Cell-based scaffold-free tissue equivalents present a limited clinical translation as consequence of the delayed extracellular matrix (ECM) deposition due to the prolonged production time in vitro. Different combinations of media supplements such as ascorbic acid, growth factors, oxygen tension among others can modulate the cell fate or the ECM synthesis. New research lines are focusing on the use of macromolecular crowders (MMCs) as media supplement for cell sheet production due to their ability to increase ECM deposition by volume exclusion effect, pro-collagenases alosteric regulation, matrix self-assembly by confinement and diffusion limitation (most probably, modulating the interaction between the ECM, MMPs and TIMPs). Herein, different molecular weights and concentrations of a natural potential MMC (Crowder-A) have been tested in equine adipose-derived stem cell (eADSC) and human dermal fibroblast (hADF) cultures in comparison with other commonly used crowders such as carrageenan and the Ficoll™ cocktail 70 KDa and 400 KDa. The eADSCs were characterized according to the current criteria for horse MSCs. Tri-lineage and FACS analysis showed eADSC osteogenic and adipogenic potentials and the presence of the markers CD29, CD44, CD90. The screening of the aforementioned Crowder-A was performed in cultures of 15,000 cells / cm² for the eADSCs and 25,000 cells / cm² for the hADFs during 3, 5, and 7 days. Non-MMC conditions were used as negative controls. Collagen type I was analysed by SDS-PAGE. Other collagen types were studied by immunocytochemistry assays. Significant increase of some ECM components was observed in some concentrations and molecular weights of the Crowder-A.