LIVER REGENERATION AND DRUG SCREENING
LIVER REGENERATION AND DRUG SCREENING
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Liver diseases have reached an all-time high limit by being ranked among the top 10 causes of death in the developing countries including India. Though liver has an innate ability to regenerate, the threshold limit to regenerate is breached when the damage to liver cells becomes greater than 65-70%. Liver transplantation an ultimate treatment faces the challenge of fewer donor availability than the recipients. Hence, recent developments are focussed to develop strategies to bridge the gap between transplantation, to generate a platform for effective drug screening and personalised medicine, and to develop regenerative therapeutics.
⚈ EXTRACORPOREAL BIOLOGICAL LIVER SUPPORT SYSTEMS FOR LIVER REGENERATION
This area focuses on the designing of a cryogel-based integrated bioartificial liver support to alleviate the conditions associated with liver failure. This is a single compact system integrating the detoxification property associated with the artificial liver devices and metabolism and biotransformation functions of the bioartificial liver devices into one single compact unit. It is a three-chambered device with first unit consisting of plasma separation unit and a subsequent activated charcoal cloth as a physical adsorbent to reduce the toxic load. The middle unit contains hepatocyte seeded cryogel disc while the lower unit contains a cellulose membrane filter. When the device was extra-corporeally connected to a rodent model of liver failure, it showed appx. 20-40% improvement in liver function parameters. Further, our objective is to improve the performance of the existing design to provide survival benefits during acute and acute-on-chronic liver failure conditions:
a) By integrating modifications in the detoxification unit for efficient removal of toxic substances present in the patient´s plasma, b) Tuning the properties of cryogel matrix supporting the cellular component for better functionality and incorporating a co-culture system, andc) Upscaling the designing parameters of the bioreactor
⚈ EXTRACORPOREAL BIOLOGICAL LIVER SUPPORT SYSTEMS FOR LIVER REGENERATION
This area focuses on the designing of a cryogel-based integrated bioartificial liver support to alleviate the conditions associated with liver failure. This is a single compact system integrating the detoxification property associated with the artificial liver devices and metabolism and biotransformation functions of the bioartificial liver devices into one single compact unit. It is a three-chambered device with first unit consisting of plasma separation unit and a subsequent activated charcoal cloth as a physical adsorbent to reduce the toxic load. The middle unit contains hepatocyte seeded cryogel disc while the lower unit contains a cellulose membrane filter. When the device was extra-corporeally connected to a rodent model of liver failure, it showed appx. 20-40% improvement in liver function parameters. Further, our objective is to improve the performance of the existing design to provide survival benefits during acute and acute-on-chronic liver failure conditions:
a) By integrating modifications in the detoxification unit for efficient removal of toxic substances present in the patient´s plasma, b) Tuning the properties of cryogel matrix supporting the cellular component for better functionality and incorporating a co-culture system, andc) Upscaling the designing parameters of the bioreactor
Kumar, A., & Damania, A. (2017). U.S. Patent Application No. 15/224,055.
⚈IN-VITRO HUMAN LIVER TISSUE 3-D MODELS AND IN-VIVO ECTOPIC MODELS FOR HIGH THROUGHPUT DRUG SCREENING AND ANALYSIS
This domain mainly targets on developing pre-clinical drug testing platform to assess toxicity and metabolism of drug. Recently developed biomimetic in-vitro systems offer advantages over conventional in-vivo models especially over non-human primate models. An optimized biomimetic system holds great potential for testing drug pharmacokinetics as it can minimize burden over higher animal models and can be cost effective as well. We mainly focus on:
This domain mainly targets on developing pre-clinical drug testing platform to assess toxicity and metabolism of drug. Recently developed biomimetic in-vitro systems offer advantages over conventional in-vivo models especially over non-human primate models. An optimized biomimetic system holds great potential for testing drug pharmacokinetics as it can minimize burden over higher animal models and can be cost effective as well. We mainly focus on:
1) Establishment of cryogel-based platform for screening of drugs and the optimization of bioprocess and chromatographic parameters. Polymeric materials in solution phase and as gel matrices are being exploited for developing the technology platforms in the form of multi-well plates and chromatographic columns. High swelling ratio, interconnected pores and tuneable properties of cryogels makes the platform leakage- and drainage- protected which allows them to be used for various applications.
2) Development of 3D human hepatocytes culture and hepatocytes co-culture with non-parenchymal cells. Here our team has combined the concept of a 3D scaffold and a spheroid culture to develop an in-vitro model to engineer liver tissue for drug screening. The proliferation and functionality of the liver cells was enhanced by culturing hepatic cells as spheroids (hepatospheres) on the poly(ethylene glycol)-alginate-gelatin (PAG) cryogel matrix using temperature-reversible soluble–insoluble polymer, poly(N-isopropylacrylamide) (poly-NIPAAm). This was also utilised to generate 3D co-cultures of parenchymal and non-parenchymal (fibroblast and endothelial) liver cells. These co-cultures showed an efficient homotypic as well as heterotypic interactions.
