Novel integrative methodology for engineering large liver tissue equivalents based on three-dimensional scaffold fabrication and cellular aggregate assembly

Abstract

A novel engineering methodology for organizing a large liver tissue equivalent was established by intergrating both 'top down' and 'bottom up' approaches. A three-dimensional (3D) scaffold was engineered comprising 43 culture chambers (volume: 11.63 cm(3)) assembled in a symmetrical pattern on 3 layers, a design which enables further scaling up of the device to a clinically significant size (volume: 500 cm(3)). In addition, an inter-connected flow channel network was designed and proved to homogenously deliver culture medium to each chamber with the same pressure drop. After fabrication using nylon-12 and a selective laser sintering process, co-cultured cellular aggregates of human hepatoma Hep G2 and TMNK-1 cells were loosely packed into the culture chambers with biodegradable poly-L-lactic acid fibre pieces for 9 days of perfusion culture. The device enabled increased hepatic function and well-maintained cell viability, demonstrating the importance of an independent medium flow supply for cell growth and function provided by the current 3D scaffold. This integrative methodology from the macro- to the micro-scale provides an efficient way of arranging engineered liver tissue with improved mass transfer, making it possible to further scale up to a construct with clinically relevant size while maintaining high per-volume-based physiological function in the near future.

Modular assembly–based approach of loosely packing co-cultured hepatic tissue elements with endothelialization for liver tissue engineering

“…The oxygen supply through the PDMS membrane was two orders of magnitude higher than that through the culture medium (41). Based on this difference, we generated high-density cultures of hepatocytes in our previous studies (36,37,39,42). Here, utilizing the same microwell device, we successfully acquired cell aggregates with a cell density of 4.0×10 6 cells/cm 2 .…”

“…In one of their later relevant studies, cell viability in the culture system decreased to less than 60% after just one day (28). Aiming to solve this problem, we proposed the idea of "looselypacked hepatic tissue elements" in our previous studies (36,37). We used biodegradable scaffold fibers and made a mixture with formed hepatic cell aggregates in a perfusion culture system, which has been proved to be a simple and effective way of maintaining the interstitial space.…”

Biomimetic Designer Scaffolds Made of D,L-Lactide-ɛ-Caprolactone Polymers by 2-Photon Polymerization

“…Innovative methods were established for generating scaffolds with defined pore sizes and pore distributions, such as fused deposition modeling, 93 selective laser sintering, 94 stereolithography (SLA), 95 and 2-PP. 96 These so-called RP techniques are manufacturing techniques in which 3D structures are generated in a LbL manufacturing process.…”

“…116 It was also demonstrated that cell response could be improved when seeding cells using perfusion bioreactors. 94…”

New Perspectives in Liver Transplantation: From Regeneration to Bioengineering

“…Despite the relatively high postoperative survival rate, there are many problems to be solved, however, including a chronic donor shortage, immune rejection, and ethical issues. Therefore, cell-based regenerative therapies and novel technologies such as liver-on-chip [4] and bioprinted liver [5] are expected to be the next-generation therapies.…”