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Microfluidic Techniques for Single-Cell Culture
CHAPTER 7
Microfluidic Techniques for Single-Cell Culture Chuan-Feng Yeh*,†, Chia-Hsien Hsu*,†,‡ *Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli, Taiwan † Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu, Taiwan ‡ Ph.D. Program in Tissue Engineering and Regenerative Medicine, National Chung Hsing University, Taichung, Taiwan
7.1 ADVANTAGES OF MICROFLUIDIC TECHNIQUES FOR SINGLE-CELL CULTURE Cell heterogeneity is always present to some level in all cell populations, thus the collective behaviors of a population of cells does not represent the behaviors of individual cells. Therefore, it is important to analyze individual single cells in order to discover mechanisms not seen by studying a bulk population of cells. Single-cell culture is a method of growing isolated single cells routinely performed to obtain single-cell-derived cell clones for both basic research and therapeutically applications. Besides forming clones, culturing individually isolated single cells can also help understand the metabolic, migration and differentiation heterogeneity in cell populations (Vermeulen et al., 2008; Sato et al., 2016). Despite single-cell culture being essential and widely used, single-cell culture is still a challenging task for many laboratories due to the difficulty of manipulating and observing single cells. Microfluidic techniques have emerged as a useful tool for single-cell culture applications. In comparison to conventional petri dishes and well plates, culturing cells in microfluidic devices offers several advantages: (1)
The microdevices can more precisely manipulate single cells and control the cell culture conditions, thus making it useful for providing better-controlled experimental conditions. For example, microfluidic channel can be used to generate stable chemical gradients for studying
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(2) (3)
single-cell chemotaxis at high temporal and/or spatial resolution (Chen et al., 2015a). The miniaturized devices can increase the throughput and reduce reagent/cell consumptions of experiments due to their small sizes. Microfluidic devices are amenable to integration with microelectromechanical systems (MEMS) technology to form lab-ona-chip devices, which allow for combining single-cell culture and analysis in one device. Besides making cell-culture devices, microfluidic techniques can be used as a helper tool to prepare cells for single-cell culture with conventional culture dishes and well plates for applications where single-cell suspension preparation is difficult.
This chapter will describe and compare state-of-the-art microfluidic techniques for single-cell preparation, separation, and culture.
7.2 CELL CLUSTER DISSOCIATION BY MICROFLUIDIC CHIP Having a cell suspension containing individually separated single cells is a prerequisite for performing single-cell culture experiments. For tissues and cells that are cultured in cluster/aggregate format, a cell dissociation step is required to break the cells into individual cells to prepare the single-cell suspension. During cell dissociation process, the extracellular matrix and cell-cell junctions must be disrupted by enzyme or mechanical force. Using enzymatic chemicals such as trypsin, elastase and hyaluronidase to destroy the cell-matrix adhesion and cellcell junctions is simple to perform. However, the amount and type of enzyme to be used need to be carefully chosen in order to not damage the cells or cause cell surface protein expression loss (Garg et al., 2014). Mechanical methods including shaking, pipetting, vortexing and sonication can also be used for cell dissociation. These physical methods however are relatively more laborious, and result in lower cell viability compared to enzymatic methods. And more importantly, it is very difficult if not impossible for pure mechanical methods to produce cell suspension containing a high percentage of viable dissociated single cells. Microfluidic techniques have been demonstrated as a useful tool for single-cell suspension preparation. Owing to the ability of microfluidics to precisely control a small volume of fluid under laminar flow condition, the generated mechanical force during cell dissociation is more controllable and reproducible, resulting in increased viable single-cell ratio in the prepared cell suspension. Some example of microfluidic cell dissociation techniques are described below. In order to reduce contamination risk and to scale up the production of neural stem cells for clinical uses, a “biogrid” device was developed for neurosphere dissociation (Wallman et al., 2011). The device contains a silicon substrate with grids whose edges were 20 μm thick with 200 μm edge to edge spacing
7.2
Cell Cluster Dissociation by Microfluidic Chip
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FIG. 7.1 Microfabricated devices for cell cluster dissociation. (A) Top photograph is a microfabricated 3-in. silicon wafer. The photograph inset shows the dissociating grids before wafer dicing. In the bottom photograph, individual microgrids are assembled into the device adapter before being used for cell dissociation (Wallman et al., 2011). (B) The left-hand photograph shows the single channel “μ-CDC” chip fabricated via soft lithography and PDMS. On the right is a scanning electron micrograph showing the microfabricated pillars. Neurospheres passing through the micropillar array (white arrow ) are mechanically dissociated into single cells (Lin et al., 2013). (C) Photograph of fabricated device for dissociating small tumor tissue via alternating constriction and expansion regions (Qiu et al., 2015). (A) Reproduced with permission from Cytotherapy. (B) Reproduced with permission from Analytical Chemistry. (C) Reproduced with permission from Lab on Chip.
(Fig. 7.1A) (Wallman et al., 2011). This device is easy to use that it can be attached to a syringe in which neurosphere suspension is loaded and can be pushed out to go through the biogrid to mechanically break neurospheres whose size are bigger than the grid-to-grid spacing. Another microfluidic device containing an array of micropillars for neurosphere dissociation has also been reported (Lin et al., 2013). The micropillars are each 50 μm wide and 167 μm tall with 20 μm spacing between adjacent pillars (Fig. 7.1B). A neurosphere suspension can be injected into the device via the device’s inlet with a syringe pump. Neurospheres were dissociated into single cells by passage through the pillar array at 3–15 mL/min flow rate. The dissociated cells are collected from the device’s outlet. It is worth mentioning that the chip could achieve high single-cell dissociation yield (91%–95%) at high single-cell viability (80%–85%), which could not be produced by the conventional trituration method (50% viability). Microfluidics-based methods could also help improve cell recovery after cell dissociation process for rare samples such as tissues aspirated from needle biopsy. A microfluidic device containing branching channels has been demonstrated for rare sample dissociation (Qiu et al., 2015). The device was capable of dissociating tumor tissue equal of size less than 1 mm into single cells (Fig. 7.1C). The dissociation is achieved by using shear forces generated by flowing cell suspension through microchannel which has repeated constriction and expansion sections. The tumor cell dissociation with this device could achieve the same cell yields but showed a significant increase
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in the single-cell population, from 60% to 90% when compared to a control dissociation method utilizing trypsin-EDTA, vortexing, and pipetting. The device dissociation could also be completed within 10 min. In summary, microfluidic techniques represent an attractive tool for cell aggregates dissociation for preparing single-cell suspension, as performing cell dissociation in microfluidic systems is easy to scale-up and can have higher productivity. Moreover, being able to handle cells in a closed microchannel system can also reduce contamination risk. Because of the tightly controlled and laminar flow environments, microfluidic techniques are able to reduce cell damage increase single-cell yield and reproducibility. Requiring smaller sample volumes also makes microfluidic devices useful for handling limited clinical samples. Lastly, the ability of microfluidic technique to improve the outcome of nonenzymatic single-cell dissociation is of great importance from a regulatory standpoint. The nonenzymatic methods are also useful for researchers in obtaining single cells that maintain endogenous surface markers.
