We measured dissolved O2 (DO) levels noninvasively in both acellular and cell-encapsulated hydrogels at the bottom of hydrogels using commercially available sensor patches (Oxygen Sensor Spot; SP-Pst3) and a multichannel fiber-optic oxygen meter (OXY-4 mini) from Presens (Regensburg, Germany). To measure O2 levels at the bottom of hydrogels, the hydrogels were added on top of the sensors, which were immobilized in each well of a 96-well plate. All experiments were conducted in a controlled environment at 37°C and 5% CO2 in a standard incubator.

O2 gradients were measured in preformed cell-encapsulated hydrogels at specified time points (continuous gradient measurements are not possible with our O2 sensors). Commercially available needle-type oxygen microsensors (Oxygen Microsensor; NTH-Pst1) and a microfiber optic oxygen transmitter (Microx TX3) from PreSens were used to measure O2 gradients. Sensors were calibrated using atmospheric and anoxic (N2 flush) conditions at 37°C. O2 sensors were precisely controlled using a Manual Micromanipulator MM (PreSens). Starting at the bottom of the hydrogel, measurements were recorded (when the reading stabilized) every 250 μm within the hydrogel and every 500 μm or 1 mm within the media.

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