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A Microfl uidic Hydrogel Capable of Cell Preservation without Perfusion Culture under Cell-Based Assay Conditions
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By Yan Xu , * Kae Sato , Kazuma Mawatari , Tomohiro Konno , Kihoon Jang , Kazuhiko Ishihara , and Takehiko Kitamori *
The interest, applications, and need for living cells have been dramatically expanding in the fi elds of medicine, pharmacy, environment, and defense. [ 1–3 ] Cell-based applications, such as drug discoveries, toxin screenings, and cell diagnostics are conventionally performed on cell culture dishes or micro-plates. Microfl uidic chip systems with micro/nanostructured geometries and microfl uidic channel networks that precisely manage the fl uids and soluble factors can be used to organize cells in dimensions that are comparable to those in vivo. [ 2 , 4 ] In addition, microfl uidic chip systems possess unique advantages over conventional analytical tools including minute reagent consumption, high throughput analysis, and rapid detection. [ 5 , 6 ] Owing to these merits of microfl uidics, recently developed cell-based applications are progressing toward a more controllable and reproducible process on microfl uidic chips. [ 2 , 4 , 7 ]
A possible impediment to the widespread use of microfl uidic chips for routine cell-based applications, however, is the lack of feasible approaches to the preservation of cells on the chip, espe-cially under normal assay conditions. In most cell-based micro-fl uidic systems, to sustain cell survival, a continuous supply of the medium fl uid via a tubing system with an external syringe pump (i.e., perfusion culture) is essential. [ 8–10 ] Obviously, without thorough off-chip support such as highly equipped facilities and highly trained personnel, the chip systems are diffi cult to manage, even in a laboratory. Conversely, some researchers have tried to develop on-chip cryopreservation techniques. [ 11 , 12 ] Never-theless, the extreme low temperature conditions required render these techniques incapable of working under normal assay conditions. For miniaturization, integration, and fi nal commer-cialization of cell-based applications on-chip, approaches for the preservation of living cells on-chip under normal assay condi-tions are vital and necessary. Unfortunately, to our knowledge, there are no reports on such kind of techniques.
© 2010 WILEY-VCH Verlag GAdv. Mater. 2010, 22, 3017–3021
DOI: 10.1002/adma.201000006
[∗] Dr. Y. Xu , Dr. K. Sato , Dr. K. Mawatari , Dr. K. Jang , Prof. T. Kitamori Department of Applied ChemistrySchool of EngineeringThe University of Tokyo7-3-1, Hongo, Bunkyo, Tokyo 113-8656 (Japan) E-mail: [email protected] ; [email protected] Dr. T. Konno , Prof. K. Ishihara Department of Materials EngineeringSchool of EngineeringThe University of Tokyo7-3-1 Hongo, Bunkyo, Tokyo 113-8656 (Japan)
As the development of cell encapsulation and tissue engi-neering technologies progresses, hydrogels have been utilized to encapsulate cells in microfl uidic channels. [ 13–16 ] Most cell-encapsulating hydrogels show the merits of spatial distribution of cells and benefi cial properties as tissue-engineering scaffolds under microfl uidic conditions, but have potential limitations when they are used to preserve cells. For example, cells encap-sulated in the hydrogels exhibit either low viability or high pro-liferation after a short period of encapsulation. Once a cell is encapsulated in hydrogel, it is usually irreversible, and the cells are likely to be affected by the toxic photoinitiator or the radia-tion treatment widely applied in the encapsulation.
This communication addresses a phospholipid polymer hydrogel, not only capable of encapsulation of cells spontane-ously and reversibly without causing any adverse physical effects to the cell, such as photo- and thermal-treatments, but also capable of preservation of cells on-chip without perfusion cul-ture under cell-based assay conditions, representing characteris-tics of maintenance of cell viability, restraint of cell proliferation, and minimization of cellular function loss over a period of days and weeks. Presented herein is a promising hydrogel material for on-chip cell preservation, which would bring about a new method for cell-based applications to be performed on microfl u-idic chips in a more fl exible, intuitive, and cheaper fashion.
