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Contract Services     Page   1  |  2  |  3  |  4  

  • Stem Cell Studies and

    Toxicity Testing

  • Predictive Residual Toxicity

  •  Drug-Drug Interaction

  • OxyFLOW™

  • ComparaTOX™

Contract Services for Stem Cell Studies and Toxicity Testing


Their ability to self renew and their high proliferation potential and ability make stem cells, regardless of their source, prime targets for many drugs, compounds and other agents. Indeed, stem cells are probably the most sensitive of all cells to insults. If the stem cells are damaged, their ability to perform their normal functions, may not only be impeded, but may result in the development of a cancerous state. For the continuously proliferating systems of the body, eradication of the stem cell compartment by toxic agents will lead to eradication of the system altogether and a domino effect that could be fatal. Therefore knowledge of stem cell properties and characteristics from different cell systems will provide more predictive information on the limits of the system and to how the system will respond to insults.

HemoGenix® has considerable expertise and experience with different stem cell systems and developing assays for those systems. It might be of interest to the reader that metabolism, cell functionality and proliferation occur in a daily or circadian rhythm. Circadian rhythms are seen in virtually all proliferating cells and stem cells are no exception. From a drug administration viewpoint, the time at which a drug is given to a patient will determine the efficacy and the extent of toxicity not only to the cells being targeted but also to the normal cells. The assay platforms developed by HemoGenix® for contract services, HALO®, LUMENESC™ and STEMAssays™, were not only developed for analyzing toxicity, but have many other applications, including chronbiology and chronotoxicology.


Assays on Stem Cell Systems

The first assay platform specifically developed to detect, measure and analyze stem cell toxicity was HALO®. Launched in March 2002, the HALO® Platform was originally designed with high throughput capability to detect toxicity to the lympho-hematopoietic system. Following that came the LUMENESC™ platform to detect toxicity to the mesenchymal stem/stromal cell system. STEMAssays™ is third platform and include STEMClone™. The recent explosion of primary and stem cell lines has meant that development of standardized and validated assays specific for different stem cell populations lags behind the identification of the stem cells. With numerous ES and iPS cell lines available, generalized maintenance and stimulatory culture conditions can vary widely. Similarly, the maintenance, regulatory and differentiation conditions of primary stem cells from different sources will also vary widely. The philosophy underlying the STEMAssays™ Platform was that regardless of how stem cells are cultured, a common readout could be used that allowed results not only to be compared from one experiment to another, but from one stem cell type to another. The concept and principles of bioluminomics™ achieve that end with remarkable reliability and reproducibility. This means that the greatest attention can be paid to the in vitro culture of different stem cells and how they respond to drug and other compounds and insults.

The ability to culture stem cells under different, but optimal conditions for the goal of the study may involve culture not under suspension or adherent conditions, but under clonal conditions. Indeed, sometimes it is necessary to know whether the cells being studied can be defined as stem cells by their ability to grow under secondary or even tertiary re-plating conditions. Proliferation potential as defined by the slope of the cell dose response (and is a measurement of potency) also helps define cells as stem cells. For clonal growth, STEMClone™ is used. This methylcellulose assay system is analogous to CAMEO™-96 for lympho-hematopoietic cells, in that not only is cell clonality determined, but also the ability to proliferate and differentiate. Thus, stem cell toxicity can be assessed under varying conditions.


Stem Cell Predictive Residual Toxicity (PRT)

When the potential toxicity of a drug or compound is initially assessed, it may be noticed that not all of the stem cells are affected; a proportion of stem cells are either resistant or are unaffected by the treatment. Potential change in drug or compound sensitivity and the ability of the residual cells to repopulate and/or expand after one or more treatments are important factors in understanding the response and toxicity to the stem cell system. For this reason, HemoGenix® developed Predictive Residual Toxicity Assays, highly specialized assays for stem cell systems.  


Multiplexing Stem Cell Assays

The ability to obtain as much information as possible about the response of a specific stem cell population is dependent not on a single assay, but on multiple assays using the same sample. This is called assay multiplexing and all HemoGenix® bioluminomics™ assays have been designed with multiplexing capability. This means, for example, that it is not only possible to determine if a drug or other agent is cytotoxic, but the mechanism underlying cytotoxicity. It may be necessary to determine cell number or perform phenotypic or gene expression analysis on the same samples. All this and many other parameters can be performed in parallel with any of the STEMAssays™.


More Information

It is obvious that the possibilities for stem cell studies and toxicity testing are too numerous to mention. To discuss our contract services in this or any other area, please contact HemoGenix® and speak to our CSO. We will be happy to discuss the needs and goals of any study under complete confidentiality. 

