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About Contract Assay Services
Learn more about CAS at STEMCELL Technologies - how we began, what we’ve done and where we can go with you.
Contact us at firstname.lastname@example.org to learn more about how we can help you meet your goals.
Our Assay Services Workflow
- One-on-one client consultation
- Preparation of a proposal that clearly defines project scope, timeline and cost
- Experimental execution and data analysis
- Preparation of final report
Contract Assay Services at STEMCELL Technologies
We combine the power of specialized STEMCELL Technologies media and reagents with the practical knowledge of our scientists to provide standardized and customized assay services. You can choose from a portfolio of characterized assays using pre-qualified primary stem cells or discuss your individual needs with our in-house experts. Since 2000, Contract Assay Services has performed such studies for over 120 pharmaceutical, biotechnology, government and academic life science organizations worldwide. We provide exceptional service through frequent communication, quality products and unparalleled expertise.
Why Use Contract Assay Services?
- Our high standards for methods, materials, processes and customer communication are evident in the loyalty of our returning clientele.
- As your eyes and ears in the lab, we place a priority on communication with our clients throughout the study process.
- STEMCELL Technologies is a world leader in the development of industry-standard products for stem, progenitor and other primary cells.
- We work directly with the scientists who develop the specialized STEMCELL products used in our assays.
Explore These Resources
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 oncan 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 to measure potential toxic effects of candidate therapeutics, including small molecule compounds and biologics. , 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.
Contact us at email@example.com for a more in-depth discussion on how each of the assays in the sections below can be modified to meet your specific goals.
Hematopoietic Toxicity Assessment
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.
1. Pessina A et al. (2003) Toxicol Sci 75:355-367
2. Pessina A et al. (2009) Toxicol In Vitro 23(1):194-200
The Hematopoietic CFU Assay as Performed by Contract Assay Services:
- Yields clinically predictive information, allowing for better planning and a reduction in in vivo studies
- Uses physiologically relevant primary cells from human, non-human primate, mouse, rat and dog (Table 1)
- Assesses both the proliferation and differentiation of erythroid (BFU-E), myeloid (CFU-GM) and megakaryocyte (CFU-Mk) progenitors
- Provides both quantitative (colony number) and qualitative (cell and colony morphology) data
- Can be used to determine dose response curves, including IC50 and IC90 values (Figure 2)
Table 1. Comparison of Myelotoxicity (CFU-GM) Response to Different Compounds in Human, Mouse, Canine and Rat Models to Drive Selection of Relevant In Vivo Model
Figure 1. Dose Response Curves and IC50 values for Human BM-Derived Erythroid and Myeloid Progenitors Incubated with 5-Fluorouracil
Figure 2. Dose Response Curves and IC50 and IC90 Values Showing Effect of Test Article 1 (TA1) Alone and in Combination with Chemotherapeutics Cisplatin (Cis), Temozolomide (TMZ) and Small Molecule (SN38) on Human BM Myeloid (CFU-GM) Progenitor Growth
Liquid Culture-Based Hematotoxicity Assays as Performed by Contract Assay Services:
- Uses physiologically relevant primary stem cells from human cord blood or bone marrow
- Assesses both the proliferation and differentiation of erythroid, myeloid and megakaryocyte progenitors
- Generally correlative with the CFU assay (Figure 3)
- Provides a higher-throughput capacity for toxicity screening
- Can be used to determine dose-response curves, including IC50 and IC90 values (Figure 4)
- Offers flexibility as test compounds may be added to the culture or cells may be collected from it at different points during culture, allowing effects on progenitor cells at different stages of differentiation to be examined.
Figure 3. Correlation Between IC50 Values for Six Drugs Measured Using the CFU-GM Assay and the 96-Well Plate Liquid Culture-Based Myeloid Cell Hematotoxicity Assay
Human BM CD34+ cells were cultured in colony-forming unit - granulocyte/macrophage (CFU-GM) assays and in liquid culture-based hematotoxicity assays for myeloid cells (HemaTox™ Myeloid Kit). IC50 values generated using each assay were plotted on the X and Y axes and shown to be highly correlated with a correlation coefficient, R2, of 0.91.
