In Vitro Immunotoxicity Testing
MSC Toxicity and
Mesenchymal Stem Cell Studies
Cell Systems Studies and
In vitro immunotoxicity testing is usually performed as a two tier approach. Bioavailability is usually ascertained, but low concentrations of the compound does not necessarily indicate lack of immunotoxicity.
Myelotoxicity is usually considered the first line of in vitro immunotoxicity testing using a colony-forming cell (CAMEO™-4 or CAMEO™-96) assay or HALO®. Myelotoxicity is toxicity to the bone marrow and does not implicate any specific cell type. The cell population normally considered for myelotoxicity is the granulocyte-macrophage colony-forming cell (GM-CFC) population. As its name implies, this population detects progenitor cells of the granulocyte-macrophage lineage. Detection of this population will provide information on whether a compound or drug will cause neutropenia, but will not provide any information regarding potential toxicity to the other hematopoietic lineages (erythropoietic, megakaryopoietic) and certainly no information on whether the stem cells are affected. More importantly, no information will be obtained as to whether there might be an affect on the T- and B-lymphocyte cell lineages, which are relevant to in vitro immunotoxicity. This is because damage to one or both of these lineages will affect the production of functionally mature T- and B-lymphocytes. The T- and B-lymphocytes are derived from the T-CFC and B-CFC progenitor cells, respectively. These, in turn, are themselves derived from lympho-hematopoietic stem cells present in the bone marrow. The primitive stem cell population that occurs at the point where lymphopoiesis diverges from hematopoiesis is called the High Proliferative Potential - Stem and Progenitor Cell (HPP-SP). Damage to this stem cell population will affect both hematopoiesis and lymphopoiesis.
Therefore, to initially investigate immunotoxicity, it is necessary to assess the effects of compounds and/or drug on 3 primary cell populations:
Responses to one or all of these cell populations can be performed using the following assays:
Both CAMEO™-96 and HALO™-Tox HT are validated assays.
Compounds and/or drugs are tested with postive and negative controls in a dose-dependent manner to estimate IC values.
The second tier of in vitro immunotoxicity testing involves specific assays that measure a number of different T- and B-cell properties and parameters. These include the following assays for innate and aquired immunity.
The above assays are usually perform on human peripheral blood cells. Purified populations can also be employed (e.g. CD4, CD8, NK). In addition, different animal species can also be used upon request. However, for some assays, species-specific reagents may not be available.
If an assay is not listed, please contact HemoGenix to see if it might be available or can be developed and validated for a specific study.
Mesenchymal stem/stromal cells can be obtained from numerous primary sources, for example, bone marrow and umbilical cord blood. As stated above and elsewhere on this website, the MSCs represent a heterogenous, fibroblastoid-like cell population that are usually assayed using the Colony-Forming Unit-Fibroblast (CFU-F) in vitro culture technique. This is a cumbersome and subjective assay to perform that cannot be properly validated. Its very low throughput capability does not lend itself to modern toxicology requirements. As a result, HemoGenix® developed the MSCGlo™-Tox HT Platform (formerly LUMENESC™), a bioluminomics™ assay platform that has high throughput and multiplexing capability. Available with 96- or 384-well plates, MSCGlo™-96 Tox or MSCGlo™-384 HT allows the quantitative and reliable assay of MSCs in response to virtually any drug, compound or other agent that might damage the MSC system. The graphs show the exponential increase, measured using MSCGlo™, of human bone marrow MSCs when passaged and expanded over a 44 day period. After 44 days of MSC culture, MSCGlo™ can detect a linear dose range from at least 500 MSC/well to 50,000 MSC/well. On days 28, 36 and 44, phenotypic analysis of the MSCs detected by multiplexing with MSCGlo™ demonstrates how the MSCs change with time in culture.
To demonstrate how drugs, in particular anti-cancer drugs, can affect MSCs, consider the effect of doxorubicin (Adriamycin) on human bone marrow MSCs shown in the graph. The MSCs were tested at two different cell doses. In both cases, there was significant cytotoxicity.
Drug-drug interaction can activate or inhibit CYP450 enzymes. Although occuring primarily in hepatocytes as the cells responsible for detoxification, drug interactions can occur in many other cell type. Although little, if anything, is known regarding the response of MSCs to drug interactions, drug-drug interaction assays can be performed in a similar manner to those for the lympho-hematopoietic stem cells using HALO®-DDI. For more information, please contact HemoGenix® directly.
