Alginate

mucin-alginate beads were used for small intestinal simulations and prepared by dripping a 5×-boiled mucin-alginate solution [50 g L⁻¹ mucin (Carl Roth), 12 g L⁻¹ agar (VWR), 12 g L⁻¹ alginate (Carl Roth) and 2.22 ml L⁻¹ 10 M NaOH (Chem-Lab)] into crosslinking solution containing 7.6 g L⁻¹ CaCl₂.2H₂O (VWR). This approach was implemented as the small alginate beads allowed for sterile sampling of colonized beads and addition of fresh beads via a 50 ml-syringe with catheter tip (Novolab) connected to an inlet port. Sterility of such handlings was a prerequisite as one worked with a synthetic consortium in multiple ileal simulations. In contrast, for the colonic microbiota, the conventional approach using mucin-covered microcosms was used as previously described by Van den Abbeele et al. (2012) (link). A buffer comprising (g L⁻¹) K₂HPO₄ (8.8; Chem-Lab) and KH₂PO₄ (6.8; Chem-Lab) was used to rinse luminal content from mucosal samples. Half of the mucus-alginate beads and mucin-covered microcosms were replaced every 2 days.

The ink was prepared according to the protocol of Schmid et al. (2021) [7 (link)]. The ink consists of 3 wt.% gelatin (Sigma-Aldrich, St. Louis, MO, USA), 0.5 wt.% alginate (alginate PH 176, JRS Pharma GmbH &Co. KG), and 0.1 wt.% HA (high molecular weight hyaluronic acid 1–2 MDA, CarboSynth Ltd., Compton, UK), all dissolved in PBS. 0.3 g of gelatin, 0.05 g of alginate, and 0.01 g of HA were weighed and dissolved in a beaker containing 10 mL of PBS at 37 °C on a magnetic stirrer for 1.5 h. The beaker was covered with a sealing film (PARAFILM^®M, 100 mm, 75 m, Roth, Karlsruhe, Germany).