The hydrogel is composed of polyvinyl alcohol (PVA) and a water-soluble phospholipid polymer, poly (2-methacryloyloxye-thyl phosphorylcholine (MPC)- co - n -butyl methacrylate (BMA)- co - p -vinylphenylboronic acid (VPBA)) (referred to as PMBV, Figure 1A ), which was previously designed for cell encapsula-tion in bulk. [ 17 ] The PMBV was synthesized by a conventional radical polymerization (see Supporting Information (SI), Part 1). The MPC units in the PMBV contain phosphorycholine (PC) moi-eties which are major components of the cell membrane and have been shown to signifi cantly inhibit nonspecifi c interactions with various proteins, platelets, and cells; hence the polymers forming MPC units (i.e., MPC polymers) possess excellent biocompat-ibility when transferring from bulk to micro/nanoscale. [ 18–20 ] The phosphorylcholine-containing polymers have been referred to as phospholipid polymers, [ 18–20 ] so the PMBV is also described as a phospholipid polymer in this study. In PMBV, the mole fraction of the MPC units is 60%, which makes PMBV water-soluble, as the MPC polymers having more than 30% MPC units in mole frac-tion are readily water-soluble. [ 21–23 ] Accordingly, the PMBV can be dissolved in water and in various cell culture media, which is favo-rable for microfl uidic devices designed to handle cells. The PMBV/PVA hydrogel spontaneously forms in an aqueous medium when
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Figure 1 . Overview of the microfl uidic hydrogel PMBV/PVA for cell preservation. A) Chemical structure of PMBV; a / b / c = 60/30/10. B) Mechanism of PMBV/PVA hydrogel formation. C) A picture of the two-chamber chip fabricated on glass substrates. D) Schematic diagram of cell preservation in the PMBV/PVA hydrogel formed in the two-chamber chip.
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PMBV meets PVA, because of the covalently cross-linking mech-anism between the phenylboronic acid moiety in the side chain of PMBV and the hydroxyl groups in PVA (Figure 1B ). [ 24 ] When a sugar solution (such as D -glucose, D -galactose, and D -fructose) is added, the hydrogel is gradually dissociated and reversibly changes to a solution state, owing to a reaction effect between the hydroxyl groups of the sugar molecule and the phenyboronic acid moiety of the PMBV. [ 17 , 25 ] Because both the formation and the dis-sociation of the PMBV/PVA hydrogel are under fl uidic conditions, they can be easily handled and controlled in the chip by microfl u-idics. Through consideration of these features, the PMBV/PVA hydrogel was used to encapsulate living cells on-chip.
The chip (Figure 1C ) was fabricated on glass substrates (see SI, Part 2). It is composed of two sets of cell-container chambers and accessorial introducing channels, and thereby two inde-pendent experiments can be performed at one time under the same conditions. The chambers are 5.0 mm in radius and approximately 400 μ m in depth ( D ), and each is approximately 30 μ L in total volume. All channels are 700 μ m in width and 200 μ m in depth, which are sizes designed to facilitate the transportation of cells.
After delivering 2.5 wt% PVA solution (prepared in 1 × phos-phate buffer saline (PBS)) and cell suspension in a culture medium containing 5.0 wt% PMBV (with a ratio of PMBV
© 2010 WILEY-VCH Verlag G
(cells)/PVA = 3/1, v/v) through the introducing microchannels into the cell-container chamber, the cells can be encapsulated in the PMBV/PVA hydrogel, which has formed in the chamber (Figure 1D ). The gelation of two water-soluble polymers depends on their concentration and mixing ratio. The optimal concen-tration (i.e., 5.0 wt% PMBV and 2.5 wt% PVA) and mixing ratio (i.e., PMBV/PVA = 3/1, v/v) obtained through serial gela-tion experiments with a 96-well microplate were applied to the hydrogel formation on the chip. Although the volume and D of the chamber of the chip are much smaller than those of the well of the microplate (see SI, Part 2), the experiments confi rmed that the gelation condition also worked in the chip. It is considered that the gelation (crosslinking) kinetics is controlled by the dif-fusion of both polymer chains in an aqueous medium. In bulk, the reactive gelation time estimated by a dynamic viscoelasticity measurement was quite lengthy, so a gentle shaking is usually applied to accelerate the gelation. [ 17 ] In the chip, as the feature sizes (especially depth, D Chamber(Chip) : D Well (96–well microplate) = 1 : 23) are greatly scaled down, the distances for molecule diffusion are signifi cantly shortened. Consequently, the PMBV and the PVA molecules can adequately crosslink in a shorter time. The hydrogel could form immediately in the chip without shaking after the introduction of the polymer solutions. In addition, there were no adverse effects found resulting from the involvement
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of the cells on the hydrogel formation in the chip. The cell-encapsulating hydrogel formed within minutes in the chip.
