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resulting in stabilized liver functions for several weeks in vitro. Studies have focused on using these models to investigate cell responses to drugs and other stimuli (for example, viruses and cell differentiation cues) to predict clinical outcomes. Gregory H. Underhill, PhD, from the department of bioengineering at the University of Illinois at Urbana-Champaign and Salman R. Khetani, PhD, from the department of bioengineering at the University of Illinois in Chicago presented a comprehensive review of the these advances in bioengineered liver models in Cellular and Molecular Gastroenterology and Hepatology (doi: 10.1016/j.jcmgh.2017.11.012).
Drug-induced liver injury (DILI) is a leading cause of drug attrition in the United States, with some marketed drugs causing cell necrosis, hepatitis, cholestasis, fibrosis, or a mixture of injury types. Although the Food and Drug Administration requires preclinical drug testing in animal models, differences in species-specific drug metabolism pathways and human genetics may result in inadequate identification of potential for human DILI. Some bioengineered liver models for in vitro studies are based on tissue engineering using high-throughput microarrays, protein micropatterning, microfluidics, specialized plates, biomaterial scaffolds, and bioprinting.
High-throughput cell microarrays enable systematic analysis of a large number of drugs or compounds at a relatively low cost. Several culture platforms have been developed using multiple sources of liver cells, including cancerous and immortalized cell lines. These platforms show enhanced capabilities to evaluate combinatorial effects of multiple signals with independent control of biochemical and biomechanical cues. For instance, a microchip platform for transducing 3-D liver cell cultures with genes for drug metabolism enzymes featuring 532 reaction vessels (micropillars and corresponding microwells) was able to provide information about certain enzyme combinations that led to drug toxicity in cells. The high-throughput cell microarrays are, however, primarily dependent on imaging-based readouts and have a limited ability to investigate cell responses to gradients of microenvironmental signals.
Liver development, physiology, and pathophysiology are dependent on homotypic and heterotypic interactions between parenchymal and nonparenchymal cells (NPCs). Cocultures with both liver- and nonliver-derived NPC types, in vitro, can induce liver functions transiently and have proven useful for investigating host responses to sepsis, mutagenesis, xenobiotic metabolism and toxicity, response to oxidative stress, lipid metabolism, and induction of the acute-phase response. Micropatterned cocultures (MPCCs) are designed to allow the use of different NPC types without significantly altering hepatocyte homotypic interactions. Cell-cell interactions can be precisely controlled to allow for stable functions for up to 4-6 weeks, whereas more randomly distributed cocultures have limited stability. Unlike randomly distributed cocultures, MPCCs can be infected with HBV, HCV, and malaria. Potential limitations of MPCCs include the requirement for specialized equipment and devices for patterning collagen for hepatocyte attachment.
Randomly distributed spheroids or organoids enable 3-D establishment of homotypic cell-cell interactions surrounded by an extracellular matrix. The spheroids can be further cocultured with NPCs that facilitate heterotypic cell-cell interactions and allow the evaluation of outcomes resulting from drugs and other stimuli. Hepatic spheroids maintain major liver functions for several weeks and have proven to be compatible with multiple applications within the drug development pipeline.
These spheroids showed greater sensitivity in identifying known hepatotoxic drugs than did short-term primary human hepatocyte (PHH) monolayers. PHHs secreted liver proteins, such as albumin, transferrin, and fibrinogen, and showed cytochrome-P450 activities for 77-90 days when cultured on a nylon scaffold containing a mixture of liver NPCs and PHHs.
Nanopillar plates can be used to create induced pluripotent stem cell–derived human hepatocyte-like cell (iHep) spheroids; although these spheroids showed some potential for initial drug toxicity screening, they had lower overall sensitivity than conventional PHH monolayers, which suggests that further maturation of iHeps is likely required.
Potential limitations of randomly distributed spheroids include necrosis of cells in the center of larger spheroids and the requirement for expensive confocal microscopy for high-content imaging of entire spheroid cultures. To overcome the limitation of disorganized cell type interactions over time within the randomly distributed spheroids/organoids, bioprinted human liver organoids are designed to allow precise control of cell placement.
