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

Mika Pietilä,Kaarina Lähteenmäki,Marko Närhi,Maarit Lönnroth,Petri Lehenkari,Hannu-Ville Leskelä,Jaana Mättö,Siri Lehtonen,Katrina Nordström

doi:10.1186/scrt144

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.

Highlights

One 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
To determine the period of time that the bacterial contamination needs to induce total dissipation of human bone marrow-derived mesenchymal stem cell (BMMSC) even with the lowest bacterial load, we first decided to monitor the infection of BMMSCs for 24 hours
The results show that P. aeruginosa PA01 caused total destruction of BMMSCs within 24 hours of infection (Figure 1b)

Summary

Introduction

One 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. Even though the incidents of microbial contamination of cell products seem to be rare, the risk of contamination is a critical issue since the consequences to patient health may be unpredictable and even devastating [10,11,12,13,14,15,16]. Despite the presence of macrophages, osteoblast-like cells lost the race of growth area on the biomaterial surface in the presence of S. aureus or P. aeruginosa, but the cells survived about 48 hours in the presence of S. epidermidis. These findings highlight the importance of microbial risk management in the prevention of biomaterial-associated infections

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