The preservations of mouse fi broblast cell line L929 cells and human arterial endothelial cells (HAECs) were investigated. Both fi broblast cells and endothelial cells are widely used as model cells in many biology studies, especially toxin screen-ings and tissue engineering. [ 26 , 27 ] In the chamber, L929 cells dispersed inside the hydrogel and exhibited round geometry after encapsulation ( Figure 2A ). In conventional dish culture, l929 cells tend to gradually adhere on the surface of the dish after seeding, and the cell morphology then becomes hetero-geneous with diverse appearances depending on their location and activity in contrast, L929 cells preserved in the PMBV/PVA hydrogel in the chip remained round during the entire period (See SI, Part 3). In the case of preserved HAECs, a similar round cell morphology was observed. Adherent cells such as L929 and HAECs, when attaching, spreading, and fl attening to the sur-face tend to grow quickly. In contrast, when cells are round, the cell growth is restrained. The number of L929 cells preserved in the hydrogel in the chip did not increase throughout the 18-day preservation period (by comparing the different panels of Figure 2A–D ), indicating that the proliferation rate was quite low for cells preserved in PMBV/PVA hydrogel in the chip.
© 2010 WILEY-VCH Verlag GmAdv. Mater. 2010, 22, 3017–3021
Figure 2 . Evaluation of cell viability. Upper: A) A phase contrast microscoplated in the PMBV/PVA hydrogel in the chip; and fl uorescence images of livrespectively, in which green fl uorescence indicates live cells and red fl uoresferent initial densities preserved in the PMBV/PVA hydrogel in the chip for F) preserved in the hydrogel in the chip (blue columns) and G) cultured indensity of 1.0 × 10 6 mL − 1 . ND: not detected. Data are mean ± SD, n > 3. Sca
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The viability of the cells preserved in the hydrogel in the chip was investigated using live/dead assays according to a protocol described in the Experimental Section. The calculated viabili-ties were very high: while the 7-day preservation of HAEC with an initial cell density of 1.6 × 10 5 mL − 1 had 93.9% viability, the 8-day preservation of L929 with an initial density of 1.8 × 10 5 mL − 1 had 92.1% viability (see SI, Part 3). The preservations of L929 cells with a lower initial cell density (1.8 × 10 4 mL − 1 ) and a higher initial cell density (1.0 × 10 6 mL − 1 ) were performed, and the results are shown in Figure 2E . The 8-day viabilities of both cases were not only as high as that of the preservation with the initial density of 1.8 × 10 5 mL − 1 , but also in a required viability level of commercial cell cryopreservation solutions at − 196 ° C (e.g., about 90% viability, Cell Banker produced by Mitsubishi Chemical Medicine, Tokyo, Japan). This reveals that the PMBV/PVA hydrogel has a capacity to preserve cells with a wide range of cell density in the chip, even a considerably high density of 1.0 × 10 6 mL − 1 . As a cell preservation method, the preservation of high density cells is expected. Therefore, the initial cell den-sity of 1.0 × 10 6 mL − 1 was used in the following investigations.
The variation of cell viability with times varying from 4 to 18 days was further investigated. Viabilities of L929 cells pre-served in the hydrogel in the chip (Figure 2F ) and cultured in
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ic image of L929 cells (density: 1.0 × 10 6 mL − 1 ) right after being encapsu-e/dead assays of the L929 cells preserved for B) 4, C) 12, and D) 18 days, cence indicates dead cells. Lower: E) Viabilities of the L929 cells with dif-
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Figure 3 . Comparative toxin screenings using L929 preserved in the hydrogel in the chip (initial density 1.0 × 10 6 mL − 1 ) for 8 days (blue columns) versus L929 cultured in medium in a 96-well microplate for 2 days with a density of approximate 1.0 × 10 6 mL − 1 (green columns): A) saponin, (0.01% and 0.10%, w/w), B) triton X-100 (0.01% and 0.10%, w/w), C) methanol (7% and 70%, v/v), and D) PBS as a control. Low and High Conc. notations on the x -axis represent high and low concentrations for each toxin. Data are mean ± SD, n > 3.
