Some Facts You Should Know About Hepatocyte Culture

Hepatocytes are the main cells that are present in the liver (~70% of total volume) . And you may wonder if hepatocytes have regenerative properties. Hepatocytes, often bi-nucleated and forming sheets in liver tissue, are crucial for various metabolic functions.

Human primary hepatocytes are the closest model to the human liver. They closely mimic the hepatic biochemical functions, particularly drug metabolism when cultured under chemically defined conditions. Primary hepatocytes, as differentiated cells, are invaluable for in vitro studies. Hepatocytes accurately reflect human liver responses to chemicals that are proven as an important tool for investigating drug metabolism, bioactivation, and assessing potential hepatotoxicity of new drugs in humans.

Factors should be considered for the sustainability of hepatocytes 

The suitability of liver samples from different sources varies, influenced by factors such as warm and cold ischemia during procurement, as well as intrinsic characteristics like sex, age, liver pathology, and xenobiotic treatment.

Comparative analyses of hepatocyte cultures from various liver tissue samples demonstrate differences in cell yield, viability, and drug biotransformation capability.

The two-step collagenase perfusion method remains the most effective technique for isolating high yields of viable hepatocytes from human liver samples, retaining typical hepatic functions, including drug-metabolizing enzymes, when cultured.

Surgical biopsies are preferred for obtaining high-quality hepatocytes, highlighting the importance of tissue preservation and processing methods in maintaining cell viability and functionality. While isolated primary human hepatocytes are considered the gold standard for developing in vitro liver cell culture models, maintaining their long-term cultures and functional proliferation in vitro has proven challenging.

Overcoming the Hurdles in Maintaining Human Hepatic Cells Culture  

Hepatocyte isolation and culture face hurdles due to interindividual differences, varied sample types, isolation procedures, and technical limitations. Researchers worldwide have attempted to optimize hepatocyte isolation and culture protocols to sustain their growth in vitro for downstream applications. To address this, some have turned to immortalized cell lines like HepaRG, although these cell lines do not fully replicate hepatocytes’ native functions.

These innovations hold promise for overcoming challenges in hepatocyte culture and enhancing their utility in various research and therapeutic applications.

• Initially, mechanical methods, such as shaking with glass beads and filtering through cheesecloth, were used for hepatocyte isolation but resulted in low yields (5-10%).
• Enzymatic perfusion techniques, utilizing collagenase and hyaluronidase, are in use to improve hepatocyte yield and integrity by perfusing the liver via the portal vein.
• An alternative method involved slicing rat liver tissues followed by collagenase digestion, which yielded intact and viable cells, offering another approach to hepatocyte isolation.
• Afterward, researchers refined the enzymatic method with a two-step perfusion procedure. The first step disrupted desmosomes forming tight cell junctions using a Ca2+-free buffer, followed by a second perfusion with a collagenase buffer containing Ca2+.

These advancements in hepatocyte isolation techniques significantly improved yield and quality, facilitating research in drug metabolism and hepatotoxicity assessment.

This two-step procedure, introduced in 1976, represented a significant breakthrough in isolating primary rat hepatocytes. The initial perfusion disrupts desmosomes irreversibly, while the subsequent perfusion with collagenase breaks down ECM proteins and cell-matrix contacts.

Isolating human hepatocytes 

Isolation of hepatocytes from resected liver tissues follows a similar process to whole liver perfusion. High flow-capacity perfusion devices with a perfusion rate of up to 1–2 L/min are in use for isolating hepatocytes from adult human livers. Multiple perfusion cannulae and ligation of bypassing vessels maximize perfusion efficiency, enhancing the yield and viability of hepatocytes.

A developed methodology is in use to isolate human hepatocytes from resected healthy liver tissue, which is not suitable for perfusion. This method utilizes similar buffers to the two-step perfusion method(EDTA and collagenase IV) and involves lysing red blood cells (RBCs) following tissue digestion to prevent contamination of RBCs in hepatocyte preparations. Despite using smaller liver tissue pieces, this method reports to obtain over 65% viable hepatocytes.

Various collagenases testing for hepatocyte isolation

Class I collagenase enzymes are more stable and exhibit greater activity towards insoluble collagen, while class II enzymes attack a higher number of smaller peptides. A combination of both classes is crucial, with high amounts of class II collagenases proving advantageous for hepatocyte isolation.

