Optical 4D oxygen mapping of microperfused tissue models with tunable in vivo-like 3D oxygen microenvironments

Abstract

Sufficient and controllable oxygen supply is essential for in vitro 3D cell and tissue culture at high cell densities, which calls for volumetric in situ oxygen analysis methods to quantitatively assess the oxygen distribution. This paper presents a general approach for accurate and precise non-contact 3D mapping of oxygen tension in high cell-density cultures via embedded commercially available oxygen microsensor beads read out by confocal phosphorescence lifetime microscopy (PLIM). Optimal acquisition conditions and data analysis procedures are established and implemented in a publicly available software package. The versatility of the established method is first demonstrated in model-assisted fluidic design of microperfused 3D printed hydrogel culture chips with the aim of full culture oxygenation, and subsequently for monitoring and maintenance of physiologically relevant spatial and temporal oxygen gradients in the 3D printed chips controlled by static or dynamic flow conditions during 3D culture.

Embedded 3D Printing in Self‐Healing Annealable Composites for Precise Patterning of Functionally Mature Human Neural Constructs

“…Oxygen Microsensor Integration and 3D Oxygen Mapping: The technical aspects of the 3D oxygen sensor method, along with sensor integration into cell laden hydrogels, microscopy read-out, and signal processing have been described in detail previously. [41] Here, a new application of the method is presented to embedded to 3D extrusion printed tissue constructs. Briefly, oxygen sensor microbeads (CPOx Beads Red, Colibri Photonics GmbH) were embedded in SHAPE printing support either by direct extrusion printing of beads in along a preprogrammed path (before cells were printed) at a concentration of 2 mg mL −1 or by mixing beads with the cellular ink at a concentration of 0.7 mg mL −1 .…”

“…This recently developed method allows 4D mapping of oxygen tension in cellular microenvironments using confocal phosphorescence lifetime microscopy. [41] Oxygen sensitive microbeads can either be directly extrusion-printed in the support or mixed with the cellular ink during printing (Figure 5A). SHAPE hydrogel support keeps microbeads in place after embedding thus allowing for controlled 3D distribution of oxygen microsensors.…”

Embedded 3D printing in self-healing annealable composites for precise patterning of functionally mature human neural constructs

“…We integrated a non-invasive optical method for oxygen sensing based on quenching of phosphorescence using microbead sensors embedded in and around the 3D printed cellular construct. This recently developed method allows 4D mapping of oxygen tension in cellular microenvironments using confocal phosphorescence lifetime microscopy [34] . Oxygen sensitive microbeads can either be directly extrusion-printed in the support or mixed with the cellular ink during printing (Fig.…”

“…The technical aspects of the 3D oxygen sensor method, along with sensor integration into cell laden hydrogels, microscopy read-out, and signal processing have been described in detail previously [34] .…”