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Dulbecco’s modifi ed Eagle’s medium (DMEM) in a 96-well microplate (Figure 2G ) were compared. The initial cell densities of all samples used in comparative investigations were the same 1.0 × 10 6 mL − 1 . Figure 2B–D show representative merged live/dead images of the L929 cells preserved in the hydrogel in the chip for 4, 12, and 18 days, respectively. In the case of the 4-day preservation, few dead cells were found. Accordingly, the cal-culated viability of the 4-day preservation was 88.3%, a viability close to that of cells cultured in medium in a 96-well micro-plate for 4 days. A high viability (87.6%) was maintained after 8 days of preservation, which is almost equal to that of the 4-day preservation. Although a few more dead cells were observed in the 12-day preservation, the percentage of live cells (70.7%) was still quite high. Even after 18 days of preservation, the per-centage of live cells was still higher than 50%. These results indicate that the PMBV/PVA hydrogel possesses the capa-bility to preserve cells in chip for a longer period than 8 days. In contrast, cells cultured in medium in a 96-well microplate only maintained a 56.7% viability after 8 days, and were entirely dead after 12 days because of cell overproliferation and the fact that the medium completely evaporated. We consider that the PMBV/PVA hydrogel in the chip provides a benefi cial cytocom-patible microenvironment, so that cells can stay alive for a long period of time while maintaining a low growth rate (for details, please see SI, Part 4).
Toxin screenings were performed using the cells preserved in the hydrogel in the chip (initial density: 1.0 × 10 6 mL − 1 , 8-day preservation) in comparison with the cells cultured in medium in a 96-well microplate (2 day culture to a density of 1.0 × 10 6 mL − 1 ). The samples were directly exposed to a panel of typ-ical cytotoxic agent solutions with different concentrations. As shown in Figure 3 , cytotoxicity responses of cells preserved in the hydrogel in the chip varied with the toxin concentration. Cell death of 100% was observed with all 3 toxins at higher concen-trations (saponin and triton X-100: 0.1% (w/w); methanol: 70% (v/v)), while lower concentrations of the 3 toxins (saponin and triton X-100: 0.01% (w/w); methanol: 7% (v/v)) resulted in the percentage of dead cells being between 26% and 43%. In each case of 3 toxins with different concentrations, no big difference in the cytotoxicity response (the percentage of dead cells after toxin exposure) was found between the use of cells preserved in the hydrogel in the chip and the use of cells cultured in medium in the microplate. This suggests that the cells preserved in the hydrogel in the chip maintained activity as functional as, and exhibited as high resolution to different toxins with different concentration as, the cells conventionally cultured in medium.
The characteristics of the PMBV/PVA hydrogel in spon-taneous encapsulation and mild preservation of living cells on-chip offer unique advantages and powerful potentials for cell-based applications as follows. Firstly, various conventional off-chip continuous medium supports (i.e., perfusion culture) for cell maintenance are not required, making the system sim-pler, safer, more independent, and easier to realize miniaturi-zation, integration, mobilization, and fi nal commercialization. Secondly, because of the fact that very few sample quantities are required in a chip assay, the cost for culture and assay agents is greatly reduced. Most importantly, all these advantages make it possible to perform cell-based applications in less strict condi-tion requirements than usual, which could possibly expand the
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application of cell-based applications to extreme conditions and environments, such as war zones, during terror threats, and in the case of environmental disasters. Therefore, we believe that the microfl uidic hydrogel and the preservation of cells in mild conditions would establish a revolutionary fl exibility for cell-based applications.
