A high-throughput microfluidic diploid yeast long-term culturing (DYLC) chip capable of bud reorientation and concerted daughter dissection for replicative lifespan determination | Journal of Nanobiotechnology

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Design concept

The high-throughput microfluidic DYLC chip was designed to capture single budding yeast cells, reliably retain mother cells, and effectively remove daughter cells so as to perform long-term culturing and microscopic monitoring for later-on image analysis to determine yeast RLS. Figure 1 shows the micrographs and schematics of the microfluidic chip, which simply consists of an 8-µm-high PDMS microchannel bonded on a glass substrate. The microchannel features an inlet for cell suspension and medium infusion, 53 cylindrical posts, 1100 “leaky bowl”-shaped traps formatted in an array, and an outlet for waste collection (Fig. 1A). The posts, with the diameter of 30 μm, were designed to support the ceiling of the wide microfluidic channel from collapse during chip bonding. In order to prevent the whole channel clogging among the dense traps, the trapping array was patterned into 5 subarrays with a spacing of 100 μm in between (Fig. 1B). Each subarray contains 220 traps (10 rows × 22 columns) with optimized distances of 30 and 34 μm between adjacent columns and rows, respectively. Traps in each column are aligned to the middle of spacings between two neighboring traps in adjacent columns. Such setting of the trapping array ensures both efficient trapping and enough growing space of yeast cells. Each trap (outer profile: 15 μm × 7 μm) comprises two pillars facing each other, together forming a “bowl”-shaped wide opening (width: 8 μm) upstream and a “leaky” orifice (width: 3 μm) downstream. The length of the wide opening was optimized to 5 μm to ensure the yeast trapping in single-cell resolution, and the length of the orifice was set to 2 μm to minimize the structural squeezing or mechanical stress on newborn buds during their growth. Once yeast suspension was infused into the microchannel, cells were dragged to be docked inside the empty “bowls” by hydrodynamic forces (Fig. 1C). Since the height (8 μm) of each trap is larger than the diameter of budding yeast cells (4 ~ 6 μm), immobilized single cells with small buds have sufficient space vertically to rotate in the “bowls” under hydrodynamic forces, and buds could be ultimately orientated and clamped in the orifice downstream when growing bigger (Fig. 1D). After the completion of cytokinesis, the matured buds (i.e., daughter cells) could be automatically detached by hydrodynamic shearing forces whereas mother cells were stably retained in their traps. Therefore, the immobilized yeast cells during their entire lifespans could undergo consistent hydraulic microenvironments, such as the hydrodynamic forces in cell growth, mother retention and daughter removal. In particular, for accurate RLS determination with the diploid budding yeast, which features random budding spots on mother cells, the delicate “leaky bowl”-shaped traps with the optimal dimensions could thus provide the merits of free mother rotation in the traps, bud reorientation to the narrow orifices and concerted daughter dissection.

Fig. 1
figure 1

Overview of the microfluidic DYLC chip. A Photograph of the PDMS microchannel including 53 cylindrical posts and a cell-trap array. B Micrograph of the array patterning into 5 subarrays with a gap of 100 μm in between. Each subarray contains 10 × 22 traps. Distances between columns and rows are 30 and 34 μm, respectively. Traps are aligned to the middle of the spacings between two neighboring traps in adjacent columns. Insert is a SEM micrograph of the trap array. Scale bar is 10 μm. C A schematic cartoon showing the cell loading and trapping processes in the array. D 3D schematics showing the geometric dimension of the “leaky bowl”-shaped trap, the hydrodynamic bud rotation and the concerted daughter dissection. The trap features a 7 μm × 15 μm × 8 μm outer profile in xyz-dimension, with a 5 μm × 8 μm wide opening upstream and a 2 μm × 3 μm narrow orifice downstream. The bud of immobilized mother cell tends to be rotated into the narrow opening downstream and then removed under the hydrodynamic shearing forces

CFD simulation of fluid field in the cell-trap array

To better understand the cell-trapping process and therefore optimize the geometric arrangement of cell traps, the fluid field distribution in the array was calculated in CFD simulation under three different settings of the distance between adjacent rows (dr) and the misalignment in rows (dm). Contour plots in Fig. 2A show the flow velocity distributions across the 5 × 5 cell-trap array in the xy-plane. Compared to the flow velocity distribution in setting 2, the region with high value of flow velocity in setting 1 is almost interconnected so that cells are prone to bypass traps and flow along this region towards downstream. In setting 2, dissected daughters from upstream tend to be captured by traps downstream, while in setting 3 they are inclined to make a detour around traps downstream due to larger dr. Moreover, for practical considerations, slightly larger dr in setting 3 could reduce the occurrence of channel clogging caused by abnormally large cells when approaching apoptosis. Hence, the distance between adjacent rows and the misalignment in rows of the cell-trap array were suggested to be 34 μm and 17 μm, respectively.