7.3 SINGLE-CELL ISOLATION AND CULTURE ON MICROFLUIDIC CHIP To start a single-cell culture experiment, individually isolated cells must be first prepared in a cell culture device. Despite being a routinely performed task in many laboratories, single-cell isolation still represents a major challenge in single-cell culture. Conventionally, this cell isolation task is commonly performed by using limiting dilution, micromanipulation or fluorescenceactivated cell sorting (FACS) technique. Isolation of individual cells by limiting dilution is achieved by using hand-held pipettes or pipetting robots to split a diluted cell suspension into small aliquots. Depending on the cell number in the diluted cell suspension, the probability of having 0, 1, 2, 3, or more cells in an aliquot can be changed. The method is simple to carry out, but relatively low efficiency due to statistical nature. Micromanipulation relies on using a micropipette to manually select and pick individual single cells. In order to ensure high confidence level of single-cell isolation, the procedure is generally performed with manual identification under a microscope, therefore the throughput is very limited. Isolating single-cell by using FACS is high-throughput and automated. However, FACS machines are expensive and not suitable for handling small sample volumes. Furthermore, in comparison to limiting dilution and micromanipulation, FACS isolation is more likely to cause cell damage. Microfluidic techniques are able to overcome the hurdles encountered by current single-cell isolation methods through miniaturization, microfluidics, and integration with MEMS technology. To date, many microfluidic techniques have been utilized to facilitate manipulation, observation, validation, and culture of single cells (summarized in Table 7.1). These techniques are categorized as described in the following sections.
7.3
Single-Cell Isolation and Culture on Microfluidic Chip
Table 7.1 Comparison of Various Microfluidic Techniques for Single-Cell Culture. Isolation Approaches
Culture Condition
Microwells (Rettig and Folch, 2005; Choi et al., 2014; Lin et al., 2015)
Microtable (Pai et al., 2010) Micropallets (CoxMuranami et al., 2016) Microlateral chambers (Shen et al., 2015; Zhang et al., 2016; Chen et al., 2016a)
Clonal Transfer
Major Advantage
Major Disadvantage
Compartmental wells
No (Rettig and Folch, 2005; Lin et al., 2015) Yes (Choi et al., 2014)
Unable to transfer colonies (Rettig and Folch, 2005; Lin et al., 2015) Pooled colonies (Choi et al., 2014)
Compartmental pallets, tables
Yes (Pai et al., 2010; CoxMuranami et al., 2016)
Easy manipulation (Rettig and Folch, 2005; Choi et al., 2014; Lin et al., 2015) High throughput (Rettig and Folch, 2005; Choi et al., 2014; Lin et al., 2015) Easy fabrication (Rettig and Folch, 2005; Lin et al., 2015) High efficiency in obtaining colonies (Pai et al., 2010; CoxMuranami et al., 2016)
Compartmental chambers
No (Shen et al., 2015; Zhang et al., 2016; Chen et al., 2016a)
High throughput (Shen et al., 2015; Zhang et al., 2016; Chen et al., 2016a)
Cell traps (Chung et al., 2014; Chen et al., 2015b, 2016b; Cheng et al., 2016)
Compartmental chambers
No (Chung et al., 2014; Chen et al., 2015b, 2016b; Cheng et al., 2016)
Microvalves (Zheng et al., 2012; Sikorski et al., 2015; Matsumura et al., 2014)
Individual chambers possible/under medium perfusion possible
No (Zheng et al., 2012; Sikorski et al., 2015) Yes (Matsumura et al., 2014)
High throughput (Chung et al., 2014; Chen et al., 2015b, 2016b; Cheng et al., 2016) High single-cell efficiency (Chung et al., 2014; Chen et al., 2015b, 2016b; Cheng et al., 2016) Scaling and automation (Cheng et al., 2016) Controllable microenvironments (Zheng et al., 2012; Sikorski et al., 2015; Matsumura et al., 2014)
Special laser equipment requirement (Pai et al., 2010; Cox-Muranami et al., 2016) Unable to transfer colonies (Shen et al., 2015; Zhang et al., 2016; Chen et al., 2016a) Low single-cell efficiency (Shen et al., 2015; Zhang et al., 2016; Chen et al., 2016a) Unable to transfer colonies (Chung et al., 2014; Chen et al., 2015b, 2016b; Cheng et al., 2016) Difficult fabrication (Chung et al., 2014; Chen et al., 2015b, 2016b; Cheng et al., 2016) Difficult fabrication (Zheng et al., 2012; Sikorski et al., 2015; Matsumura et al., 2014) Complex setting for experiments (Zheng et al., 2012; Sikorski et al., 2015; Matsumura et al., 2014)
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7.3.1
Microwells
Microfluidic devices containing miniaturized wells have been developed to overcome the difficulty of single-cell compartmentalization and identification for single-cell analysis and culture applications. A polydimethylsiloxane (PDMS) microwell device (Fig. 7.2A) was developed for single-cell isolation of fibroblast (as model of adherent cells) and rat basophilic leukemia cells (as model of suspension cells) (Rettig and Folch, 2005). The single-cell isolation efficiency is dependent on the microwells’ dimensions and cell seeding density. This microfluidic device provides a simple yet high-efficient approach to isolate and analyze single cells but is not applicable to single-cell culture due to the limited space of the microwells. In order to culture single cells and allow for retrieval of single-cell colonies, a hemispherical microwell device was developed (Fig. 7.2B) (Choi et al., 2014). This device utilized a more complex threelayer microchannel structure to capture and isolate single cells in microwells. This device was able to achieve high single-cell capture efficiency (>90%) and was suitable for long-term culture and retrieval of colonies. Another microfluidic device, termed “dual well device” has also been developed for highefficiency single-cell culture (Fig. 7.2B) (Lin et al., 2015). The advantages of this device lies in the fact that it utilizes a “dual-well” design to allow for simple device fabrication and operation (Fig. 7.2C). The device could achieve high single-cell isolation efficiency (77%) and its single-cell culture applicability was demonstrated with mouse neural stem cell differentiation, cancer cell proliferation and single-cell colony formation assay on chip. This device provides a simple approach to analyze single-cell heterogeneity during cell culture.
