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Reversible Monolayer/Spheroid Cell Culture Switching by UCST-Type Thermoresponsive Ureido Polymers Naohiko Shimada,† Minako Saito,† Sayaka Shukuri,† Sotaro Kuroyanagi,† Thasaneeya Kuboki,§ Satoru Kidoaki,§ Takeharu Nagai,‡ and Atsushi Maruyama*,† †

Department of Life Science and Technology, Tokyo Institute of Technology, 4259 B-57, Nagatsuta, Yokohama 226-8501, Japan Laboratory of Biomedical and Biophysical Chemistry, Institute for Materials Chemistry and Engineering, Kyushu University, Motooka 744-CE41, Nishi-ku, Fukuoka 819-0395, Japan ‡ The Institute for Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan §

S Supporting Information *

ABSTRACT: Multicellular spheroids have been studied in the fields of oncology, stem cell biology, and tissue engineering. In this study, we found a new polymer material for thermocontrolled spheroid/monolayer cell culture switching. The polymers that have pendant ureido groups (ureido polymers) exhibited upper critical solution temperature-type phase separation behavior. Cells in monolayer culture were converted to spheroids by the addition of ureido polymers below phase separation temperature (T p). Time-lapse observations indicated that cells began to migrate and aggregate to form the spheroids to avoid contact with phaseseparated polymer (coacervates) on the surface of the culture dish. We supposed that the coacervates seemingly suppressed interaction between cell and the dish surface or extracellular matrices. By increasing culture temperature above Tp, the spheroids began to collapse into a monolayer of cells due to dissolution of the coacervates. These results indicated that cell morphology could be repeatedly switched by changing the culture temperature in the presence of ureido polymers. KEYWORDS: thermoresponsive polymers, upper critical solution temperature, ureido polymers, spheroid, temperature switching

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INTRODUCTION Three-dimensional multicellular spheroid culture resembling more physiological environment than monolayer cell culture has been studied as tissue, organ, and tumor models.1−5 The differentiation capability and potential of stem cells are enhanced in spheroid culture. Especially, mesenchymal stem cells cultured in spheroid promoted anti-inflammatory, angiogenic, and tissue reparative effects.6 These cellular functional enhancements have received increasing attention in cell therapy and regenerative medicine fields. Spheroids have been prepared by using a variety of techniques or devices such as hanging drop culture,7 culture on nonadhesive surfaces,8 or culture in rotary bioreactors.9 A novel cell culture method that permits reversible control between monolayer and spheroid cultures is demanded to refine and characterize spheroid culture. Stimulus-responsive polymers, also known as smart polymers, change physicochemical properties when subjected to an external stimulus such as a temperature change, light irradiation, or addition of a chemical. Many temperatureresponsive polymers have lower critical solution temperature (LCST)-type solution behavior near body temperature under physiologically relevant conditions.10−12 Application of these LCST polymers to biomedical devices have been studied.13−15 © XXXX American Chemical Society

There are a few polymers having upper critical solution temperatures (UCST) in aqueous solution.16−18 Polymers showing UCST-type solution behavior under physiologically relevant conditions are very rare. Recently, several groups have reported that polymers having multiple amide groups in a repeating unit, such as poly(N-acryloylglycinamide-co-Nacetylacrylamide),19 poly(N-acryloylasparaginamide),20 and poly(acrylamide-co-acrylnitrile),21 show UCST-type phase transition behavior under physiological pH and salt conditions. The polymers likely show UCST-type behavior due to strong hydrogen bonding among the amide groups. Very recently, polypeptides having P-Xn-G motifs containing a zwitterionic pair showed UCST-type solution behavior under physiologically relevant conditions.22 These UCST-polymers could be applied to cell culture devices or drug delivery carriers used under physiological conditions. We have reported that polymers having ureido groups, poly(allylurea),23,24 poly(citrulline),23 and poly(2-ureidoethyl methacrylate),25 exhibit UCST-type behavior in aqueous buffer. Solutions of the ureido polymer, poly(allylamine-co-allylurea) (PAU, Figure 1), are Received: June 22, 2016 Accepted: November 1, 2016 Published: November 1, 2016 A

