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About Contract Assay Services
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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 both 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 extensive 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 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 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 in vitro assays on Mouse intestinal organoids can also be used to assess the effects of candidate therapeutics on cellular viability.to measure the 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.
Contact us at firstname.lastname@example.org 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
- Assesses both the proliferation and differentiation of erythroid (BFU-E) and myeloid (CFU-GM) progenitors
- 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.
- 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
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
- Exhibits correlation with the CFU assay for toxicity levels of a wide range of compounds
- 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
- Provides improved sensitivity in the evaluation of anti-proliferative effects
Figure 2. HemaTox™ Assay Workflow
Figure 3. Correlation Between IC50 and IC90 Values for 43 Test Compounds Using the CFU-GM Assay and the Liquid-Based HemaTox™ Myeloid Assay
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.
AZT Case StudyAzidothymidine (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.
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.
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
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.
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 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
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.
- Ranga A et al. (2014) Drug discovery through stem cell-based organoid models. Adv Drug Deliv Rev 69-70 19-28.
- Sato T, Clevers H. (2013) Growing self-organizing mini-guts from a single intestinal stem cell: mechanism and applications. Science 340(6137) 1190-1194.
Explore our immunological tools and assay systems below and contact us at email@example.com to learn more about how we can help answer your immunological questions.
Immunophenotyping by Flow Cytometric Marker Analysis (Surface and Intracellular)
Figure 1. Flow Cytometry Analysis of Cultured Human Peripheral Blood Mononuclear Cells (PBMCs) with Gating on Viable CD4+ and CD8+ T Cells
Figure 2. Flow Cytometry Analysis of Fresh Human Regulatory T Cells in Total PBMC Sample with Gating on the CD45+ CD3+ CD4+ CD25+ and FOXP3+ Population
PBMC Activation, Proliferation and Viability Assessment
Figure 3. Human PBMC Proliferation After Activation
Human peripheral blood mononuclear cells (PBMCs) were stimulated with different concentrations of ImmunoCult™ Human CD3/CD28 T Cell Activator (Catalog # 10971) for 5 days. PBMCs were labelled with cell tracking dye, and viability was measured by gating on 7-AAD (viability dye) negative cells. Cell division and proliferation were measured by flow cytometric analysis. Each peak represents a cell division.
Quantitation of Cytokines and Growth Factors
Figure 4. Intracellular Flow Cytometric Analysis of Immune Cytokines
Activated human T cells (gated on CD4+ and CD8+ populations) were analyzed by intracellular flow cytometry for IFN-γ and TNF-α expression. After the cells were fixed and permeabilized, antibodies against the cytokine of interest were added to the cell suspension, followed by flow cytometry.
©2013 Meso Scale Discovery a division of Meso Scale Diagnostics, LLC. All rights reserved.
Figure 5. Meso Scale Discovery Multiplex Platform
Schematic representation of Meso Scale Discovery multiplex platform. STEMCELL’s Contract Assay Services (CAS) group is a certified CRO partner of Meso Scale Discovery.
T Cell Suppression Assay
Figure 6. Compounds Can Be Screened for Immunomodulatory Effects Using the Treg Cell Suppression Assay
Fresh Treg cells and PBMCs were purified from a healthy donor. Treg cells and PBMCs were co-cultured in the presence of a solvent, an immunomodulatory compound, or a negative control article and then activated for 4 days with ImmunoCult™ Human CD3/CD28 T Cell Activator in ImmunoCult™-XF T Cell Expansion Medium. (A) The suppression response at the 1:2 Treg:PBMC ratio was 45% for the solvent control, 37% for the negative control article and 89% for the immunomodulator. Co-culturing with conventional CD4+CD25- T cells showed < 10% of the suppression response (mean ± SD, n = 3, single donor). (B) Control experiments demonstrating the proliferation of responder cells in the absence of Treg cells are shown. Cells were cultured in conditions as described above. The immunomodulator alone had a minimal effect on responder cell proliferation (mean ± SD, n = 3, a representative single donor).
Macrophage and Dendritic Cell Differentiation Assessment
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 activity of biopharmaceutical cytokines. 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 be similarly evaluated in animal models. For example, the data below show the effects of erythropoietin (EPO) measured in vitro (Figure 1, Table 1) and in vivo (Figure 2, Table 2), and of 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 you can employ the CFU assay in your organization.
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 and Table 1 below.
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 Total Number of 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 to learn how to take advantage of our stem cell characterization services and assays described below.
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. (2002) Quantitation of Murine and Human Hematopoietic Stem Cells by Limiting-Dilution Analysis in Competitively Repopulated Hosts. In: Klug CA & Jordan CT (Eds.), Methods in Molecular Medicine: Hematopoietic Stem Cell Protocols (pp. 167-187). Totowa, NJ: Humana Press.
2. Miller CL & Eaves CJ. (2002) Long-Term Culture-Initiating Cell Assays for Human and Murine Cultures. In: Klug CA & Jordon CT (Eds.), Methods in Molecular Medicine: Hematopoietic Stem Cell Protocols (pp. 123-141). Totowa, NJ: Humana Press.
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.