7.3.2
Microtables/Micropallets
Another approach for separating individual cells and picking colonies is based on fabricating small table or pallet structure in microdevices (Fig. 7.3). This approach involves single-cell capture and culture on microtables/micropallets, and selective release and picking of microtables/micropallets for colony retrieval. This approach requires manual single-cell identification with microscope and a microscopic laser equipment to selectively release microtables/ micropallets. In order to increase the cell culture surface area of the microtables while reducing the laser energy required for releasing the table, a microfluidic device consists of an array of microtables each is 250 250 μm and supported by four legs was developed (Fig. 7.3A) (Pai et al., 2010). This device allowed for acquiring single-cell derived-colonies by a simple manipulation procedure, and the release of a microtable with only 10-μJ pulses laser energy. To further reduce cell damage from laser, a micropallet device containing thousands of 270 270 μm square pallets was developed (Fig. 7.3B) (Cox-Muranami et al., 2016). The device contains a gold film substrate to enhance laser absorption so the individual pallets can be released by using low-powered laser to
7.3
Single-Cell Isolation and Culture on Microfluidic Chip
(f)
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(e) Cell transferring by flipping device
(C) FIG. 7.2 Microfabricated microwells devices for single-cell isolation and culture. (A) The left-hand schematic shows the device fabrication and cell seeding procedures. The right-side photographs show trapped single cells in microwells of different diameters (Rettig and Folch, 2005). (B) Top schematic shows the device’s architecture and the operation procedure. Bottom photographs show SEM images of the hemispherical perforated microwells (Choi et al., 2014). (c) The left-hand schematic shows the operation procedure of the dual-well device. Microscopic images on the right were obtained from different steps of the operation procedure (Lin et al., 2015). (A) Reproduced with permission from Analytical Chemistry. (B) Reproduced with permission from RSC Advances. (C) Reproduced with permission from Lab on Chip.
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FIG. 7.3 Microfabricated table/pallet structures for single-cell separation and culture. (A) Left-hand schematic shows the fabricating process of the device. Right-hand microphotographs show the microtable structures. Bottom microscopic images show cell culture on microtables. (Pai et al., 2010). (B) Top schematic shows the fabrication process of the device. Bottom microphotographs show a section of the micropallet array and SEM images of a magnetic micropallet (Cox-Muranami et al., 2016). (A) Reproduced with permission from Analytical Bioanalytical Chemistry. (B) Reproduced with permission from Lab on Chip.
7.3
Single-Cell Isolation and Culture on Microfluidic Chip
retrieve cells. The cells collected from the micropallets exhibited high viability (>90%) and great recovery. The microtables/micropallets technique represents an attractive approach for single-cell culture due to its amenability to automation to achieve high-throughput operation. However, this technique is expensive due to the requirement of laser and precision positioning equipment.
7.3.3
Micro Lateral Chambers
Microchannels incorporating lateral chambers have been used to achieve highthroughput separation of single cells into compartment space. The lateral chambers are each connected to a microchannel through which cells in suspension flow to the chamber. Single-cell-in-a-chamber events are achieved by using diluted cell suspensions. A microfluidic device consists of 1400 lateral chambers was used to culture single cancer cells for tumor heterogeneity analysis (Fig. 7.4A) (Shen et al., 2015). Despite having a low single-cell efficiency (20%), the device could still grow several hundred of single-cell clones in one device. Another microfluidic device with 1500 lateral chambers has also been demonstrated to be useful for anticancer drug testing on single cells (Fig. 7.4B) (Zhang et al., 2016). The device was able to achieve about 34% highest single-cell efficiency cell suspension density and operation procedure. Micro lateral chambers have also been used for single-cell coculture. A device containing 120 lateral chambers was developed to study the interaction between stromal and single cancer cells (Fig. 7.4C) (Chen et al., 2016a). Micro lateral chambers are easy to scale up. Despite the highest achievable single-cell event ratio (i.e., the number of single-cell-containing chambers divided by the total number of the chambers), the capture efficiency is still limited by probability theory. Nonetheless, the obtainable single-cell event number from the experiments are still higher than using a conventional limiting dilution method. Micro lateral chambers however do not provide easy cell retrieval because of the difficulty in accessing cells in closed microchambers.
7.3.4
Cell Traps
A particle suspended in a fluid is subjected to hydrodynamic forces which control the movement of the particle. Hydrodynamic forces can be used to guide a cell to move in flow path leading the cell to be trapped in a small gap between two microstructures. Such microstructures capable of capturing a single-cell are termed single-cell traps, which can be incorporated in microchambers to facilitate single-cell isolation. An microfluidic device consists of a 8 8 chamber array was develop for single-cell isolation and clonal culture (Fig. 7.5A) (Chung et al., 2014). The device achieved a high single efficiency of >80% and was used to demonstrate the clonal heterogeneity of PC3 cancer cells in drug response. Another microfluidic device incorporating a U-shaped cell trap
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FIG. 7.4 Microfluidic devices with microlateral chambers for single-cell culture experiment. (A) Top schematic shows the operation of injection cell suspension into the device’s microchambers through branched microchannel. Bottom photographs show the time lapse of a single-cell entering a microchamber (Shen et al., 2015). (B) Top photo and schematic show the device and design of device, respectively. Bottom photographs show the process of a single-cell entering a microchamber (Zhang et al., 2016). (C) Top photo shows a microchamber of a microfluidic device for single-cell co-culture. The bottom photo shows co-culture of a single T47D cancer cell with stromal cells (Scale bar 100 μm) (Chen et al., 2016a). (A) Reproduced with permission from Scientific Reports. (B) Reproduced with permission from Lab on Chip. (C) Reproduced with permission from Scientific Reports.