DOI: 10.1021/acsami.6b07614 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

promoter of a pEF-1α vector. Then, full-length paxillin (1674 bp) obtained by reverse transcription and polymerase chain reaction from immortalized human mesenchymal stem cells (Health Sciences Research Resource Bank, Osaka, Japan) was fused in frame at the ECoRV site at the C-terminal of the Venus. The six alanine amino acid linker was inserted between Venus and paxillin to facilitate the protein folding. The construct was used for transfection into the 3T3 fibroblasts using Lipofectamine LTX with Plus reagent (Thermofisher Scientific, Japan), according to the manufacturer’s instruction. Stable transfection was performed using 500 μg/mL of G418 antibiotic. NIH3T3, HepG2, and HL-60 cells were obtained from the JCRB Cell Bank. NIH-3T3 and HepG2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 5% FBS. Stable Venuspaxillin-expressing NIH-3T3 was maintained in the same medium supplemented with 250 μg/mL G418, 100 U/mL penicillin, and 100 μg/mL streptomycin. HL-60 cells were maintained in RPMI 1640 containing 5% FBS. Cell Viability. NIH-3T3 cells (1 × 104 cells/well) were seeded in wells of a 96-well plate and incubated at 37 °C in DMEM containing 5% FBS. After incubation for 24 h, PAU copolymers or poly(allylamine) homopolymer (PAA) at various concentrations were added to the cells. Cells were incubated at 37 °C for 48 h. The CellTiter-Glo 3D assay (Promega) was performed according to the manufacturer’s protocol. Luminescence was recorded using an LB 942 TriStar2 (Berthold Technologies). The cell viabilities reported are averaged results from at least two independent experiments. For microscopic qualitative live/dead observations in the presence of PAU copolymer, cells were stained with calcein-AM and propidium iodide (PI). For morphology analyses, 3 × 105 NIH-3T3 cells were seeded on a culture dish (35 mm in diameter, Iwaki) and were incubated at 37 °C for 24 h. A15K93 (1 mg/mL) was added and cells were incubated at 37 °C for an additional 48 h. Cells were washed with PBS and a mixture of 1 μg/mL calcein-AM and 3 μg/mL PI in PBS was added to the cells. After incubation for 10 min at room temperature, fluorescence was observed on a fluorescence microscope (Biozero BZ-8000, Keyence). 3D Imaging of Spheroids. For 3D imaging, 3 × 105 NIH-3T3 cells seeded on a glass-bottom dish (35 mm in diameter, Matsunami) were incubated at 37 °C for 24 h. A15K93 was added to the cells. After 24 h at 37 °C, cells were fixed using 4% paraformaldehyde in PBS for 15 min and then stained with 3 μg/mL PI in PBS for 15 min. After washing twice with PBS, 4 M urea, 0.1% Triton-100, and 10% glycerol (ScaleA2 solution30) were added, and samples were incubated at 4 °C for 24 h. 3D images were obtained using a confocal laser scanning microscope (LSM 510, Carl Zeiss). Time-Lapse Observations of Spheroid Formation and Deformation. NIH-3T3 cells (3 × 105 cells) seeded on a culture dish (35 mm in diameter, Iwaki) were incubated at 37 °C for 24 h. Time-lapse images of the cells in the presence of A15k93 or A5k91 (1 mg/mL) were acquired every 30 min by using a Biozero microscope (Keyence). The cells were maintained at 37 or 25 °C, 5% CO2, and humidified with a Tokai Hit Chamber (Tokai Hit). For A15k93, the images were acquired for 52 h at 37 °C. For A5k91, the images were acquired for 1 h at 37 °C, and then temperature was gradually decreased to 25 °C. After the acquisition of images at 25 °C for 48 h, the temperature was increased to 37 °C, and images were acquired for 24 h. Estimation of Expression of Cadherin and Paxillin in Cultured Cells. NIH-3T3 cells (3 × 105 cells) were seeded on a glass-bottom dish and then incubated at 37 °C for 24 h. Cells were treated without or with 1 mg/mL A15k93 at 37 °C. After 24 h, cells were washed with PBS containing 260 mM glucose. After the washing, cells were fixed with 4% paraformaldehyde in PBS for 10 min. The cells were washed with PBS, and then treated with 0.5% Triton X-100 for 5 min. After washing twice with PBS, the cells were incubated with 1 mg/mL BSA in PBS for 1 h. The cells were treated with antipancadherin antibody (1:250 dilution, Gene Tex) for 1 h, washed twice with PBS, stained with TAMRA-labeled goat antimouse IgG(H+L) (1:100 dilution, AnaSpec) for 1 h, and then washed twice with PBS. Finally, fluorescent images of the cells were obtained using a spinningdisk confocal scanning instrument (CSU-X1, Yokogawa). For EDTA

Figure 1. Structural formula of poly(allylamine-co-allylurea) (PAU).

