Cell cycle and size sorting of mammalian cells using a microfluidic device

Abstract

Microfluidic devices can sort viable mammalian cells by size. In this study, we investigated size-based sorting of cells using flow splitting microfluidic devices based on hydrodynamic filtration for noninvasive cell cycle synchronization. Two different types of mammalian cell lines, HepG2 (human hepatocellular liver carcinoma cell line) and NIH/3T3 (mouse embryonic fibroblast cell line) were sorted by microfluidic device and its DNA contents were analyzed. Our results showed that a microfluidic device can synchronize the cell cycle after size separation. The damage-free separation of living cells in different phases of the cell cycle represents a potentially promising technology for the investigation of gene transfection and gene expression.

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Introduction

Miniaturization of systems in microfluidic devices offers potentially considerable benefits for the label-free, size-dependent separation and sorting of cells compared with a typical fluorescence-activated cell sorting (FACS)¹ involving complex systems. Microdevices equipped with FACS functions (μ-FACS) that utilize laminar profiles have been investigated² and reviewed.³ These devices usually require optical signal detection and sorting switches. There are various sorting systems for separating particles and cells, including mechanical,⁴ electric,⁵ and flow cytometric⁶ devices. Microfabricated filters with arrays of rectangular channels have been used for the separation of white and red cells in blood,⁷ and have also been used for DNA separation.⁸ Electric devices based on dielectrophoresis⁹ or electromagnetic elements¹⁰ have been used to separate charged biological cells. However, although such microdevices are powerful tools for the separation of cells, they tend to be complex and costly.

The microfluidic devices based on a poly(dimethylsiloxane) (PDMS) are low cost, easily fabricated, and can be bonded to glass substrates. Furthermore, they are transparent, have low cytotoxicity, and maintain oxygen levels in cell cultures.¹¹ Continuous and self-sorting systems for viable mammalian cells using hydrodynamic flow¹² and hydrophoresis¹³ have also been described. In addition, pinched flow fractionation (PFF) with symmetric or asymmetric side channel devices can sort particles based on hydrodynamic filtration.¹⁴ In contrast, microchannel devices employing flow splitting and recombining (FSR) have been demonstrated to sort particles in the 2.1–3.0 μm size range and to concentrate these particle by 60- to 80-fold.¹⁵ The FSR devices have also been used to sort liver primary cells with sizes of 7.2, 9.2, and 18.8 μm, and the cells thus sorted were found to have retained albumin-production ability.¹⁶

Recently, the size separation of human leukemic monocyte cell lines and cell cycle synchronization at two different phases (G0/G1 and G2/M) were demonstrated using a microfluidic device employing hydrophoresis.¹⁷ The technique commonly used to separate cells is centrifugal elutriation against the direction of flow.¹⁸ This technique is based on the different densities or masses of cells in the different phases of the cell cycle, which is attributed to the differences in nuclear size in these phases. Although certain chemicals,¹⁹ such as aphidicolin, roscovitine, and colchicine, are available for the synchronization of whole cultured cells, treatment using these chemicals causes damage to normal cells. Thus, the label-free and noninvasive sorting of cells using microfluidic devices offers a potentially enormous benefit for biological investigations. In this study, we describe the size-based sorting of HepG2 (human hepatocellular liver carcinoma cell line) and NIH/3T3 (mouse embryonic fibroblast cell line) cells at different phases of the cell cycle using the microfluidic device based on hydrodynamic filtration described by Yamada et al.¹⁶ (Fig. 1).

Fig. 1 Schematic illustration (a) and photograph (b) of the microfluidic device design.

Experimental

Cell preparation

HepG2 and NIH3T3 cells were supplied by the American Type Culture Collection. The HepG2 cells were seeded at a density of 5 × 10⁵ cells per 60-mm dish in 5 mL of Dulbecco's Modified Eagle's Medium (DMEM, high glucose; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (FBS), penicillin (100 U mL^-1), and streptomycin (100 μg mL⁻¹). The NIH3T3 cells were seeded at a density of 3 × 10⁵ per 35-mm dish in 2 mL of DMEM. After incubation at 37 °C in a humidified 5% CO₂ incubator for 48 h, the cells were treated with 0.25% trypsin/EDTA (Invitrogen, Carlsbad, CA, USA) at 37 °C for 5 min and suspended in DMEM (10% FBS). After filtration through a 40-μm mesh cell strainer (BD Bioscience, Bedford, MA, USA) to eliminate cell aggregates, the cells were cryopreserved at −80 °C in a storage solution (Cell Banker 1; Juji Field Inc., Tokyo, Japan). Prior to use, the cells were thawed quickly and resuspended in DMEM (10% FBS) at a density of 1 × 10⁶ cells mL⁻¹.

