Human peripheral blood mononuclear cells (PBMC) represent a heterogeneous menagerie of immune cells isolated from peripheral blood. While they primarily consist of lymphocytes (B-cells, T-cells and NK cells), a smaller fraction of monocytic and dendritic cells are also present. Due to the overwhelming abundance of T-cells within the PBMC population (70-85%), these cells provide a useful and appropriate model with which to study the immunological mechanisms associated with drug-mediated hypersensitivity in vitro.
The PBMC cytotoxicity assay is an in vitro based method which utilizes PBMC isolated from consenting healthy donors using Ficoll and density gradient centrifugation. The cytotoxic potential of candidate drugs towards PBMC is assessed by measuring the levels of cellular ATP depletion and LDH release. Our recently established biobank of PBMC isolated from multiple healthy donors provides the opportunity to investigate donor-dependent PBMC cytotoxicity upon drug exposure. Furthermore, follow-up studies involving other endpoints, such as drug-induced cytokine release, can be performed using the same cryopreserved donor population.
Cyprotex deliver consistent, high quality data with the flexibility to adapt protocols based on specific customer requirements.
In vitro high throughput assays utilising primary human immune cells will significantly enhance our capabilities to predict candidate drugs with potentials to cause rare but occasionally fatal hypersensitivity reactions during early stages of preclinical drug development.
|Donors||>6 donors available for multi-donor studies|
|Analysis Platform||Cellular ATP – Cytation 3 Cell Imaging Multi-Mode reader
LDH release – SpectraMax ABS absorbance microplate reader
|Test Compound Concentrations||8 point dose response curve with top concentration based on 100x Cmax or solubility limit
3 replicates per concentration*
|Compound Requirements||Maximum (dependent upon number of repeat doses) 150 µL of a DMSO* solution to achieve 200x top concentration maintained at 0.5% DMSO or equivalent amount in solid compound|
|Time Points||24-72 hour pre-incubation*|
|Quality Control||Negative control: 0.5% DMSO (vehicle)*
Positive controls: 2 appropriate compounds
|Data Delivery||Minimum effective concentration (MEC) and AC50 value for cellular ATP content and LDH release|
* other options available on request.
1 Naisbitt DJ et al., (2020). Immune dysregulation increases the incidence of delayed-type drug hypersensitivity reactions. Allergy 75(4); 781-797
2 Sullivan A et al., (2018). β-Lactam hypersensitivity involves expansion of circulating and skin-resident TH22 cells. J Allergy Clin Immunol 141(1); 235-249
3 Mennicke M et al., (2009). Fulminant liver failure after vancomycin in a sulfasalazine-induced DRESS syndrome: fatal recurrence after liver transplantation. Am J Transplant 9(9); 2197-2202
4 Gibson A et al., (2018). Genetic and nongenetic factors that may predispose individuals to allergic drug reactions. Curr Opin Allergy Clin Immunol 18(4); 325-332