Yet another bioengineered liver model is based on perfusion systems or bioreactors that enable dynamic fluid flow for nutrient and waste exchange. These so called liver-on-a-chip devices contain hepatocyte aggregates adhered to collagen-coated microchannel walls; these are then perfused at optimal flow rates both to meet the oxygen demands of the hepatocytes and deliver low shear stress to the cells that’s similar to what would be the case in vivo. Layered architectures can be created with single-chamber or multichamber, microfluidic device designs that can sustain cell functionality for 2-4 weeks.
Some of the limitations of perfusion systems include the potential binding of drugs to tubing and materials used, large dead volume requiring higher quantities of novel compounds for the treatment of cell cultures, low throughput, and washing away of built-up beneficial molecules with perfusion.
The ongoing development of more sophisticated engineering tools for manipulating cells in culture will lead to continued advances in bioengineered livers that will show improving sensitivity for the prediction of clinically relevant drug and disease outcomes.
This work was funded by National Institutes of Health grants. The author Dr. Khetani disclosed a conflict of interest with Ascendance Biotechnology, which has licensed the micropatterned coculture and related systems from Massachusetts Institute of Technology, Cambridge, and Colorado State University, Fort Collins, for commercial distribution. Dr. Underhill disclosed no conflicts.
SOURCE: Underhill GH and Khetani SR. Cell Molec Gastro Hepatol. 2017. doi: org/10.1016/j.jcmgh.2017.11.012.
Thirty to 50 new drugs are approved in the United States annually, which costs approximately $2.5 billion/drug in drug development costs. Nine out of 10 drugs never make it to market, and of those that do, adverse events affect their longevity. Hepatotoxicity is the most frequent adverse drug reaction, and drug-induced liver injury, which can lead to acute liver failure, occurs in a subset of affected patients. Understanding a drug’s risk of hepatotoxicity before patients start using it can not only save lives but also conceivably reduce the costs incurred by pharmaceutical companies, which are passed on to consumers.
However, just as we have seen with the limitations of the in vitro systems, bioartificial livers are unlikely to be successful unless they integrate the liver’s complex functions of protein synthesis, immune surveillance, energy homeostasis, and nutrient sensing. The future is bright, though, as biomedical scientists and bioengineers continue to push the envelope by advancing both in vitro and bioartificial technologies.
Rotonya Carr, MD, is an assistant professor of medicine in the division of gastroenterology at the University of Pennsylvania, Philadelphia. She receives research support from Intercept Pharmaceuticals.
Thirty to 50 new drugs are approved in the United States annually, which costs approximately $2.5 billion/drug in drug development costs. Nine out of 10 drugs never make it to market, and of those that do, adverse events affect their longevity. Hepatotoxicity is the most frequent adverse drug reaction, and drug-induced liver injury, which can lead to acute liver failure, occurs in a subset of affected patients. Understanding a drug’s risk of hepatotoxicity before patients start using it can not only save lives but also conceivably reduce the costs incurred by pharmaceutical companies, which are passed on to consumers.
However, just as we have seen with the limitations of the in vitro systems, bioartificial livers are unlikely to be successful unless they integrate the liver’s complex functions of protein synthesis, immune surveillance, energy homeostasis, and nutrient sensing. The future is bright, though, as biomedical scientists and bioengineers continue to push the envelope by advancing both in vitro and bioartificial technologies.
Rotonya Carr, MD, is an assistant professor of medicine in the division of gastroenterology at the University of Pennsylvania, Philadelphia. She receives research support from Intercept Pharmaceuticals.
Thirty to 50 new drugs are approved in the United States annually, which costs approximately $2.5 billion/drug in drug development costs. Nine out of 10 drugs never make it to market, and of those that do, adverse events affect their longevity. Hepatotoxicity is the most frequent adverse drug reaction, and drug-induced liver injury, which can lead to acute liver failure, occurs in a subset of affected patients. Understanding a drug’s risk of hepatotoxicity before patients start using it can not only save lives but also conceivably reduce the costs incurred by pharmaceutical companies, which are passed on to consumers.
