(A-8) Fabrication of hydrogel micro-channels for 3D Blood-Brain Barrier using aqueous biphasic printing

Introduction::

Traditional cell cultures lack the complexity of human tissues and organs and their responses to stimuli. To address this, three-dimensional (3D) bioprinting and micro-physiological systems have been proposed. 3D bioprinting is similar to traditional printing, using additive molding to create 3D structures by stacking thin, two-dimensional patterns. The difference lies in the printing materials: while traditional methods use metal or plastic, 3D bioprinting employs cells, biomaterials, and biomolecules from the human body. Initially used for surgical planning, 3D bioprinting now shows promise in tissue engineering and regenerative medicine. Micro-physiological systems integrate microfluidic channels and organ-on-a-chip technologies to mimic in vivo tissue structures. Organ-on-a-chip technology, such as the blood-brain barrier (BBB)-on-a-chip model, has made significant progress in emulating the dynamic neural vascular unit and modeling neurological diseases like brain tumors and Alzheimer's disease. However, poor reproducibility in chip fabrication and fluid control hinders accurate evaluation of neurotherapeutic drug permeability due to the model's multiple layers of microchannels and the presence of a porous membrane. To overcome the limitations of 3D bioprinting and micro-physiological systems, a new BBB-on-a-chip model has been developed. This model prints microchannels within a liquid matrix containing two or more cell types using a composite matrix material that undergoes phase separation in the liquid state. By combining the strengths of 3D bioprinting and micro-physiological principles, this system holds potential to overcome the challenges faced by existing models and enhance the study of the BBB for drug testing and disease modeling purposes.

Materials and Methods::

A 3D cell culture matrix phase was created using a combination of alginate, collagen, and Matrigel. Human astrocytes (HA) and brain vascular pericytes (HBVP) were mixed within a matrix phase and co-cultured in a microfluidic chip device mimicking the BBB microenvironment, while brain microvascular endothelial cells (HBMEC) were subsequently cultured by adhering to the interior of the formed channel after removal of the ink phase. The ink phase was prepared by a mixture of 1% PEO and 17.5% PEG solutions dissolved in distilled water, and gum arabic dissolved at concentrations of 20% and 40%, respectively. An automated printing system was used, which included a syringe pump, tubing, a translation stage, and motion control software to print hollow channels for infusion of media.

Cell viability within the matrix phase was evaluated using a staining solution containing Calcein-AM and propidium iodide (PI). Viable cells fluoresced green, while non-viable cells fluoresced red. The ratio of green to red fluorescence was used to quantify cell viability. Vascular permeability was assessed using Dextran Texas Red™ 10,000 MW, a fluorescently labeled dextran molecule. The medium containing the tracer was infused into the endothelium channel and diffused through the intercellular gaps into the cell-laden matrix phase scaffold. Fluorescence intensity was monitored over time to measure vascular permeability. Immunofluorescence staining was performed on the cultured cells within the device, involving fixation, permeabilization, blocking, and the application of primary and secondary antibodies to label specific cell populations.

Results, Conclusions, and Discussions::

The BBB-on-a-chip model involves two matrices with liquid-liquid phase separation, created by mixing aqueous solutions of two incompatible polymers. The ink phase, which is printed inside the matrix phase to form microchannels, comprises a polymer that possesses a comparable specific gravity to that of the matrix phase. Human astrocytes (HA), human brain vascular pericytes (HBVP), and human brain microvascular endothelial cells (HBMEC) are cultured together in the matrix phase to mimic the BBB. The 3D microchannels created by ATPP can be used for adding cell culture media or diluting drug concentrations, making the BBB-on-a-chip suitable for drug screening experiments.

The optimal volume of the matrix phase in the reservoir is ~130 µL, while the microchannels printed inside have a diameter of 500 µm and a volume of ~1.96 µL. Plastic chips are fabricated via injection molding to create molds for the chip structure, and a metal microneedle is used for printing the ink phase. A guide hole structure is added to prevent the microchannel from moving or breaking during printing. To create the matrix phase, various combinations of alginate, gelatin, Matrigel, and collagen were evaluated based on their biocompatibility, ability to mimic brain ECM properties, mechanical support, and printability. Polyethylene oxide (PEO)-polyethylene glycol (PEG) and gum arabic were tested as ink-phase polymers at various flow rates. Gum Arabic at a concentration of 20% demonstrated stable printing at a flow rate of 5 µL/min.

In summary, a 3D BBB-on-a-chip model was developed using ATPP, allowing the printing of micro-channels and the culture of different cell types to mimic the BBB. The matrix optimal phase consisted of alginate, collagen, and Matrigel, while gum arabic was an found to be an appropriate ink phase for printing the micro-channels. The model offers potential applications in drug screening and BBB development and permeability in a controlled in vitro environment.

Acknowledgements (Optional): :

References (Optional): : Teixeira, Alyne G., et al. "Emerging biotechnology applications of aqueous two‐phase systems." Advanced healthcare materials 7.6 (2018): 1701036.