Article

Organotypic liver culture models: meeting current challenges in toxicity testing.

The Institute for Chemical Safety Sciences, The Hamner Institutes for Health Sciences, Research Triangle Park, NC, USA.
Critical Reviews in Toxicology (Impact Factor: 6.25). 05/2012; 42(6):501-48. DOI: 10.3109/10408444.2012.682115
Source: PubMed

ABSTRACT Prediction of chemical-induced hepatotoxicity in humans from in vitro data continues to be a significant challenge for the pharmaceutical and chemical industries. Generally, conventional in vitro hepatic model systems (i.e. 2-D static monocultures of primary or immortalized hepatocytes) are limited by their inability to maintain histotypic and phenotypic characteristics over time in culture, including stable expression of clearance and bioactivation pathways, as well as complex adaptive responses to chemical exposure. These systems are less than ideal for longer-term toxicity evaluations and elucidation of key cellular and molecular events involved in primary and secondary adaptation to chemical exposure, or for identification of important mediators of inflammation, proliferation and apoptosis. Progress in implementing a more effective strategy for in vitro-in vivo extrapolation and human risk assessment depends on significant advances in tissue culture technology and increasing their level of biological complexity. This article describes the current and ongoing need for more relevant, organotypic in vitro surrogate systems of human liver and recent efforts to recreate the multicellular architecture and hemodynamic properties of the liver using novel culture platforms. As these systems become more widely used for chemical and drug toxicity testing, there will be a corresponding need to establish standardized testing conditions, endpoint analyses and acceptance criteria. In the future, a balanced approach between sample throughput and biological relevance should provide better in vitro tools that are complementary with animal testing and assist in conducting more predictive human risk assessment.

0 Bookmarks
 · 
148 Views
  • [Show abstract] [Hide abstract]
    ABSTRACT: The umbilical cord (UC) represents an important source of mesenchymal stem cells (MSC). These human UC-derived MSC (UC-MSC) have already been isolated using a protocol based on the migratory and plastic adhesive properties of MSC (UC-MSC-Mig). The UC-MSC-Mig isolation method, however, is difficult to standardize. Therefore, we developed an enzymatic isolation protocol (UC-MSC-Enz) to overcome the above mentioned disadvantages. First, we investigated the UC-MSC-Enz for their MSC properties. We found that UC-MSC-Enz express the MSC markers CD73, CD90 and CD105 and are able to differentiate into osteoblasts, adipocytes and chondroblasts fulfilling the MSC criteria of the International Society for Cellular Therapy. Previously we found that UC-MSC-Mig are unique among MSCs due to their significant expression of several hepatic (progenitor) markers. Therefore, we also investigated the expression of hepatic transcription factors and other hepatic markers in UC-MSC-Enz at both the mRNA and protein level. We found that the expression of hepatic transcription factors (GATA4, GATA6, SOX9 and SOX17) and hepatic markers (AFP, DPP4, CX43, DKK1, DSG2, KRT18 and KRT19) in UC-MSC-Enz was not significantly different from those of UC-MSC-Mig. Consequently, this optimized enzyme-based method represents a fast, robust and standardized way to isolate UC-MSC for a broad range of applications.
    Toxicology in Vitro 12/2014; DOI:10.1016/j.tiv.2014.12.008 · 3.21 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Liver extracellular matrix (ECM) composition, topography and biomechanical properties influence cell-matrix interactions. The ECM presents guiding cues for hepatocyte phenotype maintenance, differentiation and proliferation both in vitro and in vivo. Current understanding of such cell-guiding cues along with advancement of techniques for scaffold fabrication has led to evolution of matrices for liver tissue culture from simple porous scaffolds to more complex 3D matrices with microarchitecture similar to in vivo. Natural and synthetic polymeric biomaterials fabricated in different topographies and porous matrices have been used for hepatocyte culture. Heterotypic and homotypic cell interactions are necessary for developing an adult liver as well as an artificial liver. A high oxygen demand of hepatocytes as well as graded oxygen distribution in liver is another challenging attribute of the normal liver architecture that further adds to the complexity of engineered substrate design. A balanced interplay of cell-matrix interactions along with cell-cell interactions and adequate supply of oxygen and nutrient determines the success of an engineered substrate for liver cells. Techniques devised to incorporate these features of hepatic function and mimic liver architecture range from maintaining liver cells in mm-sized tailor-made scaffolds to a more bottoms up approach that starts from building the microscopic subunit of the whole tissue. In this review, we discuss briefly various biomaterials used for liver tissue engineering with respect to design parameters such as scaffold composition and chemistry, biomechanical properties, topography, cell-cell interactions and oxygenation.
    Hepatology International 04/2013; 8(2):185-197. DOI:10.1007/s12072-013-9503-7 · 2.47 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Due to its central role in glucose homeostasis, the liver is an important target for drug development efforts for type 2 diabetes mellitus (T2DM). Significant differences across species in liver metabolism necessitate supplementation of animal data with assays designed to assess human-relevant responses. However, isolated primary human hepatocytes (PHHs) display a rapid decline in phenotypic functions in conventional monolayer formats. Co-cultivation of PHHs with specific stromal cells, especially in micropatterned configurations, can stabilize some liver functions for ~4 weeks in vitro. However, it remains unclear whether co-culture approaches can stabilize glucose metabolism that can be modulated with hormones in PHHs. Thus, here we compared commonly employed conventional culture formats and previously developed micropatterned co-cultures (MPCCs) of cryopreserved PHHs and stromal fibroblasts for mRNA expression of key glucose metabolism genes (i.e. PCK1), and sensitivity of gluconeogenesis to prototypical hormones, insulin and glucagon. We found that only MPCCs displayed high expression of all transcripts tested for at least 2 weeks, and robust gluconeogenesis with responsiveness to hormones for at least 3 weeks in vitro. Furthermore, MPCCs displayed glycogen storage and lysis, which could be modulated with hormones under the appropriate feeding and fasting states, respectively. Finally, we utilized MPCCs in proof-of-concept experiments where we tested gluconeogenesis inhibitors and evaluated the effects of stimulation with high levels of glucose as in T2DM. Gluconeogenesis in MPCCs was decreased after stimulation with drugs (i.e. metformin) and the PHHs accumulated significant amount of lipids following incubation with excess glucose (i.e. 340% in 50 mM glucose relative to physiologic 5 mM glucose controls). In conclusion, MPCCs provide a platform to study glucose metabolism and hormonal responsiveness in cryopreserved PHHs from multiple donors for several weeks in vitro. This model is also useful to study the effects of drugs and over-nutrition for applications in T2DM.
    Tissue Engineering Part C Methods 12/2014; DOI:10.1089/ten.TEC.2014.0512 · 4.64 Impact Factor

Full-text (2 Sources)

Download
57 Downloads
Available from
May 30, 2014