However, just as we have seen with the limitations of the in vitro systems, bioartificial livers are unlikely to be successful unless they integrate the liver’s complex functions of protein synthesis, immune surveillance, energy homeostasis, and nutrient sensing. The future is bright, though, as biomedical scientists and bioengineers continue to push the envelope by advancing both in vitro and bioartificial technologies.
Rotonya Carr, MD, is an assistant professor of medicine in the division of gastroenterology at the University of Pennsylvania, Philadelphia. She receives research support from Intercept Pharmaceuticals.
resulting in stabilized liver functions for several weeks in vitro. Studies have focused on using these models to investigate cell responses to drugs and other stimuli (for example, viruses and cell differentiation cues) to predict clinical outcomes. Gregory H. Underhill, PhD, from the department of bioengineering at the University of Illinois at Urbana-Champaign and Salman R. Khetani, PhD, from the department of bioengineering at the University of Illinois in Chicago presented a comprehensive review of the these advances in bioengineered liver models in Cellular and Molecular Gastroenterology and Hepatology (doi: 10.1016/j.jcmgh.2017.11.012).
Drug-induced liver injury (DILI) is a leading cause of drug attrition in the United States, with some marketed drugs causing cell necrosis, hepatitis, cholestasis, fibrosis, or a mixture of injury types. Although the Food and Drug Administration requires preclinical drug testing in animal models, differences in species-specific drug metabolism pathways and human genetics may result in inadequate identification of potential for human DILI. Some bioengineered liver models for in vitro studies are based on tissue engineering using high-throughput microarrays, protein micropatterning, microfluidics, specialized plates, biomaterial scaffolds, and bioprinting.
High-throughput cell microarrays enable systematic analysis of a large number of drugs or compounds at a relatively low cost. Several culture platforms have been developed using multiple sources of liver cells, including cancerous and immortalized cell lines. These platforms show enhanced capabilities to evaluate combinatorial effects of multiple signals with independent control of biochemical and biomechanical cues. For instance, a microchip platform for transducing 3-D liver cell cultures with genes for drug metabolism enzymes featuring 532 reaction vessels (micropillars and corresponding microwells) was able to provide information about certain enzyme combinations that led to drug toxicity in cells. The high-throughput cell microarrays are, however, primarily dependent on imaging-based readouts and have a limited ability to investigate cell responses to gradients of microenvironmental signals.
Liver development, physiology, and pathophysiology are dependent on homotypic and heterotypic interactions between parenchymal and nonparenchymal cells (NPCs). Cocultures with both liver- and nonliver-derived NPC types, in vitro, can induce liver functions transiently and have proven useful for investigating host responses to sepsis, mutagenesis, xenobiotic metabolism and toxicity, response to oxidative stress, lipid metabolism, and induction of the acute-phase response. Micropatterned cocultures (MPCCs) are designed to allow the use of different NPC types without significantly altering hepatocyte homotypic interactions. Cell-cell interactions can be precisely controlled to allow for stable functions for up to 4-6 weeks, whereas more randomly distributed cocultures have limited stability. Unlike randomly distributed cocultures, MPCCs can be infected with HBV, HCV, and malaria. Potential limitations of MPCCs include the requirement for specialized equipment and devices for patterning collagen for hepatocyte attachment.
Randomly distributed spheroids or organoids enable 3-D establishment of homotypic cell-cell interactions surrounded by an extracellular matrix. The spheroids can be further cocultured with NPCs that facilitate heterotypic cell-cell interactions and allow the evaluation of outcomes resulting from drugs and other stimuli. Hepatic spheroids maintain major liver functions for several weeks and have proven to be compatible with multiple applications within the drug development pipeline.
