While Tissue Engineering (TE) and Regenerative Medicine (RM) have experienced substantial growth over the past decade and have incorporated technologies from various domains to overcome the scientific community’s boundaries, these fields are still struggling with some persisting challenges.
Notably, 3D bioprinting, derived from material additive manufacturing, has made significant progress, achieving unprecedented tissue and organ engineering complexity 1,2. However, the maturation strategies, encompassing duration, culture media, and incorporating mechanical/hydrodynamic stimulation, remain in their infancy concerning the potential clinical applications of these approaches and present urgent issues that need attention 2.
Maturation protocols are developed with small-scale tissues, often below cm scale, within non-fully controlled physicochemical environments. While studies are conducted in incubators with regulated temperatures, the pH is buffered, and oxygen supply to the tissues is not controlled. To address this, dynamic cultures using perfused bioreactors are essential to respond to cues influencing tissue development and enhancing tissue maturation. Consequently, numerous studies have focused on optimising nutritive flow within cultured tissues 3,4. Adequate oxygen and nutritional element distributions are crucial for cultivating tissues larger than centimeters to prevent internal cell necrosis. The development of in-house-developed bioreactors of various shapes is the most common solution. Few studies have explored the integration of sensors into cultivation vessels to monitor the tissue's physicochemical environment 4,5. Unfortunately, these studies are often case-specific, employing in-house developed equipment and vessels, and lack comprehensive knowledge of regulation protocols.
Contrastingly, cell-culture bioprocessing in pharmaceutical environments benefits from the presence of bioreactors that regulate the cellular environment. Over the past 50 years, commercial equipment, and protocols with control-loop parameters (PID control) have been successfully implemented 6. Advanced cell culture processes now involve meticulous monitoring of the cell physicochemical environment. Additionally, spectroscopic technologies, developed over the past two decades, offer insights into and control over the biochemical composition of culture media, including cell nutrients and by-product metabolites 7,8. Consequently, these technologies and expertise are ready to be transferred to the TE and RM fields.
Developing appropriate tools to monitor tissue maturation steps and ultimately qualify tissue quality is foreseen as an impactful step for TE and RM deployment. Indeed, standard qualification of tissue quality targets their cellularity, the tissue’s organisation, and functions.
However, existing reference characterisation methods are offline and involve destructive assays, such as histology or immunohistochemistry9. These methods necessitate paraffin embedding and tissue slicing. Cellularity and viability are often observed through microscopy imaging with or without labelling. In parallel, various imaging modalities, including standard fluorescence or confocal microscopy, light-sheet-based fluorescence microscopy 10, tomography (PET—positron emission tomography, OPT—optical projection tomography) 11, and their combination with spectroscopic tools like Raman 10, have been evaluated for in vitro produced tissue but are limited in scale and depth of analysis. Tissue transparisation allows for greater depth of up to 300 µm, but it is a destructive practice 9,13. In these cases, the analysed tissue volumes typically range from 10 µm³ to 7 cm³, significantly lower than the targeted tissue sizes in RM 11,12.
The targeted tissue volumes in RM are substantial, often exceeding cubic centimetres, such as kidneys (560 cm³)13 or livers (900 cm³) 14, and with thickness above 500 µm like in the case of full-thickness skin15. Therefore, the scientific community’s primary challenge is assessing large and dense objects, and its ultimate goal is to provide analytical tools implemented close to or on the tissue cultivation vessels for real-time monitoring of tissue fate. Various approaches have been tested or considered, but none have been entirely satisfactory. Qualification and characterisation tools must align with in vivo measurement techniques. Magnetic Resonance Imaging (MRI) stands out as a reference tool for both in vivo and in vitro tissue imaging13,16,17. Indeed, MRI offers high spatial resolution from 2 mm3 in the clinical routine down to 300 µm3 in dedicated MRI systems and a high imaging depth of 0.1 mm up to 40 cm 9,13. MRI has been employed for fluid velocity mapping in an in vivo tumour environment. To reach a resolution of 133 mm3 its capabilities was enhanced using contrast agents 17. Overall, there is a tradeoff between spatial resolution and penetration depth of the imaging modality. MRI can image living tissues at a multiscale level down to an ensemble of cells (30 cells, i.e., the scale of a small spheroid) up to the organ scale 18.
Recently, in-house protocols were developed to implement and transfer bioprocess principles to the tissue culture and monitoring. Off-line assays have characterised tissue maturation timelines, including cell growth and metabolic activity profiles 19,20. The current work explores non-destructive monitoring and qualification tools for controlling the tissue's physicochemical environment, composition, and evolution. The focus is on targeting internal tissue structure, nutrient and oxygen flow paths within 3D architectures, and the metabolic state of the tissue in a non-destructive, non-contact manner to propose qualification tools for late-stage tissue assessment pre-implantation.