With the suggested geometric setting of the array, particle trajectory tracing was then simulated to investigate the process of cell trapping before and after the traps upstream were docked with cells (Fig. 2B). Due to the low flow resistance through the orifices of the “leaky bowl”-shaped traps, suspended cells could be initially dragged towards the empty traps under hydrodynamic forces. Once the wide openings were occupied, drastic increase of flow resistance in traps rendered subsequent cells bypassing these traps and flowing towards the empty traps downstream (Additional file 2: Video S1). Such dynamic process of single-cell trapping was observed in experiments (Additional file 3: Video S2). A budded cell, rotating with the medium stream, was immobilized in an empty trap, and then cell suspension from upstream bypassed this occupied trap successfully. Therefore, the simulation results demonstrated that cells in the loading process could rapidly fill the array by evading the occupied traps and giving preference for empty ones.

Fig. 2
figure 2

CFD simulations of fluid field in a 5 × 5 cell-trap array. A Flow velocity distribution across the xy-plane (4 μm above the bottom) of the array with three geometric settings (dr: 30 μm, 30 and 34 μm; and dm: 10 μm, 15 and 17 μm, respectively). B Particle trajectory tracing before and after cell immobilization in the traps upstream. Trajectories indicate the flow path of cells during the loading process. The array was set to 34 μm (dr) and 17 μm (dm)

Optimization of trapping efficiency and long-term maintenance of budding yeast cells

The efficiency and reliability of single-cell immobilization plays a vital role in sample collection for high-throughput imaging and data analysis. With the optimal design (dr = 34 μm and dm = 17 μm) of the cell-trap array, the cell-trapping efficiency was investigated in experiment. After cell loading, an initial trapping rate could reach 70.5% on average. The trapping rate could be raised by increasing the concentration of cell suspension and the duration of cell loading. However, a higher trapping efficiency in the beginning of the experiments was not demanded since empty traps could be filled with daughter cells removed from traps upstream in the next several hours during cell culturing (Fig. 3A). Within the first 4 h of yeast culturing, the cell-trapping efficiency could be raised from 70.5 to 92.3% (Fig. 3B), thereby providing a high-throughput platform for yeast immobilization. With the ability of self-filling among the staggered traps, low concentration of cell suspension was preferred for single-cell immobilization.

Next, we investigated the capability of single-cell maintenance in traps that differed in the length of the “bowl”-shaped wide opening (i.e., 4 μm, 5 and 6 μm). In the experiment with 4-µm-long “bowl” of traps, large mother cells, when approaching apoptosis, could be dragged away by unrotated buds that were located upstream (Fig. 3C). Such loss of mother cells could dramatically cut down the number of samples for RLS determination. In the experiment with 6-µm-long “bowls”, which provide sufficient space to dock two small yeast cells, traps occupied with single yeast cells could capture extra cells coming from the upstream (Fig. 3D). The extra cell trapping could affect the imaging quality and identification of original mother cells in long-term cell culturing. Figure 3E shows the percentages of occupied traps that were occurred with mother loss and extra cell capture during 0 to 4 h and 24 to 28 h after cell loading. We can see that the mother-missing rate gradually decreases with the increase of “bowl” length, while the extra-trapping rate rises up. Therefore, the length of wide-opening was optimized to 5 μm for the sake of stability in single-cell retention.

Fig. 3
figure 3

Optimization of trapping efficiency and long-term maintenance of budding yeast cells. A Time-lapse images showing the filling of empty traps at 0 h, 2 and 4 h after cell loading (time mark: “hour: minute”, the same below). Blue arrows indicate empty traps, while yellow ones indicate traps that were docked with daughter cells from upstream. Scale bar is 50 μm. B Trapping rate increasing within the first 4 h after cell loading (3 independent runs). C Two samples of mother cells being dragged away by unrotated buds in the 4-µm-long “bowl”. Scale bar is 10 μm. D Two samples of capturing extra cells in the 6-µm-long “bowl”. Scale bar is 10 μm. E Percentage of occupied traps occurring with mother loss and extra cell capture from 0 to 4 h and from 24 to 28 h after cell loading (3 independent runs, 220 traps per run). Scale bar is 10 μm

CFD simulation and experimental characterization of bud rotation for concerted daughter dissection

A haploid budding yeast always forms new buds adjacent to the scars of previous daughters [19, 20]. Thus, haploid yeast that initially sprouted downstream in the microfluidic traps could undergo successive removal of daughters in following generations [14, 15]. However, diploid budding yeasts, which forms new buds either adjacent or opposite to the previous bud site, would have high risks of being dragged away when buds are towards the upstream of traps. Hence, the microfluidic DYLC chip was designed to ensure concerted daughter removal of diploid budding yeast cells within consecutive generations by taking the advantages of bud reorientation into the unified orifices downstream.

