Toxicity Testing

With Contract Assay Services

Toxicity is a major cause of attrition in therapeutic drug development and a key consideration when selecting candidate drugs for advancement through the development pipeline. Expanding preclinical testing to incorporate assays that are better at predicting potential toxicities earlier in the 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 in vitro assays on hematopoietic stem and progenitor cells to measure the 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. Mouse intestinal organoids can also be used to assess the effects of candidate therapeutics on cellular viability.

Contact us for a more in-depth discussion on how each of the assays in the sections below can be modified to meet your specific goals.

Colony-Forming Unit (CFU) Assay

We can assess the hematotoxicity of candidate therapeutics on erythroid, myeloid, and megakaryocyte progenitors using standardized and custom-designed colony-forming unit (CFU) assays (also known as the colony-forming cell (CFC) assay).

Advantages of the CFU Assay

  • Yields clinically predictive information1,2, allowing for better planning and fewer in vivo studies
  • Uses primary hematopoietic cells from human, non-human primate, mouse, rat or dog, to assist in selection of appropriate animal models
  • Table 1. Comparison of Myeloid (CFU-GM) IC50 Values Across Animal Species
    Table of myelotoxicity response (IC50 values) to different compounds in human, mouse, canine, and rat cells plated in the colony-forming unit (CFU) assay.

  • Assesses both the proliferation and differentiation of erythroid (BFU-E) and myeloid (CFU-GM) progenitors
  • Dose-response curves and IC50 values for human bone marrow derived erythroid and myeloid progenitors incubated with 5-fluorouracil
    Figure 1. Dose Response Curves and IC50 values for Human BM-Derived Erythroid and Myeloid Progenitors Incubated with 5-Fluorouracil
  1. Pessina A et al. (2003) Application of the CFU-GM Assay to Predict Acute Drug-Induced Neutropenia: An International Blind Trial to Validate a Prediction Model for the Maximum Tolerated Dose (MTD) of Myelosuppressive Xenobiotics. Toxicological sciences 75(2) 355-367.
  2. Pessina A et al. (2009) Application of human CFU-Mk assay to predict potential thrombocytoxicity of drugs. Toxicol In Vitro 23(1) 194-200.

HemaTox - Liquid Culture-Based Hematotoxicity Assays

HemaTox™assays can assess the toxicity of drugs on the growth and lineage-specific differentiation of human CD34+ hematopoietic stem and progenitor cells (HSPCs) into one of three specific progenitor cell lineages (erythroid, myeloid, or megakaryocyte).

Advantages of HemaTox™ Liquid Medium-based Assays

  • Assesses both proliferation and differentiation of erythroid, myeloid and megataryocyte progenitors in < 10 days
  • Allows high-throughput testing of compounds in 96-well format
  • Schematic of liquid-culture based hematotoxicity assay workflow for assessment of erythroid, myeloid, megakaryocyte progenitor proliferation and differentiation

    Figure 2. HemaTox™ Assay Workflow

  • Exhibits correlation with the CFU assay for toxicity levels of a wide range of compounds

  • Correlation graphs of IC<sub>50</sub> and IC<sub>90</sub> values generated from CFU-GM assay and the HemaTox™ myeloid assay.

    Figure 3. Correlation Between IC50 and IC90 Values for 43 Test Compounds Using the CFU-GM Assay and the Liquid-Based HemaTox™ Myeloid Assay

  • Allows high flexibility as test compounds may be added to the culture at different time points, allowing the effects on progenitor cells at different stages of differentiation to be examined

  • Dose response curves of myeloid progenitors exposed to 5-fluorouracil in different treatment methods.

    Figure 4. Dose-response Curves of Different 5-FU Treatment Regimens

    CD34+ cells were exposed to 5-Fluorouracil continuously for the entire duration of the culture (red, continuous), transiently for 24 hours on day 0 followed by washout of the drug (black, 24-Hour treatment, day 0), and transiently for 24 hours on day 6 (blue, 24-Hour treatment, day 6), when committed myeloid progenitors were already present. Data is presented as average growth, as a percentage of control, obtained from triplicate culture wells derived from a single representative donor. Error bars represent standard deviation.

