Artificial Cells and HepG2 Cells in 3D‐Bioprinted Arrangements

Artificial cells are engineered units with cell‐like functions for different purposes including acting as supportive elements for mammalian cells. Artificial cells with minimal liver‐like function are made of alginate and equipped with metalloporphyrins that mimic the enzyme activity of a member of the cytochrome P450 family namely CYP1A2. The artificial cells are employed to enhance the dealkylation activity within 3D bioprinted structures composed of HepG2 cells and these artificial cells. This enhancement is monitored through the conversion of resorufin ethyl ether to resorufin. HepG2 cell aggregates are 3D bioprinted using an alginate/gelatin methacryloyl ink, resulting in the successful proliferation of the HepG2 cells. The composite ink made of an alginate/gelatin liquid phase with an increasing amount of artificial cells is characterized. The CYP1A2‐like activity of artificial cells is preserved over at least 35 days, where 6 nM resorufin is produced in 8 h. Composite inks made of artificial cells and HepG2 cell aggregates in a liquid phase are used for 3D bioprinting. The HepG2 cells proliferate over 35 days, and the structure has boosted CYP1A2 activity. The integration of artificial cells and their living counterparts into larger 3D semi‐synthetic tissues is a step towards exploring bottom‐up synthetic biology in tissue engineering.


Introduction
Mammalian tissue and organs are complex multicellular assemblies with mammalian cells as the essential building blocks.Despite the cells' complexity and the exquisite intricacy of specific DOI: 10.1002/adhm.202303699functions, there are certain characteristics that define the most common traits in cells, namely compartmentalization, growth and division, information processing, energy transduction, and adaptability. [1]The fascinating nature of living cells has led to artificial cells that imitate their living role models.A common strategy in bottom-up cell mimicry involves replicating specific cellular traits individually, rather than attempting to emulate the entire complexity of a living cell.This approach addresses the difficulty of recreating intricate cell-mimetic behavior.A wide range of structural and functional concepts have been explored for the assembly of artificial cells, including giant unilamellar vesicles (GUVs), [2] polymersomes, [3] proteinosomes, [4] coacervate microdroplets, [5] and hydrogels [6] with the aim to mimic specific characteristics of mammalian cells such as encapsulated catalysis, [7,8] energy generation, [9][10][11] or communication, [12][13][14][15][16] among others as reviewed elsewhere. [17,18]Artificial cells have also been explored as a simplistic model to elucidate complex cell biological function, for example, conductive pathways in 3D printed water-in-oil droplets [19] or collective behaviors in proteinosome-based protocells, [20] to get insight into the origin of life as reviewed elsewhere, [21,22] or to support their living counterparts.In the latter case, a feedback loop is required to ensure a beneficial interaction between artificial cells and different types of living cells including bacteria, fungi, and mammalian cells.For instance, artificial cells have demonstrated a positive impact on bacterial quorum sensing. [23,24]In a recent example, Alcinesio et al. demonstrated chemical signaling between 3D-printed synthetic (sender) and biological (receiver) building blocks using encapsulated bacterial cells. [25]Additionally, luciferase-generated bioluminescence was employed to activate photoconidiation in fungal cells. [26]Complementary, artificial cells started to be integrated with mammalian cells, as recently highlighted in more detail by van Hest and coworkers [27] or Elani. [28]The efforts become of particular interest when considering alternative approaches in regenerative medicine and tissue engineering.[34][35][36][37][38][39][40][41] Scheme 1. a) Semi-synthetic tissue is obtained by 3D printing of a composite bioink consisting of HepG2 cell aggregates and artificial cells as the solid phase and alginate/gelatin methacryloyl (GelMA) as the liquid phase.HepG2 cells express CYP1A2 enzymes that convert resorufin ethyl ether (REE) to resorufin, while b) alginate-based artificial cells are equipped with metalloporphyrins (MP) that mimic the catalytic activity of CYP1A2.CYP1A2 is an enzyme from the cytochrome P450 family, which is responsible for metabolizing various substances, medications, and environmental toxins, in the liver.
Nevertheless, despite the substantial advancements, the interaction between artificial cells and mammalian cells remains predominantly one-sided, occurring between two distinct populations.A crucial advancement for the integration of artificial cells with mammalian cells toward the development of functional semi-synthetic tissue involves not only contemplating the nature of interactions between the two cell populations but also considering their extracellular environment and spatial arrangement.This entails mimicking the geometric requirements observed at the tissue level.3D bioprinting offers unique opportunities in this context as recently outlined in several reviews, [42][43][44] in particular when considering the fabrication of more complex 3D bioprinted tissues both with regards to geometry and use of different cell types.However, although artificial cells only have been 3D printed, as reviewed elsewhere, [45,46] the potential of introducing artificial cells in tissue engineering scaffolds was recently highlighted by Sümbelli et al. with a particular focus on the potential of using artificial cells within tissue engineering scaffolds for improved spatiotemporal control over cell-matrix interactions on the microscale. [47]The tissue engineering scaffolds are commonly fabricated using 3D bioprinting for improved geometric control, while artificial cells hold potential in engineering the microscopic scale in close proximity to mammalian cells.To the best of our knowledge, however, 3D bioprinting with artificial and mammalian cells has never been demonstrated.
Here, we demonstrated that a composite bioink consisting of alginate-based artificial cells with CYP1A2-like activity, an artificial enzyme that mimics the catalytic activity of this specific enzyme from the cytochrome P450 family, and HepG2 cell aggregates mixed with a liquid phase can be 3D bioprinted into a semi-synthetic tissue with boosted CYP1A2 activity (Scheme 1).The artificial cells were made of alginate microgels equipped with liposomes that were loaded with metalloporphyrins (MPs), which exhibit CYP1A2-like activity and thus represented a sub-compartmentalized artificial cell recapitulating a minimal hepatocyte-like activity.The aim was to demonstrate that these types of artificial cells could be incorporated in a tissue-like structure together with mammalian cells, thereby bridging the gap towards more fully integrated systems, while demonstrating a supportive function.Specifically, we i) determined that a mixture of alginate and gelatin methacryloyl (GelMA) was a suitable liquid phase, which could be mixed with HepG2 cell aggregates for 3D bioprinting with subsequent HepG2 cell proliferation, ii) illustrated that a printable composite ink could be made of either alginate-based microgels or alginate-based microgels and HepG2 cell aggregates, both with an alginate and GelMA liquid phase, iii) followed the proliferation of the HepG2 cells in the prints, and iv) confirmed that the CYP1A2 activity was boosted in 3D printed semi-synthetic tissue overtime when metalloporphyrin-loaded artificial cells with dealkylation function were used instead of the microgels.

