Toxicity is a major cause of attrition in therapeutic drug development and is a key factor in decision-making around the advancement of candidate drugs through the development pipeline. The expansion of preclinical testing to incorporate assays that better predict potential toxicities earlier in the drug development process has obvious advantages for the selection of successful lead candidates. In vitro testing on primary cells can allow investigators to preview in vivo responses, thus facilitating design of better dosing strategies and optimization of animal models in preclinical testing, as well as Phase I clinical trials.
We specialize in performing the in vitro colony-forming unit (CFU) assay on hematopoietic stem and progenitor cells to measure potential toxic effects of candidate therapeutics, including small molecule compounds and biologics. Mesenchymal stem and progenitor cells, which generate key components of bone, fat and cartilage, may also be tested in the colony-forming unit - fibroblast (CFU-F) assay to predict possible cytotoxic effects. In addition, potential immunomodulation caused by candidate therapeutics can be evaluated through in vitro and in vivo assays.
Hematopoietic Toxicity Assessment
Human hematopoietic stem and progenitor cells (HSPCs) may be isolated from bone marrow, cord blood or peripheral blood. They have the capacity to differentiate into all mature hematopoietic cells including erythroid, myeloid and megakaryocyte lineages. One of the best examples of an in vitro assay used in hematotoxicity testing is the culture of primary human hematopoietic cells in the colony-forming unit (CFU) assay (also known as a colony-forming cell (CFC) assay). The in vitro CFU assay may be used to predict in vivo bone marrow toxicity and clinical cytopenia (anemia, neutropenia, and thrombocytopenia).
Our scientists have the expertise to assess possible hematotoxicity due to the action of candidate therapeutics, alone or in combination, on erythroid, myeloid and megakaryocyte progenitors using standardized and custom-designed CFU assays. These in vitro studies can be performed using human, non-human primate, mouse, rat and dog cells, which can assist in the selection of appropriate animal models for pharmaceutical development. Clinically relevant results have been shown through the addition of candidate therapeutics directly to cells in a CFU assay, allowing the determination of the maximum tolerated dose (myeloid)1 or Cmax (megakaryocyte)2 using these in vitro assays.
In addition to the CFU assay, we also offer higher-throughput assays for liquid culture-based hematotoxicity testing in a 96-well plate format. Small molecule compounds or biologics may be assessed for their effects on the expansion and lineage-specific differentiation of CD34+ cells into erythroid, myeloid and megakaryocyte progenitor cells. The readout for this assay includes viability phenotyping using flow cytometry but it may be customized depending on your needs. The toxicity levels of molecular entities observed in this assay generally correlate with those observed in the CFU assay, with the added benefit of increased screening capacity in addition to flexibility in treatment schedules.
Mesenchymal Toxicity Assessment
Mesenchymal stem cells (MSCs), under the appropriate conditions, can differentiate into cells that make up adipose tissue, cartilage, bone and muscle. Drug candidates destined for the tissue engineering market can be assessed for effects on the differentiation potential, as well as stimulatory or inhibitory effects on mesenchymal stem and progenitor cells, using the colony-forming cell - fibroblast (CFU-F) assay (Figure 4). The effects of compounds on the size and density of the colony can also be evaluated.
The CFU-F Assay as Performed by Contract Assay Services:
Measures the effects of a test article on progenitor frequency
Assesses proliferative or expansion potential of progenitors (size and morphology of colonies)
Quantitates mesenchymal progenitors in bone marrow
Immunotoxicity and Immune Profiling
The immune system can be the target of many chemicals, including environmental contaminants and therapeutic drugs, leading to potentially adverse effects. Although evaluation of immune function following in vivo exposure is the most relevant model to predict patient response, it is increasingly desirable to limit the use of animals and use human cells to maximize the relevance of testing. Recommendations from the ECVAM workshop on “The Use of In Vitro Systems for evaluating Immunotoxicity” suggests that in vitro testing for direct immunotoxicity can be done in a tiered approach, the first tier measuring myelotoxicity using the CFU-GM assay.1
In addition to performing the CFU-GM assay with test articles of interest, CAS can assist in designing and optimizing experiments to evaluate aspects of immunotoxicity that result from altered activity of the immune system. Flow cytometry and immune profiling may be used to assess for various immunological effects, where cells from primary human or mouse sources are isolated, enriched and used in the appropriate in vitro assay to assess effects of test articles on immune pathways, such as pro- or anti-inflammatory responses. Custom assays can be designed and optimized to answer your particular question regarding the immunomodulation of your test articles.
Contract Assay Services can customize:
· Flow cytometric analysis and immune profiling of B cells, various T cell subsets, NK cells, dendritic cells and macrophages/monocytes
· ELISA assays to quantify immune effector molecules (e.g. cytokines and immunoglobulins)
· Cytometric bead analysis (for simultaneous analysis of multiple analytes)
· Chemotactic assays
· Cell proliferation assays, including T cell proliferation assays (mitogen or Anti-CD3-stimulated)
Intestinal Organoid Models for Toxicity Assessment
3D culture systems have the potential to increase the physiological relevance of basic research into biological mechanisms, bridging the gap between high-throughput in vitro screening methods and large in vivo studies during drug development.1,2 Organoids generated from the intestinal epithelium are thought to recapitulate numerous features of the adult intestine in vivo, including self-renewal and differentiation pathways, cell types present and cellular organization within the epithelium. Together these characteristics create a culture system that is a powerful tool for investigating potential toxicity to the intestinal epithelium due to the action of candidate therapeutics.