These spheroids showed greater sensitivity in identifying known hepatotoxic drugs than did short-term primary human hepatocyte (PHH) monolayers. PHHs secreted liver proteins, such as albumin, transferrin, and fibrinogen, and showed cytochrome-P450 activities for 77-90 days when cultured on a nylon scaffold containing a mixture of liver NPCs and PHHs.
Nanopillar plates can be used to create induced pluripotent stem cell–derived human hepatocyte-like cell (iHep) spheroids; although these spheroids showed some potential for initial drug toxicity screening, they had lower overall sensitivity than conventional PHH monolayers, which suggests that further maturation of iHeps is likely required.
Potential limitations of randomly distributed spheroids include necrosis of cells in the center of larger spheroids and the requirement for expensive confocal microscopy for high-content imaging of entire spheroid cultures. To overcome the limitation of disorganized cell type interactions over time within the randomly distributed spheroids/organoids, bioprinted human liver organoids are designed to allow precise control of cell placement.
Yet another bioengineered liver model is based on perfusion systems or bioreactors that enable dynamic fluid flow for nutrient and waste exchange. These so called liver-on-a-chip devices contain hepatocyte aggregates adhered to collagen-coated microchannel walls; these are then perfused at optimal flow rates both to meet the oxygen demands of the hepatocytes and deliver low shear stress to the cells that’s similar to what would be the case in vivo. Layered architectures can be created with single-chamber or multichamber, microfluidic device designs that can sustain cell functionality for 2-4 weeks.
Some of the limitations of perfusion systems include the potential binding of drugs to tubing and materials used, large dead volume requiring higher quantities of novel compounds for the treatment of cell cultures, low throughput, and washing away of built-up beneficial molecules with perfusion.
The ongoing development of more sophisticated engineering tools for manipulating cells in culture will lead to continued advances in bioengineered livers that will show improving sensitivity for the prediction of clinically relevant drug and disease outcomes.
This work was funded by National Institutes of Health grants. The author Dr. Khetani disclosed a conflict of interest with Ascendance Biotechnology, which has licensed the micropatterned coculture and related systems from Massachusetts Institute of Technology, Cambridge, and Colorado State University, Fort Collins, for commercial distribution. Dr. Underhill disclosed no conflicts.
SOURCE: Underhill GH and Khetani SR. Cell Molec Gastro Hepatol. 2017. doi: org/10.1016/j.jcmgh.2017.11.012.
resulting in stabilized liver functions for several weeks in vitro. Studies have focused on using these models to investigate cell responses to drugs and other stimuli (for example, viruses and cell differentiation cues) to predict clinical outcomes. Gregory H. Underhill, PhD, from the department of bioengineering at the University of Illinois at Urbana-Champaign and Salman R. Khetani, PhD, from the department of bioengineering at the University of Illinois in Chicago presented a comprehensive review of the these advances in bioengineered liver models in Cellular and Molecular Gastroenterology and Hepatology (doi: 10.1016/j.jcmgh.2017.11.012).
Drug-induced liver injury (DILI) is a leading cause of drug attrition in the United States, with some marketed drugs causing cell necrosis, hepatitis, cholestasis, fibrosis, or a mixture of injury types. Although the Food and Drug Administration requires preclinical drug testing in animal models, differences in species-specific drug metabolism pathways and human genetics may result in inadequate identification of potential for human DILI. Some bioengineered liver models for in vitro studies are based on tissue engineering using high-throughput microarrays, protein micropatterning, microfluidics, specialized plates, biomaterial scaffolds, and bioprinting.
High-throughput cell microarrays enable systematic analysis of a large number of drugs or compounds at a relatively low cost. Several culture platforms have been developed using multiple sources of liver cells, including cancerous and immortalized cell lines. These platforms show enhanced capabilities to evaluate combinatorial effects of multiple signals with independent control of biochemical and biomechanical cues. For instance, a microchip platform for transducing 3-D liver cell cultures with genes for drug metabolism enzymes featuring 532 reaction vessels (micropillars and corresponding microwells) was able to provide information about certain enzyme combinations that led to drug toxicity in cells. The high-throughput cell microarrays are, however, primarily dependent on imaging-based readouts and have a limited ability to investigate cell responses to gradients of microenvironmental signals.