Results and Discussion
[50][51] Composite bioinks are promising alternatives to enhance the existing bioink options.These composite bioinks are made of a crosslinkable liquid phase and nano-or microparticles.In other words, by combining chemically diverse building blocks or incorporating materials in different states (such as a liquid phase or pre-crosslinked microgels), composite bioinks provide a wide range of possibilities for advancements.A popular example is cellulose nanoparticles and fibers, which have a versatile range of applications. [52]Inorganic particles, such as amine-modified silica nanoparticles, have been shown to improve the printability of oxidized alginate by dynamic covalent cross-linking. [53]Also, Laponite nanoparticles not only improved the printability but also prolonged the drug release in the alginate-methylcellulose solution phase. [54]Micron-sized hydrogel-based particles have shown a similar ability to improve printability in composite ink.For instance, pure gelatin composite inks allowed for the printing of complex 3D structures without additives, [55] or norbornene-modified hyaluronic acid microgels have been used to show that the mixing ratio of particles and solution phase, as well as the stiffness of the particles, greatly affected the printability of the composite ink. [56]1.3D Bioprinting of HepG2 Cell Aggregates 3D-printed semi-synthetic liver tissue is relevant due to the need for extracorporeal liver support devices that can support patients with acute liver failure, which can replace the function of the failing tissue to support spontaneous recovery.[57] The 3D bioprinting of semi-synthetic tissue that supports selected liver functions presents a rapid and facile method to obtain supportive devices.The fabrication requires three aspects to be considered -the type of carrier ink, the HepG2 cell aggregates, and the bioprinting of the combination thereof.
The first step towards the 3D bioprinting of semi-synthetic tissue using composite ink was the bioprinting of the liquid phase ink, and ensuring the subsequent proliferation of the mammalian cells, HepG2 cell aggregates in this case.

Liquid Phase
Identifying a suitable liquid phase ink for the mammalian cells is essential to obtain a 3D bioprinted structure.The liquid phase ink has to offer both appropriate mechanical properties for bioprinting (i.e., viscosity, gelation, surface tension, and shear-thinning behavior) and a suitable environment for cells to proliferate.Combinations of alginate and gelatin-derived polymers such as the photocrosslinkable GelMA have previously been used as hybrid bioinks, [58,59] offering a combination of both physical (facile gelation) and physiological (presence of cell attachment sites) beneficial properties.Inspired by Ouyang et al., [58] different liquid phase ink compositions of alginate/GelMA were printed in a standard 10 × 10 mm grid lattice, followed by UV crosslinking, and their printability was compared using bright field microscopy (Table S1 and Figure S1a, Supporting Information).The alginate fraction was composed of a 1:1 w/w mixture of low (M w < 75 kDa) and high (M w > 200 kDa) molecular weight sodium alginates, with high -L-guluronate (G block) content (>60%).
The alginate concentration was kept constant (1.5% w/v), while the concentration of GelMA was varied, and the liquid phase ink is referred to as LPI x where x indicated the GelMA concentration in % w/v.As expected, the extrusion pressure and the extent of filament irregularities increased with increasing GelMA concentrations.The irregular morphology of the LPI 7.5 and LPI 10 prints indicates that the gelation occurred too rapidly resulting in inhomogeneous filament deposition.However, structurally stable two-and three-layered constructs were obtained when using LPI 7.5 and LPI 10 .The printed LPI 5 filaments tended to fuse in the two-and three-layered constructs suggesting the gelation was too slow.Nonetheless, LPI 5 was chosen for all subsequent efforts since additional components were planned to be added to the ink that were expected to overcome the shortcomings of pristine LPI 5 including the slow mechanical recovery after high strain (500%), the low storage modulus (160 ± 30 Pa), and the relatively high yield strain of 25% (Figure S2a,b and Table S2, Supporting Information).
Upon printing, the structures were immediately UV crosslinked for 1 min.Both a 1-layer and 3-layer LPI 5 structure were visualized using confocal laser scanning microscopy (CLSM, Figure S1b, Supporting Information), and an increase in height by a few microns was observed in 3D projections of z-stacks of the prints.Additionally, prints made with 1 layer and 3 layers were imaged using scanning electron microscopy (SEM, Figure S1c, Supporting Information), illustrating that the polymers were evenly distributed in the printed lines with alignment in the printing direction (Figure S1c-i, Supporting Information) or the post-flow in multilayered structures (Figure S1c-ii, Supporting Information).

HepG2 Cell Aggregates
We chose to use aggregates of HepG2 cells for the efforts here.[62] Cell aggregates show a closer resemblance to complex tissue-like organizations than single cells, including HepG2 cells, in terms of viability, phenotype preservation, and proliferation in matrices. [63]Different methods can be used to obtain cell aggregates as recently discussed in detail by Klak et al. [64] We chose to exploit the spontaneous cell aggregate formation on a non-adhesive surface to get a high yield in the short time required for 3D bioprinting.To this end, 6-well plates were coated with alginate to render the surface non-adhesive, and different numbers of HepG2 cells were seeded per well and incubated for 24, 48, or 72 h to allow for aggregate formation followed by the evaluation of their yield and size.The alginate coating resulted in HepG2 cell aggregates in all cases in comparison to uncoated surfaces (Figure S3, Supporting Information).Visual inspections suggested that the optimal yield and monodispersity of HepG2 cell aggregates was obtained when seeding 600 000 cells per well (62 500 cells per cm 2 ) with a 24-h incubation time (Figure 1a).The cell aggregates were sieved through a 100 μm cell strainer to remove over-sized cell aggregates and the average diameter was measured as 100 ± 50 μm with a broad size distribution (Figure 1b).Further, 20 ± 20 cells were counted on average per aggregate when the nuclei of the HepG2 cells were stained with Hoechst 33342 (Figure 1c).The viability of the HepG2 cells in the aggregates after 24 h was high when evaluating CLSM images of Calcein-AM and ethidium homodimer (EthD-I) (live/dead) stained cells (Figure 1d).Finally, the CYP1A2 activity of the HepG2 cells in the aggregates was compared to HepG2 cells cultured on 2D tissue culture polystyrene by monitoring the dealkylation of non-fluorescent resorufin ethyl ether (REE) into the fluorescent resorufin.CYP1A2 is one of the dominant enzymes of the CYP450 family that are involved in the phase I metabolism of the liver, [65] and is therefore an essential feature in the current effort.HepG2 cells are well known to have a limited inherent CYP1A2 activity compared to primary hepatocytes, [66] but the expression can be induced by treatment with the AhR agonist -napthoflavone (BNF). [67]The monitored production of resorufin by the HepG2 cells was normalized to the number of cells by quantifying the double-stranded DNA (dsDNA, Figure S4, Supporting Information).As expected, HepG2 cells that were incubated with BNF had a ≈2× and ≈3× higher resorufin production in 2D cultures and in the cell aggregates, respectively, compared to the pristine HepG2 cells (Figure 1ei).Further, the resorufin production by HepG2 cells in aggregates was ≈5× higher compared to the cells cultured in 2D, suggesting a higher CYP1A2 expression.In addition, RT-qPCR was performed to determine the expression of CYP1A2 mRNA by the HepG2 cells.The expected relative increase in CYP1A2 expression in BNF-induced HepG2 cells was observed compared to the non-induced HepG2 cells.A ≈21× and an ≈18× increase in CYP1A2 expression was found for BNF-treated HepG2 cells in 2D culture and in aggregates, respectively.Further, the expression level of induced HepG2 cells in aggregates was significantly higher (≈1.7×) than in 2D culture.Taken together, these results confirmed that the HepG2 cell aggregates provided a beneficial culturing condition as previously reported for hepatocytes such as HepaRG [68][69][70] or primary human hepatocytes [66,71] when grown in spheroids.However, the present method of aggregate formation by rendering surfaces non-adhesive with alginate, rather than producing single spheroids in, for example, ultra-low attachment wells, presents an easy means of scaling up the production of aggregates.

