Fibrosis is a pathological accumulation of excessive collagen that underlies many of the most common diseases,

representing dysfunction of the essential processes of normal tissue healing. Fibrosis research aims

to limit this response without ameliorating the essential role of fibrogenesis in organ function. However, the

absence of a realistic in vitro model has hindered investigation into mechanisms and potential interventions

because the standard 2D monolayer culture of fibroblasts has limited applicability. We sought to develop

and optimize fibrosis spheroids: a scaffold-free three-dimensional human fibroblast-macrophage spheroid

system representing an improved benchtop model of human fibrosis. We created, characterized and optimized

human fibroblast-only spheroids, demonstrating increased collagen deposition compared to monolayer

fibroblasts, while spheroids larger than 300 μm suffered from progressively increasing apoptosis. Next,

we improved the spheroid system with the addition of human macrophages to more precisely recapitulate

the environment during fibrogenesis, creating a hybrid spheroid system with different ratios of fibroblasts

and macrophages ranging from 2 : 1 to 64 : 1. We found that in the hybrid spheroids (particularly the 16 : 1

[F16] ratio) more fibroblasts were activated, with greater macrophage polarization towards a pro-inflammatory

M1 phenotype. Hybrid spheroids containing higher ratios of macrophages showed greater macrophage

heterogeneity and less fibrogenesis, while low macrophage ratios limited macrophage-induced effects and

yielded less collagen deposition. The F16 group also had the highest expression levels of fibrosis-related

genes (Col-1a1, Col-3a1 and TGF-β) and inflammation-related genes (TNF, IL1β and IL6). IF staining demonstrated

that F16 spheroids had the highest levels of αSMA, collagen-1 and collagen-3 deposition among all

groups as well as formation of a dense collagen rim surrounding the spheroid. Future studies exploring the

greater fibrotic activity of F16 spheroids may provide new mechanistic insights into diseases involving excessive

fibrotic activity. Microtissue fibrosis models capable of achieving greater clinical fidelity have the potential

to combine the relevance of animal models with the scale, cost and throughput of in vitro testing.