Researchers from Korea University have now developed a networked concave microwell array that enables effective construction of 3D multicellular spheroids. Connecting each microwell with its neighbouring wells enables diffusion of oxygen, nutrients and cytokines for inter-cellular signalling. The team investigated the performance of this networked system using rat hepatocytes, which produce cytokines during metabolism (Biofabrication 10 015001).

"Every cell in an organism connects to each other to actively communicate, a process termed signalling," explained Dong-Hwee Kim, head of the Applied Mechanobiology Group at Korea University. "Cells in a spheroid are directly in contact with other cells and, likewise, spheroids in a microwell array should communicate each other. In our system, cytokines secreted by spheroids diffused better than in a conventional system, revealing better in vivo relevance."

Accelerated diffusion

Kim and colleagues fabricated their "networked chip" from polydimethylsiloxane, which is transparent, gas permeable and easy to mould at low cost. The chip comprised connected concave microwell arrays, 500 µm in diameter, 380 µm in depth and with 195 microwells/cm2. To ensure sufficient oxygen and nutrient supply, all microwells were mutually connected with six neighbouring wells via 100 µm channels.

The team tested the performance of the networked chip in comparison with an un-networked control microwell array. First, they compared the diffusion rates of a cytokine in the two arrays. Such cytokines, which are secreted from individual spheroids, enhance the cellular function and viability in cultured cells.

Time-lapse fluorescence imaging of the tracer FITC-dextran placed in the microwells revealed that, as expected, it diffused more rapidly in the networked than in the control microwells. The researchers also performed computational analysis of molecular diffusion, which showed that diffusive propagation from a source spheroid to a neighbouring spheroid was 1.5 times faster in the networked than the control chip.

These results indicated that molecular diffusion was accelerated in the networked chip due to the physical path through which the molecules could diffuse to neighbouring microwells.

Cellular viability

To determine the impact of networked microwells on cellular morphology and viability, the team seeded primary rat hepatocytes into microwells in the two arrays. Cells spontaneously aggregated in the bottom of the wells and spheroids appeared within a day or two. By day 13, however, they observed many detached cells in the control chip, but not in the networked chip.

The researchers next used Live and Dead assay to assess cell viability. They found that the number of wells with healthy hepatocyte spheroids was considerably enhanced in the networked chip compared to the control (98.6% versus 77.9%). The average viability was also enhanced (89.3% versus 78.7%). CCK-8 assay and immunofluorescent analysis further confirmed the improved cellular viability in networked spheroids compared with isolated spheroids.

In addition, while both chips showed decreasing spheroid diameter over time, due to dead and/or detached cells, the diameter of spheroids in the networked chip was statistically larger than those in the control chip after day 11. This suggests that networked microwells could maintain the 3D morphology of the spheroids longer than conventional un-networked chips.

Hepatocyte functionality

As the networked chip improved spheroid stability and cell viability, the team investigated whether it also enhanced the main functionality of hepatocytes – secreting albumin, the most abundant blood plasma protein. They found that, on day seven, hepatocyte spheroids in the networked chip showed higher expression of albumin than spheroids in the control chip, and did so for two weeks.

"Maintenance of viability and functionality is a significant index in tissue engineering," Kim explained. "Hepatocytes in our system expressed a higher level of albumin, which implies that our system provided a better cell culture condition for long-term maintenance."

Overall, the results demonstrated that hepatocyte spheroids cultured in the networked microwells showed higher cell viability, prolonged morphological stability and enhanced albumin secretion than spheroids formed in isolated microwells, and that cytokine transport was accelerated in the networked chip.

"This networked chip could be used as a high-throughput cellular screening platform to study cell-cell interactions in response to multiple cytokines, as well as in vivo mimicking of customized 3D cell culture systems to regenerate damaged tissues in vitro," Kim told medicalphysicsweb.