August 10, 2021
by Aarati Ranade
Every time we perform an experiment with hepatocytes, I am curious to see how the culture looks over time after plating the cells. I carefully place the cell culture plate under the microscope. I carefully observe through the microscope eyepiece. The transition from round cells to formation of flat, hexagonal cells with one or two prominent nuclei with cell borders nicely touching each other to form a carpet-like monolayer amuses me. Yet, looking at this flat, two-dimensional structure, I feel “this hepatocyte monolayer culture looks great, but maybe this will not be enough to answer many questions about liver, especially considering the structural and functional complexity of the whole organ”
The scientific quest of “mimicking in vivo liver microstructure and simulating pathophysiology” is the driving force for generating three-dimensional in vitro liver models. The highly complex structure of the liver and multiple key functions this organ performs make the task of 3D liver model generation very challenging and necessary, at the same time.
Complex Organization of Cell Types in the Liver
The liver is a highly vascularized organ perfused by a dual blood supply – with arterial blood (via the hepatic artery) and venous blood (via the portal vein). After exchange of nutrients and oxygen, blood from the liver is collected into the central vein. Also, the bile ducts collect bile, which is then concentrated in the gall bladder. The hepatic lobule, a roughly hexagonal unit consisting of parenchymal cells (hepatocytes) and nonparenchymal cells (Kupffer, stellate, sinusoidal endothelial, and cholangiocytes) is the basic structural unit of the liver. It extends between the central vein and portal triad (hepatic artery, portal vein and bile ducts).
Cells within the liver have well-defined functions, and liver responses to any acute or chronic external stimuli are based on a cumulative response of various types of hepatic cells. The sinusoid is lined with a layer of endothelial cells, which regulate nutrient and xenobiotic transport, and a layer of hepatocytes. The stellate cells reside in the space between the sinusoidal endothelial cells and the hepatocytes, which is called the space of Disse. Kupffer cells are the resident macrophages that reside in the sinusoid. The oxygen-rich arterial blood from the hepatic artery mixes with the venous blood via the portal vein, which is low in oxygen saturation but rich with hormones and nutrients from the gastrointestinal tract. The mixed blood supply travels along the liver sinusoid to the central vein, generating a unique, complex environment.
Hepatocytes use high amounts of oxygen and are involved in the secretion and metabolism of several molecules, and thus the environment within the sinusoid is dynamic, driven by hepatocyte metabolism. In addition, the transport of nutrients and oxygen from the liver sinusoid occurs through the endothelial cells and the space of Disse, creating a unique environment whose physiologic responses are driven by the mass transport occurring within the microarchitecture of the liver sinusoid.
This complexity in liver architecture demands more complex 3D in vitro models for better recapitulation of in vivo structure and response.
Need for 3D Models
To improve the long-term phenotype maintenance of hepatocytes in culture, a vast number of 3D culture models has been developed in recent years. Maintenance of robust hepatic functions, physiological expression levels of drug metabolizing enzymes and transporters, and prevention of rapid dedifferentiation of hepatocytes observed in 2D models are key factors considered in the development of 3D hepatocyte models for assessment of drug hepatotoxicity. This is important because toxicity of most compounds manifests with a delayed onset, and drug metabolizing enzymes are among the first to be lost during dedifferentiation.
The efforts towards generation of 3D in vitro models are mainly driven by:
1) providing a structure suitable for mimicking in vivo microarchitecture
2) simulating liver pathophysiology in an in vivo-like microenvironment
3) providing a rapid, easy, consistent, and high throughput process for screening diverse molecules using a small number of human cells
Current 3D liver models
Currently available organotypic 3D liver models can be categorized as:
a) scaffold-free spheroid in multi-well formats
b) scaffold-free spheroid in perfused bioreactor format
c) scaffold-based spheroid culture models
d) micropatterned format
e) hollow fiber bioreactors
f) perfused liver chips
g) 3D bioprinting
A scaffold-free approach to establishing 3D hepatocyte cultures is based on the self-aggregation of cells in suspension leading to formation of spheroids in the absence of any substrate. These spheroid cultures can be established either by seeding the cells in low-attachment culture vessels or by the hanging drop method. Studies published using the scaffold-free approach include formation of spheroids from human liver cell lines (HepG2, HepaRG and Huh-7), primary human hepatocytes and NPCs, primary rat hepatocytes, and induced pluripotent stem cells (iPSC)-derived hepatocytes.
Scaffold-based culture models use a variety of natural or synthetic scaffolds that can facilitate the formation and maintenance of cell-cell contacts, cell polarity, and tissue organization. The material of choice for spheroid formation is an important factor; it should exhibit minimal leaching into culture medium and should not interact with the media components or chemicals used in the study. Oxygen concentrations in the medium can influence the phenotype of hepatic cells. Thus, the gas permeability properties of scaffold material are important in ensuring that cells are exposed to relevant oxygen levels. The stiffness and topography of substratum material also affect the molecular phenotype of cultured hepatocytes via mechanosensing pathways. Previous studies have reported use of alginate, collagen, nanofibrous poly-L-lactic acid (PLLA) or polystyrene as scaffold material for spheroid formation either using encapsulation or emulsion droplet microfluidic methods. Carbohydrate polymer sodium-alginate offers the advantages of biocompatibility, chemical and mechanical stability, and permeability for living cells; its hydrated 3D network also allows cells to interact with each other. Alginate encapsulation has been shown to improve viability and metabolic capacity of HepG2 and HepaRG spheroids. Recent study has reported assessment of drug induced hepatotoxicity in a simple paper-based scaffold array model generated by co-culturing human-induced hepatocytes (hiHeps) with human umbilical vein endothelial cells (HUVECs).
Our team developed a rat in vitro 3D model using the alginate encapsulation method and we are in the process of developing a human 3D model for assessing toxicity of chemicals.
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