2.1. Preparation and Characterization of HepaRG Cell-Laden Hydrogels Containing Varying Amounts of Extracellular Matrix (ECM)
The first aim of our study was to identify the optimal composition of a bioink to maintain HepaRG cultivation in a printed 3D construct. Although being of non-human origin, the suitability of alginate [19
] and gelatin [16
] for the fabrication of 3D scaffolds and bioprinting has been well established [33
]. Our previous results for the 3D printing of lung epithelial cells showed favorable characteristics of blends containing alginate and gelatin in terms of printability, cell viability and preservation of the shape of the generated constructs during culture [35
]. Therefore, the same basic bioink formulation consisting of 2% (w
) alginate and 3% (w
) gelatin was used to print mature HepaRG cells. This basic bioink was supplemented with varying amounts of human ECM. In the present study, we used lung-derived hECM for practical reasons. No substantial differences were reported for the composition of lung and liver ECM [36
]. Therefore, we carried out this proof of concept study using lung-derived hECM in order to determine suitable hECM concentrations for extrusion-based bioprinting of HepaRG-laden hydrogels. Concentrations of 0, 0.25, 0.5, 1 and 2 mg/mL of decellularized hECM were added to the alginate/gelatin blend to determine the concentrations which are most beneficial in terms of improving biocompatibility and attachment of the cells to the matrix. The bioink contained 7 × 106
mature HepaRG cells/mL, so that each construct should contain slightly less than 1 × 106
HepaRG cells. In fact, when we lysed the constructs and counted the cells by trypan blue staining as previously described [35
], we recovered approximately 750,000 cells from a model. We used a rectangular construct (length 1 cm × width 1 cm × height 0.1 cm) with regularly spaced pores laid out in a grid pattern for the experiments (Figure 1
The bioinks used in the present study were of good printability and the shape of the constructs was highly reproducible (Supplementary Figure S1
). Furthermore, the 3D models were stable during the cultivation time of seven days.
The spatial distribution of the mature HepaRG cells in the 3D printed constructs was analyzed by fluorescence microscopy. To visualize the cells, nuclei were stained with Hoechst stain and the 3D distribution was recorded with the Z-stack tool, which creates a projection of the transmitted light, following one and seven days in culture. Although this method is of limited resolution, it provides an overview over the cell distribution in the 3D printed construct. One day after printing, the HepaRG cells were well distributed throughout the printed constructs, with no obvious differences between the tested ECM concentrations (Figure 2
A, upper row). At day seven after printing, the spatial distribution of the HepaRG cells was less homogenous and the cells tended to sediment in all constructs (Figure 2
A, lower row); however, the bioink containing 1 mg/mL hECM was superior in maintaining spatial distribution of the cells compared to the other concentrations tested. Sections from the top, middle and bottom of the Z-stacks are shown in the Supplementary Figure S2
Next, cell viability was qualitatively evaluated by staining living (calcein-AM, green) and dead (ethidium homodimer-1, red) cells after one and seven days of culture, followed by microscopic analysis. As obvious in Figure 2
B (upper row), after one day of culture, cell viability was high in all bioink conditions except for 2 mg/mL hECM. This concentration resulted in a greater number of ethidium homodimer-1 positive, i.e., dead, cells compared to the other hECM concentrations. After seven days in culture (Figure 2
B, lower row), the number of dead cells increased only slightly under all tested hECM conditions. Again, like after one day, the addition of 2 mg/mL hECM was detrimental as the percentage of dead cells was comparatively high. For constructs printed with bioinks containing 0.5 or 1 mg/mL hECM, the fraction of dead cells was also slightly lower than for those containing less hECM. While no differences in the number of calcein-AM positive, i.e., living, cells were detected after one day, seven days of culture with no or only 0.25 mg/mL hECM resulted in slightly reduced numbers of living HepaRG cells compared to 0.5 and 1 mg/mL hECM. Therefore, we concluded that hECM concentrations greater than 0.25 mg/mL and less than 2 mg/mL are best suited for cell viability.
The metabolic activity of the bioprinted mature HepaRG cells was determined by quantification of the reduction of the tetrazolium salt XTT by dehydrogenase enzymes after one and seven days in culture (Figure 2
C). Consistent with the results from the microscopic evaluation of the cell staining, measurement of the metabolic activity of the bioprinted HepaRG revealed that 2 mg/mL hECM are unfavorable for cultivation of HepaRG cells, resulting in reduced metabolic activity levels. Even though slight differences regarding the enzymatic activity between the different hECM concentrations could be measured on days one and seven of culture, no significant decreases between day one and seven were detected at a given concentration of hECM.
