In this study, we successfully demonstrated the stereolithographic printing principle of a complex hepatic tissue construct with an intrinsic hollow channel system. The results build a foundation for future detailed characterization and development of a functional liver model with possible applications in metabolic and toxicologic assays. The translation of a 3D digital drawing into a highly-structured, cell-laden hydrogel-based organoid was successfully performed (Figure 2
). Right after printing, some cells were loosely attached to the printed model due to the high cell density within the bioinks. These cells can easily be washed away, and only the designed structure with the incorporated cells remains. Multiple tissue constructs, printed at the same time, showed a high equality in morphology and cell distribution constituting an accurate representation of the intended design for the tissue model, thus reproducible printing of organoids was demonstrated. Furthermore, apoptotic cells could only be found in the positive control after two weeks of cultivation (Figure 3
). Proliferating cells were found both at day zero and after two weeks in culture demonstrating a high viability for cultivation over at least 14 days. More Ki67 positive cells were observed at day 0 compared to day 14, which indicates a loss of proliferative capacity due to cell differentiation. In this study, we compared HepaRG cells cultivated in monolayer with the same cells incorporated in the printed lobular constructs. For both cultivation methods, cells were matured over two weeks in monolayer. Afterwards one half of the cell culture was printed and cultivated in 3D, the other half remained in the monolayer culture. Unfortunately, we had to exclude stellate cells from the monolayer controls, as these cultures appeared unstable over 14-day cultivation, contracting into an elliptic random construct, whereas monolayers without stellate cells remained stable over the two week cultivation period. Both monolayer and printed tissue cultivations were performed over 14 days. Overall gene expression of selected markers was found to be higher in the printed tissues compared to the monolayer cultures, confirming the direct effect of the three-dimensional cultivation on the cells’ biology (Figure 5
). In the printed liver equivalents, the tight junction protein ZO-1 was found to be stably expressed over 14 days of cultivation. At day 14, its standard deviation dropped to a minimum. As cells expressed tight junctions when in contact with each other, and more organoids showed a higher ZO-1 expression at day 14 compared to day 0, these results suggest some cell proliferation within the hydrogel. One of the models already showed maximal expression on day zero, maintaining this level over the entire cultivation time. Since the cells are pre-differentiated over 14 days in monolayers, detached, suspended in the bioink and then printed in high cell density, tight junction protein expression might remain high from the first day on, but this assumption requires further experiments. MRP2 expression was found to increase in monolayers, whereas there was a slight decrease in expression levels found in printed tissues (Figure 5
). In monolayer cultures, the HepaRGs proliferate until complete confluency, differentiating and forming bile ducts with transporter expression. In the printed tissues, cells are incorporated in a hydrogel at a given concentration. Thus, the cells are surrounded by the printed matrix, but not all cells interact with each other. Cells that expressed MRP2 in the confluent monolayer prior to printing might not have any cell–cell contact after printing within the gelatin hydrogel. This might explain the decrease in MRP2 expression in our printed tissues over 14 days of cultivation. An increase in bioink cell concentration in future organoids might support the expression of MRP2 as more cells stay in contact with each other, supporting the formation of bile ducts and the main bile acid transporters [36
]. There was a high difference in protein expression at day zero, comparing printed tissue and monolayers, although the cells were pre-differentiated equally in both experiments. Albumin expression was found to be two-fold higher and CYP3A4 expression was found to be even 20-fold higher in monolayers, than in printed tissues, at day zero (Figure 5
). This difference can be explained by the procedure used to take the RNA samples, as different cell handling results in metabolic alterations [44
]. For the monolayers, RNA samples were taken by lysing the attached cells directly from the tissue culture flask so that no changes in expression were expected. For printed tissues, cells were detached, mixed with bioink, resulting in a single-cell-suspension, and printed within the hydrogels. Right after the printing process, the tissue constructs were lysed to extract the RNA. The cells remained in suspension before the bioprinting process, thus downregulating liver-specific gene expression (albumin, CYP3A4, MRP2). Only ZO-1 was not affected by this phenomenon in all printed tissues. Nevertheless, this procedure was chosen to investigate the actual changes in gene expression from day zero to day 14. Monolayer controls lose albumin and CYP3A4 expression over the two weeks cultivation period. Usually, HepaRG functionality in monolayers is maintained by adding DMSO after the pre-differentiation. As the printed tissues are cultivated without DMSO, monolayer cultures were also kept without DMSO, resulting in a loss of hepatic function. The printed tissues, however, maintained hepatic functionality under these native conditions.
Protein expression was verified by immunohistology, being in accordance with the results from the qPCR experiments (Figure 6
). Some morphological deformations were observed in cryosections due to the freezing and cutting procedure of the tissues. As some sections appeared to be squeezed in one direction (e.g., y-direction, Figure 6
a) these deformations were most likely introduced by the cutting procedure. In Figure 6
a,e, a part of the channel structure is visible (black linear area within the staining). Not all channels are visible at once due to the cutting angle. An accumulation of vimentin positive cells (stellate cells) was observed at the bottom edge of the tissue section (Figure 6
a). As HepaRGs and SteCs are homogenously mixed in the bioinks, and SteCs were found homogenously distributed throughout the printed constructs at day zero, this accumulation suggests the proliferation or migration of stellate cells at this spot. As cells at the edges of the printed tissues might not be fully incorporated in the printed matrix, they have more space to proliferate and populate the construct surface. Cytokeratin 8/18-positive cells within the same staining suggest some proliferative hepatocytes, representing an active tissue with regenerative capabilities.
Metabolic analyses revealed the production of small amounts of lactate (Figure 4
c) in accordance with glucose consumption (Figure 4
a). These results suggest oxygen limitation so that glucose is converted to lactate. The oxygen limitation might be a result of the medium amount used for cultivation. In this study, the printed tissues were cultivated in a 24-well format with 1 mL of medium. Manufacturers like Greiner Bio-one suggest using only 0.5 mL of medium in this format. The higher liquid level limits the amount of oxygen at the bottom of the culture well, where the organoid can be found [45
]. In future experiments, medium levels will need to be adapted and cultivation under perfusion will help to alleviate this problem. As LDH levels appeared to stay stable after the first two days (Figure 4
, center), the printed tissues stay viable. Metabolic data suggest homeostasis, as no significant increases or drops in neither glucose consumption nor lactate concentration were visible.
In future experiments, the printed liver organoid will be cultivated in a multi-organ-chip platform, which facilitates the in- and efflux of oxygen, carbon dioxide, nutrients and metabolites [46
]. Co-cultivation with other organ models, such as pancreatic islets, neural tissue or skin, might pose a promising strategy in testing and optimizing the printed liver equivalents at their current state [4
]. As the channel system allows for perfusion (Figure 2
d,e and the supplementary video
), adding endothelial cells to form tight homogenous channel walls is a crucial extension to the development of this organ model for physiologic perfusion experiments. In native tissue, substances need to pass endothelial barriers before reaching biological active hepatocytes [49
], thus, the addition of endothelial cells will support hepatic polarization, leading to a higher biological activity and a more detailed physiology [50