Periodic Reporting for period 4 - ArtHep (Hepatocytes-Like Microreactors for Liver Tissue Engineering)
Periodo di rendicontazione: 2023-11-01 al 2025-04-30
ArtHep is to assemble hepatic-like tissue, consisting of biological and synthetic entities, mimicking the core structure elements and key functions of the liver as an alternative in tissue engineering. This project aims to help to bridge between the low number of organ donors and the large number patients with liver failure, which possess an increasingly pressing challenge for the society.
The overall objectives are the engineering of enzyme-mimics, which can perform core biocatalytic conversions similar to the liver; the assembly of subunits either carrying the enzyme-mimics or exhibiting inherent biocatalytic activity, the design of cell-like assemblies (microreactors) and their integration with living mammalian cells into 3D liver-like tissue.
The overall objectives are the engineering of enzyme-mimics, which can perform core biocatalytic conversions similar to the liver; the assembly of subunits either carrying the enzyme-mimics or exhibiting inherent biocatalytic activity, the design of cell-like assemblies (microreactors) and their integration with living mammalian cells into 3D liver-like tissue.
ArtHep aimed at the bottom-up assembly of artificial cells with hepatic-like structural and functional elements as well as their interaction with mammalian cells. During the ArtHep project, we published two review articles that not only provided a coherent summary of recent developments in the field, but also offered our perspective on future directions.1 2
We considered the synthesis of liver relevant catalysts with focus on antioxidant properties and CYP450-imitating molecules. We synthesized derivatives of the salen manganese complex EUK as superoxide dismutase- and catalase-like artificial enzymes that were either conjugated to lipids3 as well as integrated in either subunits4 or artificial cells as support against reactive oxygen species (ROS)5. Complementary, we synthesized metalloporphyrins that mimicked the CYP450 activity and employed these catalysts in subunits and in artificial cells.6
Hybrid vesicles made of amphiphilic block copolymers and lipids were considered as scaffold for either subunits or artificial cells due to their unique and tunable features as outlined in our tutorial review.7 We explored a variety of hydrophobic8-10 and hydrophilic blocks11 with the aim of creating homogeneous hybrid membranes that mimic features of mammalian cells or support the assembly of cytoskeleton-like networks,12 providing an alternative to hydrogel-based artificial cells.
ATP is an essential molecule in living cells. Therefore, we successfully equipped hydrogel-based artificial cells with mitochondria purified from hepatocytes that produced ATP for up to 6 h to obtain minimal systems that might with due development allow for the investigation of complex biological questions in controlled environments.13
The successful integration of hydrogel-based artificial cells with hepatocytes is primarily driven by the surface coating of the artificial cells as long as their mechanical properties are within the range of liver tissue. We demonstrated that poly(L lysine) coatings allowed for integration.14 However, we proposed cell membrane vesicles as the nature-like approach for this purpose as summarized in our review.15 Interestingly, cell membrane vesicle cloaked artificial cells not only showed improved integration but also enhanced proliferation of the mammalian cells.16 Particularly important is the observation that the extracellular support of artificial cells equipped with EUK improved the viability of the hepatocytes as well as allowed them to preserve intracellular homeostasis.
Using 3D bioprinting, we fabricated cm-scale semi-synthetic liver like tissue with augmented CYP450 activity using a composite ink of artificial cells and mammalian cells in a liquid phase.17 In parallel, we explored the use of granular hydrogel ink with the aim to increase the number of artificial cells in the semi-synthetic tissue.18 Re-designing of artificial cells to produce NO19 was the first step within ArtHep to implement a pre-vasculature system in the semi-synthetic tissue where the endothelial cells will be supported in addition to the hepatocytes.