Figure 4. Reproducibility of Liquid Culture-Based Hematotoxicity Assay Results Between Experiments and Using Different CD34+ Cell Preparations
Dose-response curves were generated from titrations of 5-FU added to human CB CD34+ cells isolated from 3 donors and cultured with the liquid culture-based hematotoxicity assays for (A,D) erythroid and (B,E) myeloid cells, as well as (C,F) megakaryocytes. Three to five separate experiments were performed with cells from each donor. 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 normalized to the percentages (%) of maximum cell growth without drug. Despite differences in absolute cell counts, curves are reproducible when normalized within each experiment. (D, E, F) Tables showing IC50 values generated for 5-FU in culture with each kit including standard deviation (SD) and the coefficient of variation (% CV) across experiments.
Mesenchymal Toxicity Assessment
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 5. 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 (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).
Immunotoxicity and Immune Profiling
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.
1. Gennari A et al. (2005) J Immunotoxicol 2(2):61-83
Contract Assay Services can customize:
- Flow cytometric analysis and immune profiling of , various , , and
- 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
Contract Assay Services offers an in vitro mouse intestinal organoid-based assay to assess the effect of candidate therapeutics on cell viability via measurement of intracellular ATP.
1. Ranga et al. Drug Discovery Through Stem Cell-Based Organoid Models. Adv Drug Deliv Rev 69-70C: 19-28, 2014
2. Sato T & Clevers H. Growing Self-Organizing Mini-Guts from a Single Intestinal Stem Cell: Mechanism and Applications. Science 340(6137):1190-94, 2013
Biopharmaceutical and Biosimilar Assessment
Biopharmaceutical drugs, also known as biologics, have become an essential part of modern pharmacotherapy and include examples such as biological proteins, cytokines, hormones, monoclonal antibodies, vaccines, and cell- and tissue-based therapies. Biopharmaceuticals may include compounds such as biosimilars, molecules meant to be an improved version of a previously developed drug on which the patent has expired.
The hematopoietic colony-forming cell (CFC) or colony-forming unit (CFU) assay can be used to test the biosimilar activity of biopharmaceutical cytokines, such as erythropoietin (EPO) both in vitro (Figure 1, Table 1) and in vivo (Figure 2, Table 2), or granulocyte-stimulating factor (G-CSF) (Figure 3). Hematopoietic progenitors are isolated from human bone marrow, and plated in a CFU assay to observe the direct stimulatory effects of test articles on progenitor growth and morphology. The effects of these cytokines can also be evaluated in animal models.
Contact us at firstname.lastname@example.org to learn more about how we can help you and your organization reach your goals.
Example Data from Previous Studies
Figure 1. The Effect of EPO on Erythroid Colony Growth in CFU Assays of Human Bone Marrow Cells
Human bone marrow mononuclear cells were isolated from three individual patients and plated in CFU assays using MethoCult™ medium, in the presence of decreasing concentrations of Control erythropoietin (EPO). The percentage of erythroid colonies in samples treated with decreasing concentrations of EPO-based biosimilars can be compared to the number of colonies supported by an optimal concentration of Control EPO, as determined by this graph.
Table 1. EC50 Values for the Effect of EPO on Erythroid Colony Growth in CFU Assays of Human Bone Marrow Cells
Figure 2. Mobilization of Erythroid Progenitors and Increased Erythropoiesis in Mice Treated with EPO
Wild type BDF1 mice were treated with EPO on days 0 and 1. On day 4 the mice were sacrificed and the progenitor content of the spleens was assessed by plating cells in a CFU assay and counting the number of erythroid (BFU-E) progenitors per spleen. The percentage of reticulocytes were also measured by flow cytometry (Table 2).
Table 2. The Number of BFU-E Measured by a CFU Assay and Percentage of Reticulocytes as Measured by Flow Cytometry per Spleen Isolated from Mice Treated with EPO
Figure 3. The Number of Total CFU Observed in the PB of Mice Following Treatment With G-CSF
C3H/HeN mice were treated with 5 µg rhG-CSF per day on days 0 - 2. Eighteen hours after the last G-CSF injection mice were sacrificed and the peripheral blood (PB) was collected. The progenitor content of in the PB of each mouse was then assessed in a CFU assay. Mice treated with G-CSF showed a 10-fold increase in the number of CFUs per mL of PB when compared to vehicle control.
