In vitro long term differentiation and functionality of three-dimensional bioprinted primary human hepatocytes: application for in vivo engraftment

Abstract

In recent decades, 3D in vitro cultures of primary human hepatocytes (PHH) have been increasingly developed to establish models capable of faithfully mimicking main liver functions. The use of 3D bioprinting, capable of recreating structures composed of cells embedded in matrix with controlled microarchitectures, is an emergent key feature for tissue engineering. In this work, we used an extrusion-based system to print PHH in a methacrylated gelatin matrix (GelMa). PHH bioprinted in GelMa rapidly organized into polarized hollow spheroids and were viable for at least 28 days of culture. These PHH were highly differentiated with maintenance of liver differentiation genes over time, as demonstrated by transcriptomic analysis and functional approaches. The cells were polarized with localization of apico/canalicular regions, and displayed activities of phase I and II biotransformation enzymes that could be regulated by inducers. Furthermore, the implantation of the bioprinted structures in mice demonstrated their capability to vascularize, and their ability to maintain human hepatic specific functions for at least 28 days was illustrated by albumin secretion and debrisoquine metabolism. This model could hold great promise for human liver tissue generation and its use in future biotechnological developments.

Gelatin-Based Hybrid Hydrogel Scaffolds: Toward Physicochemical Liver Mimicry

“…Other 3D-constructs involve the use of hydrogel scaffolds (e.g., alginate dialdehyde-gelatin, gelatin methacryloyl, and collagen/chitosan), in combination with primary human hepatocytes (PHH), , primary rat hepatocytes, or hepatic cell lines. , Those constructs exhibit a decreasing viability and functionality over time, leading to a limited or discontinued secretion of important metabolic enzymes that render them inappropriate toward hepatocyte support, mainly due to inadequate resemblance with the native liver’s physicochemical properties. , …”

Advancements of 3D bioprinting in regenerative medicine: Exploring cell sources for organ fabrication

“…However, their practical application has been hindered by the limited availability of suitable donor sources and the challenges associated with in vitro culture and expansion. Freshly isolated primary cells are often delicate and vulnerable to the stresses imposed by temperature, pressure, and shear forces during 3D bioprinting, leading to high mortality rates [ [192] , [193] , [194] ].…”

“…By harnessing the unique characteristics of primary cells, several kinds of primary cells had been utilized in the 3D bioprinting of tissues and organs ( Table 4 ) [ 176 , [203] , [204] , [205] , [206] , [207] , [208] ]. Primary human hepatocytes and hepatocellular carcinoma cells were applied in constructing the liver and liver cancer models [ 194 , 209 ]. Primary fibroblasts and intestinal cells have also been employed in the generation of skin tissues and intestinal models [ 192 , 193 , 210 ].…”

“… Cell types Bioprinting techniques Bioink materials Tissues or Organs Ref. Human mesenchymal stem cells (hMSCs) Extrusion-based Alginate- and silk-based Smart dual scaffold [ 23 ] Advantages: High safety Stable functionality Patient-specific Excellent in vivo integration capability Disadvantages: Limited cell sources High cost Weak proliferation ability High heterogeneity hMSCs Femtosecond-laser-assisted Tunicate cellulose nanofibrils & Alginate-based Cell-laden corneal tissue [ 176 ] Human primary dermal fibroblasts Inkjet ECM-like BioInk Soft tissue models [ 192 ] Primary normal human fibroblasts (NHF) Magnetic-based Collagen Skin tissue [ 193 ] Primary human hepatocytes Extrusion-based GelMa Liver [ 194 ] Primary Skeletal Muscle Progenitor Cells Extrusion-based GelMA Skeletal muscle [ 200 ] Primary human osteoblasts Extrusion-based GelMA Osteoblasts scaffold [ 203 ] Primary human keratocytes Extrusion-based Collagen-based Corneal stromal tissue …”

Hybrid biofabrication of neurosecretory structures as a model for neurosecretion

“…Irrespective of the type of bioprinting technology, the purpose is to deposit cells, biomaterials, and cytokines with high precision and to accurately control the spatial geometry, density, and controllable distribution of loaded cells to realize 3D construction of a 3D-bioprinted structure[ 12 , 13 ]. 3D bioprinting tissues has been reported, including 3D-bioprinted liver tissue[ 14 - 16 ], glomerulus[ 17 ], myocardial[ 18 ], brain[ 19 ], and skeletal muscle[ 20 ] and has been shown to maintain cell bioactivity, tissue morphological structure, and organoid function. The tissue-like structures produced by the organisms also showed varying degrees of biological functions.…”