Published articles during ArtHep:
1. Qian, X., et al., Cell mimicry as a bottom-up strategy for hierarchical engineering of nature-inspired entities. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol., 2021. 13, e1683
2. Westensee, I.N. P. de Dios Andres, and B. Städler, From Single-Compartment Artificial Cells to Tissue-Like Materials. Adv. Mater. Technol., 2024. 9, 2301804
3. Westensee, I.N. et al., Engineered Lipids for Intracellular Reactive Oxygen Species Scavenging in Steatotic Hepatocytes. Small, 2024. 20, 2400816
4. Ade, C., et al., Polymer Micelles vs Polymer–Lipid Hybrid Vesicles: A Comparison Using RAW 264.7 Cells. Biomacromolecules, 2022. 23, 1052
5. Westensee, I.N. et al., Antioxidant Microgels Support Peroxide-Challenged Hepatic Cells. Adv. Biol., 2024. 8, 2300547
6. Qian, X., et al., Enzyme Mimic Facilitated Artificial Cell to Mammalian Cell Signal Transfer. Angew. Chem. Int. Ed., 2021. 60, 18704
7. Brodszkij, E. and B. Städler, Advances in block copolymer-phospholipid hybrid vesicles: from physical–chemical properties to applications. Chem. Sci., 2024. 15, 10724
8. Abild Meyer, C., et al., Astrocytes in Paper Chips and Their Interaction with Hybrid Vesicles. Adv. Biol., 2023. 7, 2200209
9. Brodszkij, E., et al., Poly(Sitosterol)-Based Hydrophobic Blocks in Amphiphilic Block Copolymers for the Assembly of Hybrid Vesicles. Small, 2024. 20, 2401934
10. Brodszkij, E., et al., Membrane composition of polymer-lipid hybrid vesicles. Appl. Mater. Today, 2022. 29, 101549
11. Spanjers, J.M. et al., On the assembly of zwitterionic block copolymers with phospholipids. Eur. Polym. J., 2022. 180, 111612
12. De Dios Andres, P., et al., Distinct Network Morphologies from In Situ Polymerization of Microtubules in Giant Polymer-Lipid Hybrid Vesicles. Adv. Biol., 2025. 9, 2400601
13. Westensee, I.N. et al., Mitochondria Encapsulation in Hydrogel-Based Artificial Cells as ATP Producing Subunits. Small, 2021. 17, 2007959
14. Westensee, I.N. and B. Städler, Artificial cells eavesdropping on HepG2 cells. Interface Focus, 2023. 13, 20230007
15. Spanjers, J.M. and B. Städler, Cell Membrane Coated Particles. Adv. Biosyst., 2020. 4, 2000174
16. De Dios Andres, P., et al., Cell Membrane Vesicle Camouflaged Artificial Cells. Adv. Funct. Mater. n/a, 2424504
17. Westensee, I.N. et al., Artificial Cells and HepG2 Cells in 3D-Bioprinted Arrangements. Adv. Healthc. Mater., 2024. 13, 2303699
18. Pendlmayr, S., et al., Light-activated 3D printed fish-like actuator. J. Appl. Polym. Sci., 2024. 141, e55746
19. Qian, X., et al., Nitric oxide producing artificial enzymes based on metalloporphyrins. Mater. Today Chem., 2022. 23, 100743
We considered the synthesis of liver relevant catalysts with focus on antioxidant properties and CYP450-imitating molecules. We synthesized derivatives of the salen manganese complex EUK as superoxide dismutase- and catalase-like artificial enzymes that were either conjugated to lipids3 as well as integrated in either subunits4 or artificial cells as support against reactive oxygen species (ROS)5. Complementary, we synthesized metalloporphyrins that mimicked the CYP450 activity and employed these catalysts in subunits and in artificial cells.6
Hybrid vesicles made of amphiphilic block copolymers and lipids were considered as scaffold for either subunits or artificial cells due to their unique and tunable features as outlined in our tutorial review.7 We explored a variety of hydrophobic8-10 and hydrophilic blocks11 with the aim of creating homogeneous hybrid membranes that mimic features of mammalian cells or support the assembly of cytoskeleton-like networks,12 providing an alternative to hydrogel-based artificial cells.
ATP is an essential molecule in living cells. Therefore, we successfully equipped hydrogel-based artificial cells with mitochondria purified from hepatocytes that produced ATP for up to 6 h to obtain minimal systems that might with due development allow for the investigation of complex biological questions in controlled environments.13
The successful integration of hydrogel-based artificial cells with hepatocytes is primarily driven by the surface coating of the artificial cells as long as their mechanical properties are within the range of liver tissue. We demonstrated that poly(L lysine) coatings allowed for integration.14 However, we proposed cell membrane vesicles as the nature-like approach for this purpose as summarized in our review.15 Interestingly, cell membrane vesicle cloaked artificial cells not only showed improved integration but also enhanced proliferation of the mammalian cells.16 Particularly important is the observation that the extracellular support of artificial cells equipped with EUK improved the viability of the hepatocytes as well as allowed them to preserve intracellular homeostasis.