 

Contract Services for Predictive Residual Toxicity (PRT)


Residual toxicity is primarily important for stem cells and the stem cell compartment. A drug or other insult may affect stem cells in different ways. The effect may be felt on stem cells that are quiescent as well as those in cell cycle and proliferating. Small molecules can enter quiescent stem cells. When called upon to enter the cell cycle and proliferate, the effect of the agent may partially or completely inhibit this function. After treatment of quiescent or proliferating stem cell populations, it may be noticed that not all of the stem cells are affected. This may be due to resistance to the agent or perhaps a proportion of the stem cells were in a phase in which they were unaffected by the treatment. The importance of this residual stem cell population lies in the fact that these stem could repopulate or replenish the system if allowed to do so. However, changes to the residual stem cells may have occurred. These could include potential changes in drug or compound sensitivity and/or the ability of the residual stem cells to expand after one or more treatments. These are important factors in understanding the response and toxicity to stem cells. For this reason, HemoGenix® developed Predictive Residual Toxicity Assays that are highly specialized assays for stem cell systems.


Measuring Residual Toxicity to Stem Cells

There are several permutations, but the basic methodology occurs as follows:

  1. Perform a drug or compound dose response on one or more stem cell populations within the stem cell compartment in a primary in vitro cell culture system.
  2. Analyze the results to determine if residual stem cells remain after the first treatment.
  3. Remove cells treated with different doses from the primary cell culture and perform a secondary, re-plating culture of the treated cells.
  4. Analyze the secondary response for changes in dose sensitivity and expansion potential.

 

 

HALOPRT

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Assays to Determine Predictive Residual Toxicity (PRT)

Performing Predictive Residual Toxicity assays depends primarily on an understanding of the possible response of the stem cell population(s) in question and how the stem cell population(s) can be detected. These assays are available for lympho-hematopoietic stem cells using the HALO®-96 PRT assay and for other stem cell systems using the STEMGlo™-96 PRT assay. For ex vivo primary explanted cells, XVPrime™-PRT is available. To perform a PRT assay, it may be necessary to carry out some preliminary studies.

An example of a PRT assay is shown. In this example, residual toxicity is determined for Daunorubicin. In the first, primary culture, a Daunorubicin dose response is performed for both the primitive lympho-hematopoietic (HPP-SP) and more mature hematopoietic (CFC-GEMM) stem cells. After secondary re-plating of the cells from the HPP-SP dose response, the results shown in the right-hand graph were obtained. In this case, both the HPP-SP 1 and HPP-SP 2 primitive stem cell populations were detected. The HPP-SP 2 assay detects the ability of the HPP-SP 1 cells to expand. Since Daunorubicin results in eradication of the both primitive and mature stem cell populations at high doses (left graph), it is clear that few if any residual cells will remain as depicted in the right-hand graph. However, it is also clear that the HPP-SP2 stem cell population has increased its sensitivity to the drug at all IC values. 

Contract Services for Drug-Drug Interaction Studies and Toxicity


Drug-drug interaction (DDI) studies are usually restricted to whether a drug or compound induces or inhibits one or more enzymes of the Cytochrome P450 (CYP450) system in the mitochondria of hepatocytes. The effect can be an unwanted augmentation or decrease in the dose of a drug administered to a patient, that can lead to severe side effects. However, hepatocytes are not the only cells endowed with a CYP450 system. All cells possess this system, although not all cells will possess the same CYP450 enzymes. For example, lympho-hematopoietic cells in the bone marrow are quite capable of metabolizing 5-fluorouracil in vitro to cause stem cell toxicity and a hypocellular bone marrow. If cells outside the liver can metabolize drugs and other compounds, it follows that potential drug-drug interactions can occur in other organs and tissues at the local level and could have local cellular effects.


How is Drug-Drug Interaction Detected at the Cellular Level?

The usual method of detecting DDI is to perform assays that will demonstrate either an induction or inhibition of CYP450 enzymes. At HemoGenix®, 1A2, 2C9, 3A4, 2C19 and 2D6 as well as other CYP450 isoforms are detected. However, this is essentially at the sub-cellular level and provides no information regarding potential effects seen at the cellular level on specific cell populations. HemoGenix® has developed assays specifically designed to determine whether two or more drugs cause affects at the cellular level. The assays developed for drug-drug interaction studies are usually performed on stem cell populations:

  • HALO®-384 DDI for lympho-hematopoietic stem cells.
  • MSCGlo®-384 DDI for mesenchymal stem/stromal cells.
  • STEMGlo™-DDI for other primary stem cells and stem cell lines.
  • HepatoGlo™-DDI for hepatocytes.


When evaluated individually, a drug or other compound, may demonstrate one of four primary responses:

  1. Little, or no effect.
  2. A stimulatory or potentiation effect. 
  3. Partial inhibitory or cytotoxic effect.
  4. Cytotoxic effect.


The form of the dose response curve produced will indicate the type of response. However, when two drugs or compounds are titrated against each other, the response can be quite surprising and different to that expected. Drug-drug interaction will often be indicated by a U-shaped dose response curve, indicating that at a specific dose, the effect of one drug or compound is counteracting the effect of the other. Examples for Cimetidine and Warfarin and Cyclosporine and Verapamil are provided by navigating to Products: HALO®: HALO®-384 DDI.