was also used for high-throughput single-cell trapping and clonal culture (Fig. 7.5B) (Chen et al., 2015b). This device contains 528 chambers and has high single-cell isolation efficiencies on different cell types (78.9%–89.8%). Another microfluidic device, containing 1024 micro-chambers for growing single-cell-derived cell spheroids has also been reported (Fig. 7.5C) (Chen et al., 2016b). To prevent the isolated cells from attaching to the chamber surface, polyHEMA was used to treat the surface. This device also has high singlecell isolation efficiencies on different cell types (71%–84%). And finally, the same design concept was used in another device for single-cell-derived tumor spheroids formation (Fig. 7.5D) (Cheng et al., 2016). This device contains an impressive number of 12,800 single-cell traps and could reliably isolate 800 single cells per device. The single-cell-trap technique represents a useful method
FIG. 7.5 Single-cell isolation by single-cell traps. (A) The schematic shows a single-cell being hydrodynamically guided to a small gap structure in a microchamber (Chung et al., 2014). (B) Top schematic shows the design of a high-throughput microfluidic devices containing an array of single-cell trap-containing microchambers. Bottom image shows the detailed structure of the microchamber (Chen et al., 2015b). (C) Left photo shows single-cells trapped by the cell traps in a microchamber array. The right-hand schematic shows the design the microchamber (Chen et al., 2016b). (D) The image shows an array of microchambers and an enlarged portion of the array, showing three single cells being isolated in three adjacent microchambers (Cheng et al., 2016). (A) Reproduced with permission from Biomicrofluidics (B), (C) and (D) Reproduced with permission from Lab on Chip.
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to enhance single-cell isolation in microfluidic devices. But since it requires the use of hydrodynamic forces to guide cells to the traps, microfluidic devices incorporating this technique require good flow rate control during operation.
7.3.5
Microvalves
Microvalves are important components in microfluidic system. They are used for routing fluid as well as for active control of fluid compartmentation. For example, a microfluidic containing 45 microchambers uses micro-valves to create fully isolate microchambers in which cross-talk between neighboring microchambers is eliminated (Fig. 7.6A) (Zheng et al., 2012). The micro-valves were opened to replace the culture medium every 4 h as to supply nutrition and remove metabolic waste. Another microfluidic device also used integrated micro-valves to create 160 individually separable microchambers to culture individual human embryonic stem cell clones (Fig. 7.6B) (Sikorski et al., 2015). The device offers a useful tool to quantify cell proliferation, observe cell colony morphology and analyze the heterogeneity of the OCT4 expression among the cell colonies. Another microfluidic device containing 27 culture chambers and pneumatically controllable micro-valves has also been developed for single-cell cloning and expansion (Fig. 7.6C) (Matsumura et al., 2014). This device was utilized for long-term culture of single human induced pluripotent stem cells. Integrating micro-valves into microfluidic devices, allows for precise and on-demand fluid control but makes the devices more complex to fabricate and operate.
7.4
CONCLUSIONS AND FUTURE OUTLOOKS
Microfluidic techniques have emerged as a useful tool for single-cell culture applications owing to their ability to overcome obstacles encountered by conventional methods. However, there are still challenges which need to be removed in order to make microfluidic techniques more accessible and adaptable for single-cell culture applications. First, most of reported devices are made of PDMS, which although being cell culture-compatible, has physical and chemical properties different from that of petri-dish. Therefore cell culture protocols that work with conventional culture dishes may need to be adjusted to obtain optimal outcome and cell behaviors normally seen in cells cultured in pertri dishes. Or alternatively, the microfluidic devices may need to be made of the same plastic materials used for petri-dish. However, some of the current designs may not be easily translated into plastic devices due to potential difficulties in device fabrication and operation. Second, most of the current microfluidic devices require the use of external equipment (e.g., fluid pump, pneumatic pump, pinch valves, controller, etc.) to operate, and cannot be easily setup by a regular biology laboratory personnel. This has become a major
Isolation valve
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FIG. 7.6 Microfluidic devices incorporating microvalves for fluid routing and compartmentation. (A) A microfluidic device integrated with microvalves was used to isolate individual single cells and provide compartmentalized space for cell clonal growth (Zheng et al., 2012). (B) A microfluidic device with a complex microvalves system for single-cell culture experiment (Sikorski et al., 2015). (C) A microfluidic device using microvalves to separate single cells in its microchannel for single-cell culture (Matsumura et al., 2014). (A) Reproduced with permission from Science China Chemistry. (B) Reproduced with permission from Biotechnology Journal. (C) Reproduced with permission from Biochemical and Biophysical Research Communications.
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hurdle in the dissemination of microfluidic techniques to common biological laboratories. To overcome this problem, the operation and setup of current microfluidic techniques for single-cell culture should be further simplified. And last, current microfluidic tools are not easily accessible to biological laboratories due to the fact that very few of the reported techniques are commercialized. This problem could be solved when more techniques are translated into products and the cost of the devices are also brought down.
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Pai, J.H., Xu, W., Sims, C.E., Allbritton, N.L., 2010. Microtable arrays for culture and isolation of cell colonies. Anal. Bioanal. Chem. 398 (6), 2595–2604. Qiu, X.L., De Jesus, J., Pennell, M., Troiani, M., Haun, J.B., 2015. Microfluidic device for mechanical dissociation of cancer cell aggregates into single cells. Lab Chip 15 (1), 339–350. Rettig, J.R., Folch, A., 2005. Large-scale single-cell trapping and imaging using microwell arrays. Anal. Chem. 77 (17), 5628–5634. Sato, S., Rancourt, A., Sato, Y., Satoh, M.S., 2016. Single-cell lineage tracking analysis reveals that an established cell line comprises putative cancer stem cells and their heterogeneous progeny. Sci. Rep. 6. Shen, F.M., Zhu, L., Ye, H., Yang, Y.J., Pang, D.W., Zhang, Z.L., 2015. A high throughput microchamber array device for single cell clonal cultivation and tumor heterogeneity analysis. Sci. Rep. 5. Sikorski, D.J., Caron, N.J., VanInsberghe, M., Zahn, H., Eaves, C.J., Piret, J.M., Hansen, C.L., 2015. Clonal analysis of individual human embryonic stem cell differentiation patterns in microfluidic cultures. Biotechnol. J. 10 (10), 1546–1554. Vermeulen, L., Todaro, M., de Sousa Mello, F., Sprick, M.R., Kemper, K., Perez Alea, M., Richel, D.J., Stassi, G., Medema, J.P., 2008. Single-cell cloning of colon cancer stem cells reveals a multilineage differentiation capacity. Proc. Natl. Acad. Sci. U. S. A. 105 (36), 13427–13432. Wallman, L., Akesson, E., Ceric, D., Andersson, P.H., Day, K., Hovatta, O., Falci, S., Laurell, T., Sundstrom, E., 2011. Biogrid—a microfluidic device for large-scale enzyme-free dissociation of stem cell aggregates. Lab Chip 11 (19), 3241–3248. Zhang, Z.X., Chen, Y.C., Cheng, Y.H., Luan, Y., Yoon, E., 2016. Microfluidics 3D gel-island chip for single cell isolation and lineage-dependent drug responses study. Lab Chip 16 (13), 2504–2512. Zheng, C.H., Chen, G.E., Pang, Y.H., Huang, Y.Y., 2012. An integrated microfluidic device for longterm culture of isolated single mammalian cells. Sci. China Chem. 55 (4), 502–507.