Using 3D bioprinting, we fabricated cm-scale semi-synthetic liver like tissue with augmented CYP450 activity using a composite ink of artificial cells and mammalian cells in a liquid phase.17 In parallel, we explored the use of granular hydrogel ink with the aim to increase the number of artificial cells in the semi-synthetic tissue.18 Re-designing of artificial cells to produce NO19 was the first step within ArtHep to implement a pre-vasculature system in the semi-synthetic tissue where the endothelial cells will be supported in addition to the hepatocytes.
Published articles during ArtHep:
1. Qian, X., et al., Cell mimicry as a bottom-up strategy for hierarchical engineering of nature-inspired entities. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol., 2021. 13, e1683
2. Westensee, I.N. P. de Dios Andres, and B. Städler, From Single-Compartment Artificial Cells to Tissue-Like Materials. Adv. Mater. Technol., 2024. 9, 2301804
3. Westensee, I.N. et al., Engineered Lipids for Intracellular Reactive Oxygen Species Scavenging in Steatotic Hepatocytes. Small, 2024. 20, 2400816
4. Ade, C., et al., Polymer Micelles vs Polymer–Lipid Hybrid Vesicles: A Comparison Using RAW 264.7 Cells. Biomacromolecules, 2022. 23, 1052
5. Westensee, I.N. et al., Antioxidant Microgels Support Peroxide-Challenged Hepatic Cells. Adv. Biol., 2024. 8, 2300547
6. Qian, X., et al., Enzyme Mimic Facilitated Artificial Cell to Mammalian Cell Signal Transfer. Angew. Chem. Int. Ed., 2021. 60, 18704
7. Brodszkij, E. and B. Städler, Advances in block copolymer-phospholipid hybrid vesicles: from physical–chemical properties to applications. Chem. Sci., 2024. 15, 10724
8. Abild Meyer, C., et al., Astrocytes in Paper Chips and Their Interaction with Hybrid Vesicles. Adv. Biol., 2023. 7, 2200209
9. Brodszkij, E., et al., Poly(Sitosterol)-Based Hydrophobic Blocks in Amphiphilic Block Copolymers for the Assembly of Hybrid Vesicles. Small, 2024. 20, 2401934
10. Brodszkij, E., et al., Membrane composition of polymer-lipid hybrid vesicles. Appl. Mater. Today, 2022. 29, 101549
11. Spanjers, J.M. et al., On the assembly of zwitterionic block copolymers with phospholipids. Eur. Polym. J., 2022. 180, 111612
12. De Dios Andres, P., et al., Distinct Network Morphologies from In Situ Polymerization of Microtubules in Giant Polymer-Lipid Hybrid Vesicles. Adv. Biol., 2025. 9, 2400601
13. Westensee, I.N. et al., Mitochondria Encapsulation in Hydrogel-Based Artificial Cells as ATP Producing Subunits. Small, 2021. 17, 2007959
14. Westensee, I.N. and B. Städler, Artificial cells eavesdropping on HepG2 cells. Interface Focus, 2023. 13, 20230007
15. Spanjers, J.M. and B. Städler, Cell Membrane Coated Particles. Adv. Biosyst., 2020. 4, 2000174
16. De Dios Andres, P., et al., Cell Membrane Vesicle Camouflaged Artificial Cells. Adv. Funct. Mater. n/a, 2424504
17. Westensee, I.N. et al., Artificial Cells and HepG2 Cells in 3D-Bioprinted Arrangements. Adv. Healthc. Mater., 2024. 13, 2303699
18. Pendlmayr, S., et al., Light-activated 3D printed fish-like actuator. J. Appl. Polym. Sci., 2024. 141, e55746
19. Qian, X., et al., Nitric oxide producing artificial enzymes based on metalloporphyrins. Mater. Today Chem., 2022. 23, 100743
The catalytic subunits as well as the surface modification goes well beyond the state of the art in bottom-up synthetic biology using synthetic tools to facilitate integration with the natural world. Hybrid systems between artificial cells and mammalian cells only start to emerge, and our level of integration with bio-related function is cutting edge. The creation of centimeter-sized viable semi-synthetic tissue using 3D bioprinting that remained functional for at least 28 days, was a significant achievement in itself. However, what was truly unexpected was how closely these large constructs replicated the performance and physiological state of living tissue, outperforming both 2D cell cultures and cell aggregates.