Firstly, bud reorientation of immobilized budding yeast under continuous fluidic perfusion was theoretically analyzed by numerical simulation of hydrodynamic forces on a single cell docked in a trap (Additional file 1: Fig. S1A). When the bud was set to 0 (i.e., along -x direction) or 45 degree, the hydrodynamic force on bud points obliquely upwards, showing the motional tendency of the bud. After the bud rotated over 90 degree (i.e., along + z direction), the force points obliquely downwards, indicating the bud to be orientated into the orifice. At 180 degree (i.e., along + x direction), the z-component of the hydrodynamic force on the bud is negative, meaning that the reoriented bud could be stably maintained in the orifice. Therefore, the simulation result shows the feasibility of bud rotation from upstream to downstream in the orifice under hydrodynamic forces. Moreover, with the increase of bud diameter from 2 to 4 μm, the x-component of hydrodynamic forces gradually rises up and dominates during the bud growth (Figure S1B), suggesting that larger bud could be retained in the orifice more stably till the consequent daughter dissection had occurred after the completion of cytokinesis. Also, larger bud possesses higher hydrodynamic force for dragging the bud downstream to accomplish daughter detachment.

Secondly, occurrences of bud rotation in experiment were verified by time-lapse images, especially with the mother cells that had two buds during a certain time period (Fig. 4A). In the case of Cell 1, the small bud (2nd bud) sprouted next to the unseparated daughter (1st daughter), and the 2nd bud remained in the narrow orifice downstream after the separation of 1st daughter. The 3rd bud successfully oriented into the orifice following the same path as the 2nd one did. The cases of Cell 2 and Cell 3 demonstrate the reorientation of new buds that sprouted on opposite and random sites of previous daughters, respectively. We could clearly observe that newborn buds (2nd buds) appeared before the removal of daughters (1st daughters). In the next images, the buds were oriented into the orifice downstream and meanwhile the mature daughters disappeared. Afterwards, the buds remained downstream throughout their division periods, followed by their dissection and the reorientation of new progenies (3rd buds) into the orifice. Furthermore, Videos S3 and S4 show the dynamic rotation and reorientation of immobilized budding yeast cell. Initially, the mother cell with a tiny bud was freely rotating in the “bowl”-shaped trap. With the bud growth, the cell stopped rotating with its bud clamped in the narrow orifice and swayed under the continuous medium perfusion. As the bud grew larger, the bud became motionless until it was dissected after the completion of cytokinesis (Additional file 4: Video S3). Right after the removal of the mature progeny, the following bud was immediately rotated into to the orifice (Additional file 5: Video S4).

Thirdly, the rotation rates of cells immobilized in two trap arrays that differed in height (7 μm vs. 8 μm) were compared in terms of the initial six generations of buds. The percentage of successful reorientation towards the downstream in traps with the height of 7 and 8 μm were 64.67% and 81.12%, respectively (Fig. 4B). The result indicated that 8-µm-high traps provided sufficient space for cell rotation and consequently concerted daughter dissection. Video S5 recorded the whole lifespan of an immobilized budding yeast cell. We could observe the successful orientation of buds towards the orifice downstream followed by concerted daughter dissection in the orifice for the 25 generations. However, bud rotation occurred less as the mother cell aged. This phenomenon could be attributed to following reasons: One is that the size increase of the senescent mother severely restricted the mobility of buds in traps; the other one is that the delayed detachment of daughter cells rendered the new buds growing too large to be rotated.

Therefore, the above experimental results demonstrated that the “leaky bowl”-shaped yeast traps enable efficient cell rotation by hydrodynamic forces, and cells could subsequently undergo the concerted processes of bud reorientation and daughter dissection in successive division cycles until apoptosis.

Fig. 4
figure 4

Characterization of yeast cell rotation and but reorientation in traps. A Time-lapse images showing the rotation of newborn buds, which were sprouted in adjacent, opposite and random spots regarding previous ones in three consecutive generations. Trap height is 8 μm. Scale bar is 10 μm. B Cell rotation rate in the initial 6 generations. Cells were immobilized in traps with the height of 7 μm versus 8 μm (3 independent runs, 220 traps per run)

