Monitoring mitochondrial inner membrane potential for detecting early changes in viability of bacterium-infected human bone marrow-derived mesenchymal stem cells

Abstract

IntroductionOne of the most challenging safety issues in the manufacture of cell based medicinal products is the control of microbial risk as cell-based products cannot undergo terminal sterilization. Accordingly, sensitive and reliable methods for detection of microbial contamination are called for. As mitochondrial function has been shown to correlate with the viability and functionality of human mesenchymal stem cells (hMSCs) we have studied the use of a mitochondrial inner membrane potential sensitive dye for detecting changes in the function of mitochondria following infection by bacteria.MethodsThe effect of bacterial contamination on the viability of bone marrow-derived mesenchymal stem cells (BMMSCs) was studied. BMMSC lines were infected with three different bacterial species, namely two strains of Pseudomonas aeruginosa, three strains of Staphylococcus aureus, and three strains of Staphylococcus epidermidis. The changes in viability of the BMMSCs after bacterial infection were studied by staining with Trypan blue, by morphological analysis and by monitoring of the mitochondrial inner membrane potential.ResultsMicroscopy and viability assessment by Trypan blue staining showed that even the lowest bacterial inocula caused total dissipation of BMMSCs within 24 hours of infection, similar to the effects seen with bacterial loads which were several magnitudes higher. The first significant signs of damage induced by the pathogens became evident after 6 hours of infection. Early changes in mitochondrial inner membrane potential of BMMSCs were evident after 4 hours of infection even though no visible changes in viability of the BMMSCs could be seen.ConclusionsEven low levels of bacterial contamination can cause a significant change in the viability of BMMSCs. Moreover, monitoring the depolarization of the mitochondrial inner membrane potential may provide a rapid tool for early detection of cellular damage induced by microbial infection. Accordingly, mitochondrial analyses offer sensitive tools for quality control and monitoring of safety and efficacy of cellular therapy products.

Interaction with Intestinal Epithelial Cells Promotes an Immunosuppressive Phenotype in Lactobacillus casei

“…It is well known that proliferation of bacterial contaminants in cell cultures can be extremely efficient. In our previous study, we have shown that infection of host cell cultures with Staphylococcus and Pseudomonas species leads to at least five log increase in bacterial numbers within 24 hours [37] . On the contrary to pathogenic bacteria, proliferation of L. casei in cell culture conditions was moderate, and the number of bacteria remained at approximately 10 8 cfu/ml even during prolonged incubation (data not shown).…”

Pioglitazone Protects Compression-Mediated Apoptosis in Nucleus Pulposus Mesenchymal Stem Cells by Suppressing Oxidative Stress

“…Mitochondrion dysfunction plays an important role in cell death [44]. Mitochondria, as a key regulator of cellular processes acting as cellular oxygen sensors [45], not only produce adenosine triphosphate (ATP) but also regulate cell proliferation and death [46].…”

The effect of temperature on the viability of human mesenchymal stem cells

“…This has raised hopes of alternative stem cell therapies for the treatment of a number of degenerative conditions [14]. It was proposed that the addition of hMSCs onto allograft could restore the integrity of bone structure and enhance new bone formation.…”

“…Previous studies show that human mesenchymal stem cells (hMSCs), most commonly derived from bone marrow, can be proliferated and differentiated into bone lineage using tissue engineering techniques [ 11 - 13 ]. This has raised hopes of alternative stem cell therapies for the treatment of a number of degenerative conditions [ 14 ]. It was proposed that the addition of hMSCs onto allograft could restore the integrity of bone structure and enhance new bone formation.…”