Bioink: HepG2 Cell Aggregates and Liquid Phase Ink LPI 5
Finally, the HepG2 cell aggregates were mixed with LPI 5 (Hep-LPI 5 ) to evaluate the printability of this ink, and the performance of the HepG2 cells in the printed structure.The HepG2 cell aggregates were grown in alginate-coated T75 flasks to increase their yield.HepG2 cell aggregates from three T75 flasks were harvested, centrifuged, and resuspended in media and mixed with LPI 5 in a 1:4 ratio (cell aggregates: LPI 5 (v/v)) to obtain 2.5 mL ink.Two-layered structures were printed with similar parameters as for pristine LPI 5 , followed by UV crosslinking with 0.05% w/v lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), which have previously been shown not to have adverse effects on cell viability. [72,73]The prints were incubated in media at 37 °C in 5% CO 2 for up to 5 weeks, and the two-layered prints were imaged after 1, 4, 7, 14, 21, 28, and 35 days using bright field microscopy (Figure 2a).Importantly, the integrity of the two-layered structures was preserved, and the diameters of the HepG2 cell aggregates visibly grew in the 5 weeks.CLSM of the live/dead stained HepG2 cells with Hoechst 33342 marked nuclei in the printed Hep-LPI 5 filaments showed an increasing number of viable HepG2 cells over time (Figure 2b-i).Further, two-layered structures were printed using rhodamine-labeled GelMA (GelMA Rho ) when making LPI 5 to illustrate that part of the HepG2 cells were present within the hydrogel network, while others grew spheroidlike at the filament interface, that is, as mushroom-like cell aggregates that were anchored to the filaments (Figure 2b-ii).It should be noted that a granular structure of the filaments was revealed by the inhomogeneous fluorescent signal originating from GelMA Rho , indicating a limited mixing of alginate and GelMA Rho , which could be amplified in the presence of the relatively hydrophobic rhodamine.Additionally, bright spots of rhodamine were observed, likely inside of the HepG2 cells, suggesting a degree of degradation and internalization of GelMA Rho by the HepG2 cells.
In addition to visualization of the proliferating cells, the total amount of dsDNA of the HepG2 cells in the two-layered structures grown for up to 35 days was determined at different time points (Figure 2c).The concentration of dsDNA was correlated to the number of cells by quantifying the amount of dsDNA in a known number of cells.The number of HepG2 cells increased over the 35 days from ≈100 × 10 3 to ≈1200 × 10 3 cells.The growth rate was linear for the first 14 days (80 ± 30 × 10 3 cells per day) and then slowed down.We attribute this observation to the fact that large aggregates were observed to grow out of the filaments and detach from the printed structures.This could potentially be prevented by printing a non-cell-laden outer layer of LPI 5 to provide a substrate for the growing cells.
Further, it was confirmed that the CYP1A2 activity of the HepG2 cells in these printed structures was preserved, illustrated by the ability of the HepG2 cells to convert REE into re-sorufin overtime (Figure 2d), here in prints made 4 days prior.BNF-induced HepG2 cells had a statistically significant higher resorufin production as expected, and after 1 h, ≈8 pM resorufin was produced per 10 3 BNF-induced HepG2 cells, while noninduced HepG2 cells only made ≈2.5 pM resorufin per 10 3 cells.It should be noted that pristine HepG2 cell aggregates before printing had a ≈3× higher resorufin production (Figure 1e-i), probably due to diffusion limitations of substrate/product in the prints or the HepG2 cells did not fully recover from the printing process.

3D Printing of Composite Ink Consisting of Alginate-Based Microgels in LPI 5
In the next step, we determined the most suitable amount of alginate-based microgels that could be added to LPI 5 to obtain a printable composite ink.Alginate was chosen for this purpose since this type of hydrogel has previously been shown to allow for the assembly of non-degradable artificial cells, which were required when the printing of semi-synthetic tissue was the target.The addition of microgels to the liquid phase has been shown to improve the shear-thinning properties of the composite ink, that is, when the extrusion pressure was removed, the more solid microgels helped to stop the movement of the liquid phase and thereby re-formed the overall ink into a stable semi-solid state. [56,74,75]We made microgels from alginate (15 mg mL −1 ) using an Encapsulator B-390 and crosslinked them in a 0.1 M CaCl 2 solution , resulting in microgels with a mean diameter of 100 ± 60 μm (Figure 3a).The microgels were washed with buffer (10 mM HEPES, 150 mM NaCl, pH 7.4), which had no impact on their morphology or size (Figure S5, Supporting Information) and are referred to as AC 0 .Composition inks (Figure 3b) were obtained by mixing LPI 5 with different amounts of AC 0 , resulting in CInk k:m , where k:m denoted the AC 0 to liquid phase ratio in w/v.
First, the rheological properties of CInk k:m were determined (Figure 3c and Figure S2, Supporting Information).Compared to the LPI 5 , the storage modulus (G') decreased for CInk 1:5 from ≈150 to ≈100 Pa.Furthermore, G' increased for CInk 1:2 , CInk 1:1 , and CInk 2:1 to ≈350, ≈650, and ≈550 Pa, respectively.The increase in G' resulted from the incorporation of the crosslinked AC 0 into the liquid phase (Figure S2a, Supporting Information).These G' values were all lower than the natural liver tissue, which is around 1000 Pa for porcine liver. [76]However, during the UVcuring, it was expected that the G' increased to comparable values of the liver.The yield point, where G' is no longer linear, decreased with increasing amount of AC 0 in CInk k:m .The flow point, the cross-over between G' and the loss modulus (G''), was the same for LPI 5 and CInk 1:2 , but it decreased for the rest of the CInk k:m (Table S2 and Figure S2c, Supporting Information) using the definition by Schwab et al. for these two parameters. [77]hese results showed that the AC 0 acted as a lubricant, that is, the material yielded at lower strains but the flow behavior remained dominated by the liquid phase until CInk 1:1 at which the liquid phase becomes too diluted to retrain the flow point.The recovery of G' and G'' after high strain (500%) for CInk 1:5 , CInk 1:2 , and CInk 1:1 had comparable speeds but CInk 2:1 showed a slower recovery (Figure 3c).A decrease in recovery from LPI 5 (83%) to CInk 2:1 (43%) was observed for the first recovery cycle and was most likely due to the reorganization of the AC 0 in the material (Table S2, Supporting Information).This explanation was supported by the fact that the recovery for the second cycle was comparable for LPI 5 , but higher for CInk 2:1 (80%), assuming that the re-arranged AC 0 facilitated the recovery.In addition, CInk 1:5 and CInk 1:2 had a 74% and 62% recovery in the first cycle, respectively, and a similar recovery of above 90% for the second cycle, again suggesting that the difference in recovery was due to the initial re-organization of AC 0 .All inks showed shear thinning behavior during flow testing due to the linear relation between the viscosity and shear rate (Figure S2d, Supporting Information). [78]aken together, CInk 1:1 was a stiffer ink with similar flow and recovery properties than LPI 5 but with anticipated better structural integrity after extrusion.
Second, the printability of CInk k:m was evaluated by printing grid lattices with up to three layers and visualized by bright field microscopy (Figure 3d-i).As expected from the rheology data (yield point), no increase in extrusion pressure was required for higher AC 0 concentrations, illustrating the shear-thinning character of CInk k:m (Table S3, Supporting Information).However, the resulting filament shape and homogeneity decreased for CInk 1:1 and CInk 2:1 compared to composite inks with lower AC 0 content due to the decreased yield point, that is, the gravitational force was enough to deform the extruded lines together with the lower recovery ability.The increased stiffness of CInk 1:2 compared to CInk 1:5 seemed to result in less sagging of the filament.Further, SEM was used to image the filaments in the one-layered prints in more detail (Figure 3d-ii).The filaments had increasing amounts of cavities (porosity) with increasing amounts of AC 0 present in CInk k:m (Figure S6, Supporting Information).Therefore, we attributed the origin of the cavities to AC 0 due to their low alginate content, that is, their structural contribution to the filaments was lost as a result of lyophilization of the printed structures.CLSM images of the prints using GelMA Rho when making CInk k:m showed the presence of increasing amount of AC 0 in the filaments for prints made of CInk 1:5 to CInk 2:1 (Figure 3d-iii).The filament thickness in the center increased from ≈230 μm for CInk 1:5 to ≈380 μm for CInk 2:1 (Figure 3d-iii), while the printing of two layers led to more pronounced increases in height with higher AC 0 content (Figure S7, Supporting Information).
Taking all these observations into consideration, CInk 1:2 was chosen for further experiments due to good filament homogeneity, extrudability, and fidelity.

