Results

3.4 Systematic comparison of single-cell cultivation technologies 

97 with _, [h] as the time for doubling of the cell number.

  Figure 3.22: Design and functional principles of the compared single-cell cultivation systems from a macroscopic and microscopic point of view. (A) Operating principle of the nDEP for single-cell isolation and trapping, with cells guided into the electrode cage by funnel electrodes under continuous perfusion (a1). Convective mass transfer is dominant in nDEP system (a2). (B) Illustration of PDMS-based MGC chip with single-cell seeding and cultivation (b1). Mass transfer in MGC in the cell cultivation is mainly driven by diffusion (b2). (C) Casted sandwich agarose pad with a layer of solidified growth medium between two glass cover slides. Cells are located between agarose pad and bottom glass cover slide (c1).

Mass transfer in agarose pads is exclusively facilitated by passive diffusion. (D) Key numbers and characteristics of nDEP, MGC and agarose pads.

The third technology constitute agarose pads, where single-cells are entrapped between a layer of semi-solid growth medium and a glass cover slide (Figure 3.22C c1).

Cells are randomly spread on the agarose pad by applying an appropriately diluted cell suspension. As with MGC, cells are also forced to grow in one focal plane. Numerous

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Resulting from the respective cell trapping principle, the three systems offer inherently different degrees of control over the extracellular environment. This comparison yielded a distinct application profile for each of the three cultivation technologies.

nDEP is particularly suitable for the targeted isolation of certain phenotypes from a population and allows for retrieving cells afterwards. Furthermore, nDEP also works for all types of unicellular microorganisms. Another characteristic of nDEP is the free levitation and rotation, which excludes cell-surface interaction and allows the observation of the cells from multiple perspectives.

In terms of the number of cells that can be analyzed in parallel, the use of nDEP is typically restricted to the analysis of a single to few cells. The maximum number of cells that can be trapped inside a 20 µm x 20 µm x 20 µm electrode cage depends on cell dimensions. For bacteria and smaller yeast species, up to 30 cells can be retained in the field cage. Quantification of growth is restricted to a maximum of eight cells.

For higher throughput single-cell cultivations and analysis, MGC is well suited. 200 cultivation chambers can be observed in one experiment; hence highly parallelized analysis can be performed. Micropopulations of up to 1000 cells can be grown in a single cultivation chamber with the dimension of 60 µm x 60 µm x 1 µm. A cultivation chamber with a maximum volume of 10 pL, can be seen in Figure 3.22B. Cells are forced to grow in a monolayer, which allows for automated image acquisition. However, the cell isolation process is stochastic and neither enables a targeted isolation of specific phenotypes nor a subsequent release of cultivated cells.

Agarose pads allow parallelized high-throughput analyses with cells growing in one focal plane and allow to simultaneously follow the development from single cells to microcolonies consisting of several hundred cells. Cell isolation relies on stochastic distribution of cells and confinement is largely independent of the cell dimension and type.

II) Mass transfer

Mass transfer with nDEP systems is dominated by convective flow and eddy diffusion as a result of direct cell perfusion. This provides a steady supply of nutrients and also guarantees fast removal of secreted metabolites from the extracellular microenvironment. Fluctuations in concentration in the direct surrounding of the cell are therefore minimized [66]. Further, a rapid and accurate chemical perturbation of the cells is given.

Similar to nDEP, mass transfer in MGC relies on a combination of convective and diffusive mass transfer. Each microchamber is connected to 10 x deeper supply channels which are continuously flushed with fresh medium, allowing for swift diffusion of nutrients into the cultivation area (Chapter 3.3).

In contrast to nDEP and MGC, agarose pads exhibit a static environment, where mass transfer is exclusively facilitated by diffusion. Here, nutrients can deplete locally when the consumption rate is higher than the rate of resupply by diffusion. Moreover, produced metabolites may accumulate in the direct vicinity of the cell. Combination of both, agarose and microfluidic based medium supply has been reported as well [115, 139].

The trapping principle and mode of medium supply are illustrated in Figure 3.22A.

III) Platform design, setup and periphery

There are substantial differences in design, fabrication and periphery of the respective cultivation technologies. For operating nDEP devices, a rather complex periphery is required, consisting of a radio frequency generator that drives the electrodes and a temperature control system using peltier elements for cooling. The nDEP system consists of the actual microfluidic glass chip mounted to a support plate, which also includes contact pads for connecting the generator to the chip electrodes). Rapid and reliable connection of the external fluidics to the microchannel structures is realized with a customized pressure-based fluidic block [66]. This fluidic block also acts as a cooling block for transferring heat from the peltier elements. The chip fabrication process is comparably complex and involves several lithography and etching steps for creating channel structures and electrode geometries. The fabrication process yields robust microfluidic chips that can be thoroughly cleaned after cultivation experiments, which allows for a repeated use of the chips [66].

In contrast, MGC fabrication process and periphery is rather simple. The microfluidic chip consists of a glass plate that adheres to a PDMS slap. Because of the cheap materials and the simply molding process, MGC systems are disposed after usage.

The MGC structures are created by soft lithography, which enables creating multiple chips from one mold. Fluidic connections are established by punching holes. Temperature of the chip and surrounding periphery is controlled by an incubation system.

