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Development of scalable microfluidic device for drug testing using humanized adipose tissue spheroids

Periodic Reporting for period 1 - SMD-SPH (Development of scalable microfluidic device for drug testing using humanized adipose tissue spheroids)

Reporting period: 2021-12-01 to 2023-11-30

In regenerative medicine, advanced therapeutic products derived from tissue engineering technologies and are critical for more accurate medical innovation. Tissue engineered scaffold-free 3D models, including organoids, exhibit functional hallmarks of human native tissues improving the search for biomarkers, drug testing/development and toxicology models, while supporting the development of alternative methods to animal use, as stated by the Directive (2010/63/ EU) established the European Centre for the validation of alternative methods (ECVAM).
To replace animal testing, it is important to develop microphysiological systems and ‘body-on-chip’ approaches at a reasonable cost. Yet, most current bioreactors in tissue engineering are expensive, designed for organ transplant and poorly designed for miniaturization and scale-up. Furthermore, organoid production faces critical challenges such as small-scale, high costs and reproducibility. Currently, the scientific literature has been arguing that the convergence of organoids and microfluidic technologies to overcome these limitations. Specifically, microfluidics technologies can work as bioreactors, reducing labor hours and cost by supporting automated systems and low volume of reagents.
The main objective of this study was to develop a microfluidic bioreactor for organoid mass production. To reached this, we accomplish the following specific objectives:
- a bioreactor design containing hundreds of individual chambers for organoids;
- 3D simulations to analyze the microfluidic flow and the nutrients reaching the organoids inside the individual chambers;
- microfabrication of the bioreactor prototype based on hot embossing technology (Sublym) using a biocompatible polymer (Flexdym). Sublym and Flexdym are registered technologies from Eden Tech.
- seeding of human adipose-tissue derived stem/stromal cells (ASCs) in the microchannels of the bioreactor to form individual organoids inside each individual chamber;
- viability and adipogenesis assays to attest the functionality of ASC organoids.
A bioreactor that forms and cultivates ASC organoids was engineered based on organoid-on-a-chip and microfluidics technologies. The prototype consisted of a microfluidic chip made of Flexdym with 192 individualized organoids chambers containing one inlet and one outlet (Figure 1). The 3D simulation analysis demonstrated that the configuration of the microchannels and the shape of the chambers were appropriate to provide enough nutrients in a homogenized way. The microfabrication process was considered successful since the fluidic test showed a homogeneous distribution of the fluid by the microchannels and chambers (no bubbles was observed). The ASC suspension was seeded through the inlet and cells were decanted inside the individualized chambers forming one organoid per chamber. ASC organoids were maintained for until two weeks to stimulate the accumulation of lipid droplets inside cells (fat formation). ASC organoids remained viable and showing lipid droplets during the culture time.
This project meets the convergence of microfluidics and organoids offering a reliable alternative for drug and toxicology assays, besides offer a scalable and reproductible system for fat organoids mass production. The next step is to incorporate a controllable perfusion system, as for example, using peristaltic pumps. More importantly, some technological advances are worth highlighting, such as:
- the bioreactor was made from flexdym, a thermoplastic, opening a perspective of a large-scale production based on injection molding;
- cell suspension was seeded using a syringe through a unique inlet, opening a perspective of cell seeding automation;
- the cost of fat organoids production represents only 15% compared with traditional cell culture plates.
Main steps of the bioreactor prototype based on organoid-on-a-chip and microfluidics technologies.