3D Bioprinting using HepG2 Cell Aggregates and Alginate Microgel-Containing Composite Ink
Following on, 25% v/v of AC 0 was replaced with HepG2 cell aggregates in the composite ink, resulting in a k:m ratio of 1:3 and is referred to as Hep-CInk 1:3 , as a first step towards printing semi-synthetic tissue.We printed two-layered grid lattice structures using Hep-CInk 1:3 and the HepG2 cells were left to proliferate for up to 35 days (Figure 4a).The higher extrusion pressure required during the printing process of Hep-CInk 1:3 (≈38 kPa) compared to Hep-LPI 5 (≈18 kPa) did not result in any visible change in the growth and proliferation of viable cells over 35 days (Figure 4b-i).In contrast to HepG2 cells printed in LPI 5 that mostly remained clustered in aggregates, the proliferating HepG2 cells in structures printed with Hep-CInk 1:3 distributed and spread more freely, surrounding the AC 0 s and losing their original aggregated morphology.AC 0 s were found to be evenly distributed throughout the ink after 14 days when mixing GelMA Rho in Hep-CInk 1:3 (Figure 4b-ii, and Figure S8, Supporting Information).The filaments were densely populated with HepG2 cells throughout after 35 days (Figure 4b-iii).
The proliferation rate of the HepG2 cells (40 ± 20 × 10 3 cells per day), which was estimated by quantifying the dsDNA, was not significantly lower compared to HepG2 cells printed with LPI 5 (80 ± 30 × 10 3 cells per day), again showing linear growth for the first 14 days followed by an apparent slower growth (Figure 4c).However, we again attributed this latter aspect to the fact that HepG2 cell aggregates grew out of the filament in a mushroomlike shape, which eventually detached and were removed during the media changes.
In contrast to HepG2 cells printed in LPI 5 with GelMA Rho , the granular appearance of the filament was not observed, indicating that the AC 0 s led to an improved mixing of alginate and GelMA in the ink (Figure 4b-ii, inset).

3D Bioprinting of Metalloporphyrin-Loaded Artificial Cells with HepG2 Cell Aggregates
Thus far, AC 0 only offered structural benefits to the composite ink and the resulting prints.Consequently, the aim was to add activity  to AC 0 that boosted a liver-relevant function in the printed semisynthetic tissue.We chose to implement a cytochrome P450related activity due to its importance in liver performance, and the documented limited function thereof in HepG2 cells, [66] thereby potentially partly mitigating a shortcoming of this widely used cell line.Thus, the incorporation of CYP450 activity was chosen as a proof-of-concept to illustrate that artificial cells can be designed to boost a specific hepatic function.In addition, artificial cells can act as a generalizable approach for introducing activity on the cellular scale in 3D bioprinted scaffolds.Currently, the aim is to fundamentally show that functionalized hydrogel-based artificial cells can be integrated with mammalian cells in 3D bioprinted structures.In the future, this approach of boosting compromised biotransformation functions of hepatocytes using minimal artificial cells is envisioned to be applicable to fabricating semi-synthetic tissue for potential use in regenerative medicine or as an active component in extracorporeal liver support devices.We have previously assembled and characterized alginate-based artificial cells that could be functionalized with metalloporphyrins, which are enzyme mimics of the CYP450 enzyme family. [11]In other words, metallopor-phyrins have been designed drawing inspiration from the hemethiolate monooxygenases (the CYP450 enzymes), whereof heme is an iron-coordinated metalloporphyrin.Specifically, the selected metalloporphyrin here could imitate the catalytic activity of CYP1A2, a major CYP450 enzyme in the liver (≈13%), [79] which is responsible for metabolizing several widely used clinical drugs (such as tacrine, clozapine, tizanidine [80] ) and endogenous compounds (such as steroids [80] and bilirubin [81] ).Further, CYP1A2 activities have been shown to be ≈14× and ≈4× higher in primary human hepatocytes and HepaRG cells, respectively, compared to HepG2 cells. [82]Therefore, the CYP1A2 activity represents a compromised function in the latter case that is beneficial to boost using artificial cells.The conversion of REE to resorufin was chosen, as it is a common model substrate for CYP1A2, to demonstrate a liver-specific cell-mimetic function in the artificial cells, an approach which in the future can be expanded to include more directly relevant therapeutic functions.Here, we assembled artificial cells following our previously published protocol resulting in MP-functionalized artificial cells (AC MP ). [8]Briefly, MPs were first encapsulated in liposomes made from the phospholipid 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) using the , or 8 h given as μ ± .The prints were treated with BNF to induce CYP1A2 expression.The resorufin production was monitored on prints that have been cultured for i) 4 or ii) 14 days after printing.The statistical significance used to compare the means was determined using a two-sample t-test (*p < 0.05, n = 3).rehydration method, followed by encapsulation in the alginate microgels.
First, printed structures of a composite ink of AC MP and LPI 5 were made to evaluate the resorufin production 4 or 14 days after printing when stored in a cell medium at 37 °C (Figure S9, Supporting Information).There was no loss of activity over the 14day period, illustrated by the constant 6 nM resorufin produced by AC MP over 8 h.
Then, a composite ink was made by replacing AC 0 with AC MP in Hep-CInk 1:3 resulting in Hep-CInk MP .This ink was used to print two-layered structures.The CYP1A2 activity of the HepG2 cells in these prints was then recorded after 4 and 14 days of incubation by monitoring the conversion of REE into resorufin for up to 8 h following the REE addition of the media.In addition, the HepG2 cells in the prints were BNF-induced 48 h before the assessment of the resorufin production.Prints made of Hep-CInk 1:3 were used as controls for the resorufin production by the HepG2 cells only (Figure 5a).
After 4 days of incubation, the resorufin production by BNFinduced HepG2 cells in prints made of Hep-CInk 1:3 was comparable to structures printed with Hep-LPI 5 after 2 h (Figure 5b-i, and Figure S10, Supporting Information).As expected, the BNFinduced HepG2 cells had a ≈2× higher resorufin production 8 h after REE addition compared to pristine (non-induced) HepG2 cells (Figure 5b-i).Notably, prints of Hep-CInk MP led to a significantly higher resorufin production of ≈6 nM after 8 h for both structures with BNF-induced and pristine HepG2 cells, compared to prints of Hep-CInk 1:3 .This increase was in agreement with the expected contribution from the AC MP in the printed structures, demonstrating the potential of 3D printed semisynthetic tissue that has boosted CYP1A2 activity after 4 days in culture.
Further, the impact of the AC MP on overall resorufin production after 14 days in culture was evaluated when the HepG2 cell aggregates grew over the course of this time (Figure 5).Again, resorufin production in the prints was monitored after 3, 6, or 8 h (Figure 5b-ii).An expected overall increase in resorufin production due to the proliferation of HepG2 cells was observed, and BNF treatment led to ≈1.5× higher resorufin production.The added effect of ≈6 nM resorufin from AC MP was less pronounced at this time point, but there was still a significant difference between the Hep-CInk 1:3 and Hep-CInk MP prints, illustrating that the MPs were still present and active.Additionally, RT-qPCR was performed to confirm that the expression of CYP1A2 mRNA from the Hep-CInk 1:3 and Hep-CInk MP prints were comparable after 14 days in culture.First, it was found that the CYP1A2 expression was ≈6× higher in induced Hep-CInk 1:3 prints (Figure S11, Supporting Information).Second, there was no significant difference in the expression of CYP1A2 between Hep-CInk 1:3 and Hep-CInk MP prints.Thus, the boosted resorufin production could be attributed to the presence of AC MP and not to an unexpected effect on the CYP1A2 expression of the HepG2 cells.Further, the CYP1A2 activity of Hep-CInk 1:3 and Hep-CInk MP prints was confirmed after 35 days in culture (Figure S12, Supporting Information).However, the difference between the two prints was no longer significant due to the high number of HepG2 cells present compared to AC MP .Therefore, the impact of the AC MP was most beneficial in the first 14 days of the culture, before the CYP1A2-activity of the proliferating HepG2 cells dominated.It should be noted that the presence of MPs did not affect the growth and viability of the HepG2 cells in the prints after 35 days as shown with bright field microscopy, CLSM, and DNA quantification (Figure S13, Supporting Information).

