Unveiling the Power of Hepatocytes: The Cornerstone of Liver Research and Innova

1. Unveiling the Power of Hepatocytes: The Cornerstone of Liver Research and Innaovation Liver is largest gland of the body. Hepatocytes are primary functional cells of liver, constitute approximately 70% of liver’s mass. Hepatocytes play a crucial role in metabolism (carbohydrate, lipid, drug) detoxification and albumin secretion, clotting factors and complements. Hepatocytes are widely used to study liver diseases, metabolism and drug safety assessment. Hepatocytes are either primary cells isolated from tissue biopsies, cell lines or can be derived from Induced Pluripotent Stem Cells (iPSCs). Figure 1: Illustration of different hepatic lines used for in vitro studies. Image resource: PMID: 32106230

2. Primary Hepatocytes Primary hepatocytes are directly isolated from liver tissue biopsies or cadaver, either human or animals. Primary hepatocytes are gold standard for drug metabolism drug-drug interaction, enzyme activity for CYP3A4, CYP2B6, and CYP1A2, hepatotoxicity studies etc. Hepatotoxicity is one of the major challenges during drug development. Primary hepatocytes closely mimic in vivo microenvironment of liver cells in terms of functionality. Isolation of primary hepatocytes from liver, maintenance and expansion are challenging. Recovery after cryopreservation usually results in low cell count. Utility of primary hepatocytes is limited by its low proliferative capacity and lose of liver-specific functions in vitro conditions and donor-to donor variability. Primary hepatocytes are amenable to high-throughput drug screening and toxicity assays. Figure 2: Schematic representation of isolation of hepatocytes from human liver biopsy. Image resource: PMID: 26381499 Hepatic Cell lines Hepatic Cell lines are immortalized cells that can proliferate indefinitely in vitro conditions, providing unlimited resource of hepatocytes. Hepatic cell lines are easy-to culture. Although limited by altered phenotype, genetic expression and liver-specific functions on prolonged culture. Hepatic cell lines are amenable to high-throughput drug screening and toxicity assays. Commonly used hepatic cell lines are: HepG2: HepG2 is an immortal hepatic cell line, derived in 1975 from a Caucasian male with a well-differentiated hepatocellular carcinoma. Huh7: Huh7 is an immortal hepatic cell line, derived in 1982 from hepatoma tissue of Japanese male. Huh7 cell line is widely used in hepatitis C research. Hep3B: Hep3B cell line is derived from an 8-year-old child with liver cancer. Hep3B is commonly used for studying hepatocarcinoma and Hepatitis B virus. Hep3B is hepatitis B virus positive and tumorigenic.

3. HepaRG: HepaRG cell line is an immortalized hepatic cell line that retains many characteristics of primary human hepatocytes. HepaRG cell line is derived from a female with hepatocarcinoma and chronic hepatitis C infection. HepaRG cells express multiple phase 1 and Phase 2 drug metabolizing enzymes and nuclear receptor at same level as of primary hepatocytes. HepaRG is suitable for assessing metabolic stability of drug compounds. Fa2N-4: Fa2N-4 immortalized human hepatocyte cell line, which is useful to evaluate the induction of major cytochrome P450s (CYPs), UDP-glucuronosyltransferases (UGTs), and P-gp, and are a suitable test system for CYP induction screening and lysosomal sequestration/trapping. PLC/PRF/5 (Alexander Cells): PLC/PRF/5 is human hepatoma cell line, derived from African male with hepatoma in 1975. Notably it produces hepatitis b surface antigen. PLC/PRF/5 cell line is widely used in liver cancer research, infectious disease research, and sexually transmitted disease research. WEDNESDAY NOVEMBER 27, 2019 DINNER STARTS AT 6PM THLE-2 & THLE-3: THLE-2 & THLE-3 are immortalized Primary normal liver epithelial cells derived by infection with SV-40 large T-antigen gene. These cell lines are commonly used for hepatotoxicity studies. iPSC-derived hepatocytes: iPSC-derived hepatocytes are generated by differentiating iPSCs into hepatocyte-like cells. iPSCs are derived by reprogramming somatic cells like skin fibroblast, Peripheral Blood Mononuclear Cells (PBMCs) etc. iPSCs-derived hepatocytes are renewable resource of hepatocyte-like cells. iPSCs derived from patients are used for disease modelling, drug efficacy testing and generation of personalised hepatocytes. Utility of iPSC-derived hepatocytes are limited by generation of immature hepatocytes, exhibits lower functional protein expression and metabolic activity. Quality of hepatocytes varies with hepatic differentiation protocols. Achieving robust hepatic differentiation protocol and fully functional hepatocytes is a major challenge (Chitrangi et al, 2023; PMID: 37479967). 473 CHARLES COURT  LITHONIA, GA 30038 FOR MORE INFO CONTACT MARIA: 202-555-0167 | MARIA@EMAIL.COM Figure 3: Schematic representation of hepatic differentiation of iPSCs. Image resource: PMID: 35598806