Liver development, physiology, and pathophysiology are dependent on homotypic and heterotypic interactions between parenchymal and nonparenchymal cells (NPCs). Cocultures with both liver- and nonliver-derived NPC types, in vitro, can induce liver functions transiently and have proven useful for investigating host responses to sepsis, mutagenesis, xenobiotic metabolism and toxicity, response to oxidative stress, lipid metabolism, and induction of the acute-phase response. Micropatterned cocultures (MPCCs) are designed to allow the use of different NPC types without significantly altering hepatocyte homotypic interactions. Cell-cell interactions can be precisely controlled to allow for stable functions for up to 4-6 weeks, whereas more randomly distributed cocultures have limited stability. Unlike randomly distributed cocultures, MPCCs can be infected with HBV, HCV, and malaria. Potential limitations of MPCCs include the requirement for specialized equipment and devices for patterning collagen for hepatocyte attachment.
Randomly distributed spheroids or organoids enable 3-D establishment of homotypic cell-cell interactions surrounded by an extracellular matrix. The spheroids can be further cocultured with NPCs that facilitate heterotypic cell-cell interactions and allow the evaluation of outcomes resulting from drugs and other stimuli. Hepatic spheroids maintain major liver functions for several weeks and have proven to be compatible with multiple applications within the drug development pipeline.
These spheroids showed greater sensitivity in identifying known hepatotoxic drugs than did short-term primary human hepatocyte (PHH) monolayers. PHHs secreted liver proteins, such as albumin, transferrin, and fibrinogen, and showed cytochrome-P450 activities for 77-90 days when cultured on a nylon scaffold containing a mixture of liver NPCs and PHHs.
Nanopillar plates can be used to create induced pluripotent stem cell–derived human hepatocyte-like cell (iHep) spheroids; although these spheroids showed some potential for initial drug toxicity screening, they had lower overall sensitivity than conventional PHH monolayers, which suggests that further maturation of iHeps is likely required.
Potential limitations of randomly distributed spheroids include necrosis of cells in the center of larger spheroids and the requirement for expensive confocal microscopy for high-content imaging of entire spheroid cultures. To overcome the limitation of disorganized cell type interactions over time within the randomly distributed spheroids/organoids, bioprinted human liver organoids are designed to allow precise control of cell placement.
Yet another bioengineered liver model is based on perfusion systems or bioreactors that enable dynamic fluid flow for nutrient and waste exchange. These so called liver-on-a-chip devices contain hepatocyte aggregates adhered to collagen-coated microchannel walls; these are then perfused at optimal flow rates both to meet the oxygen demands of the hepatocytes and deliver low shear stress to the cells that’s similar to what would be the case in vivo. Layered architectures can be created with single-chamber or multichamber, microfluidic device designs that can sustain cell functionality for 2-4 weeks.
Some of the limitations of perfusion systems include the potential binding of drugs to tubing and materials used, large dead volume requiring higher quantities of novel compounds for the treatment of cell cultures, low throughput, and washing away of built-up beneficial molecules with perfusion.
The ongoing development of more sophisticated engineering tools for manipulating cells in culture will lead to continued advances in bioengineered livers that will show improving sensitivity for the prediction of clinically relevant drug and disease outcomes.
This work was funded by National Institutes of Health grants. The author Dr. Khetani disclosed a conflict of interest with Ascendance Biotechnology, which has licensed the micropatterned coculture and related systems from Massachusetts Institute of Technology, Cambridge, and Colorado State University, Fort Collins, for commercial distribution. Dr. Underhill disclosed no conflicts.
SOURCE: Underhill GH and Khetani SR. Cell Molec Gastro Hepatol. 2017. doi: org/10.1016/j.jcmgh.2017.11.012.
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