## Materials and Methods  
### Production of Microcarrier Prototypes  
MCs were manufactured through the water in oil in water (W\({}_{1}\)/O/W\({}_{2}\)) double emulsion solvent evaporation method with a foaming agent, according to a modified version of the process defined by [PERSON] et al. [25] to obtain a functionalized MC surface with hydrophilic components (e.g., gelatin). MCs surface functionalization is crucial since the cell culture will be conducted in a defined xeno- and serum-free medium. The cells cannot properly attach to nude MCs (data not shown). Initially, a double emulsion volume of 310.5 mL was used before scaled-up to a final manufacturing process volume of 2066 mL. All the further development efforts to optimize the parameters were conducted using this final volume. Poly(D,l-lactide-co-glycolide) (PLGA) (lactide:glycolide ratio 50:50), Purasorb PDLG 5002 (Mw 17 KDa), Purasorb PDLG 5004 (Mw 44 KDa), Purasorb PDLG 5010 (Mw 153 KDa), Purasorb PLC 7015 and Purasorb PLC 9517 (copolymer of L-lactide and \(\epsilon\)-caprolactone) were purchased from Corbion Purac. PLGA with Mw 7 KDa (5050 DLG 1A) was purchased from Evonik. Polycaprolactonees (PCL) with Mw 14 KDa and Mw 80 KDa were purchased from Sigma-Aldrich. Polyvinyl alcohol (PVA, 87-89% hydrolyzed, Mw 31 KDa) and methylene chloride were purchased from Merck. The bovine and porcine gelatine was a generous gift from Italgelatine (Santa Vittoria d'Alba, Italy), and ammonium hydrogen carbonate was purchased from Applichem. Several of the manufacturing process parameters that affect different MC characteristics were analyzed. The parameters that were evaluated and mainly affected MCs morphology are as follows: (I) starting polymer raw material, (II) gas foaming agent (NH\({}_{4}\)HCO\({}_{3}\)) concentration, (III) PVA concentration, and (IV) polymer concentration in the hydrophobic phase (O), and (V) primary emulsion (W\({}_{1}\)/O) and secondary emulsion (W\({}_{1}\)/O/W\({}_{2}\)) stirring speeds, which affect the MCs PSD. Furthermore, surface functionalization was attempted using: collagen peptides and gelatin from different sources (fish, bovine, and porcine). Of course, changes in morphology might lead to a consequent change in PSD. Different functionalization molecules also lead to morphological and particle dimension changes, thus creating interconnections between the different characteristics. (I) Several polymers that have biodegradability and resorbability features and are FDA approved were evaluated. The three main polymers tested were: PLGA (50:50 lactic acid/glycolic acid ratio) with different molecular weights (7 KDa, 17 KDa, 44 KDa, and 153 KDa), PCL (14 KDaand 80 kDa), and two copolymers of lactic acid and caprolactone with different monomer compositions. Specifically, PLC 7015 (30% caprolactone and 70% lactic acid) and PLC 9517 (5% caprolactone and 95% lactic acid) were used. In general, PCL and both PLC (7015 and 9517) tend to be less prone to porous formation during MC manufacturing processes, while PLGA tends to form perfectly porous structures (Figure 1). This tendency to form a porous surface is reduced when the polymer's molecular weight increases, independently from the used material (PLGA or PCL). For identical foaming agent concentrations, a higher polymer molecular weight results in a less porous surface (data not shown). Based on this understanding, PLGA with a molecular weight of 17 kDa was selected. (II) The gas foaming agent utilized was NH\({}_{4}\)HCO\({}_{3}\). This was included in the aqueous phase of the primary emulsion and spontaneously produced ammonia and carbon dioxide gas bubbles during solvent evaporation. This phenomenon resulted in the formation of open pores in the interior region and the MC's surface. As already shown by [PERSON] et al. (2015), increasing the gas foaming agent's concentration increases the pores' number and size.