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Microfluidic Techniques for Single-Cell Culture Chuan-Feng Yeh*,†, Chia-Hsien Hsu*,†,‡ *Institute of Biomedical Engineering and Nanomedicine, National Health Research Institutes, Miaoli, Taiwan † Institute of Nanoengineering and Microsystems, National Tsing Hua University, Hsinchu, Taiwan ‡ Ph.D. Program in Tissue Engineering and Regenerative Medicine, National Chung Hsing University, Taichung, Taiwan
7.1 ADVANTAGES OF MICROFLUIDIC TECHNIQUES FOR SINGLE-CELL CULTURE Cell heterogeneity is always present to some level in all cell populations, thus the collective behaviors of a population of cells does not represent the behaviors of individual cells. Therefore, it is important to analyze individual single cells in order to discover mechanisms not seen by studying a bulk population of cells. Single-cell culture is a method of growing isolated single cells routinely performed to obtain single-cell-derived cell clones for both basic research and therapeutically applications. Besides forming clones, culturing individually isolated single cells can also help understand the metabolic, migration and differentiation heterogeneity in cell populations (Vermeulen et al., 2008; Sato et al., 2016). Despite single-cell culture being essential and widely used, single-cell culture is still a challenging task for many laboratories due to the difficulty of manipulating and observing single cells. Microfluidic techniques have emerged as a useful tool for single-cell culture applications. In comparison to conventional petri dishes and well plates, culturing cells in microfluidic devices offers several advantages: (1)
The microdevices can more precisely manipulate single cells and control the cell culture conditions, thus making it useful for providing better-controlled experimental conditions. For example, microfluidic channel can be used to generate stable chemical gradients for studying
Single-Cell Omics. https://doi.org/10.1016/B978-0-12-814919-5.00007-5 © 2019 Elsevier Inc. All rights reserved.
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(2) (3)
single-cell chemotaxis at high temporal and/or spatial resolution (Chen et al., 2015a). The miniaturized devices can increase the throughput and reduce reagent/cell consumptions of experiments due to their small sizes. Microfluidic devices are amenable to integration with microelectromechanical systems (MEMS) technology to form lab-ona-chip devices, which allow for combining single-cell culture and analysis in one device. Besides making cell-culture devices, microfluidic techniques can be used as a helper tool to prepare cells for single-cell culture with conventional culture dishes and well plates for applications where single-cell suspension preparation is difficult.
This chapter will describe and compare state-of-the-art microfluidic techniques for single-cell preparation, separation, and culture.
7.2 CELL CLUSTER DISSOCIATION BY MICROFLUIDIC CHIP Having a cell suspension containing individually separated single cells is a prerequisite for performing single-cell culture experiments. For tissues and cells that are cultured in cluster/aggregate format, a cell dissociation step is required to break the cells into individual cells to prepare the single-cell suspension. During cell dissociation process, the extracellular matrix and cell-cell junctions must be disrupted by enzyme or mechanical force. Using enzymatic chemicals such as trypsin, elastase and hyaluronidase to destroy the cell-matrix adhesion and cellcell junctions is simple to perform. However, the amount and type of enzyme to be used need to be carefully chosen in order to not damage the cells or cause cell surface protein expression loss (Garg et al., 2014). Mechanical methods including shaking, pipetting, vortexing and sonication can also be used for cell dissociation. These physical methods however are relatively more laborious, and result in lower cell viability compared to enzymatic methods. And more importantly, it is very difficult if not impossible for pure mechanical methods to produce cell suspension containing a high percentage of viable dissociated single cells. Microfluidic techniques have been demonstrated as a useful tool for single-cell suspension preparation. Owing to the ability of microfluidics to precisely control a small volume of fluid under laminar flow condition, the generated mechanical force during cell dissociation is more controllable and reproducible, resulting in increased viable single-cell ratio in the prepared cell suspension. Some example of microfluidic cell dissociation techniques are described below. In order to reduce contamination risk and to scale up the production of neural stem cells for clinical uses, a “biogrid” device was developed for neurosphere dissociation (Wallman et al., 2011). The device contains a silicon substrate with grids whose edges were 20 μm thick with 200 μm edge to edge spacing
7.2
Cell Cluster Dissociation by Microfluidic Chip
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FIG. 7.1 Microfabricated devices for cell cluster dissociation. (A) Top photograph is a microfabricated 3-in. silicon wafer. The photograph inset shows the dissociating grids before wafer dicing. In the bottom photograph, individual microgrids are assembled into the device adapter before being used for cell dissociation (Wallman et al., 2011). (B) The left-hand photograph shows the single channel “μ-CDC” chip fabricated via soft lithography and PDMS. On the right is a scanning electron micrograph showing the microfabricated pillars. Neurospheres passing through the micropillar array (white arrow ) are mechanically dissociated into single cells (Lin et al., 2013). (C) Photograph of fabricated device for dissociating small tumor tissue via alternating constriction and expansion regions (Qiu et al., 2015). (A) Reproduced with permission from Cytotherapy. (B) Reproduced with permission from Analytical Chemistry. (C) Reproduced with permission from Lab on Chip.
