ADME / Tox >> In Vitro Toxicity >> Cytotoxicity

Evaluate potential organ-toxicity early in discovery using human primary cells in vitro

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Toxicity testing is mandated by regulatory agencies for IND submission and is typically performed during the preclinical phase of discovery. However, uncovering toxicity at such a late stage means that considerable investment and time has been lost, jeopardizing the project. Eurofins has developed a reliable high-throughput model of multiplexed toxicity testing which provides a quick and cost-effective option suitable for early liability assessment, when changing lead series has minimum impact on the project. By utilizing imaging expertise with five human primary cell lines, we can provide a sensitive in vitro cell-based method to aid in lead selection and optimization by identifying potential tissue-relevant toxicity earlier in the drug development process.

Our organ toxicity services employ primary cells derived from human donor tissue that are purified and cryopreserved. Primary cells such as the Normal Human Dermal Fibroblasts (NHDF), Human Renal Proximal Tubular Epithelial Cells (HRPTEpiC), Human Umbilical Vein Endothelial Cells (HUVEC) and Human Umbilical Mesenchymal Stem Cells (HUMSC) included in this panel are able to propagate in for a short number of passages before they stop dividing. These cells are banked at a low passage number and proliferate in culture pre- and post-plating. The Human Primary Hepatocytes (HPH) are plated directly from thawed cryovials and do not proliferate in culture.

Our assays employ high-resolution cell imaging to provide more sensitive detection of cell viability determined by proliferation inhibition or cell loss. Our services can be run with a single cell type, the pre-validated primary cell panel, or cell lines chosen through collaboration with our expert scientists who have experience running cytotoxicity assays with hundreds of human cell lines.

Advantages of Eurofins' Organ-Toxicity in Human Primary Cell Panel:

  • Pre-validated human cells harvested and purified from single donor source are used for reproducible assays
    • Normal Human Dermal Fibroblasts (NHDF)
    • Human Renal Proximal Tubular Epithelial Cells (HRPTEpiC)
    • Human Umbilical Vein Endothelial Cells (HUVEC)
    • Human Umbilical Mesenchymal Stem Cells (HUMSC)
    • Human Primary Hepatocytes (HPH)
  • Flexible project scope from single cell type to full cell panel
  • Low compound requirement - as little as 300 μl of a 20 mM solution is needed for testing at a final assay concentration of 100 μM
  • Relevant reference compound and vehicle controls are included on each plate

 

Organ-toxicity using human primary cell panel


We utilize two different assays to provide mechanistic information on a test compound's organ specific toxicity. Our cytotoxicity assay measures cell viability, apoptosis and mitosis, while our cellular stress assay measures cell viability and cellular stress.

 

Cytotoxicity assessment with human primary cells undefined

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A three parameter multiplexed study using five primary human cell types is used to assessment compound induced cytotoxicity in vitro. A decrease in cell viability is a very sensitive marker to detect general toxicity. Activation of caspase 3 indicates an apoptotic mechanism. Phosphorylation of histone H3, a marker for mitotic block, reveals a defect in cell cycle regulation.

 

Features of Eurofins' cytotoxicity assessment:

  • High Content Analysis, cytotoxicity parameters:
    • Cell viability - decrease in DAPI (nuclear dye) stained cells
    • Apoptosis - increased staining with an anti-active caspase antibody
    • Mitotic block - increased staining with an anti-phospho-histone-H3 antibody

 

Cellular stress assessment with human primary cells undefined

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A two parameter multiplexed study using five primary human cell types is used to assessment compound induced cellular stress in vitro. A decrease in cell viability is a very sensitive marker to detect general toxicity. Heat shock protein 27 (HSP 27) is expressed in response to cellular stress in an attempt to block the apoptosis pathway. Kidney injury molecule-1 (KIM-1) is a well-accepted marker of renal proximal tubule injury and is used as a third parameter studies using HRPTEpiC cells.

 

Features of Eurofins' cellular stress assessment:

  • High Content Analysis, cellular stress parameters:
    • Cell viability - decrease in DAPI (nuclear dye) stained cells
    • Cellular stress - increased staining using an anti-HSP27 antibody
    • Kidney injury: increased staining using an anti-KIM-1 antibody

 

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Figure 1. Cellular stress in five human primary cell types as demonstrated by a decrease in viable cells (black) and increases in HSP27 expression (blue) and, for HRPTEpiC cells, an increase in KIM-1 expression (red)

 

Explore our human primary cell panel

Human Primary Hepatocytes (HPH) undefined

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Cryopreserved human primary hepatocytes have been shown to be a useful cellular reagent for assessment of drug metabolism, hepatic toxicity, and inhibitory drug-drug interaction potential. The high quality plateable hepatocytes used for cytotoxicity screening are of a single donor origin. They are well characterized for in vivo-like enzyme expression levels, proper cell morphology, and retention of viability post plating.

 

 

Normal Human Dermal Fibroblasts (NHDF-neo) undefined

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Normal human dermal fibroblasts (NHDF) are generated from the dermis of normal neonatal and human adult skin. Fibroblasts are present in all connective tissues. They secrete a non-rigid extracellular matrix that is rich in type I and/or type III collagen. In response to inflammatory stimuli dermal fibroblasts secrete large quantities of hyaluronan a chief component of cellular matrix. Dermal fibroblasts change phenotype during wound healing from migratory, repopulating to contractile, matrix-reassembling cell population.

 

Human Renal Proximal Tubular Epithelial Cells (HRPTEpiC) undefined

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Human renal proximal tubular epithelial cells (HRPTEpiC) are a critical component of normal renal function. Their primary function is to reabsorb glucose and amino acids from the glomerular filtrate and allow the excretion of non-nutritional waste in the urine. The HRPTEpiC produce cytokines and chemokines involved in mediating inflammation and via production of IL-8 have been shown to direct leukocyte chemotaxis in acute inflammation. Expression of IL-2R alpha and MHC class II antigens in HRPTEpiC post kidney transplantation and glomerulonephritis has implicated these cells in the pathogenesis of immune renal injury.

 

Human Umbilical Vein Endothelial Cells (HUVEC) undefined

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Human Umbilical Vein Endothelial Cells (HUVEC) are derived from normal human umbilical veins. Endothelial cells play a pivotal role in numerous aspects of vascular biology, including atherosclerosis, as a selective blood barrier, thrombosis and fibrinolysis, inflammation, angiogenesis, control of blood pressure and are responsive to cytokine stimulation in the expression of cell adhesion molecules. The signaling protein ERK5 is crucial for normal endothelial cell function maintenance. A hallmark of vascular disease is endothelial dysfunction which can lead to the formation of potentially fatal thrombi caused by platelet aggregation and adhesion to the subendothelium. Endothelial dysfunction also has a causative link to inflammatory disease such as rheumatoid arthritis and systemic lupus erythematosus.

 

Human Umbilical Mesenchymal Stem Cells (HUMSC) undefined

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Human mesenchymal stem cells are a well characterized adult stem cell line that has been differentiated into a diverse array of mature cells with therapeutic potential for use in replacement of damaged tissue. Isolated from Wharton's jelly of umbilical cords, human umbilical mesenchymal stem cells (HUMSC) can be transformed via in vitro conditioned medium cell culturing and or chemical treatment into one of the following target cell types: neuronal, cardiomyocyte, adipogenic and osteogenic cell lineages. Mesenchymal stem cell markers SH2 and SH3 are present in HUMSC and matrix receptors CD44 and CD105 are expressed however, the HUMSC lack hematopoietic lineage marker CD34.