## Materials and Methods
### Production of Microcarrier Prototypes
Concentrations ranging from 0% to 15% \(w/w\) in W\({}_{1}\) were tested, ultimately leading to the selection of 0.3% \(w/w\), which provided an optimal open porous structure and an MC density that resulted in sedimentation within an adequate timeframe. (III) Two different PVA concentrations were tested in W\({}_{2}\): 0.2% and 1%. An increase in PVA concentration leads to a reduction in pores on the MC surface and, consequently, to a more spherical bead shape, keeping the PSD comparable (data not shown). The selected PVA concentration was 0.2% \(w/w\) in W\({}_{2}\). (IV) The standard polymer concentration in the hydrophobic phase of the primary emulsion, according to our protocol, was 4.3% \(w/w\). An increase in polymer concentration to 8.6% \(w/w\) led to a reduction in porosity and maintained a more spherical MC shape, suggesting that an adjustment in gas foaming agent concentration will be required to maintain the morphological characteristics of the lower polymer concentration. This approach will be followed whenever the manufacturing process needs to be scaled up. (V) The stirring of the primary emulsion was performed by a magnetic bar. At low stirring speed, the MCs are small, and they do not present the effects of the gas foaming agent. High mixing power rates are required to stabilize the emulsion and obtain the desired bead morphology and dimension. In general terms, a stable primary emulsion results in a more homogeneous MC preparation. In the absence of the gas foaming agent, the incorporation of air bubbles in the primary emulsion leads to the formation of open porous MCs in the interior region. The final stirring speed of W\({}_{1}\)/O was 1400 rpm. In terms of W\({}_{2}\), a higher overhead stirrer speed results in smaller MCs since the size of the W\({}_{1}\)/O droplet is reduced by the introduced energy. Therefore, the selected speed was 700 rpm during the secondary emulsion formation phase and 300 rpm during the overnight evaporation step. During the manufacturing process, several functionalizations were evaluated to develop a ready to use a product that does not require post-manufacturing coatings. Different protein sources were evaluated, including gelatin and collagen peptides of different animal origins. The difference between gelatin and collagen peptides is mainly the amino acid chains' length, with the collagen peptides having shorter chains. This difference affects their solubility and ability to form a gel at room temperature. The presence of these proteins positively affects the stability of the primary emulsion. Gelatin was successfully incorporated into the MC matrix and surface. More difficulties were encountered with collagen peptides, as demonstrated by Bicinchoninic Acid (BCA) testing on the final products (data not shown). The difference in water solubility at different temperatures and the smaller molecular weight might explain the difficulty in incorporating the collagen peptides into the polymeric matrix. Bovine and porcine gelatin provide comparable results in morphology and microcarrier PSD; however, porcine gelatin is more acceptable for the medical regulatory authorities since it avoids BSE-related issues, leading to porcine gelatin being the gelatin of choice.