(Fig. 7.1A) (Wallman et al., 2011). This device is easy to use that it can be attached to a syringe in which neurosphere suspension is loaded and can be pushed out to go through the biogrid to mechanically break neurospheres whose size are bigger than the grid-to-grid spacing. Another microfluidic device containing an array of micropillars for neurosphere dissociation has also been reported (Lin et al., 2013). The micropillars are each 50 μm wide and 167 μm tall with 20 μm spacing between adjacent pillars (Fig. 7.1B). A neurosphere suspension can be injected into the device via the device’s inlet with a syringe pump. Neurospheres were dissociated into single cells by passage through the pillar array at 3–15 mL/min flow rate. The dissociated cells are collected from the device’s outlet. It is worth mentioning that the chip could achieve high single-cell dissociation yield (91%–95%) at high single-cell viability (80%–85%), which could not be produced by the conventional trituration method (50% viability). Microfluidics-based methods could also help improve cell recovery after cell dissociation process for rare samples such as tissues aspirated from needle biopsy. A microfluidic device containing branching channels has been demonstrated for rare sample dissociation (Qiu et al., 2015). The device was capable of dissociating tumor tissue equal of size less than 1 mm into single cells (Fig. 7.1C). The dissociation is achieved by using shear forces generated by flowing cell suspension through microchannel which has repeated constriction and expansion sections. The tumor cell dissociation with this device could achieve the same cell yields but showed a significant increase
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in the single-cell population, from 60% to 90% when compared to a control dissociation method utilizing trypsin-EDTA, vortexing, and pipetting. The device dissociation could also be completed within 10 min. In summary, microfluidic techniques represent an attractive tool for cell aggregates dissociation for preparing single-cell suspension, as performing cell dissociation in microfluidic systems is easy to scale-up and can have higher productivity. Moreover, being able to handle cells in a closed microchannel system can also reduce contamination risk. Because of the tightly controlled and laminar flow environments, microfluidic techniques are able to reduce cell damage increase single-cell yield and reproducibility. Requiring smaller sample volumes also makes microfluidic devices useful for handling limited clinical samples. Lastly, the ability of microfluidic technique to improve the outcome of nonenzymatic single-cell dissociation is of great importance from a regulatory standpoint. The nonenzymatic methods are also useful for researchers in obtaining single cells that maintain endogenous surface markers.
7.3 SINGLE-CELL ISOLATION AND CULTURE ON MICROFLUIDIC CHIP To start a single-cell culture experiment, individually isolated cells must be first prepared in a cell culture device. Despite being a routinely performed task in many laboratories, single-cell isolation still represents a major challenge in single-cell culture. Conventionally, this cell isolation task is commonly performed by using limiting dilution, micromanipulation or fluorescenceactivated cell sorting (FACS) technique. Isolation of individual cells by limiting dilution is achieved by using hand-held pipettes or pipetting robots to split a diluted cell suspension into small aliquots. Depending on the cell number in the diluted cell suspension, the probability of having 0, 1, 2, 3, or more cells in an aliquot can be changed. The method is simple to carry out, but relatively low efficiency due to statistical nature. Micromanipulation relies on using a micropipette to manually select and pick individual single cells. In order to ensure high confidence level of single-cell isolation, the procedure is generally performed with manual identification under a microscope, therefore the throughput is very limited. Isolating single-cell by using FACS is high-throughput and automated. However, FACS machines are expensive and not suitable for handling small sample volumes. Furthermore, in comparison to limiting dilution and micromanipulation, FACS isolation is more likely to cause cell damage. Microfluidic techniques are able to overcome the hurdles encountered by current single-cell isolation methods through miniaturization, microfluidics, and integration with MEMS technology. To date, many microfluidic techniques have been utilized to facilitate manipulation, observation, validation, and culture of single cells (summarized in Table 7.1). These techniques are categorized as described in the following sections.
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Single-Cell Isolation and Culture on Microfluidic Chip
Table 7.1 Comparison of Various Microfluidic Techniques for Single-Cell Culture. Isolation Approaches
Culture Condition
Microwells (Rettig and Folch, 2005; Choi et al., 2014; Lin et al., 2015)
Microtable (Pai et al., 2010) Micropallets (CoxMuranami et al., 2016) Microlateral chambers (Shen et al., 2015; Zhang et al., 2016; Chen et al., 2016a)
Clonal Transfer
Major Advantage
Major Disadvantage
Compartmental wells
No (Rettig and Folch, 2005; Lin et al., 2015) Yes (Choi et al., 2014)
Unable to transfer colonies (Rettig and Folch, 2005; Lin et al., 2015) Pooled colonies (Choi et al., 2014)
Compartmental pallets, tables
Yes (Pai et al., 2010; CoxMuranami et al., 2016)
Easy manipulation (Rettig and Folch, 2005; Choi et al., 2014; Lin et al., 2015) High throughput (Rettig and Folch, 2005; Choi et al., 2014; Lin et al., 2015) Easy fabrication (Rettig and Folch, 2005; Lin et al., 2015) High efficiency in obtaining colonies (Pai et al., 2010; CoxMuranami et al., 2016)
Compartmental chambers
No (Shen et al., 2015; Zhang et al., 2016; Chen et al., 2016a)
High throughput (Shen et al., 2015; Zhang et al., 2016; Chen et al., 2016a)
Cell traps (Chung et al., 2014; Chen et al., 2015b, 2016b; Cheng et al., 2016)
Compartmental chambers
No (Chung et al., 2014; Chen et al., 2015b, 2016b; Cheng et al., 2016)
Microvalves (Zheng et al., 2012; Sikorski et al., 2015; Matsumura et al., 2014)
Individual chambers possible/under medium perfusion possible
No (Zheng et al., 2012; Sikorski et al., 2015) Yes (Matsumura et al., 2014)
High throughput (Chung et al., 2014; Chen et al., 2015b, 2016b; Cheng et al., 2016) High single-cell efficiency (Chung et al., 2014; Chen et al., 2015b, 2016b; Cheng et al., 2016) Scaling and automation (Cheng et al., 2016) Controllable microenvironments (Zheng et al., 2012; Sikorski et al., 2015; Matsumura et al., 2014)
Special laser equipment requirement (Pai et al., 2010; Cox-Muranami et al., 2016) Unable to transfer colonies (Shen et al., 2015; Zhang et al., 2016; Chen et al., 2016a) Low single-cell efficiency (Shen et al., 2015; Zhang et al., 2016; Chen et al., 2016a) Unable to transfer colonies (Chung et al., 2014; Chen et al., 2015b, 2016b; Cheng et al., 2016) Difficult fabrication (Chung et al., 2014; Chen et al., 2015b, 2016b; Cheng et al., 2016) Difficult fabrication (Zheng et al., 2012; Sikorski et al., 2015; Matsumura et al., 2014) Complex setting for experiments (Zheng et al., 2012; Sikorski et al., 2015; Matsumura et al., 2014)
