregates may be useful components of artificial organs and mammalian cell bioreactors, but many cells do not naturally aggregate. In a previous r e ~o r t , ~ we described a method for promoting neural cell aggregation by addition of water-soluble conjugates of cell adhesion peptides, containing the three amino acid sequence Arg-Gly-Asp (RGD), and poly(ethy1ene glycol) (PEG). Here, we examined the mechanism of conjugate-induced aggregation using fibroblasts and a variety PEG-peptide conjugates. Aggregation was monitored during rotation culture of fibroblasts in the presence of unconjugated GRGDY and PEG; monofunctional (PEG-GRGDY) and bifunctional (GRGDY-PEG-GRGDY) conjugates; and bifunctional conjugates produced with a similar, but non-cell-binding, peptide (GRGEY-PEG-GRGEY ). GRGDY-PEG-GRGDY conjugates induced rapid and pronounced fibroblast aggregation that was dose-dependent: at the highest concentration tested (5 mg/mL GRGDY-PEG-GRGDY), cell aggregates were produced more quickly (-1 h) and were significantly larger at 24 h (mean radius -66 p m ) than at slightly lower concentrations (1.7 and 3.3 mg/mL). Aggregation with GRGDY-PEG-GRGDY was completely inhibited by dissolved GRGDY (1.7 mg/mL). Neither unmodified GRGDY, unmodified PEG, PEG-GRGDY, nor GRGEY-PEG-GRGEY conjugates led to significant aggregation. The extent of aggregation depended on PEG molecular weight: conjugates with 3400 Mw PEG produced aggregates with significantly larger mean radius than conjugates with 20,000 Mw PEG. When 1 N-8Afibroblasts, genetically engineered to produce recombinant nerve growth factor (NGF), were aggregated with GRGDY-PEG-GRGDY, aggregated cells produced more NGF per cell than nonaggregated cells. Aggregation of cells may lead to improved cell function, such as the increase in NGF production observed here, which could be useful in large-scale cell culture and construction of artificial organs or tissue transplants for tissue engineering. 0