Primary hepatocytes have a full complement of enzymes used for metabolizing drugs and other compounds. The donor-to-donor variability might be considered a disadvantage, but provides a better representation of the in vivo situation than the use of cell lines. The ability of hepatocytes to metabolize drugs and detoxify harmful compounds is primarily due to the presence of the cytochrome P450 system (CYP450). The inhibition or induction of enzyme isoforms of this system is also responsible for potential drug-drug interactions. The CYP450 system is contained within the mitochondria of the cells and the integrity of the mitochondria, and indeed the cell overall, is dependent upon the ability of the mitochondria to produce intracellular ATP (iATP). It therefore follows that the ability of the hepatocytes to function properly is, in part, dependent upon the mitochondria to produce sufficient energy in the form of iATP. Although impaired mitochondrial function can be detected by the inability of the cells to reduce a terazolium salt (MTT), the most sensitive indicator would be the measurement of iATP itself.
Consistant with the HemoGenix® paradigm of providing trustworthy assays, the company incorporated its proven bioluminomics™ concept and principles to the field of hepatotoxicity by extending its contract services and producing assay kits that can be employed in this important area of early drug screening.
The HepatoGLO™-HT Platform can be used for fresh or cryopreserved primary human or animal hepatocytes as well as hepatocyte cell lines. HepatoGLO™-HT contract services use either 96- and 384-well plate formats. Hepatocytes are usually incubated for 4-24 hours (depending on the study) with the test compound(s). All human HepatoGLO™-HT contract service assays include the necessary vehicle controls as well as a positive control, usually perhexiline, and a negative control, theophylline. In contrast to most hepatocyte assays, HepatoGLO™-HT also includes an external ATP standard and high and low controls for calibration and assay standardization, thereby allowing results to be compared over time. For examples of hepatotoxicity, please navigate to PRODUCTS: XVPrime™: HepatoGLO™-HT. The HepatoGLO™-HT Platform is also used for drug-drug interaction at the cellular hepatocyte level. Together with CYP450 information that can also be included in hepatotoxicity studies, HemoGenix® can provide more information than most contract service organizations.
HepatoGLO™-HT, like all other biolumnomics™ assays from HemoGenix® has multiplexing capability so that other assays performed on hepatocytes can be setup and performed in parallel. Multiplexing assays are described on a previous page, but the combination of HepatoGLO™-HT with cytochrome P450 enzyme determination is probably the most important.
In addition to studies and toxicity testing on the lympho-hematopoietic and mesenchymal stem cell systems and hepatocytes, HemoGenix® also performs contract studies on numerous other cell systems. These cell systems may be derived from various species (although not all may be available) and include organs and tissues such as:
From these organs and tissues, cell types that may be available include:
Primary cells from these cell systems are usually used in their early proliferating state. In addition, transformed cell lines can also be used. Different cell types require different culture reagents and conditions. The primary assay platform for these studies is LumiSTEM™-Tox HT.
XVPrime™-Tox HT is a bioluminomics™ assay platform developed by HemoGenix® for in vitro toxicity testing for ex vivo primary explanted cells. Contract services utilizing the XVPrime™-Tox HT Platform will use either a 96- or 384-well plate format, depending on the size and type of studies being performed. Specific culture reagents and conditions will be applied to the cell type being analyzed, regardless of whether primary cells or cell lines are used. As with all bioluminomics™ assays, XVPrime™-Tox HT is calibrated and fully standardized prior to processing the samples being tested. For all contract services, the necessary basic controls are included as well as positive and negative compound controls.
Performing additional assays on the same cell sample and under the same conditions in parallel with LumiSTEM™-Tox HT can provide a wealth of information. Some of the assays that can be multiplexed with LumiSTEM™-Tox HT are listed on a previous page. Specialized assays may also be performed.
Part of the contract service may involve further characterization of the cells being used as target cells. Sometimes, cells are best tested under clonal conditions rather than in suspension or adherence. In such cases,XVPrime-Clone™ is used. XVPrime-Clone™ is a methylcellulose clonal assay that allows both proliferation and differentiation to be studied using the same assay platform. XVPrime-Clone™ is analogous to CAMEO™-96 for lympho-hematopoietic stem and progenitor cells. Please see Contract Services for Stem Cell Studies on the next page for more information.