separated into two liquid phases (polymer-rich coacervate and polymer-poor phase) below the phase separation temperature (Tp).23 The Tp can be controlled from 5 to 65 °C by changing ureido content or molecular weight.23 Some UCST-polymers such as zwitterionoc polymers, poly[3-dimethyl (methacryloyloxyethyl) ammonium propanesulfonate], showed coacevation at lower salt concentration.26 The coacervate was used as a matrix to catch and release DNA.27 No application of UCST polymers forming coacervates to cellular engineering was described. An aqueous two-phase separation system has been employed to form spheroids from cell suspension. Spheroid of hepatocytes was formed by suspending cells in the two phase-separated liquid phases containing methacrylic acid/ methacrylate copolymer.28 Recently, Han et al. reported a simple spheroid generation method using an aqueous twophase separation system of dextran/PEG solution.29 Thermoresponsive control between monolayer and spheroid cultures has not been described yet. In this paper, we describe reversible monolayer/spheroid cell culture switching achieved with a thermoresponsive ureido polymer solution that separates into two liquid phases below a phase transition temperature. Monolayer-to-spheroid and spheroid-to-monolayer morphological changes of cultured cell were successfully controlled.

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EXPERIMENTAL METHODS

Materials. PAUs, A15k87, A15k90, A15k93, and A5k91 (see Table 1) were prepared by ureido modification to poly(allylamine) hydro-

Table 1. Tp Values of PAUs Used in This Study under Cell Culture Conditiona polymer code

ureido content/%

Tp/°C

A15k87b A15k90b A15k93b A5K91c

87 90 93 91

62 53 45 37

DMEM with 5 vol % FBS. bMw of PAA: 1.5 × 104. cMw of PAA: 5 × 103.

a

chloride (PAA) (Mw: 1.5 × 104 and 5 × 103) as we previously reported.23 In brief, potassium cyanate (amount dependent on desired degree of modification) was added to PAA in water. The mixtures were incubated at 50 °C for 24 h. The polymers were purified by dialysis and then lyophilized. Resulting polymers were characterized by 1 H NMR. Transmittance Measurements of Ureido Polymers. The copolymers were dissolved in DMEM containing phenol red with 5 vol % fetal calf serum (FBS). Thermal transmittance change at 700 nm of the 1 mg/mL copolymer solution in a 10 mm quartz cell was measured on a Shimadzu UV-1650PC UV−visible spectrophotometer equipped with a Peltier temperature controller at scanning rate 1 °C/ min from 70 to 5 °C. Cell Culture. Stable Venus-paxillin-expressing NIH-3T3 cells were generated. The gene encoding Venus fluorescent protein was cloned into BamHI and EcoRI restriction sites downstream of EF-1α B

DOI: 10.1021/acsami.6b07614 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

ACS Applied Materials & Interfaces treatment, NIH-3T3 spheroids induced by treatment with 1 mg/mL A15k93 were incubated with 10 mM EDTA in PBS 37 °C for 1 h prior to microscopic observations. Venus-paxillin-expressing NIH-3T3 (3 × 105 cells) were seeded on glass-bottom dishes and then incubated at 37 °C for 24 h. Localization of paxillin in living cells treated without or with A15k93 (1 mg/mL) for 48 h at 37 °C was observed using spinning-disk confocal microscopy without fixation. Estimation of Albumin Secretion from Spheroids. HepG2 cells (1 × 104 cells/well) seeded in wells of 96-well plates were incubated without or with A15k93 (1 mg/mL) at 37 °C for 1 to 7 days in DMEM containing 5% FBS. Albumin secretion from the cells was estimated by ELISA (Human Albumin ELISA Quantitation kit, Funakoshi). The number of the cells was determined by MTT assay (Biotium) after treatment with 0.25% trypsin-EDTA (Gibco) at 37 °C for 10 min (mixed by pipetting every 2−3 min during the incubation). Absorbance at 570 nm was measured on a SpectraMax 340PC (Molecular Devices).

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RESULTS AND DISCUSSION Cell Morphological Change by Addition of Ureido Polymers. We prepared PAU copolymers showing UCST-type

Figure 4. Morphology of NIH-3T3 cells cultured in DMEM supplemented with 5% FBS (A−D) without or (E−H) with 1 mg/ mL A15k93 at 37 °C for 48 h. The cells were stained with (B,F) calceinAM and (C,G) PI. In panel E, edges of cell aggregates are shown with red lines. Scale bars indicate 50 μm.

Figure 2. Transmittance curves of PAUs in DMEM with 5% FBS. [PAU] = 1 mg/mL.