Conclusion
Artificial cells equipped with MPs, thereby exhibiting a minimal hepatocyte-like function by mimicking the catalytic activity of a CYP450 enzyme, were incorporated in 3D bioprints with HepG2 cells to demonstrate the incorporation of artificial cells with biological cells in a tissue-like environment.The 3D prints of AC MP and HepG2 cell aggregates showed enhanced resorufin ethyl ether conversion for up to 14 days compared to prints with AC 0 , illustrating that the synthetic units supported their living counterpart by enhancing the overall enzymatic activity of the semi-synthetic assembly.This successful effort is an advancement towards the fabrication of bionic tissue.While the present effort illustrated artificial cells that were active in the presence of HepG2 cells using a model substrate, there was no direct interaction between the artificial and mammalian cells.Consequently, the next steps will include interactive artificial cells for reciprocal signal exchanges with beneficial impact on the mammalian cells.The integration of multiple cell types in tissue-relevant geometric arrangements, or the addition of multiple enzyme-like activities from the CYP450 family, are relevant aspects that will be targeted.
Cell Culture: The human liver cancer cell line HepG2 was purchased from the European Collection of Cell Cultures.HepG2 cells were cultured in glucose-free DMEM supplemented with 10% FBS, 6 mM L-glutamine, 1 mM sodium pyruvate, 10 mM galactose, 100 μg mL −1 streptomycin, and 100 U mL −1 penicillin, which is referred to as galactose media.
Cell Aggregate Formation: The surfaces of a 6-well plate or T75 flask were coated with alginate to render them non-adhesive for cell aggregate formation.A 15 mg mL −1 alginate (Merck) solution was sterile filtered using 0.2 μm filters (Minisart NY). 2 mL was added to each well of a 6-well plate, and 15 mL was added to a T75 flask, and was left for 5 min.Then, leftover alginate was aspirated, leaving a thin coating and 2 or 15 mL 0.1 M CaCl 2 was gently added to the 6-well plate or T75 culture flask, respectively.The alginate coating was left to crosslink for 3 min, and the CaCl 2 was aspirated.In the 6-well plate, 300 000 cells mL −1 (high), 150 000 cells mL −1 (medium), and 75 000 cells mL −1 (low) stock were prepared and 2 mL was added per well.The aggregates were incubated for 24, 48, or 72 h at 37°C and 5% CO 2 and imaged using bright field microscopy (each condition was prepared in duplicate).
In the T75 flask, 15 mL of 300 000 cells mL −1 solution was added to each T75 flask for a total of 4.5 × 10 6 cells per flask.The cell aggregates formed overnight at 37 °C and 5% CO 2 .For bioink fabrication, the cell aggregates from three T75 flasks were harvested and sieved through a 100 μm cell strainer (Corning) to remove aggregates >100 μm, followed by centrifugation at 130 g for 7 min at 4 °C.The supernatant was removed until 500 μL remained.The cell pellet was re-suspended, transferred to a 1 mL syringe, and stored at room temperature until the solution was ready for further mixing to make bioinks.
Gelatin Methacryloyl (GelMA) Fabrication: GelMA was synthesized according to a reported protocol with some slight modifications. [83]Briefly, gelatin from porcine skin (type A) was dissolved in PBS in a round bottom flask to a final concentration of 100 mg mL −1 and incubated in a fume hood at 50 °C at 500 rpm (using a magnetic stirring bar) until the solution became transparent.Subsequently, 0.6 mg of methacrylic anhydride was added dropwise per 1 mg of gelatin while stirring at 700 rpm.After addition of the methacrylic anhydride, the solution was incubated for 1 h while stirring at 700 rpm.Thereafter, the reaction solution was poured into 50 mL falcon tubes and centrifuged at 3260 g for 3 min after which the supernatant was transferred to a 500 mL glass beaker.The reaction was quenched via the addition of ddH 2 O water until 2× the original volume was reached.This solution was incubated at 50 °C while stirring until the solution was homogeneous.The obtained mixture was transferred to dialysis tubes (12-14 kDa) and dialyzed against ddH 2 O water for 5 days in a large 5 L glass beaker.The dialysis solution was exchanged twice per day.After dialysis, the contents of the dialysis tubing were transferred into 50 mL falcon tubes and lyophilized for 5-7 days.
Liquid Phase Ink (LPI x ) Preparation: UP MVG and UP VLVG alginate (Pronova) were mixed 1:1 (w/w) and dissolved overnight on a rotary mixer in HEPES buffer for a final concentration of 30 mg mL −1 .Then, GelMA and 1.0 mg mL −1 LAP were dissolved in HEPES buffer in a flat bottom glass vial at 60 °C under rotation for at least 1 h.The alginate and GelMA solution were mixed in a 1:1 (v/v) ratio and incubated at 60 °C under stirring for at least 1 h to form a homogeneous solution, resulting in alginate/GelMA liquid phase inks referred to as LPI 10 , LPI 7.5, and LPI 5 , respectively.The details can be found in Table S4, Supporting Information.
Hep-LPI 5 Preparation: GelMA was dissolved in HEPES to a final concentration of 125 mg mL −1 (12.5% w/v) and alginate to a final concentration of 37.5 mg mL −1 (3.75% w/v) before mixing them in a 1:1 ratio (v/v), for a final concentration of 18.75 mg mL −1 (1.875% w/v) alginate and 62.5 mg mL −1 (6.25% w/v) GelMA. 2 mL of this solution was transferred to a 3 mL syringe and stored in a 60 °C stove until further handling.Cell aggregates from three T75 culture flasks were harvested as described above, and the resulting 500 μL cell aggregate solution in a 1 mL syringe was mixed with 2 mL alginate/GelMA solution in a 1:4 v/v ratio, by connecting the syringes via a female-female luer lock.The cell aggregate solution in the 1 mL syringe was pushed into the 3 mL syringe, and the 1 mL syringe was then replaced by a 5 mL syringe to improve further mixing.The solutions were mixed by pushing the 5 and 3 mL syringes back and forth ≈20 times each way.The resulting Hep-LPI 5 bioink (1.5% w/v alginate and 5% w/v GelMA) was transferred from the 3 mL syringe into a printing cartridge, wrapped in aluminum foil to protect from light, and stored at 4 °C until printing (see section on 3D printing).
Metalloporphyrin Loading in Liposomes: The water-soluble metalloporphyrin (MP) was encapsulated in 1,2-dioleoyl-sn-glycero-3phosphocholine (DOPC) liposomes, according to a previously published protocol, in which the MP was referred to as MP 9 . [8]lginate Microgel AC 0 Formation: 37.5 mg UP MVG alginate was mixed with 37.5 mg UP VLVG alginate (Pronova) and dissolved overnight in 5 mL HEPES buffer for a final alginate concentration of 15 mg mL −1 .Alternatively, the alginate was dissolved in 4 mL overnight, and the day after 1 mL of the MP-loaded liposome solution was added.The resulting alginate solutions were loaded into a 20 mL syringe and connected to an Encapsulator B-390 (Buchi).An 80 μm inner and a 200 μm outer nozzle were used to form the microgels.The syringe was fixed onto a syringe pump and injected at a speed of 1.1 mL min −1 .A frequency of 1300 Hz, a pressure of 0.945 mbar, a voltage of 800 V, and an airflow of 0.5 normal liters min −1 was used during microgel fabrication.The cross-linked particles were collected in a glass beaker containing 0.1 M CaCl 2 under stirring and were collected in a 40 μm cell strainer (Corning).The non-loaded microgels are referred to as AC 0 and the MP-loaded microgels are referred to as AC MP .The alginate microgels were visualized using an inverted Olympus microscope (IX81), and the AC 0 size distribution was assessed from the images using ImageJ.The size distributions were fitted with a Gaussian function, and at least 680 particles were measured.AC 0 diameters stored in CaCl 2 were measured from two independent repeats, and AC 0 washed with HEPES were measured from 4 independent repeats.
Composite Ink (CInk k:m ) Preparation: The CInk k:m s were made by mixing LPI 5 with varying amounts of microgels.Rhodamine B-labeled GelMA (GelMA Rho ) was added for visualization.First, a 30 mg mL −1 1:1 v/v UP MVG:UP VLVG alginate solution was prepared and left to dissolve overnight.Two solutions were made; a solution of 190 mg mL −1 GelMA and 1 mg mL −1 LAP, and a 10 mg mL −1 GelMA Rho solution in HEPES were prepared by stirring at 100 rpm at 60 °C for a few hours.The two solutions were mixed 1:1 resulting in a solution of 0.5 mg mL −1 LAP, 95 mg mL −1 GelMA, and 5 mg mL −1 GelMA Rho .The alginate and GelMA solutions were mixed 1:1 v/v to obtain Rho-labeled LPI 5 .AC 0 were rinsed thoroughly with HEPES buffer in a 40 μm cell strainer, and 250, 500, 750, or 1000 mg (wet) microgels were weighed off in 2 mL Eppendorf tubes.The tubes were centrifuged at 4.5k rpm (Eppendorf Minispin) for 3 min to ensure no remaining supernatant was present.LPI 5 Rho were added to the tubes using a high viscosity pipette containing AC 0 , yielding CInk k:m (k = microgel and m = LPI 5 in w/v ratio).The detailed compositions can be found in Table S4, Supporting Information.The solutions were thoroughly mixed while keeping at ≈60 °C.The warm CInk k:m was transferred to cartridges and placed at 4°C until printing (see section on 3D bioprinting).
Preparation of Hep-CInk 1:3 : The Hep-CInk was made of a 1:3 m/v of AC 0 to LPI (1.5% w/v alginate and 5% w/v GelMA), containing HepG2 cell aggregates as outlined above to yield Hep-CInk 1:3 , with a final composition of 1.5% w/v alginate, 5% w/v GelMA, and 25% v/v AC 0 or AC MP .The detailed compositions can be found in Table S4, Supporting Information.
3D Printing: After transferring 2.5 mL of ink to a cartridge and leaving it to incubate at 4 °C for 30 min on a rotary mixer, the cartridges were placed in the bioprinter (BioX TM , CELLINK) and left to incubate for 30 min in the printhead at 20 or 25 °C before printing (Table S1, Supporting Information).A 22G nozzle was installed to the cartridges and the print bed temperature was set to 8 °C.The structures were printed in each well of a 6-well plate.
Printing parameters for LPI x and CInk k:m prints (without HepG2 cells) are listed in Tables S1 and S3, Supporting Information, respectively, and the printing parameters for Hep-LPI 5 , Hep-CInk 1:3 and Hep-CInk MP are listed in Table S5, Supporting Information.The prints were UV crosslinked (405 nm, 16 mW cm −1 ) for 60 s.Prints without HepG2 cells were stored at 4 °C in HEPES buffer.Prints with HepG2 cells were put in 4 mL galactose media, and the prints were cultured at 37 °C and 5% CO 2 followed by characterization on different days up to 35 days.
Rheology of LPI 5 and CInk k:m : A parallel plate geometry (Ø 25 mm) with a 1 mm (10× particle size) gap height, at 20 °C in a humid environment was used on an MCR 501 (Anton Paar, Switzerland) rheometer for all experiments.The non-cross-linked LPI 5 was loaded to the bottom plate at ≈30 °C followed by cooling down to 4 °C for 30 min before it was then heated to 20 °C for 30 min, to ensure the same physical cross-linking of the GelMA as in the following printing conditions.The following experiments were performed back to back with inspiration from; [84] a time sweep for 2 min at 1% strain and 0.1 Hz, a recovery test of two cycles with high shear strain (500%) and low shear strain (1%) for 2 min each at 1 Hz, a new time sweep for relaxation of the material, a strain sweep from 1-500% at 1 Hz, and finally a flow sweep (hear ramp) from 0.01-100 s −1 .