  • Provides improved sensitivity in the evaluation of anti-proliferative effects

AZT Case Study

Azidothymidine (AZT) is an antiviral nucleoside analog that targets viral polymerases but can also inhibit cellular polymerases, leading to decreased cell proliferation and ultimately the suppression of hematopoiesis, resulting in anemia and neutropenia. Traditional CFU assays primarily quantitate the effects of a drug on colony numbers and not effects on colony size that may result from inhibited cell proliferation. The inability to quantify changes in colony size may explain why AZT, well known to perturb hematopoiesis in patients, does not exhibit high toxicity in CFU assays. In contrast, HemaTox™ assays can detect changes in both cell differentiation, by assessing the expression of cell surface markers used to distinguish between specific cell populations, and cell proliferation, by absolute cell counts.
Photos of anti-proliferative effect of AZT on erythroid (BFU-E) colony size.

Figure 5. Strong Anti-Proliferative Effects of AZT can be Qualitatively Observed in the CFU Assay by Changes in Colony Size

Shown are erythroid (BFU-E) colonies at 10X magnification in a CFU assay after 14 days of culture in the absence and increasing presence of AZT.

Dose response curves of erythroid and myeloid progenitor cells treated with AZT in CFU and HemaTox assays.

Figure 6. HemaTox™ Assays Show Greater Toxicity of AZT when Compared with CFU Assays

Representative dose-response curves for AZT based on colony numbers (black, CFU) and lineage-specific cell numbers (red, HemaTox™). The table shows the average IC50 values from three to five multiple donor lots and four to seven independent experiments.

  • Provides high reproducibility with pre-qualified primary stem cells from human cord blood or bone marrow
  • Dose-response curves of human CD34<sup>+</sup> cells treated with 5-FU show high reproducibility of the HemaTox assay.

    Figure 7. Inter-Donor and Inter-Assay Reproducibility of 5-FU Dose-Response Curves

    Dose-response curves were generated from titrations of 5-Fluorouracil added to human CD34+ cells from five to seven donor lots in HemaTox™ (A) Myeloid, (B) Erythroid and (C) Megakaryocyte assays. In each assay, similar IC50 values were obtained with cells from different donors and in different experiments with cells from the same donor. Shown are values (% of control growth) normalized to the number of cells in the solvent control cultures.

Read more about Services for Predicting Hematotoxicity in Drug Development with HemaTox™ Assays

Mesenchymal Toxicity Assessment

Mesenchymal stem cells (MSCs), under the appropriate conditions, can differentiate into cells that make up adipose tissue, cartilage, bone and muscle. Using the colony-forming cell-fibroblast (CFU-F) assay, drug candidates destined for the tissue engineering market can be assessed for stimulatory or inhibitory effects on mesenchymal stem and progenitor cells, as well as for effects on the differentiation potential of these cells. The effects of compounds on colony size and density 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

Figure 8. The Presence of an Inhibitory Compound Changes the Morphology of Human Bone Marrow-Derived CFU-F Colonies

Shown are Colony-Forming Unit - Fibroblast (CFU-F) assays containing MSCs plated (A) in the absence and (B) in the presence of an inhibitory compound. Notable differences in morphology include fewer cells and a more scattered distribution in the culture containing (B) the inhibitory compound. Colony numbers are also reduced in the presence of an inhibitory compound (data not shown).

Intestinal Organoid Models for Toxicity Assessment

The physiological relevance of cell-based assays can potentially be increased through the use of 3D culture systems, 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 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 the potential toxicity of candidate therapeutics to the intestinal epithelium.

Contract Assay Services offers an in vitro intestinal organoid-based assay to assess the effect of candidate therapeutics on cell viability via measurement of intracellular ATP. Read more about Development of a 96-well Assay for Assessing Cell Viability in Mouse Small Intestinal-Derived Organoids After Treatment with Cytotoxic Compounds.

Learn more about organoid-based assays for intestinal toxicity testing >

  1. Ranga A et al. (2014) Drug discovery through stem cell-based organoid models. Adv Drug Deliv Rev 69-70 19-28.
  2. Sato T, Clevers H. (2013) Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340(6137) 1190-1194.

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Our Assay Services Workflow

  1. One-on-one client consultation.
  2. Preparation of a proposal that clearly defines project scope, timeline and cost.
  3. Experimental execution and data analysis.
  4. Preparation of final report.