1 Motivation and background
3.4 Systematic comparison of single-cell cultivation technologies
3.4.5 Discussion
the cell division as a result from the snapping postfission movement (snapping division) [250, 251]. The snapping division involves a sudden swing, during which the outer membrane of the two emerging cell poles is still interconnected at their proximal ends.
This feature is particularly well suited for evaluating the effect of spatial restriction or an applied external force on the cells during the cultivation in the respective cultivation system. For a quantitative description, the angular arrangement of the cells immediately after the division event was used.
With an average angle of 73.2° ± 6.6 (CV= 9.1%) the division angle of cells cultured in nDEP is 22% smaller than of cells cultured with the MGC system with an average angle of 96.1° ± 30.5 (CV= 31.7%) (Figure 3.26 G-I). A tendency towards higher division angles was observed for cells cultivated with agarose pads, with cells exhibiting an average division angle of 107.8° ± 22.2 (CV= 20.6%). The frequency distribution of division angles was very sharp and normally distributed with the nDEP system. A broader distribution of division angles was observed with the MGC system and agarose pads.
Division angles were broadly distributed in a more random fashion compared to the nDEP system, which may result from an inhomogeneous adhesion of the cells to adjacent surfaces and local variations in the extent of the spatial constriction.
3.4 Systematic comparison of single-cell cultivation technologies
107 Biological discussion
However, besides these technological characteristics, inherent influences of the cultivation technology itself on cellular physiology of C. glutamicum could be clearly demonstrated. The analysis of growth revealed highly consistent specific volume growth rates of single-cells and micropopulations for all three investigated systems despite distinct differences in trapping principle and the type of environment (nDEP: levitation – liquid medium – continuous perfusion; MGC: surface contact – liquid medium – mainly diffusion; agarose pad: surface adhesion/embedding – semi-solid medium – diffusion).
Throughout all performed growth experiments, the measured specific growth rates exceeded those of populations cultivated with bulk systems like shake flasks [73]. This indicated that during cultivations of at least 2 h, growth conditions were provided that allowed the cells to exploit their maximal biological capacity in terms of growth rate. We deduce from these results that stress resulting from adaptation to changing environmental conditions, which inevitably occurs in bulk cultivations and was previously shown to impair growth, is minimized in all three systems [18, 252]. Only such state of equilibrium between the cell and its extracellular environment enables an unbiased analysis of the true response of cellular physiology to exogenic stimuli.
In addition to elevated and robust specific volume growth rates, none of the cultivated single cells showed a detectable lag-phase after introduction into the cultivation devices. This observation was especially surprising for agarose pad cultivations, since recent population-based studies reported that a change from liquid medium to semi-solid medium involves stress resulting in temporary growth arrest in bacteria [253]. However, despite differences of agarose pads and agar plates in terms of cell exposure to ambient air, our data on single-cell growth suggest to study this phenomenon in detail. Our results clearly demonstrate the superiority of the employed single-cell cultivation systems in comparison to bulk approaches when it comes to an accurate description of fundamental biological parameters. Our work also introduces a reliable and universal method for the quantitative description of specific growth rates at a single-cell level, which is of high practical relevance for biological single-cell analyses.
A further important implication of our work is that the dielectrophoretic force during nDEP trapping does not affect cellular physiology of C. glutamicum with the chosen trapping parameters. The origin for previously reported adverse effects of nDEP trapping on cellular viability [254] is thus to be sought elsewhere, for example in insufficient compensation of temperature effects induced by resistive joule heating.
However, in contrast to the consistent volume growth rates, division rates of cells grown on agarose pads significantly deviated from nDEP and MGC cultivations. Analysis of cellular morphology during this study disclosed significant irregularities during cultivation of C. glutamicum on agarose pads. Differences in division rate could be clearly assigned to elongation on agarose pads. Cells continually inclined towards an elongated cell shape, while cells grown with nDEP and MGC did not show such behavior. The process of cell elongation in C. glutamicum was shown to be induced by multiple stresses,
for example, DNA damage or nutrient starvation during stationary phase. It has been speculated that an alteration of the usually tightly regulated cell morphology represents a protective strategy of many microorganisms against antibiotic activity, solvent stress or starvation [255]. Remarkably, specific volume growth rates of single cells were steady and not influenced by cell elongation, whereas division rates were significantly reduced.
A further remarkable observation was the tendency of C. glutamicum towards asymmetric division on agarose pads, since several studies revealed that C. glutamicum exhibits a symmetric type of cell division [40]. Asymmetry in cell division points to a disturbance in chromosome segregation, which is responsible for the tightly controlled midcell division positioning [256].
Even though reasons for the elongation and deviating division symmetry of C. glutamicum on agarose pads cannot be unambiguously disclosed on the basis of our data, it can be yet narrowed down to two central aspects. Both, the static environment which is influenced by the metabolic activity of the cell as well as tight embedding of cells in between the agarose layer and glass cover slip clearly distinguish agarose pads from the other two systems.
We therefore utilized the snapping movement of C. glutamicum upon cell division as a three dimensional sensor in order to evaluate the extent of spatial restriction of the cells for each of the cultivation systems. Significant spatial restriction, resulting in increased snapping angles after cell division, suggests a strong spatial restriction of cells embedded in the agarose pads. Since influences of the extracellular environment would be manifested in decreased specific growth rates, we speculate that the spatial restriction of the cells on agarose pads is the origin of stress that triggers elongation. In comparison to nDEP trapping, an increased division angle distribution could be also observed for cells cultivated with the MGC system. This indicates an elevated degree of spatial restriction in the MGC system in comparison to agarose pads, which is however sufficiently low to prevent elongation.
To our knowledge, this work represents the first systematic characterization and evaluation of single-cell cultivation technologies. We have demonstrated that the cultivation technology itself influences fundamental cellular characteristics, e.g., division rate and morphology. Our results imply the need of thoroughly characterizing the cultivation technology to be used in terms of inherent influences on cellular physiology.
Furthermore, we developed a universal and straightforward method that allows deriving specific volume growth rates of single cells from any time-lapse microscopy data. In terms of versatility and practicability, this method is comparable to measurements of optical density at the bulk level. We therefore anticipate that growth analysis may be included as a tool for quantitative single-cell analysis by default to enable comparison with growth rates obtained at the population level.
3.4 Systematic comparison of single-cell cultivation technologies
109 Performed the experiments: A.G. performed MGC experiments
C.D. performed nDEP and agarose pad experiments Analyzed the data: A.G. analyzed MGC experiments
C.D. analyzed nDEP and agarose pad experiments Wrote the manuscript: A.G.:
Introduction
Material and methods of MGC experiments
Parts of the systematic evaluation
Results of MGC
Technical comparison in the Discussion C.D.:
Abstract
Material and methods of nDEP/agarose pad
Determination of volumetric/division growth rates
Parts of the systematic evaluation
Results of nDEP and agarose pad
Biological part of the Discussion
Figures: A.G. created Figure 3.21, 3.22, 3.23 and 3.24 (A+B) C.D. created Figure 3.24(C-I), 3.25, 3.26 and 3.27 Both authors finally edited and proofed the text.
111