A 3D Biohybrid Real-Scale Model of the Brain Cancer Microenvironment for Advanced In Vitro Testing

Abstract

The modeling of the pathological microenvironment of the central nervous system (CNS) represents a disrupting approach for drug screening for advanced therapies against tumors and neuronal disorders. The in vitro investigations of the crossing and diffusion of drugs through the blood-brain barrier (BBB) are still not completely reliable, due to technological limits in the replication of 3D microstructures that can faithfully mimic the in vivo scenario. Here, an innovative 1:1 scale 3D-printed realistic biohybrid model of the brain tumor microenvironment, with both luminal and parenchyma compartments, is presented. The dynamically controllable microfluidic device, fabricated through two-photon lithography, enables the triple co-culture of hCMEC/D3 cells, forming the internal biohybrid endothelium of the capillaries, of astrocytes, and of magnetically-driven spheroids of U87 glioblastoma cells. Tumor spheroids are obtained from culturing glioblas-toma cells inside 3D microcages loaded with superparamagnetic iron oxide nanoparticles (SPIONs). The system proves to be capable in hindering dextran diffusion through the bioinspired BBB, while allowing chemotherapy-loaded nanocarriers to cross it. The proper formation of the selective barrier and the good performance of the anti-tumor treatment demonstrate that the proposed device can be successfully exploited as a realistic in vitro model for high-throughput drug screening in CNS diseases.

Keywords: biohybrid microfluidic systems; biomimetics; blood–brain barrier; glioblastoma; two-photon lithography.

Figure 1

a) 3D depiction of the microfluidic device for the modeling of the brain tumor microenvironment; b) scheme of the TPL fabrication of the microfluidic system mimicking the BBB.

Figure 2

Views of the microfluidic device: a) perspective view; b) top view of the capillaries array; c) top view of the entire microfluidic device; d) detailed design of the porous microtube; and e) main geometrical parameters of the design.

Figure 3

a) SEM image of the top view of the microfluidic device; b) optical image of the capillary array; c) detail of the link between the capillaries and the joint (SEM image); d) detail of the porous microtubes (SEM image); and e) high magnification of a microtube, showing the surface micro-roughness (SEM image).

Figure 4

a) Optical image of the endothelial cells seeded inside the capillaries; b) section views of the biohybrid capillary acquired at the confocal microscope: red represents endothelial hCMEC/D3 cells, green represents the microfabricated capillary; c) 3D reconstruction of the biohybrid capillary; d) extratubular concentration graph of the fluorescent dextran monitored during time-lapse fluorescence imaging in the presence and without endothelial hCMEC/D3 cells inside the porous microtubes; fluorescence images of e,f) TRITC-dextran in the extratubular region pumped in the micro-fluidic system without cells and g,h) in the biohybrid microsystem with hCMEC/D3 cells, acquired at time t = 0 and t = 700 s.

Figure 5

a) 3D rendering of the magnetic microcage; b) SEM image of the fabricated microcage; c) fluorescence image of the array of tumor spheroids before the detachment from the glass substrate; d) confocal microscopy acquisition of a tumor spheroid (U87 cells seeded into the microcage): red represents F-actin, blue represents nuclei; e) confocal microscopy image of the biohybrid GB model: red represents the microtubes with endothelial hCMEC/D3 cells, yellow represents astrocytes, green represents spheroids of U87 GB cells; f) fluorescence time-lapse images of the fluorescent nutlin-loaded nanocarriers dispersion pumped in the selected microtube; g) intratubular fluorescence of nutlin-loaded nanocarriers flow monitored during time-lapse fluorescence imaging, in the presence of endothelial hCMEC/D3 cells inside the porous microtubes and astrocytes outside; h) control spheroid and i) treated tumor spheroid subjected to LIVE/DEAD assay for quantitative analysis of the effect of the antitumor treatment: green represents calcein, red represents EthD-1, blue represents nuclei, and white represents nutlin-loaded nanocarriers.