Contract Services for Oxidative DNA Damage Using OxyFLOW™


A number of disease or pathological states can be attributed to or involve oxidative DNA damage. These include, but are not limited to:

  • Arthritis
  • Stroke
  • Ischemia-reperfusion therapy
  • Neurodegenerative disorders
  • Inflammation
  • Crohn's disease
  • Multiple sclerosis
  • Teratogenesis
  • Carcinogenesis


In addition, a number of biological processes have mechanisms related to or directly involved with oxidative DNA damage. These include:

  • Aging
  • Normal metabolic activity
  • Drug therapy
  • Oxidative stress
  • Nutrient deficiency
  • Genotoxicity
  • Radiation
  • Xenobiotics


Oxygen free radicals are highly active species that cause significant damage to DNA. One of the products is 8-oxoguanine. OxyFLOW™ is a flow cytometric assay for cell suspensions derived from non-adherent or adherent cells that detect 8-oxoguanine adducts as a result of oxidative DNA damage. A fluorescein isothiocyanate (FITC) - conjugated 8-oxoguanine binding protein is used to detect the presence of these adducts. 

 

 

OxyFLOWMeB

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OxyFLOW™ - Assay Principle

The target cells are prepared into a single cell suspension. The cells are first subjected to permeation followed by incubation with the FITC-conjugated binding protein. The binding protein enters the cells and binds to 8-oxoguanine adducts. After washing the cells, they are suspended in buffer and analyzed by flow cytometry. Target cells are also used to perform the methylene blue standard curve which is performed in parallel. By incubating cells with methylene blue and exposing them to bright light, single-standed DNA damage occurs. With increasing doses of methylene blue, there is an increase in fluorescence intensity, indicating a concomitant increase in oxidative DNA damage. At high doses, the fluorescence intensity decrease. This is an indication of cell death due to oxidative DNA damage. An example of a methylene blue standard curve is shown in the diagram to the left.

 

 

OxyFLOW&HALO

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Multiplexing OxyFLOW™ with HALO®-96 Tox

Etoposide causes single- and double-stranded DNA breaks. The diagram to the left shows the result of treating normal human peripheral blood with etoposide in a dose-dependent manner. The mononuclear cells were incubated for 4 or 7 days with etoposide. A aliquot from each dose was used to determine the presence of oxidative DNA damage using OxyFLOW™. A second aliquot was cultured to determine the effect of etoposide on peripheral blood in vitro multipotential stem cells (CFC-GEMM). A dimethylsulphoxide (DMSO) vehicle was used to dissolve the test compound. The results from OxyFLOW™ demonstrate that DMSO itself, can produce a significant toxic effect on the cells. When the dose of etoposide increases, there is an increase in the fluorescence intensity up to 1µM. This corresponds to the levels of intracellular ATP seen up to 1µM. Doses greater than 1µM cause a decrease in fluorescence intensity that correlates with the decrease in intracellular ATP levels indicating cytotoxicity. At the highest dose (0.1mM) of etoposide, few cells remain to be detected using OxyFLOW® and this again correlates with the very low intracellular ATP levels found using HALO®. This is not only an excellent example of assay multiplexing, but clearly demonstrates that HALO®-96 Tox can be used as a cytotoxic assay and that the mechanism of action of etoposide can be elucidated with OxyFLOW™. 

ComparaTOX™-1: In Vitro Comparative Toxicity Contract Services

What is ComparaTOX™?
 
ComparaTOX is a unique, high throughput in vitro cytotoxicity screening platform that directly compares the response of cell types from different organs, tissues and species to drugs and other compounds using a single ATP bioluminescence readout technology.

Advantages of using ComparaTOX™-1 to Rank Toxicity of Compounds
 
  • ComparTOX™ is used to detect potential toxicity in an array of different cell types derived from most cell systems and up to five different species.
  • The assay platform uses the most sensitive ATP bioluminescence readout technology to measure potential cytotoxicity.
  • ComparaTOX™-1 is built on Bioluminomics™ Technology that incorporates assay calibration and standardization to directly compare the response of different cell types using a single readout.
  • ComparaTOX™-1 allows the ranking and direct comparison of IC and Area Under the Curve (AUC) values.
  • Incorporates high throughput capability (Z Factor > 0.7) using either 96- or 384-well plates formats.
  • The assay platform is designed specifically for proliferating or high metabolic rat cells.
  • Both adherent and non-adherent cell types can be used.
  • Each cell type is cultured in its own specific growth medium.
  • Applicable to 3D in vitro cultures.
  • A 3Rs Assay Platform: Helps Reductioin, Refinement, and Replacement for animal testing.
  • Validated assay readout according to FDA Bioanalytical Method Guidelines.
  • Available as GLP or non-GLP contract services.
  • Fast turnaround time: Usually within 7 days or less depending on cell type and species.

For more information on ComparTOX™ Contract Services, please download the ComparaTOX™ Information Flyer or contact HemoGenix® to discuss how ComparaTOX™ can be used yo achieve the goals of comparative toxicology.