Scanning Electron Microscopy (SEM):
The printed structures made with LPI x or CInk k:m were frozen in dry ice directly after printing and subsequently lyophilized before being imaged by SEM.The samples were coated with a thin layer of platinum (6 nm) before visualization.Images were collected on an FEI SEM (Nova Nano600) operating at an acceleration voltage of 5 kV and at a working distance of 5 mm using an ETD detector.
Bright Field Microscopy: A complete 6-well plate with printed structures was imaged using bright field microscopy at days 1, 4, 7, and 14 after printing (day 0) using a 4× magnification objective.Three images were taken per print.
CYP450 Activity of 2D or 3D HepG2 Cell Cultures: 300 000 HepG2 cells mL −1 were cultured in non-coated wells (2D) or alginate-coated wells (3D) of 6-well plates, and 0.5% v/v of a 2 mM -napthoflavone (BNF) in DMSO solution was added to the wells during seeding for a final concentration of 10 μM BNF in 2 mL media to induce the CYP1A2 expression in the HepG2 cells.After 24 h, another 0.5% v/v of 2 mM BNF was added.48 h after seeding, 10 μL 1 mM resorufin ethyl ether (REE) was added to each well for a final concentration of 5 μM and were placed on a rotary shaker in an incubator (37 °C and 5% CO 2 ) to allow for the conversion of REE to resorufin.At different time points,100 μL supernatant was removed from each well and was transferred to a black 96-well plate.The fluorescence was monitored at  ex/em = 570/585 nm.
CYP450 Activity of Hep-LI 5 or Hep-CInk 1:3 Prints: The printed structures were transferred to galactose-supplemented phenol red-free DMEM on day 2 after printing 0.5% v/v of a 2 mM BNF was added to each well for a final concentration of 10 μM BNF in 3 mL media after 24 and 48 h (on days 3 and 4).On day 4, 2985 μL of new media and 15 μL of 1 mM REE were added to each well (final REE concentration of 5 μM).The cells were placed on a rotary shaker in an incubator (37 °C and 5% CO 2 ) to allow for the conversion of REE to resorufin.100 μL supernatant was removed from each well and was transferred to a black 96-well plate after 3, 6, and 8 h.A solution of 5 μM REE without HepG2 cells was used as background.The fluorescence was monitored at  ex/em = 570/585 nm.
RNA Isolation from 2D or 3D Cultured HEPG2 Cells: HepG2 cells cultured on alginate-coated or non-coated wells of a 6-well plate were harvested after induction for 48 h (as described above), centrifuged at 300 g for 5 min at 4 °C, and washed 1× with PBS.The PBS was removed and the RNA was isolated using the RNeasy Mini Kit following the protocol.Briefly, 350 μL RLT buffer was added to the pellets and the cells were disrupted by vortexing and pipetting.350 μL 70% EtOH was added to each tube and was mixed thoroughly.Then, 700 μL was transferred to the RNeasy Mini spin column and the protocol was followed including DNAse digestion.For the final centrifugation step, 50 μL RNAsefree water was used to elute the RNA.The yield was measured using a Nanodrop 2000.
Quantification of CYP1A2 mRNA from 2D or 3D Cultured HEPG2 Cells: After RNA isolation, reverse transcription was performed using the High-Capacity cDNA Reverse Transcription Kit.Each sample was diluted to 600 ng in 14.2 μL DEPC water and was mixed with 10 μL reaction mix (2 μL 10× RT Buffer, 2 μL 10× RT random primers, 0.8 μL 25× dNTP stock solution (100 mM), 1 μL MultiScribe Reverse Transcriptase (50 U μL −1 )) in PCR tube strips on ice.Reverse transcription was performed on a thermocycler.Then, the 20 μL of each sample was diluted 10× by adding 180 μL NFW.Reverse transcription quantitative PCR (RT-qPCR) was performed in triplicates on a 96-well reaction plate in final volumes of 10 μL.Each reaction well contained 2 μL sample, 5 μL 2× Power SYBRGreen PCR Master Mix, 0.5 μL of forward primers, 0.5 μL reverse primers, and 2 μL NFW for a final concentration of 0.5 μM of forward and reverse primers.CYP1A2 and glyceraldehyde 3-phosphate dehydrogenase (GADPH) expression were amplified using a QuantStudio TM Real-Time PCR system with the following primer sequences (5′ to 3′).
CYP1A2 Forward: GTCAATGACATCTTTGGAGCAG CYP1A2 Reverse: CCTGCCAATCACAGTGTCC GADPH Forward: AGCCACATCGCTCAGACAC GADPH Reverse: GCCCAATACGACCAAATCC The sequences of CYP1A2 primers were previously reported by Déri et al. [85] Changes in the CYP1A2 expression were determined using the 2 −ΔΔCt method using GADPH as an internal standard, and is represented as fold-change relative to 2D cultured, non-BNF treated HepG2 cells.
RNA Isolation and Quantification of CYP1A2 mRNA from Hep-CInk 1:3 and Hep-CInk MP Prints: Induced or pristine prints were incubated with 350 μL of RLT buffer and vortexed prior to homogenization with a 26 G needle (5×).Then, the samples were centrifuged for 5 min at 6000 rpm and the supernatant was collected for RNA extraction.RNA was isolated using the RNeasy Mini Kit (Qiagen) as described above.Single-strand cDNA was synthesized from a total RNA of 100 ng using a High-Capacity cDNA Reverse Transcription kit.After reverse transcription, each sample was diluted 10×.RT-qPCR was performed in triplicates on a 96-well plate in a final volume of 20 μL containing 10 μL 2× Power SYBRGreen PCR Master, 1 μL forward primers (0.5 μM final concentration), 1 μL reverse primers (0.5 μM final concentration), and 8 μL sample.The primers were validated with a Taqman assay.The changes in the CYP1A2 expression were determined using the 2 −ΔΔCt method using GAPDH as an internal standard to correct for differences in starting mRNA concentrations.The data was represented as fold-change relative to non-induced Hep-CInk 1:3 prints.Hoechst 33342 Staining of Cell Aggregates: 2 mL Hoechst 333342 staining solution (100 μM) was added to the cell pellet obtained from cell aggregates in a T75 flask or prints.After 15 min incubation at RT, images were taken using CLSM (Zeiss LSM 700, AxioObserver; EC Plan-Neofluar 20×/0.50M27) using  ex = 405 nm.
DNA Quantification: A cell digestion solution was prepared in PBS with a final concentration of 125 μg mL −1 papain, 0.242 mg mL −1 Lcysteine, and 2 mM EDTA. 1 mL of the digestion solution was added to either 50 μL of cell pellet obtained from cell aggregates in a T75 flask, or prints collected in duplicate on days 1, 4, 7, and 14 and stored at −80 °C.Solutions were placed in a 65 °C oven overnight.Then, the digested solution was removed from the oven, vortexed, and placed back into the oven for 30 min.The solution was centrifuged at 130 g for 7 min to pellet undigested material.PicoGreen reagent (Invitrogen) was diluted 200× in a Tris-EDTA buffer (10 mM Tris-HCl, 1 mM EDTA in PBS, pH 7.5), while protected from light, to make a PicoGreen working solution.A dsDNA standard was prepared by diluting a 100 μg mL −1 stock 50× in Tris-EDTA buffer to a final concentration of 2 μg mL −1 .The digested cell aggregate samples were diluted 10× in Tris-EDTA buffer, while the digested prints were not diluted.Both the dsDNA standard and samples were diluted in a 1:1 serial dilution in a final volume of 100 μL.Tris-EDTA buffer was used as a background control.100 μL PicoGreen working solution was added to each well and carefully mixed.After 2-5 min incubation at RT, the fluorescence intensity was measured in a plate reader (PerkinElmer Enspire 2300) at  ex/em = 480/520 nm.
Statistical Analysis: Data were pre-processed by evaluating outliers, and normalization was performed when necessary as indicated in the figure text.Data were represented as μ ±  unless otherwise stated.All experiments were performed in three independent biological repeats (n = 3) unless otherwise stated.The statistical significance used to compare the means was generally determined using either a one-way analysis of variance (one-way ANOVA) followed by a Tukey's multiple comparison posthoc test, or a two-sample t-test, using p < 0.05.The statistical analysis related to each experiment was specified further in the figure texts.Origin data analysis software was used for the statistical analysis.