Fabrication of the microfluidic device

The microchannel design of the device used in the present study followed that of Yamada's microdevice,¹⁶ as shown in Fig. 1(a). The microfluidic device was fabricated using a conventional molding process for PDMS using a negatively patterned mold master.¹⁸ Briefly, a precise Cr-photomask for the microchannels was fabricated using a maskless lithography system (DL-1000; NanoSystem Solutions, Tokyo, Japan). The photomask was then exposed to UV light using a mask aligner to produce the structure of the SU-8 50 photoresist (MicroChem, Newton, MA, USA) on a silicon wafer. The microchannels were formed by replicating the SU-8 master using PDMS prepolymer (Sylgard 184 silicone elastomer kit; Dow Corning, Midland, MI, USA). After the inlet and outlet holes were punched, the PDMS chip was bonded to a glass slide by O₂ plasma treatment (Fig. 1(b)). The depth of the microchannels was optimized at 80–90 μm to enable the high-speed processing of cell separation without cell stacking.

Cell size separation

Two polytetrafluoroethylene (PTFE) tubes (1.0 mm outer diameter, 0.5 mm inner diameter) were connected to Inlets 1 and 2. The length of the tube connected to Inlet 1 was 2.6 m and was capable of holding 0.5 mL cell suspension. Two microsyringes (Hamilton, Reno, NV, USA) were filled with 1 mL of DMEM (10% FBS+). Before introducing the cell suspensions, the microchannels were filled with Milli-Q water as a means of degassing.²⁰ The cell suspensions and DMEM were introduced at a flow rate of 50 μL min⁻¹ for 10 min using a syringe pump (ESP-64; Eicom, Kyoto, Japan).

Measurement of cell size

The cells recovered from each outlet were suspended in a 4-well plate and observed using an inverted microscope (Eclipse TE300; Nikon Corp., Tokyo, Japan). The cell sizes were calculated from the photographs.

Cell cycle analysis

In order to estimate the populations of separated cells in different phases of the cell cycle, flow cytometric analyses of the DNA content using propidium iodide (PI) staining was performed.²¹ The cells were centrifuged, washed with Dulbecco's phosphate-buffered saline (PBS), and fixed in 1 mL of the PBS-ethanol (3 : 7, v/v) solution on ice for 30 min. After centrifugation, the cell pellets were incubated in 0.1 mL of a solution containing 50 μg mL⁻¹ of PI, 0.1 μg of RNase A, and 0.1% of Triton-X 100 for 30 min at 37 °C. The cell suspensions were diluted to 0.5 mL with PBS and analyzed using a FACSCalibur flow cytometer (Becton-Dickinson, San Jose, CA, USA). The populations of cells in each cycle phase were calculated using cell cycle analysis software (ModiFit LT; Becton Dickinson) with the FL-W gating for doublet discrimination. The cytometric histograms were analyzed by the Gaussian fitting.

Results and discussion

We investigated the size distribution of 5 × 10⁵ HepG2 cells separated using the microfluidic device. The cells were introduced into the channel of the microfluidic device at a flow rate of 50 μL min⁻¹ for 10 min, and were separated by hydrodynamic filtration according to the principle described by Yamada and Seki.¹⁵ The hydrodynamic filtration is based on a laminar flow profile intersected with different volumetric ratio of different flow rates from the Inlets 1 and 2, and the ratio determines the maximum cell size that can pass through side channels. The cells introduced are flowing near side walls and can be separated into the outlets according to the cell sizes.

After separation, the cells were recovered from three different outlets (2, 3, and 4) using a micropipette. Fig. 2 shows microphotographs of the HepG2 cells recovered from each outlet. The cells were successfully separated on the basis of their size. Notably, the cells recovered from Outlet 4 were smaller than those recovered from the other two outlets. The cells thus separated using the microfluidic device appeared to have maintained a normal morphology without incurring any physical damage.

Fig. 2 Microscopic images of sorted HepG2 cells. Without separation (a), Outlet 2 (b), Outlet 3 (c), and Outlet 4 (d). The bars represent 50 μm.