Type IV collagenase, has lower tryptic activity levels to limit damage to membrane proteins and receptors while maintaining normal to above-normal collagenase activity, resulting in a good yield of hepatocytes.

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Collagenase P, initially developed for isolating pancreatic islet cells, is suitable for human liver tissues, particularly in mildly fibrotic cases. When supplemented with 10% fetal calf serum (FCS), collagenase P-containing perfusion solutions enable prolonged but mild tissue digestion, yielding high amounts of both parenchymal and non-parenchymal cells from a single liver tissue.

Cryopreserved human hepatocytes

Isolating the hepatocytes is a crucial step, but maintaining their viability is also important. For that purpose, the freezing and thawing process is performed. However these processes sometimes damage the cells to avoid cryoprotectant solutions such as DMSO, glycerol, and ethylene glycol for use after isolating hepatocytes.  After adding cryoprotectant in hepatocyte suspension, they are set in cryobags. The hepatocyte suspension goes in cryovials or cryobags and placed in a controlled-rate freezer. Controlled-rate freezing gradually reduces the temperature of the cell suspension, typically at a rate of 1-2°C per minute, to minimize the formation of ice crystals and cell damage. This step is critical for preserving cell viability and functionality.

Process of hepatocyte culture

Thawing Your Cells:

• Avoid leaving hepatocytes in the cryopreservation medium for extended periods to maintain viability. Immediately transfer them from liquid nitrogen to a 37°C water bath.
• Refrain from removing small aliquots from the vial to prevent repeated freezing and thawing cycles, which can stress and damage the cells.

Seeding Your Cells:

• Seed hepatocytes at the optimal density to achieve a confluent monolayer. Refer to the certificate of analysis (CoA) for the recommended seeding density of each cell lot.
• Distribute cells evenly by manually moving the plate in a T-shape and verify distribution under a microscope.
• Accurate cell counting is crucial, as both under-seeding and over-seeding can affect sample quality. Compare your cell count to the provided values in the CoA.

Culture Methods:

• For plateable primary hepatocytes, 2D configurations involve culturing on collagen-coated surfaces for extended periods. Sandwich cultures between collagen layers maintain hepatic functions over weeks.
• To mimic the liver microenvironment and prevent hepatocyte dedifferentiation, consider 3D configurations. Generate hepatocyte spheroids using low-attachment plates or hanging drop cultures.
• BeCytes hepatocytes are 3D spheroid and can self-assemble using manufacturer’s protocol, ultra-low attachment plates, and 3D plating and maintenance medium.

Maintaining Your Cells in Culture:

• Avoid splitting primary hepatocytes, as they are non-proliferative and highly sensitive to manipulation.

These are some key precautions you should take care of 

• Hepatocytes require proper handling: Hepatocytes are extremely fragile cells, so it’s important to handle them with care to prevent damage to cell membranes and maintain cell viability.
• Hepatocytes are most viable at low temperatures: To maintain the cold temperature, keep hepatocytes on ice or in a cold room throughout the handling process to slow down metabolic activity and preserve cell integrity. This also preserves the hepatocyte RNA and protein structure.
• Use appropriate buffers: it is also crucial to provide the hepatocytes with an environment that is good for their viability. For that purpose, use buffers that are isotonic and pH-balanced to maintain cell osmolarity and pH stability, which are essential for cell health.
• Minimize exposure to air: Limit exposure to air during cell isolation and handling to prevent oxidative stress and maintain cell viability.
• Avoid excessive centrifugation: Hepatocytes are sensitive to mechanical stress, so minimize centrifugation speed and duration to prevent cell damage.
• Optimize cell density: When plating hepatocytes for experiments, optimize cell density to ensure proper cell attachment and function while avoiding overcrowding.
• Consider cell culture media: Use appropriate culture media supplemented with essential nutrients and growth factors to support hepatocyte viability and function.
• Monitor cell morphology: Regularly monitor hepatocyte morphology under a microscope to assess cell health and detect any signs of damage or deterioration.

Sterile conditions: Work in a sterile environment and follow aseptic techniques to prevent contamination, which can compromise cell viability and experimental results.

• Follow protocols Always try to use standardized protocols for hepatocyte isolation, culture, and experimentation to ensure the reproducibility and reliability of results.