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In Vitro Mesenchymal Cell System Toxicity

  

Mesenchymal stem cell summary of toxicity performed by Hemogenix

 

Please contact HemoGenix® at info@hemogenix.com or call (719) 264-6250 for more information of mesenchymal cell system toxicity testing. Our testing services are also available through Science Exchange.

 

 MSCGlo™-Tox HT and MSCGlo™-Real Time: A MSC/MPC Toxicity Assay Platforms

 

The kinetics of mesenchymal stem/progenitor cell growth

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Analysis of mesenchymal stem/progenitor cell surface markers by flow cytometry

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MSC/MPC can be obtained from numerous primary sources, for example, bone marrow, umbilical cord blood, Wharton's gelee, adipose tissue. The MSCGlo™ assays can be used to study MSC/MPCs from any source, including iPS-derived cells. MSC/MPCs have a finite lifespan in culture. This lifespan is usually measured in the number of passages or the number of doubling times. Withime in culture, the potency and quality of the cells decreases, a fact that is particularly important when MSC/MPC are used in regenerative medicine applications.

These parameters are also important in measuring potential toxicity to the system regardless of the cell source. MSCGlo™-Real Time can be used to measuring the doubling time of MSC/MPCs and determine the growth kinetics, onset of cytotoxicity and other parameters. MSCGlo™-Tox HT is a multifacetted, standardized and validated in vitro end-point assay platform designed to measured multiple parameters of toxicity. 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.

 

Benefits of Using MSCGlo™-Tox HT and MSCGlo™-Real Time for Predictive In Vitro MSC Toxicity Testing
  • MSCGlo™-Tox HT is an end-point, lytic, ATP bioluminescence predictive toxicity assay for MSC/MPCs from any source or species.
  • MSCGlo™-Real Time is a non-lytic, bioluminescence assay for MSC/MPCs from any source or species that can help optimize culture conditions, define growth kinetics, measure doubling time and determine the onset of potential toxicity.
  • Both assays measure cell viability and proliferation/cytotoxicity. Under specific conditions, the onset of MSC/MPC differentiation into chondrocytes, adipocytes or osteoblasts can be determined.
  • Used to analyze effects to the bone marrow stroma (microenvironment).
  • Can be used with other HemoGenix® assays to study the immunomodulatory role of MSCs.
  • Both assays can replace the manual CFU-F assay.
  • Both assays have high-throughput capability using 96- or 384-well plate formats allowing ADME-Tox drug or compound screening, thereby significantly reducing unexpected results during pre-clinical testing.
  • The assays can be used at all stages of drug development.
  • Both are 3Rs Assay Platform-Reduction, Refinement, Replacement for animal testing.
  • MSCGlo™-Tox HT is part of the ComparaTOX™ 1 Platform for comparing and ranking toxicity according to cell type and species.
  • Both assays can use CRUXRUFA human platelet lysate or proprietary, high performance MSCGro™ medium with either low serum, serum-free or humanized formulations.
  • Results are usually available in 2 to 7 days depending on the assay used, source and species.
  • MSCGlo™-Tox HT has been validated according to FDA Bioanalytical Method Guidelines.
  • MSCGlo™-Tox HT standardization allows results to be compared over time and between samples.
  • Both assays have been designed for multiplexing with other assays using the same sample. The non-destructive MSCGlo™-Real Time assay actually allows cells to be phenotypically analyised by flow cytometry during culture.

 Analyzing the MSC/MPC System

Two types of assay procedures have been used to characterize and identify MSC from different sources. These include the subjective Colony-Forming Unit - Fibroblast (CFU-F) assay and flow cytometry. The CFU-F assay is, in many ways, similar to the CFC assay for hematopoietic cells. Unlike the CFC assay, the CFU-F assay requires that the cells are grown under adherent conditions, usually in large petri dishes (60-100mm in diameter). Since the MSCs represent a continuously proliferating system, it is important that when the colonies of fibroblasts are counted under an inverted microscope, cell confluency has not occured. The assay lacks standards and controls and has not be validated according to regulatory requirements. Although flow cytometry is usually used to charcterize MSCs by the expression (or lack of) membrane markers, flow cytometry provides no indication regarding the functional capability or capacity of the MSCs.

 

MSCGlo™-Tox HT is a validated MSC/MPC cytotoxicity assay that can be multiplexed with the following (and other) assays:

  • Flow cytometry to characterize the cells, perform cell cycle analysis and apoptosis.
  • Growth factor, cytokine, chemokine production and release.
  • Apoptosis detecting caspases.
  • Membrane integrity.
  • Mitochondrial dysfunction.
  • Oxidative stress.
  • Oxidative DNA damage (OxyFLOW™, see below).
  • Kinase assays.
  • Protease assays.
  • Lactate dehydrogenase assays.
  • Cell-based drug interaction.
  • Residual toxicity and drug sensitivity change assays.

 Examples of MSC/MPC Toxicity Testing

 

LUMENESCTox

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Toxicity to Mesenchymal Stem/Progenitor Cells

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. 

 

Cellular Drug-Drug Interaction

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.