Fig. 3 shows the cell size distributions of the separated and non-separated (control) HepG2 cells. Cell sizes were determined from their diameters on microphotographs using Image J software (n = 642, 381, 898, and 524 for the control, Outlet 2, Outlet 3, and Outlet 4 cells, respectively). In the control cells, there was a broad distribution of sizes (approximately 10–40 μm) with a clear peak at 15 μm. In contrast, although the HepG2 cells recovered from Outlet 2 also had a broad size distribution, there were no peaks and the cells were relatively larger than those recovered from the other outlets. The size distribution of the cells recovered from Outlet 3 was similar to that of the control cells. As noted above, the cells recovered from Outlet 4 were smaller than those recovered from the other outlets, and they exhibited a considerably narrower size distribution of approximately 13–15 μm. Another cell line, NIH/3T3 cells, could also be separated size dependently in the different outlets similar to the HepG2 cells. The microfluidic device thus facilitates a size-dependent separation of cells. These results are consistent with a previous report that describes the sorting of primary hepatocytes isolated from Wister rats.¹⁶

Fig. 3 Size distributions of sorted HepG2 cells.

Cell size is related to the phase of the cell cycle. In the M phase (mitotic phase), for example, cell volume doubles as the cell prepares to divide. The interphase includes the Gap 0 (G0), Gap 1 (G1), synthetic (S), and Gap 2 (G2) phases. During the synthesis phase, cells undergo DNA replication, which affect densities and cell sizes dependent on their cellular nuclei. Fig. 4 shows the cell cycle populations of the separated HepG2 and NIH/3T3cells recovered from each outlet as determined by flow cytometric analysis. The relative populations of cells in different phases of the cell cycle in each outlet were calculated from their DNA contents. The distribution of the control cells in three phases of the cell cycle (G0/G1, S, and G2/M) was 51.4%, 33.2%, and 15.4% for the HepG2 cells (n = 7452) and 50.7%, 19.2%, and 30.1% for the NIH/3T3 cells (n = 8719), respectively. Of the HepG2 cells in Outlet 2 (n = 864), 33.5%, 29.6%, and 36.8% were in the G0/G1, S, and G2/M phases, respectively, whereas for the NIH/3T3 cells (n = 62), the corresponding values were 29.0%, 21.0%, and 50.0%. Although the populations of cells in the S phase were similar to the control cells, the numbers of cells in the G2/M and G0/G1 phases were increased and decreased, respectively. Of the HepG2 cells (n = 3664) recovered from Outlet 3, 48.0%, 33.7%, and 18.3% were in the G0/G1, S, and G2/M phases, respectively, whereas for the NIH/3T3 cells (n = 8605) the corresponding values were 48.2%, 19.5%, and 32.2%. No differences were observed in the cell cycle populations of the control cells and those recovered from Outlet 3. Interestingly, of the HepG2 cells (n = 2374) recovered from Outlet 4, 85.9%, 10.1%, and 4.0% were in the G0/G1, S, and G2/M phases, respectively, whereas for the NIH/3T3 cells (n = 304), the corresponding values were 80.6%, 11.5%, and 7.9%. The populations of the cells in the G0/G1 phase were significantly increased compared with the control cells, whereas those in the S and G2/M phases were decreased. Both HepG2 and NIH/3T3 cells exhibited the same tendencies regarding the phases of the cell cycle. Thus, two different types of adherent mammalian cell (HepG2 and NIH/3T3 cell lines) were successfully separated depending on their size using the microfluidic device.

Fig. 4 Cell cycle populations of separated HepG2 cells (a) and NIH/3T3 cells (b).

The ratio of G0/G1 to G2/M for the non-separated HepG2 cells was approximately 3.3 : 1. In contrast, the ratio for HepG2 cells recovered from Outlet 4 increased to 21.5 : 1. Choi et al. reported that the ratio for the sorted U937 cells was 22.1 : 1 by hydrophoresis.¹⁷ Kim et al. described size-based cell separation using dielectrophoresis, and the ratio for the sorted MDA-MB-231 cells was 23 : 1.²² Our data of the HepG2 cells was similar to other reports of the size-based separation. However, the ratio of G0/G1 to G2/M for NIH/3T3 cells recovered from Outlet 4 was 10.2 : 1, which was low compared with those of the other cells. The results suggest that the cell cycle synchronization based on size separation depends on the type of cells.

This device can accordingly synchronize the phase of cell cycle based on the relationship between cell size and cell cycle phase. Moreover, our results indicate that microfluidic devices can be used to separate not only suspension cells but also adherent cells.

Conclusions

In this study, a microfluidic device based on hydrodynamic filtration was employed for sorting the viable mammalian cells with size. The separated cells clearly showed the relationship between cell size and cell cycle, indicating the cell cycle synchronization via size separation. It can be achieved with intact cells cultured in complete growth medium without the need for physical fields or inhibitory chemicals. The damage-free separation of living cells in different phases of the cell cycle represents a potentially promising technology for the investigation of gene transfection and gene expression.

Notes and references

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