The simplest technology in terms of design, fabrication and needed periphery is the agarose pad, which is usually prepared within in a short time period of only a few hours.

It is made of standard materials that are in stock in every standard bio(techno)logical laboratory. Temperature control can be performed with a microscope incubator or by simply moving the microscope system to a temperature-controlled incubation room.

Features that all three cultivation technologies have in common, as well as respective unique characteristics are illustrated in Figure 3.23.

3.4 Systematic comparison of single-cell cultivation technologies 

101   Figure 3.23: System evaluation of nDEP, MGC and agarose pads. The Venn diagram illustrates the common properties as intersections and unique properties of each method (nDEP = red, MGC = blue, agarose pad = green).

Specific volume growth rates and division rates of C. glutamicum ATCC 13032 cells at the single-cell level

We cultivated C. glutamicum starting from one cell at standard growth conditions (Figure 3.24 A-C). In general, growth immediately commenced without any detectable lag-phase upon introduction of the cells into the respective cultivation system. These growth characteristics could be observed independently of the applied cultivation technology. Measured maximal specific volume growth rates of micropopulations were consistent with a mean value of µmax of 0.6 h^-1 ± 0.03 for nDEP cultivations and 0.61 h^-1

± 0.06 for cells cultivated with the MGC. Specific volume growth rates of micropopulations cultivated with agarose pads were 0.57 h^-1 ± 0.05. All investigated micropopulations followed a strictly exponential volume increase (Appendix D.1). The frequency plots of the measured specific growth rates revealed a normal distribution for all three systems (Figure 3.24D-F).

  Figure 3.24: Cultivation of C. glutamicum with nDEP, MGC and agarose pad. (A-C) Trapped and growing cells in the center of the octupole cage by nDEP, in the MGC and on agarose pad. Image sequence of a

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In addition to volume growth, we also assessed specific cell division rates of individual cells and micropopulations (Figure 3.24 G, H, I). Surprisingly, cells displayed significantly scattered division rates with coefficient of variance (CV, PAD = 19.8%) on agarose pads. In comparison to the respective measured volume growth rates, the mean of all measured specific division rates corresponded to the mean specific volume growth rate (vPAD = 0.59 h^-1 ± 0.11). Mean specific cell division rates matched the mean specific volume growth rates (vnDEP = 0.63 h^-1 ± 0.03, vMGC = 0.59 h^-1 ± 0.05) for nDEP and MGC (Figure 3.25). However, in comparison to nDEP cultivations, a minor increase in variation of specific division rates was observed with the MGC (CV, nDEP = 4.5%, CV, MGC = 7.7%).

The observed irregularities in terms of division rate consistency indicate an inherent influence of the agarose pad technology on cellular physiology of C. glutamicum.

  Figure 3.25: Scatter plot of measured specific volume growth rates (left) and specific division rates (right) for nDEP, MGC and agarose pads.

Analysis of morphology and snapping division during single-cell cultivations of C. glutamicum

The observed irregular division rates of C. glutamicum might be caused by deviating cell morphology during cultivation.

  Figure 3.26: (A-C) Determination of cell lengths and division angles based on cytometric data. (D-F) Frequency distribution of cells length before and after cell division with the nDEP, MGC and agarose pad.

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Therefore, cell morphology was investigated in detail during cultivation. The distributions of cell lengths just before and after division for nDEP, MGC and agarose pad-based cultivations are illustrated in Figure 3.26 D-F. For nDEP and MGC cultivations average cell lengths are in good agreement with 4.68 µm ± 0.4 (CV= 8.4%) and 4.42 µm

± 0.44 (CV= 12.8%) before and 2.45 µm ± 0.21 (CV= 8.7%) and 2.67 ± 0.36 µm (CV= 13.4%) after the division event, respectively. Only 2% of the cells cultivated with MGC had lengths above 6 µm before cell division. With nDEP, all cells divided before reaching 6 µm in length. Cell length distributions follow a normal distribution with the nDEP and MGC technologies (P>0.05).

For cells grown on agarose pads, a higher tendency towards elongation in comparison to nDEP and MGC cultivations was observed. This inclination towards elongation was pronounced with 23% of the cells reaching lengths of more than 6 µm right before cell division. Average cell length before division was 5.44 µm ± 1.16 (CV= 21.6%) and 3.04 µm ± 0.75 (CV= 24.7%) after division, with both distributions exhibiting significant variance. Cell lengths distributions were heavy-sided towards increased cell lengths, rather than following a normal distribution. Cells grown on agarose pads also exhibited a stronger tendency towards asymmetric division, pointing to a disturbance in the placement of the division septum (Figure 3.27).

Figure 3.27: Division symmetry of C. glutamicum is influenced by cultivation technology. Cells tend to divide in a more asymmetric fashion on agarose pads. Division symmetry was measured as the ratio of short to long cell pole directly after the cell division event.

We concluded from these results that the physiology of cells cultivated on agarose pads is subjected to stress caused by the mode of trapping or environmental conditions.

In addition to cell lengths, we also assessed division angles of single cells directly after the division event. Cells of C. glutamicum exhibit a distinct V-formed shape after

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.