Dionysia Kefallinou, collaborating researcher of INN, defended successfully her PhD Thesis, entitled “Microfluidic devices for studying cell behavior- Bone-marrow on a Chip” on October 12, 2023.

ABSTRACT

Organs-on-chips (OoCs) is a rising field that has emerged in the last decades at the crossroads of microfluidic technology and cell biology, managing to become the epitome of biomimetic systems. They are reliable preclinical models, responding to modern challenges in the field of biomedicine and biological research, which are nowadays limited to conventional static cell cultures and animal testing. The bone marrow, due to its vital role in the human body with the process of hematopoiesis, is a key study organ. Thus, the development of bone marrow-on-a-chip (BMoC) is important for understanding the biology of the organ itself, mapping related diseases, such as multiple myeloma and leukemia, and finally, for identifying cells’ response to drugs, radiation and chemotherapy. In this context, the present PhD thesis aims to develop a bone marrow-on-a-chip device for the purely in vitro and scaffold-free generation and maintenance of the human bone marrow perivascular hematopoietic niche. 

First, all stages of the device construction have been developed, from the design to its sealing, which led to a successful and repeatable completion of the device. It consists of two cylindrical poly(dimethylsiloxane) (PDMS) microchambers, one for medium perfusion and one for cell culture, which communicate with each other through an intervening porous membrane, while the whole assembly is sealed with glass. The primary methods used for its construction were soft lithography (replica molding) and surface treatment using plasma technology. Also, a successful and reproducible PDMS membrane fabrication protocol was proposed, relying on two main aspects, i.e. chemical surface passivation, and mechanical strength enhancement. In order to simplify and speed up the device manufacturing process, the incorporation of a commercial polyethylene terephthalate (PET) polyester membrane was finally chosen.

Aiming to achieve long-term cell culture on the BMoC device, a one-step, simple method of surface modification of the PDMS-based microdevices prior to collagen coating was suggested, in order to address the inherent hydrophobicity of the material. This is air plasma pre-treatment, a step already built into the device manufacturing process at the bonding stage. Testing of mesenchymal stem cells (MSCs) culture in microchambers treated with the proposed method, a task little examined in the research community, revealed enhanced cell growth and complete surface coverage on the 5^th day, the longest in the existing literature. The abovementioned contribute to the suitability of the plasma pre-treatment method for the immobilization of collagen in PDMS-based microdevices, for the purpose of long-term culture of MSCs.

In order to optimize the operation of the microdevice under flow (active perfusion), the phenomenon and causes of bubble formation at low and high flows were studied, and improvement actions were investigated to limit it, but also to eliminate it by incorporating a commercial bubble trap. Overall, bubble formation is shown to be intrinsic to the operation of a microfluidic system under flow for long periods of time, and in particular, to the device assembly itself, meaning the device material, its components and wiring, but also, to the temperature/pressure gradients between the feed and the device.

For this reason, a passively perfused BMoC device is finally proposed. The device is embedded with the commercial PET membrane, which showed greater uniformity of MSCs growth when compared with the PDMS membrane. Further, the BMoC devices under flow and passively perfused were compared, both with the PET membrane incorporated, where a faster and uneven growth of MSCs was demonstrated in the former, and a smoother and more uniform in the latter. Due to bubble formation in microdevices under flow, passive perfusion of the BMoC was chosen to ensure a uniformly organized supportive stromal tissue before the HSCs introduction. Thus, co-culture of MSCs with hematopoietic stem cells (HSCs) in the passively perfused BMoC, purely in vitro and scaffold-freely for 3 days, demonstrated the ability of the device to maintain and proliferate HSCs, towards the generation of a hematopoietic bone marrow organoid. Finally, design results of a microdevice with three integrated cell culture microchambers are presented, for the parallel conduct of three simultaneous experiments, as part of future plans of the thesis. The geometry of the perfusion microchambers is investigated both analytically and computationally, in order to ensure the filling and equal operation of all three microchambers.