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7.3.1
Microwells
Microfluidic devices containing miniaturized wells have been developed to overcome the difficulty of single-cell compartmentalization and identification for single-cell analysis and culture applications. A polydimethylsiloxane (PDMS) microwell device (Fig. 7.2A) was developed for single-cell isolation of fibroblast (as model of adherent cells) and rat basophilic leukemia cells (as model of suspension cells) (Rettig and Folch, 2005). The single-cell isolation efficiency is dependent on the microwells’ dimensions and cell seeding density. This microfluidic device provides a simple yet high-efficient approach to isolate and analyze single cells but is not applicable to single-cell culture due to the limited space of the microwells. In order to culture single cells and allow for retrieval of single-cell colonies, a hemispherical microwell device was developed (Fig. 7.2B) (Choi et al., 2014). This device utilized a more complex threelayer microchannel structure to capture and isolate single cells in microwells. This device was able to achieve high single-cell capture efficiency (>90%) and was suitable for long-term culture and retrieval of colonies. Another microfluidic device, termed “dual well device” has also been developed for highefficiency single-cell culture (Fig. 7.2B) (Lin et al., 2015). The advantages of this device lies in the fact that it utilizes a “dual-well” design to allow for simple device fabrication and operation (Fig. 7.2C). The device could achieve high single-cell isolation efficiency (77%) and its single-cell culture applicability was demonstrated with mouse neural stem cell differentiation, cancer cell proliferation and single-cell colony formation assay on chip. This device provides a simple approach to analyze single-cell heterogeneity during cell culture.
7.3.2
Microtables/Micropallets
Another approach for separating individual cells and picking colonies is based on fabricating small table or pallet structure in microdevices (Fig. 7.3). This approach involves single-cell capture and culture on microtables/micropallets, and selective release and picking of microtables/micropallets for colony retrieval. This approach requires manual single-cell identification with microscope and a microscopic laser equipment to selectively release microtables/ micropallets. In order to increase the cell culture surface area of the microtables while reducing the laser energy required for releasing the table, a microfluidic device consists of an array of microtables each is 250 250 μm and supported by four legs was developed (Fig. 7.3A) (Pai et al., 2010). This device allowed for acquiring single-cell derived-colonies by a simple manipulation procedure, and the release of a microtable with only 10-μJ pulses laser energy. To further reduce cell damage from laser, a micropallet device containing thousands of 270 270 μm square pallets was developed (Fig. 7.3B) (Cox-Muranami et al., 2016). The device contains a gold film substrate to enhance laser absorption so the individual pallets can be released by using low-powered laser to
7.3
Single-Cell Isolation and Culture on Microfluidic Chip
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(e) Cell transferring by flipping device
(C) FIG. 7.2 Microfabricated microwells devices for single-cell isolation and culture. (A) The left-hand schematic shows the device fabrication and cell seeding procedures. The right-side photographs show trapped single cells in microwells of different diameters (Rettig and Folch, 2005). (B) Top schematic shows the device’s architecture and the operation procedure. Bottom photographs show SEM images of the hemispherical perforated microwells (Choi et al., 2014). (c) The left-hand schematic shows the operation procedure of the dual-well device. Microscopic images on the right were obtained from different steps of the operation procedure (Lin et al., 2015). (A) Reproduced with permission from Analytical Chemistry. (B) Reproduced with permission from RSC Advances. (C) Reproduced with permission from Lab on Chip.
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FIG. 7.3 Microfabricated table/pallet structures for single-cell separation and culture. (A) Left-hand schematic shows the fabricating process of the device. Right-hand microphotographs show the microtable structures. Bottom microscopic images show cell culture on microtables. (Pai et al., 2010). (B) Top schematic shows the fabrication process of the device. Bottom microphotographs show a section of the micropallet array and SEM images of a magnetic micropallet (Cox-Muranami et al., 2016). (A) Reproduced with permission from Analytical Bioanalytical Chemistry. (B) Reproduced with permission from Lab on Chip.
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Single-Cell Isolation and Culture on Microfluidic Chip
retrieve cells. The cells collected from the micropallets exhibited high viability (>90%) and great recovery. The microtables/micropallets technique represents an attractive approach for single-cell culture due to its amenability to automation to achieve high-throughput operation. However, this technique is expensive due to the requirement of laser and precision positioning equipment.
7.3.3
Micro Lateral Chambers
Microchannels incorporating lateral chambers have been used to achieve highthroughput separation of single cells into compartment space. The lateral chambers are each connected to a microchannel through which cells in suspension flow to the chamber. Single-cell-in-a-chamber events are achieved by using diluted cell suspensions. A microfluidic device consists of 1400 lateral chambers was used to culture single cancer cells for tumor heterogeneity analysis (Fig. 7.4A) (Shen et al., 2015). Despite having a low single-cell efficiency (20%), the device could still grow several hundred of single-cell clones in one device. Another microfluidic device with 1500 lateral chambers has also been demonstrated to be useful for anticancer drug testing on single cells (Fig. 7.4B) (Zhang et al., 2016). The device was able to achieve about 34% highest single-cell efficiency cell suspension density and operation procedure. Micro lateral chambers have also been used for single-cell coculture. A device containing 120 lateral chambers was developed to study the interaction between stromal and single cancer cells (Fig. 7.4C) (Chen et al., 2016a). Micro lateral chambers are easy to scale up. Despite the highest achievable single-cell event ratio (i.e., the number of single-cell-containing chambers divided by the total number of the chambers), the capture efficiency is still limited by probability theory. Nonetheless, the obtainable single-cell event number from the experiments are still higher than using a conventional limiting dilution method. Micro lateral chambers however do not provide easy cell retrieval because of the difficulty in accessing cells in closed microchambers.