Figure 1 .
Figure 1.HepG2 cell aggregates: a-i) Schematic showing the alginate-coated well plates used for HepG2 cell aggregate formation.ii) Representative bright field images of HepG2 cell aggregates grown 24, 48, or 72 h starting from low, medium, or high seeding density (15 625, 31 250, or 62 500 cells per cm 2 ).Scale bars are 1 mm.b) The size distribution of the diameters of the cell aggregates (n = 2 with >39 cells each).A log-normal function is fitted to the size distribution, and the size is given as μ*/e 2 .c) The number of cells per aggregate was counted by staining the nuclei with Hoechst 33342.A cell aggregate is outlined in the insert and the counted nuclei are marked with crosses (n = 3 with >39 cells each).A log-normal function is fitted to the size distribution, and the size is given as μ*/e 2 .d) Representative CLSM image of cell aggregates stained with live/dead stain (green: Calcein-AM and red: EthD-I).Scale bars are 200 μm.Inset is 5× zoom.e-i) The CYP1A2 activity of HepG2 cells in aggregates and culture on 2D tissue culture polystyrene is monitored by detecting the conversion of REE to resorufin given as μ ± .The production of resorufin per 10 3 HepG2 cells, which are either untreated or BNF induced, is measured over 60 min ( ex/em = 570/585 nm).The statistical significance used to compare the means was determined using a one-way analysis of variance (one-way ANOVA) followed by Tukey's multiple comparisons post hoc test (*p < 0.05, n = 3).ii) The relative CYP1A2 mRNA expression of HepG2 cells in aggregates and culture on 2D tissue culture polystyrene was quantified by RT-qPCR and is given as μ ± .The expression is normalized to non-induced cells in 2D culture.The statistical significance used to compare the means was determined using a one-way analysis of variance (one-way ANOVA) followed by Tukey's multiple comparisons post-hoc test (*p < 0.05, n = 3).