Stem Cell Characterization
Contract Assay Services can provide both phenotypic and functional assessments of your stem cell population of interest. Our scientists are experienced in designing and performing studies to evaluate the effects of novel test articles on the expansion and differentiation capacity of stem cells.
Contact us at email@example.com if you are interested in our stem cell characterization services and assays.
Hematopoietic Stem and Progenitor Cell Characterization
- Engraftment Assays
Complex in vivo assay that allows the determination of the true hematopoietic stem cell (HSC) frequency and effects of test articles on this frequency by transplanting human stem cells into the NOD/SCID mouse model1
- Long-Term Cultures - Initiating Cell (LTC-IC) Assays
Assay to determine the effect of test articles on the in vitro growth of primitive HSPCs using co-culture with stromal cells2
- More information on the LTC-IC assay
- Mobilization Assays
Evaluation of the movement in vivo of HSCs out of bone marrow into peripheral blood following treatment with agents such as Mozobil and cytokines such as G-CSF (Figure 1)
- Hematopoietic Lineage-Specific Expansion Cultures
Ex vivo expansion of HSPCs (CD34+ cells) isolated from human cord blood or bone marrow and differentiation into erythroid, megakaryocyte or myeloid progenitor cells.
- Flow Cytometry and profiling of various cell populations
Including megakaryocytes, myeloid, erythroid, T, B and NK cells
Example Data from Previous Studies
Figure 1. Mobilization of Hematopoietic Progenitors Induced by rhG-CSF and AMD3100
C3H/HeN mice were treated with 5 µg rhG-CSF per day on days 0 - 2. Sixteen hours after the last G-CSF injection, one group of 5 mice was treated with 5 mg/mL of AMD3100 (also know as Mozobil or Plerixafor). Ninety minutes after treatment with AMD3100, mice were sacrificed and peripheral blood (PB) was collected. The progenitor content in the PB of each mouse was then assessed using the CFU assay. Mice treated with rhG-CSF alone showed a 10-fold increase in the number of CFUs per mL of PB, and mice treated with rhG-CSF and AMD3100 showed a 50-fold increase in the number of CFUs per mL of PB, when compared with vehicle control.
1. Szilvassy SJ et al. Quantitation of Murine and Human Hematopoietic Stem Cells by Limiting-Dilution Analysis in Competitively Repopulated Hosts. Chapter 12 Methods in Molecular Medicine Vol 63: Stem Cell Protocols. Edited by Klug CA and Jordon CT Humana Press Inck Totowa NJ Pages 167-187.
2. Miller CL and Eaves CJ. Long-Term Culture-Initiating Cell Assays for Human and Murine Cultures. Chapter 8 Methods in Molecular Medicine Vol 63: Stem Cell Protocols. Edited by Klug CA and Jordon CT Humana Press Inc. Totowa NJ Pages 123--141.
Mesenchymal Stem Cell Characterization
- MSC expansion
- Culture media that allows expansion using high quality media options including xeno- and animal component-free formulations
- Characterization of surface markers
- Including CD90, CD105, CD73, CD34 and CD45
- In vitro differentiation into adipocytes, osteocytes and chondrocytes
- Evaluation of the ability of MSC to suppress T cells (Figure 2)
Figure 2. MSC Suppression of T Cell Proliferation
Using co-culture experiments and the CFSE dye dilution assay, we can show that passage 2 MSCs can suppress division of activated T cells at both day 3 and day 7 of culture.
Custom Stem Cell Assays
Clinical Trial Support
Samples That Can Be Assessed:
- Peripheral blood samples from clinical trials and preclinical studies, pre- and post-drug treatment
- Mobilized peripheral blood patient samples for transplantation
- Cord blood samples for banking and/or transplantation
Parameters Assessed Include:
- Total cell count and viability
- Frequency of hematopoietic stem and progenitor cells for erythroid, myeloid and megakaryocyte progenitors
- Percentage of CD34+ cells quantified by the ISHAGE1 protocol
- Flow cytometric analysis of hematopoietic and immune cells
1. Sutherland et al. (1996). J of Hematoth. 5(3):213-226.