Set-up of a pharmaceutical cell bank of Magnetospirillum gryphiswaldense MSR1 magnetotactic bacteria producing highly pure magnetosomes

Abstract

We report the successful fabrication of a pharmaceutical cellular bank (PCB) containing magnetotactic bacteria (MTB), which belong to the Magnetospirillum gryphiswaldense MSR1 species. To produce such PCB, we amplified MTB in a minimal growth medium essentially devoid of other heavy metals than iron and of CMR (Carcinogenic, mutagenic and reprotoxic) products. The PCB enabled to acclimate MTB to such minimal growth conditions and then to produce highly pure magnetosomes composed of more than 99.9% of iron. The qualification of the bank as a PCB relies first on a preserved identity of the MTB compared with the original strain, second on genetic bacterial stability observed over 100 generations or under cryo-preservation for 16 months, third on a high level of purity highlighted by an absence of contaminating microorganisms in the PCB. Furthermore, the PCB was prepared under high-cell load conditions (9.10⁸ cells/mL), allowing large-scale bacterial amplification and magnetosome production. In the future, the PCB could therefore be considered for commercial as well as research orientated applications in nanomedicine. We describe for the first-time conditions for setting-up an effective pharmaceutical cellular bank preserving over time the ability of certain specific cells, i.e. Magnetospirillum gryphiswaldense MSR1 MTB, to produce nano-minerals, i.e. magnetosomes, within a pharmaceutical setting.

Keywords: Magnetospirillum gryphiswaldense MSR1; Cell Bank; Fed-batch cultivation; Magnetosome; Magnetotactic bacterium; Minimal medium; Pharmaceutical grade nanoparticle; Scale-up.

Fig. 1

Transmission electron micrographs of a single magnetotactic bacterium originating from the CCB (a), or PCB (b). Both bacteria display a similar spirilla shape, a similar length of 1.2 µm and the presence of a chain of magnetosomes inside their organism

Fig. 2

Evolution of the biomass estimated in gram of dry cell weight (DCW) per liter of growth medium (a), and intracellular iron concentration (b), during the growth step under pH–stat fed-batch culture lasting 140 h, starting the fermentation using unfrozen MSR1 originating from a freshly obtained CCB (unfrozen CCB), unfrozen MSR1 originating from a freshly prepared PCB (unfrozen PCB), unfrozen MSR1 from the PCB stored during 16 months at − 80 °C (16 months stored PCB). The error bar originates from the cultures that were carried out in duplicate

Fig. 3

Histograms indicating the mass of each metallic element from toxic classes I, IIa, IIb and III containing for 1 g of iron contained in magnetosomes originating from CCB and PCB. Elements are regrouped in classes according to ICH-Q3D standards and green and blue values indicates the cumulative amount of each element in a given class of the mass of each metallic element containing for 1 g of iron