Controlled environment perfusion bioreactor for tissue engineering

Objectives: The field of tissue engineering (TE) aims to provide solutions for the regeneration of organs and tissues. Perfusion bioreactors are an important tool for the development of 3D TE constructs [1]. Indeed, they provide enhanced nutrient delivery and mechanical stimulation to the cells (inducing extracellular matrix deposition) and overcome diffusion limitations potentially harming cell viability and quality in large scale TE constructs. Most perfusion bioreactors studies carried out up to date were in non-controlled set ups, hindering the robustness and reproducibility of the bioprocess. In this study, we designed a new type of standalone benchtop perfusion bioreactor, independent of an incubator. This results in a reduced footprint and allows precise control of critical process parameters. The benchtop perfusion bioreactor was designed as a closed silicon tubing circuit linking the medium reservoir to a perfusion chamber containing the TE construct (scaffold + cells). The perfusion of the medium was achieved using a peristaltic pump downstream of the perfusion chamber. A bubble trap device was included at the inlet of the chamber in order to prevent contact between air bubbles and cells. The whole circuit was enclosed in a custom built cassette containing all the electronics required for bioreactor operation and environment control (temperature and concentrations of CO2, O2 and N2). Using sensor ports specifically built for the perfusion chamber, pH, dO2 and temperature could be measured in the medium flowing to the cells. In order to validate the functionality of the bioreactor, additively manufactured titanium scaffolds were seeded with human periosteum derived cells (hPDCs) at a cell density of 200.000 cells/scaffold and cultured under a 0.1 mL/min perfusion flowrate. Results: Regular sampling of the medium was performed during culture to measure glucose consumption and lactate production. The cumulative increase in consumption and production rates of these metabolites indicated cell proliferation, Live/Dead staining of the construct showed the presence of live cells. The medium sensors showed stable measurements during the culture. Changes in the applied cassette parameters (temperature, CO2 and O2) resulted in changes in medium temperature, pH and dO2 (respectively), confirming the controllability of the culture environment. After 3 weeks of culture, this process resulted in a 3D TE construct containing around 1.6 million cells (+/- standard deviation). Conclusion: This standalone benchtop perfusion bioreactor allowed the growth of hPDCs in a controlled 3D environment. These results have proven to be reproducible throughout multiple repetitions of the experiments. Significance of the work: This study goes one step further in developing robust, reproducible 3D perfusion bioprocesses for tissue engineering.status: publishe