Fd 23

A total of 25 mL of 50 mM manganese(II) nitrate hydrate (Mn(NO₃)₂∙4H₂O, 99.99%, Sigma-Aldrich, Taufkirchen, Germany) or nickel(II) nitrate hexahydrate (Ni(NO₃)₂∙6H₂O, 99.999% Sigma-Aldrich, Taufkirchen, Germany) were mixed with 25 mL of 100 mM cobalt(II) nitrate hexahydrate (Co(NO₃)₂∙6H₂O, 99.999%, Sigma-Aldrich, Taufkirchen, Germany) at 250 rpm for 30 min, as reported elsewhere [25 (link)]. Subsequently, 5.2 mL of 1.25 M NaOH solution was added dropwise into the reaction mixture under vigorous stirring at 500 rpm for 30 min until the color of the solution turned brown or dark green in case of Mn(NO₃)₂∙4H₂O and Ni(NO₃)₂∙6H₂O precursor, respectively. Afterward, the mixture was transferred into 60 mL vials and the hydrothermal process was carried out at 160 °C for 10 h in a drying oven (FD 23, Binder, Tuttlingen, Germany). The black-colored hydroxide was collected by centrifugation (3K30, Sigma, Germany) at 10,000 rpm for 3 min, washed with deionized water until attaining a pH value of 7, and stored overnight in a freeze-drying unit (L06, Dieter Piatkowski-Forschungsgeräte, Petershausen, Germany) at −50 °C and 50 mbar. Then, the powder was grounded manually in a mortar and calcinated in a tubular oven (RHTC 80-710/15, Nabertherm, Lilienthal, Germany) at 400 °C for 1 h.

For the compartmentalised coculture in 2D, Xona microfluidics commercial devices with microchannels of 900 μm (length), 10 µm (width), 5 µm (height) and spaced 50 µm apart (Xonamicrofluidics, #SND900, US) were utilised.
To prepare the devices, a 2 mm perforation was made in the SN seeding side with a punch (Miltex Instruments, #33-31, US), and a 3 mm perforation in the SkM seeding side with another punch (Miltex Instruments, #33-32, US), as shown in Fig. 2B for chambers c1 and c2, respectively. Then, the glasses (Fisher scientific, #12-542-C, US) and the Xona polydimethylsiloxane (PDMS) devices were cleaned sonicating in 100% ethanol for 15 min, drying with a nitrogen gas stream and dehydrating in a drying oven at 60 °C for at least 30 min (Binder, #FD-23). Finally, glass coverslips were permanently bonded to the PDMS pieces exposing both to 1 min under high frequency oxygen plasma (Harrick plasma, #PDC-001) at 700 mTorr, then putting both pieces together and sealing them with at least 30 min dehydration in the oven at 60 °C. At this point, devices could be stored for the beginning of the experiment.