Figure 5. (A) Time-lapse images of NIH-3T3 cells incubated with 1 mg/mL A15k93 at 37 °C for indicated times. Edges of cell aggregates are indicated by red lines. Scale bars indicate 100 μm. Time lapse movie is available at Movie S1. (B) Schematic illustrations of monolayer to spheroid morphological change by the addition of PAU coacervates.

Figure 3. Viability of NIH-3T3 cell in the presence of PAUs or PAA at concentrations from 0.1 to 1 mg/mL. Cell viabilities were determined by using the by CellTiter-Glo 3D assay after 48 h at 37 °C. Values are expressed as mean ± SD (n = 6).

suspension was separated into two transparent solutions when it was allowed to stand in a glass tube overnight below Tp (Figure S1C). In a culture dish, the fused coacervate droplets were sedimented on the dish surfaces after 48 h incubation at 37 °C (Figure S1E). These observations indicated that the PAU in culture medium underwent phase separation with coacervate formation, in the same manner observed in a buffer.23 A5k91 that has Tp = 37 °C was dissolved homogeneously in the culture medium at 37 °C but phase separated upon cooling below 37 °C. The coacervates of A5k91 sedimented on the surface of a culture dish were clearly visualized by bromophenol blue staining at 25 °C (Figure S3A). The A5k91 coacervates on

solution behavior under cell culture condition (Figure 2 and Table 1). Tp was defined as an initial point temperature where the transmittance first begins to drop. A15k87, A15k90, and A15k93 had Tp values at 62, 53, and 45 °C, respectively, and were phase-separated in the culture media at 37 °C. The medium solution containing A15k93 became transparent upon heating above Tp, and became opaque upon cooling below Tp (Figure S1A,B). The microscopic observation indicated that the opaque suspension resulted from coacervate formation (Figure S1D). The phase separated PAUs coacervates were fused with each other during incubation at 37 °C (Figure S2). The C

DOI: 10.1021/acsami.6b07614 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

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ACS Applied Materials & Interfaces

scopic observation evidenced multicellular spheroid formation of NIH-3T3 cells cultured in the presence of A15k93 (Figure S4). In the presence of A15k93, more than 80% of the cells in the culture dish formed spheroids. Spheroid formation was also observed in the presence of A15k87 and A15k90 (Figure S5). The spheroids were stained with calcein-AM and PI to assess whether cells were alive or dead. A large number of the cells were stained by calcein-AM, and very few were stained by PI (Figure 4), indicating that the cells in the spheroids were alive. A time course of the spheroid formation was revealed by time-lapse microscopic observation (Figure 5 and Movie S1). In the absence of A15k93, NIH-3T3 cells were elongated and attached on the surface of the dish. Upon addition of A15k93, coacervates appeared on the surface of the dish, and the cells began to move to avoid contact with the coacervates. The cells adhered to each other, lost pseudopodia, and eventually formed spherical aggregates. Characterization of Spheroid Induced by Ureido Polymers. Culture cells generally adhere to the extracellular matrix (ECM) on the surface of the culture dish. Focal adhesions are multiprotein complexes that link the ECM to intercellular actin proteins. To image focal adhesions, NIH-3T3 cells were engineered to stably express Venus fused to paxillin, a marker of focal adhesions. In the absence of A15k93, focal adhesions were observed in cells (Figure S6A). In contrast, no focal adhesions were observed in cells cultured in the presence of A15k93 (Figure S6B). These results suggest that PAU coacervates suppressed interactions between ECM and cells. Cadherin proteins, Ca2+ dependent cell−cell adhesion molecules, played important roles for cellular self-assembly into spheroid.35 Cadherin was expressed on membranes of cells in the spheroid in the presence of PAU (Figure S7). That the cells in the spheroids adhered to each other via cadherin was supported by our finding that spheroids dissociated upon EDTA treatment (Figure S8). Further, human promyelocytic leukemia cells (HL-60 cells), which slightly express E-

Figure 6. Albumin production from HepG2 cells incubated with 1 mg/mL A15k93 (closed circles) or without PAU (open circles) at 37 °C for the indicated number of days.

the dish were disappeared when incubation temperature was increased to 37 °C (Figure S3B). We next assessed cytotoxicity of PAU copolymers to NIH3T3 cells. While PAA homopolymer caused significant cytotoxicity, PAUs did not show apparent toxicity at 37 °C regardless of their phase states, i.e., dissolved (A5k91) or phaseseparated (A15k87, A15k90, and A15k93) (Figure 3). Effect of the addition of ureido polymers on NIH-3T3 cell morphology was explored. Morphological change31,32 including spheroid formation33,34 of NIH-3T3 cells has been extensively studied. A5k91 has a Tp at 37 °C and homogeneously dissolved in DMEM containing 5% FBS at culture temperature, 37 °C. A5k91 did not change NIH-3T3 cell morphology as will be described later. A15k93 has higher Tp than A5k91 and is phase separated at 37 °C in the culture medium. When A15K93 (1 mg/mL) preheated at 37 °C was added to NIH-3T3 cell culture and incubated at 37 °C, multicellular aggregates were formed. The cellular aggregates were observed in the spaces among the coacervates (Figure 4). Confocal scanning micro-