The two-dimensional (2D) cultured mature HepaRG monolayer, which contained a comparable number of cells as the printed constructs, showed a significantly higher metabolic activity at day one (Figure 2
C). However, metabolic activity in the monolayer culture decreased over time and was statistically no longer distinguishable on day seven of culture.
As an additional measure of metabolic activity, the release of lactate dehydrogenase (LDH) was measured to determine the cytotoxicity resulting from the different bioink conditions. Figure 2
D shows that cytotoxicity of all tested bioink conditions was comparatively low (around 10% compared to the lysis control on day one after printing). A minor increase of about 5–10% was observed for the cultivation period of seven days, which is also typical for conventional 2D cell culture systems as also included in Figure 2
D. Differences between day one and seven of culture were significant only for bioinks containing 0.25 and 2 mg/mL hECM.
The reduced viability of printed HepaRG cells at a concentration of 2 mg/mL hECM came as a surprise given the generally beneficial effects of ECM on cellular viability. The most abundant protein in the ECM is collagen [38
], which is known to modulate the mechanical properties of tissues in vivo as well as in vitro dependent on its concentration [39
]. One of the most common types of the 28 known collagens in mammals is type I collagen [41
]. Collagen I monomers undergo fibrillar collagen formation at 37 °C and neutral pH values [42
] to form hydrogels, a property which has been used in 3D bioprinting approaches [43
]. The majority of the studies used low concentrations of collagen between 1 and 4 mg/mL. Likewise, most commercially available formulations contain low concentrations of collagen. In most of these studies, only a single concentration of collagen was used, leaving open the effects of varying concentrations on cellular behavior. Cross et al. showed that higher collagen concentrations (>20 mg/mL) restricted cell migration and viability of human vein endothelial cells due to the high density of the fibrillar structures [47
]. This finding may explain our observation that the hECM concentration need to be high enough to support cell viability (≥0.5 mg/mL), but must not exceed a certain threshold of approximately 1 mg/mL to prevent detrimental effects. In addition, the stiffness of the construct should not exceed a certain level, which must still be determined, as this might negatively affect cell functionality.
2.2. Characterization of Hepatic Metabolism in HepaRG Cell-Laden Bioinks Containing Various Amounts of ECM
To assess the impact of different hECM concentrations on the hepatic metabolism of printed HepaRG cells, albumin secretion and cytochrome P450 3A4 (CYP3A4) activity were analyzed. Albumin and CYP3A4 are two of the main markers for the characterization of hepatocytes. The production of albumin reflects the synthesis capacity of healthy cells and CYP3A4 their biotransformation activity [48
]. HepaRG cells plated at low densities undergo morphological changes from an epithelial- and biliary-like phenotype to a hepatocyte-like phenotype [50
]. The addition of dimethyl sulfoxide (DMSO) supports and maintains the hepatic maturation, which is accompanied by increased metabolic activities like albumin expression and cytochrome P450 activity [29
]. One day after bioprinting of DMSO-treated mature HepaRG cells, no substantial differences in albumin secretion were observed between the different hECM concentrations tested (Figure 3
A). The amount of secreted albumin then rose over time under all conditions tested; however, the increase was only statistically significant using 0.5 and 1 mg/mL hECM. At day seven of culture, the albumin level was approximately two- to threefold higher under these conditions compared to the other bioink compositions; the difference, again, being statistically highly significant.
CYP3A4 activity increased significantly from day one to day seven for all hECM concentrations except the highest, 2 mg/mL (Figure 3
B). The differences between the CYP3A4 activity produced by the lower concentrations of hECM were not significant (Figure 3
B). HepaRG cells cultured in the 2 mg/mL hECM-containing bioink showed significantly lower CYP3A4 activity on day seven compared to bioinks with other concentrations or no hECM (Figure 3
B). In the absence of DMSO, the CYP3A4 activity is substantially lower (Supplementary Figure S3
). Considering both albumin secretion and CYP3A4 activity, alginate/gelatin-based bioinks containing hECM concentrations of 0.5 or 1 mg/mL were found to be best-suited for mature HepaRG cell bioprinting. In our experiments bioinks with 0.5 or 1 mg/mL hECM triggered the highest HepaRG albumin secretion, as well as the highest CYP3A4 activity. In contrast, bioinks with 2 mg/mL hECM do not induce the tested hepatic activities, which may result from negative effects of high collagen concentrations on the metabolic conditions, as already discussed above for the LDH and XTT measurements.