Experimental Section Cell Encapsulation in Chip : Chips, holders, capillaries, and all the
other accessories were sterilized by autoclaving before use. All solutions were sterilized with 0.2 μ m pore-size sterilizing-grade fi lters before use. The details of the setup and operation of the chip are described in the SI, Part 2. To encapsulate cells in the chip, fi rst, 5 μ L of 2.5 wt% PVA (polymerization degree, about 1500; Wako) solution (1 × Dulbecco’s PBS (D-PBS) as buffer) was delivered into the cell-container chamber through the introducing microchannel using a microsyringe pump with a withdraw mode. Then, 15 μ L of cell suspension in a culture medium (L929: DMEM supplemented with 10% fetal bovine serum; HAECs: EBM-2 supplemented with EGM-2 SingleQuots; Cambrex) containing PMBV (5.0 wt%) was introduced. After that, PMBV/PVA hydrogel which spontaneously encapsulated the cells formed in the cell-container chamber. With all inlets/outlets open, the chip was incubated at 37 ° C in a cell culture incubator (Model MCO-17A1C; Sanyo) without any additional operation.
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Viability Assays : The viability of the cells preserved in the hydrogel in the chip was investigated using live/dead assays (Live/Dead viability/cytotoxicity kit, Molecular Probes). For each sample, after calcein-AM (2 μ M )/ethidium homodimer-1 (4 μ M ) reagent mixture solution (10 μ L) was introduced to the chip, the chip was incubated at room temperature for 45 min, and then observed and analyzed using the fl uorescence mode of an inverted microscope (IX 71; Olympus) with a charge coupled device (CCD) camera (Retiga EXi; QImaging). Great care was taken to minimize the photobleaching because of excessive exposure. In a live/dead assay, green fl uorescence (calcein, excitation (Ex) : emission (Em) = ∼ 494 nm : ∼ 517 nm) is an indicator of live cells (live image), while red fl uorescence (ethidium homodimer-1, Ex:Em ∼ 528 nm : ∼ 617 nm) is an indicator of dead cells (dead image). The viability was estimated by counting the number of live and dead cells in more than 3 different areas of the chamber (i.e., more than 3 merged live/dead images) using an image processing program, ImageJ 1.40g, developed by the National Institutes of Health (NIH). The viability (or the percentage of live cells) is defi ned as the number of live cells/number of cells in total (%). The viability of cells cultured in medium in a 96-well microplate was investigated using the same protocol. Cell suspension (100 μ L) was fi rst seeded in each well. Before live/dead assays, the cells were washed 5 times with 1 × PBS (100 μ L per well), and then 1 × PBS (100 μ L) was added to each well. After that, the live/dead assay solution (50 μ L) was added to each well for the following viability assays. The same initial cell densities were applied in comparative investigations of two formats.
Toxin Screenings : After being preserved in the hydrogel in the chip for 8 days, L929 cells (initial density 1.0 × 10 6 mL − 1 ) were directly exposed to the toxin solutions. Approximately 10 μ L of toxin was injected to each chamber. The chip was then incubated in a cell culture incubator for 30 min at 37 ° C. Postexposure, the solution remaining in the chip was drained using a syringe, and then approximately 10 μ L of the live/dead assay solution (2 μ M calcein-AM/4 μ M ethidium homodimer-1) was injected to each chamber. Following incubation at room temperature for 45 min, the labelled cells preserved in the chip were observed and the percentage of dead cells (defi ned as, number of dead cells/number of cells in total (%)) was analyzed using fl uorescence microscopy and the image processing program ImageJ. As comparative experiments, toxin screening experiments were also performed with L929 cells cultured in medium in a 96-well microplate. To maintain consistency, cells were fi rst cultured for 2 days to a cell density of 1.0 × 10 6 mL − 1 , an equivalent density as that of cells preserved in the hydrogel in the chip, and then the same protocol used in chip experiments was applied. Before live/dead assays, the cells were washed 5 times with 1 × PBS (100 μ L per well), and then 100 μ L of 1 × PBS and 50 μ L of toxin solution were added to each well. After exposure, the supernatant solution in each well was removed and then 1 × PBS (100 μ L) was added to each well. Finally, the live/dead assay solution (50 μ L) was added to each well for the following viability assays.
Supporting Information Supporting Information is available online from Wiley InterScience or from the author.
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Acknowledgements This work was supported by a Grant-in-Aid for Japan Society for the Promotion of Science (JSPS) Fellows (No. 19-07374).
Received: January 1, 2010 Revised: January 27, 2010
Published online: May 25, 2010
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