Figure 2 .
Figure 2. Bioprinted HepG2 cell aggregates: a) Prints were made using HepG2 cell aggregates mixed with LPI 5 .The prints are visualized 1, 4, 7, 14, 21, 28, or 35 days after printing with bright field microscopy and representative images are shown.Scale bars are 1 mm.b-i) Representative CLSM images of prints of live/dead stained HepG2 cells (green: calcein-AM and red: EthD-I).The nuclei are stained with Hoechst 33342 (blue).Scale bar is 200 μm.ii) Representative CLSM image of a print made with GelMA Rho -containing (red) LPI 5 , and cells stained with Calcein-AM (green) and Hoechst 33342 (blue).Scale bar is 200 μm and the inset is 5× zoom.iii) 3D projection of z-stacks showing HepG2 cell aggregates growing out of the printed filaments.Scale bar is 50 μm.c) Number of cells in prints present at different incubation times based on dsDNA quantification is given as μ ±  (n = 3).d) CYP1A2 activity of HepG2 cells in prints 4 days after printing, monitored by the conversion of REE to resorufin over 2 h given as μ ± , comparing BNF-induced (48 h) cells to untreated cells (n = 3).

Figure 3 .
Figure 3. Composite Ink CInk k:m : a) Representative bright field images of AC 0 (left) and their size distribution (right).Scale bar is 500 μm.A Gaussian function is fitted to the histogram and the size is given as μ ± 2.960 particles were measured from four independent repeats.b) Schematic of the composite ink.c) Recovery graphs (1% strain for low periods and 500% strain for high periods) for the different compositions with storage modulus (G', dark blue) and loss modulus (G'', light blue).d-i) Representative bright field microscopy images of printed 1-, 2-, and 3-layered structures using CInk k:m .Scale bars are 5 mm.ii) Representative SEM images of the filaments printed using CInk k:m in one-layered structures.Scale bars are 200 μm.iii) Representative CLSM images (top) and 3D projections of z-stacks (bottom) of the filaments printed using CInk k:m containing GelMA Rho in one-layered structures.Scale bars are 200 μm.

Figure 4 .
Figure 4. Bioprinting of AC 0 and HepG2 cell aggregates.a) Two-layered prints were made using HepG2 cell aggregates in composite ink (Hep-CInk 1:2 ).Representative bright-field microscopy images of prints on different days for up to 35 days after printing.Scale bars are 1 mm.b-i) CLSM images of prints with live/dead stained HepG2 cells (green: calcein-AM and red: EthD-I).The nuclei were stained with Hoechst 33342 (blue).Scale bars are 200 μm.A close-up illustrating the HepG2 cells surrounding AC 0 s is shown for day 35.Scale bar is 50 μm.ii) Representative CLSM image of a print made with GelMA Rho -containing (red) CInk 1:2 , and cells stained with calcein-AM (green) and Hoechst 33342 (blue).The inset is 5× zoom and the scale bar is 200 μm.iii) 3D projection of z-stacks showing HepG2 cells in the printed filaments after 35 days (left) including a top view on a single plane in the middle of the filament (right).Scale bars are 100 μm.c) The number of cells present in the prints at different incubation times is based on dsDNA quantification and is given as μ ±  (n = 3).

Figure 5 .
Figure 5. a) Schematic of bioprints made with AC 0 and AC MP bioinks (Hep-CInk 1:3 and Hep-CInk MP , respectively).The conversion of REE to resorufin is catalyzed by CYP1A2 in HepG2 cell aggregates, and by MP-loaded liposomes in AC MP .b) Production of resorufin in the BNF-induced (top) or pristine (bottom) AC 0 and AC MP prints after 3, 6, or 8 h given as μ ± .The prints were treated with BNF to induce CYP1A2 expression.The resorufin production was monitored on prints that have been cultured for i) 4 or ii) 14 days after printing.The statistical significance used to compare the means was determined using a two-sample t-test (*p < 0.05, n = 3).
LIVE/DEAD Staining of Cell Aggregates: Cell aggregates were harvested from a T75 flask and washed 2× with PBS by centrifugation at 130 g for 7 min at 4 °C. 2 mL LIVE/DEAD staining solution (2 μM calcein-AM and 4 μM EthD-I staining solution in PBS) was added to the resulting cell pellet.The cells were re-suspended and transferred to a 35 mm confocal dish (VWR).After 45 min incubation at RT, images were taken using CLSM (Zeiss LSM 700, AxioObserver; EC Plan-Neofluar 20×/0.50M27) using  ex = 488 nm (calcein-AM) and  ex = 555 nm (EthD-I).