Figure 7. Temperature dependent morphological change in NIH-3T3 cells grown in DMEM and 5% FBS. (A) The cells were incubated with 1 mg/ mL A5k91 at 37 °C for 1 h. Cultures were moved to 25 °C and were photographed at (C) 6 h, (D) 12 h, (E) 24 h, and (F) 48 h after addition of A5k91. The cultures were returned to 37 °C and were photographed at (I) 3 h, (J) 6 h, (K) 12 h, and (L) 24 h. A5k91(1 mg/mL) was added to DMEM containing 5% FBS without cells at (B) 37 °C or (H) 25 °C. Scale bars indicate 100 μm. Time lapse movies are available at Movies S2 and S3. D

DOI: 10.1021/acsami.6b07614 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

ACS Applied Materials & Interfaces cadherin,36 did not form spheroids in the presence of PAU coacervates (Figure S9). Human hepatocellular carcinoma cells, HepG2 cells, also formed spheroids in the presence of A15k93 (Figure S10). It has been reported that albumin secretion by HepG2 cells is enhanced by spheroid formation.37 HepG2 cells in spheroids induced by the presence of A15k93 secreted more albumin than cells in monolayer culture did (Figure 6). Albumin secretion from the spheroid was 2-fold higher for 7 days than that in monolayer culture. The long-term enhancement of albumin secretion was previously observed for spheroids prepared using rotating wall vessels.38 Spheroid/Monolayer Switching by Temperature Change. We then explored temperature dependence of the spheroid formation. When A5k91 (Tp = 37 °C) was added to a monolayer culture of NIH-3T3 cells, no change in cell morphology was observed (Figure 7A and Movie S2). Upon cooling to 25 °C, coacervates of A5k91 appeared on the surface of the dish, and cells gradually converted to spheroidal form (Figure 7B−E and Movie S2). Upon heating again to 37 °C, the coacervates disappeared, and the spheroids began to collapse (Figure 7G and Movie S3). Finally, the cells spread on the dish surface to form a monolayer (Figure 7H−J and Movie S3). These results indicated that the monolayer to spheroid transition was reversible and able to be controlled by temperature. Furthermore, the observation strongly indicated that PAU in the coacervate state is responsible for spheroid formation.

ACKNOWLEDGMENTS

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REFERENCES

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CONCLUSIONS In this study, we demonstrated, for the first time, temperaturecontrolled switching of monolayer/spheroid culture by the added ureido polymers that showed UCST-type phase separation behavior under cell culture condition. Importantly, the ureido polymers do not adversely affect cell viability. Ureido polymers have the potential for use as tools for controlling cell−cell and cell−matrix interactions and for development of cell therapy or tissue engineering devices. ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b07614. Photographs of UCST-type behavior of A15K93 in culture media; Micrograph of coacervates; Confocal 3D images of spheroid; Spheroids induced by A15K87 or A15K0; paxillin localization; cadherin localization; spheroid in the presence of EDTA; HL-60 cells in the presence of ureido polymer; HepG2 cells in the presence of ureido polymer (PDF) Time course of the spheroid formation (MPG) Cells converting to spheroid form upon cooling to 25 °C (MPG) Cells collapsing upon heating to 37 °C (MPG)

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This work was financially supported by a Grant-in-Aid for Scientific Research on Innovative Areas ”Molecular Robotics” (No. 15H00804), “Nanomedicine Molecular Science” (No. 2306) and the Cooperative Research Program of “Network Joint Research Center for Materials and Devices” from the Ministry of Education, Culture, Sports, Science and Technology, by Center of Innovation (COI) Program, Japan Science and Technology Agency (JST), and by KAKENHI (No. 15H01807, 25350552) from Japan Society for the Promotion of Science.

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Research Article

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel&FAX: +81-45-9245840. Notes

The authors declare no competing financial interest. E

DOI: 10.1021/acsami.6b07614 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX

Research Article

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DOI: 10.1021/acsami.6b07614 ACS Appl. Mater. Interfaces XXXX, XXX, XXX−XXX