Despite the elevated albumin secretion and CYP3A4 activity in 3D models printed with bioinks containing 0.5 and 1 mg/mL hECM, even higher metabolic activity was measured for HepaRG cells cultured in conventional 2D monolayers (Figure 3
). A possible explanation is that the lower activity in 3D cultures is a result of the encapsulation of the cells in the bioink, as it is essential to safeguard the cells from pressure and shear stress occurring during the printing process [53
]. This leaves the cells surrounded by a barrier-like layer of hydrogel, comparable to a sandwich culture, which has been associated with limitations in mass transport and drug sensitivity [54
]. Consequently, it is plausible that secreted albumin and CYP3A4-formed luciferin were not completely released from the cells due to the surrounding hydrogel, or alternatively the diffusion of the CYP3A4 luminescence substrate into the hydrogel was insufficient or albumin release might have been incomplete due to interactions with the bioink material. Thus, the albumin and CYP3A4 luminescence values measured in the 3D culture may be underestimated compared to the ones in 2D cultured HepaRG cells, since a fraction of the measured metabolic parameters might not have been fully released from the hydrogel. In addition to limitations in mass transport, the encapsulation prevents cell–cell connection [35
], which influences the measured metabolic activity of hepatocytes. For instance, hepatocytes, like HepaRG cells, cultured in spheroid models show increased metabolic activity compared to conventional monolayer culture [58
]. Reasonable strategies to overcome this limitation and to further improve the bioinks presented here in this study have been published, e.g., the use of spheroids instead of single cell suspensions [60
] or modified hydrogels that are sensitive to matrix metalloproteases (MMPs) which can be used to degrade the cell encapsulation [57
]. Another approach to optimize the bioink is the inclusion of liver-derived, instead of lung-derived hECM, as it might contain non-collagenous proteins that may influence liver-specific metabolism.
2.4. Transduction of Bioprinted Liver Model with Adeno-Associated Virus (AAV) Vectors
AAV vectors are efficient tools for gene delivery without inducing any recognized pathogenicity [7
]. They are particularly well-suited for application in RNAi approaches [65
]. Pseudotyped AAV2.6 vectors (AAV2.6) display a pronounced liver tropism [66
], and so they were used in the transduction experiments. Mature HepaRG-laden constructs cultured with or without 1 mg/mL hECM were transduced with 1 × 105
AAV2.6/cell for seven days. Independent of the concentration of hECM used, high AAV vector transduction rates were observed, as well as an even spatial distribution within the printed 3D constructs seven days post-transduction. The ability of the AAV2.6 vectors to transduce mature HepaRG cells was characterized by observing the expression of the encoded marker EmGFP, which was analyzed by fluorescence microscopy. The overview images in Figure 5
A demonstrate widespread transduction of the cells in the printed constructs. Sections from the top, middle and bottom of the Z-stacks are shown in the Supplementary Figure S4
Furthermore, the functional applicability of the approach was determined by an RNAi experiment as the vectors also expressed a small hairpin RNA (shRNA) directed against hCycB, an endogenously expressed target that is well-suited to investigate silencing efficiency [67
]. In the printed 3D tissue models, transduction with AAV2.6 mediated an average knockdown of hCycB of 70–80% compared to the shRNA control, which was used for normalization to assess the knockdown efficiency (Figure 5
B). As no differences were observed for the bioinks containing no or 1 mg/mL hECM, we conclude that the addition of hECM is not detrimental to the transduction efficiency of the AAV vectors. Furthermore, the printed model and geometry including pores support widespread transduction of all cells. In a recent publication, we estimated by scanning electron microscopy that the pore size of an optimized alginate/gelatin bioink supplemented with Matrigel is about 1–2 µm [35
]. It is reasonable to assume that the alginate/gelatin bioink with hECM used here has a comparable pore size. This can explain the efficient penetration by AAV vectors we observed, since they are two orders of magnitude smaller. This contrasts with bulky spheroids that are often too dense for large particles like viral vectors to penetrate the 3D culture right to the center.