4. Characterization of Hepatocyte Hepatocytes are characterized by different method to assess its phenotypic and genotypic profile, morphology and functionality. Morphology: Hepatocytes exhibit polygonal, cobble-shaped morphology with prominent central nucleus, well defined cytoplasm and organelles like mitochondria, endoplasmic reticulum and Golgi bodies. Protein Expression: Liver-specific protein expression is analysed by immunochemistry, flowcytometry, western blot, ELISA etc in hepatocytes. Common hepatic markers are albumin, hepatocyte nuclear factor 4α (HNF4α), Cytokeratin 18 (CK18) etc. Gene Expression: Liver-specific gene expression is studied by RT-PCT or RNA sequencing in hepatocytes. Common hepatic genes are CYP450 enzymes, albumin, α1-antitrypsin etc. Albumin Secretion Assay: Albumin synthesis and secretion is one of the crucial features of hepatocytes. Albumin secreted in hepatocyte cultured spent media is generally assessed by ELISA. Cytochrome P450 (CYP450) Activity Assays: CYP450 enzyme plays a crucial role in drug metabolism. Hepatocytes are major parenchymal cells, play a crucial role in drug metabolism. Measuring CYP450 metabolic activity in hepatocytes is essential for drug toxicity and metabolic studies. CYP450 metabolic activity is generally measured by using luminescent or fluorescent substrates. Lipid Accumulation Assay: Lipid metabolism and storage is one of the major function of hepatocytes. Accumulated lipids in hepatocytes are measured by oil red O stain or by LDL (Low Density Lipid) uptake assays. Glycogen storage Assay (PAS staining): Periodic acid-Schiff reagent (PAS) are used to demonstrate the amount of glucose stored as glycogen. Bile Acid Secretion Assay: Bile acid synthesis and secretion is major function of hepatocytes. Bile secretion by hepatocytes is generally measured by colorimetric or fluorometric assays. Urea Production Assay: Hepatocytes play a critical role in converting ammonia to urea. Commonly urea production by hepatocytes is measured by colorimetric assays.

5. Applications of Hepatocytes The hepatocytes are a major parenchymal cell type of the liver and involves in many liver functions, such as detoxification, carbohydrate metabolism, lipid metabolism, secretion of albumin, clotting factors, and complements. Hepatocytes are used in different in vitro applications: Drug Metabolism and Toxicity Testing: Liver play a key role in drug metabolism and biotransformation. Primary hepatocytes are gold standard for studying drug metabolism and hepatotoxicity. Liver disease modelling: Hepatocytes are used to study liver diseases like non-alcoholic fatty liver diseases (NAFLD), hepatitis etc; which is further used for new drug discovery. iPSC- derived hepatocytes are used to model genetic liver diseases include Wilson disease, hemochromatosis, alpha-1-antitrypsin deficiency etc. Regenerative Medicine: Primary hepatocytes and iPSC-derived hepatocytes are used for liver regeneration and cell therapy. High-throughput screening: Primary hepatocytes, hepatic cell lines and iPSC-derived hepatocytes are amenable to high-throughput screening of drug and hepatotoxicity assays. Liver-on-Chip Technology: Liver-on-chips refer to the use of a small number of liver cells on the chips to simulate the liver microenvironment and ultrastructure in vivo. They hold extensive applications in multiple fields by reproducing the unique physiological functions of the liver in vitro. Conclusion: Each resource of hepatocytes- primary hepatocytes, hepatic cell lines and iPSC-derived hepatocytes- offers unique advantages and limitations. At Kosheeka, we offer high-quality hepatocytes amenable to high throughput screening, regenerative medicine and disease modelling. These hepatocytes can be used to study liver biology, development of new therapies and drug efficacy and safety studies. KOSHEEKA : PRIMARY CELLS FOR RESEARCH Thanks For reading A-102, SECTOR-5 NOIDA-201301, INDIA FOR MORE INFO CONTACT: +91- 9654321400, OR WWW.KOSHEEKA.COM