High-throughput RLS determination of diploid budding yeast

To validate the capability and reliability of the microfluidic DYLC chip in high-throughput aging study of diploid budding yeast cells, long-term monitoring of single immobilized cells for over 60 h was performed. Figure 5A shows one representative cell that continuously gave birth to new buds until its death. The corresponding data analysis of this cell was conducted to enumerate the budding and dissection events throughout its lifespan by using the sequential two-digit coding. RLS of this cell, 23 generations, was calculated from the recorded digital matrix and its converted oscillogram (Additional file 1: Fig. S2). According to the data analysis of 786 cells trapped in the initial 10 h of 3 independent experiments, we obtained the reduction in cell viability as a function of replicative ages (Fig. 5B), resulting in an average RLS of 24.29 ± 3.65 generations. Then, additional 211 cells captured after the first 10-hour running of experiments were included in RLS analysis. We redistributed all 997 cells by grouping them according to the time point of the mother-cell trapping at a 2-hour interval (Fig. 5C). The fitting curve shows statistically shortened lifespans through the whole data set. The mean RLS (24.87 generations) was highest at the first 2 h of the experiments, while the minimum (only 14.25 generations) appeared on cells captured between 44 and 46 h. Moreover, the mean RLS of cells captured after 20-hour running was 17.21, a large decrease of 29.15% compared to that in the first 10 h. This phenomenon implies that a portion of senescent mother cells after 20-hour culturing may produce daughters that could not inherit full lifespan [25]. Due to the asymmetric division between mother and daughter yeast, the shortened lifespan in daughters from old mothers has been debated at the protein level. The function of asymmetric distribution of proteins, which accumulate steadily as mother cells aged, usually keep the protein level in daughter cells lower than that in their mothers. However, as was proposed and verified that daughters born from old mother cells have a protein level comparable to middle aged mothers, the lifespan could be limited when the inherited proteins reach a proper level [26]. The downward trend of the linear fitting curve in Fig. 5C also agrees well with the reported results that lifespan reduction in daughters may accumulate progressively as the age of their mothers increased.

Fig. 5
figure 5

Long-term monitoring and RLS determination of diploid budding yeast cells in the DYLC chip. A Time-lapse images of a representative budding yeast cell in aging. Sequential two-digit codes indicate the bud appearance and daughter dissection. Scale bar is 10 μm. B Cell viability plotted as a function of the number of generations (n = 786 in 3 independent runs). C Mean RLS of cells captured within every 2 h. The dark blue line indicates a linear regression of the correlation between the mean RLS and the time of capturing cells (n = 997 cells in 3 independent runs)

Budding time interval (BTI) of diploid yeast cells throughout the lifespan

The microfluidic DYLC chip enables large scale screening of cell samples to investigate the age-related changes in BTI. Since the time duration for the first G1 phase of haploid yeast fluctuated over a tenfold range, the mean budding time interval (BTI) of the first generation of haploid yeast could show great extension than subsequent generations [27]. To investigate the dynamic BTI of diploid budding yeast cells at birth, 33 mother cells in a small size and initially showing no obvious bud were selected and analyzed. The budding time interval (BTI) was obtained from the digital matrix (Additional file 1: Fig. S2). As illustrated in Fig. 6A, mean BTI of the first generation of diploid cells exhibits an extension of 9%, compared to that of the next five generations, similar to the haploid yeast. To investigate the BTI variation upon cell apoptosis, BTI of the 33 mother cells was then calculated in death-centric perspective by aligning all data to the last generation in their lifespan. The result in Fig. 6B demonstrates that BTI of diploid yeast cells is almost steady during most of the lifespan, whereas it rises up dramatically in the last several generations.

It is commonly recognized that the stochasticity and robustness are inherent properties of living cells [28]. However, experimental results derived from conventional approaches were usually based on assumptions that cells performed uniform growth and division and the group behaviors could hide heterogeneities among individual cells. The cellular de-synchronization in diploid yeast aging was verified through 400 samples from 3 independent experiments, where cells were relatively small in size and with no obvious bud initially. For better visualization, BTIs in every 5 consecutive generations were periodically marked with 5 different colors in a kymograph. As shown in Fig. 6C, the BTI distribution remains synchronized within the first 8 to 10 generations, whereas it gradually differentiated and elongated as cells progressively approached to the end of their lives. The BTI divergence may demonstrate the heterogeneity among individual cells even under the same microenvironment. In addition, the result of diploid yeast cells may exhibit better stability than haploids, which started to de-synchronize after about 5 divisions [14]. Therefore, the consistence of the mean BTI and BTI distribution until the last few generations indicates that diploid yeast cells have a better resistance to extrinsic fluctuations and intrinsic noises, such as environmental stress, protein accumulation and DNA damage during their lifespans [29].

Fig. 6
figure 6

Budding time interval (BTI) of diploid yeast cells in replicative aging. A Mean BTI with all samples aligned to birth (n = 33). B Mean BTI with all samples aligned to death (n = 33). C Kymograph of BTI. In every 5 generations, each BTI was marked with a distinctive color, for better illustration of the synchronization and de-synchronization among individual cells. Samples were ordered by their RLS (n = 400)

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