Poster BB17 - Detecting and quantifying oxygen levels in a tissue-engineered construct in real-time

Introduction:

Introduction:
The fate of cells in a tissue-engineering bioreactor depends on the presence of nutrients and metabolites. Bioreactor systems rely on perfusion or medium changes to maintain a consistent biochemical environment. However, this method does not ensure precise monitoring or control within the tissue, where even slight variations can significantly impact cell viability and differentiation. Muscle is a highly metabolically active tissue, and precise control of oxygen levels is thus crucial during muscle tissue engineering. The goal of this project is to develop an extremely sensitive oxygen optode sensor that can be integrated with scaffolds to enable real-time monitoring of oxygen levels in tissue-engineered constructs.

Materials and

Methods: Materials and

Methods:
We developed electro-sprayed PDMS sensor beads infused with a ruthenium chloride-based complex. Sensing is based on the time constant of excitation/emission, providing accurate O2 measurements which are insensitive to changes in spot sizes, dye stability, and intensity, that plague intensity-based methods of O2 sensing. A blue LED array emits a 30 µs pulse for excitation. A fluorescence decay phase follows the end of the excitation pulse and follows an inverse exponential curve. High-speed data for the dynamics of excitation and emission are collected through a photomultiplier tube. As the O2 partial pressure increases, the decay's time constant (τ) decreases. Data are collected using a custom A/D system and analyzed offline. For this study, the sensor beads were embedded in a porous collagen scaffold, either seeded or not, with 106 human MSCs. O2 levels were measured every fifteen seconds continuously for 14 hours.

Results, Conclusions, and Discussions:

Results:
Our analysis revealed that the oxygen levels in the cell-free scaffolds remained stable at approximately 20%. In contrast, the levels in scaffolds with cells decreased to a new equilibrium level of around 6% over 14 hours. The sensor beads remained undisturbed and did not hinder cell attachment. The cells remained attached to the scaffolds and did not detach or migrate to the petri dish. Importantly, the sensor beads did not interfere with standard live-dead assays.

Conclusions:
This advanced oxygen-sensing technology has the potential to significantly contribute to the manufacturing of tissue-engineered medical products. Initial experiments allowed us to track a significant decrease in oxygen concentration in viable tissue over 14 hours. Improvements are being made to increase the resolution and repeatability.

Discussion:
The goal of this work is to allow real-time optimization and feed-back-controlled corrective interventions during the fabrication of TE constructs specifically for muscle tissue engineering applications using this unique sensitive oxygen optode sensor. This is expected to result in more efficient and successful tissue engineering constructs.

Acknowledgements (Optional): This work was funded in part by a grant from the NIH/NIBIB EB021911 (Welter)