3) Establishment of humanized ectopic liver tissue in immune-compromised mice. In this, hepatitis C virus model was used to study its infection cycle through tissue engineering strategy. The 3D liver cell co-cultures utilising the PAG cryogels and poly-NIPAAM are subjected to the permissiveness of HCV-like particles and are shown to exhibit expression of HCV glycoproteins along with hepato-specific genes. This system was implanted in nude mice successfully to establish it as a successful small rodent model system as ectopic liver tissue, which can be exploited for HCV infection studies.
Further, we intend to optimize 3D human hepatocytes spheroid culture conditions for enhanced cell viability for longer periods and improved drug screening. Also, to generate a new viral (dengue) model system to be used in studies utilizing humanized ectopic liver approach.
■Sarkar, J., Kumari, J., Tonello, J.M., Kamihira, M. and Kumar, A., (2017). Enhanced hepatic functions of genetically modified mouse hepatoma cells by spheroid culture for drug toxicity screening. Biotechnology journal, 12(10), p.1700274.
Further, we intend to optimize 3D human hepatocytes spheroid culture conditions for enhanced cell viability for longer periods and improved drug screening. Also, to generate a new viral (dengue) model system to be used in studies utilizing humanized ectopic liver approach.
■Sarkar, J., Kumari, J., Tonello, J.M., Kamihira, M. and Kumar, A., (2017). Enhanced hepatic functions of genetically modified mouse hepatoma cells by spheroid culture for drug toxicity screening. Biotechnology journal, 12(10), p.1700274.
⚈ REGENERATIVE THERAPEUTICS USING EXTRACELLULAR MATRIX AND EXOSOMES
This area has targeted its interest towards tissue engineering strategies aimed at enhancing the rate of regeneration during a liver injury. Organ bioengineering and mesenchymal stem cell (MSCs) derived fractionated secretome are coming up as promising frontiers in regenerative therapeutics. Bioengineered organ employ the utilization of decellularization- recellularization of naturally occurring cadaveric human or animal liver organs. These matrices can serve as potential hepatocyte carriers in liver tissue engineering applications. Continuous integration of the cryogel matrix and cellular infiltration was obtained within a liver failure induced rodent model; 20-60% improvement in liver function parameters suggests its potential as an alternative of liver transplantation. The future scope is to develop an implantable liver construct using the decellularized matrix with minimal mechanical disintegration of the matrix to maintain the structural integrity of the system.On the other hand, MSCs are popular candidates in regenerative medicines. The use of MSC-derived exosomes can help in relieving progression towards liver cirrhotic conditions and can also inhibit cell apoptosis/death. The liver regeneration rate increased by ~90% in the presence of exosomes as compared to 60% in the control liver failure models, as the exosomes showed a cryoprotective as well as anti-oxidative effect. In future, we intend to synthesize liver-targeted exosomes to improve the uptake efficiency by modifying them using galactosylated ligands and to assess the liver regeneration rate during the different liver fibrotic stages in the presence of exosomes.
This area has targeted its interest towards tissue engineering strategies aimed at enhancing the rate of regeneration during a liver injury. Organ bioengineering and mesenchymal stem cell (MSCs) derived fractionated secretome are coming up as promising frontiers in regenerative therapeutics. Bioengineered organ employ the utilization of decellularization- recellularization of naturally occurring cadaveric human or animal liver organs. These matrices can serve as potential hepatocyte carriers in liver tissue engineering applications. Continuous integration of the cryogel matrix and cellular infiltration was obtained within a liver failure induced rodent model; 20-60% improvement in liver function parameters suggests its potential as an alternative of liver transplantation. The future scope is to develop an implantable liver construct using the decellularized matrix with minimal mechanical disintegration of the matrix to maintain the structural integrity of the system.On the other hand, MSCs are popular candidates in regenerative medicines. The use of MSC-derived exosomes can help in relieving progression towards liver cirrhotic conditions and can also inhibit cell apoptosis/death. The liver regeneration rate increased by ~90% in the presence of exosomes as compared to 60% in the control liver failure models, as the exosomes showed a cryoprotective as well as anti-oxidative effect. In future, we intend to synthesize liver-targeted exosomes to improve the uptake efficiency by modifying them using galactosylated ligands and to assess the liver regeneration rate during the different liver fibrotic stages in the presence of exosomes.
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