7.3.4
Cell Traps
A particle suspended in a fluid is subjected to hydrodynamic forces which control the movement of the particle. Hydrodynamic forces can be used to guide a cell to move in flow path leading the cell to be trapped in a small gap between two microstructures. Such microstructures capable of capturing a single-cell are termed single-cell traps, which can be incorporated in microchambers to facilitate single-cell isolation. An microfluidic device consists of a 8 8 chamber array was develop for single-cell isolation and clonal culture (Fig. 7.5A) (Chung et al., 2014). The device achieved a high single efficiency of >80% and was used to demonstrate the clonal heterogeneity of PC3 cancer cells in drug response. Another microfluidic device incorporating a U-shaped cell trap
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FIG. 7.4 Microfluidic devices with microlateral chambers for single-cell culture experiment. (A) Top schematic shows the operation of injection cell suspension into the device’s microchambers through branched microchannel. Bottom photographs show the time lapse of a single-cell entering a microchamber (Shen et al., 2015). (B) Top photo and schematic show the device and design of device, respectively. Bottom photographs show the process of a single-cell entering a microchamber (Zhang et al., 2016). (C) Top photo shows a microchamber of a microfluidic device for single-cell co-culture. The bottom photo shows co-culture of a single T47D cancer cell with stromal cells (Scale bar 100 μm) (Chen et al., 2016a). (A) Reproduced with permission from Scientific Reports. (B) Reproduced with permission from Lab on Chip. (C) Reproduced with permission from Scientific Reports.
was also used for high-throughput single-cell trapping and clonal culture (Fig. 7.5B) (Chen et al., 2015b). This device contains 528 chambers and has high single-cell isolation efficiencies on different cell types (78.9%–89.8%). Another microfluidic device, containing 1024 micro-chambers for growing single-cell-derived cell spheroids has also been reported (Fig. 7.5C) (Chen et al., 2016b). To prevent the isolated cells from attaching to the chamber surface, polyHEMA was used to treat the surface. This device also has high singlecell isolation efficiencies on different cell types (71%–84%). And finally, the same design concept was used in another device for single-cell-derived tumor spheroids formation (Fig. 7.5D) (Cheng et al., 2016). This device contains an impressive number of 12,800 single-cell traps and could reliably isolate 800 single cells per device. The single-cell-trap technique represents a useful method
FIG. 7.5 Single-cell isolation by single-cell traps. (A) The schematic shows a single-cell being hydrodynamically guided to a small gap structure in a microchamber (Chung et al., 2014). (B) Top schematic shows the design of a high-throughput microfluidic devices containing an array of single-cell trap-containing microchambers. Bottom image shows the detailed structure of the microchamber (Chen et al., 2015b). (C) Left photo shows single-cells trapped by the cell traps in a microchamber array. The right-hand schematic shows the design the microchamber (Chen et al., 2016b). (D) The image shows an array of microchambers and an enlarged portion of the array, showing three single cells being isolated in three adjacent microchambers (Cheng et al., 2016). (A) Reproduced with permission from Biomicrofluidics (B), (C) and (D) Reproduced with permission from Lab on Chip.
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to enhance single-cell isolation in microfluidic devices. But since it requires the use of hydrodynamic forces to guide cells to the traps, microfluidic devices incorporating this technique require good flow rate control during operation.
7.3.5
Microvalves
Microvalves are important components in microfluidic system. They are used for routing fluid as well as for active control of fluid compartmentation. For example, a microfluidic containing 45 microchambers uses micro-valves to create fully isolate microchambers in which cross-talk between neighboring microchambers is eliminated (Fig. 7.6A) (Zheng et al., 2012). The micro-valves were opened to replace the culture medium every 4 h as to supply nutrition and remove metabolic waste. Another microfluidic device also used integrated micro-valves to create 160 individually separable microchambers to culture individual human embryonic stem cell clones (Fig. 7.6B) (Sikorski et al., 2015). The device offers a useful tool to quantify cell proliferation, observe cell colony morphology and analyze the heterogeneity of the OCT4 expression among the cell colonies. Another microfluidic device containing 27 culture chambers and pneumatically controllable micro-valves has also been developed for single-cell cloning and expansion (Fig. 7.6C) (Matsumura et al., 2014). This device was utilized for long-term culture of single human induced pluripotent stem cells. Integrating micro-valves into microfluidic devices, allows for precise and on-demand fluid control but makes the devices more complex to fabricate and operate.
7.4
CONCLUSIONS AND FUTURE OUTLOOKS
Microfluidic techniques have emerged as a useful tool for single-cell culture applications owing to their ability to overcome obstacles encountered by conventional methods. However, there are still challenges which need to be removed in order to make microfluidic techniques more accessible and adaptable for single-cell culture applications. First, most of reported devices are made of PDMS, which although being cell culture-compatible, has physical and chemical properties different from that of petri-dish. Therefore cell culture protocols that work with conventional culture dishes may need to be adjusted to obtain optimal outcome and cell behaviors normally seen in cells cultured in pertri dishes. Or alternatively, the microfluidic devices may need to be made of the same plastic materials used for petri-dish. However, some of the current designs may not be easily translated into plastic devices due to potential difficulties in device fabrication and operation. Second, most of the current microfluidic devices require the use of external equipment (e.g., fluid pump, pneumatic pump, pinch valves, controller, etc.) to operate, and cannot be easily setup by a regular biology laboratory personnel. This has become a major
Isolation valve
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PDMS Glass : Medium flow
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PDMS Glass : Medium flow
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FIG. 7.6 Microfluidic devices incorporating microvalves for fluid routing and compartmentation. (A) A microfluidic device integrated with microvalves was used to isolate individual single cells and provide compartmentalized space for cell clonal growth (Zheng et al., 2012). (B) A microfluidic device with a complex microvalves system for single-cell culture experiment (Sikorski et al., 2015). (C) A microfluidic device using microvalves to separate single cells in its microchannel for single-cell culture (Matsumura et al., 2014). (A) Reproduced with permission from Science China Chemistry. (B) Reproduced with permission from Biotechnology Journal. (C) Reproduced with permission from Biochemical and Biophysical Research Communications.
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Microfluidic Techniques for Single-Cell Culture
hurdle in the dissemination of microfluidic techniques to common biological laboratories. To overcome this problem, the operation and setup of current microfluidic techniques for single-cell culture should be further simplified. And last, current microfluidic tools are not easily accessible to biological laboratories due to the fact that very few of the reported techniques are commercialized. This problem could be solved when more techniques are translated into products and the cost of the devices are also brought down.
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