Efficient Cell and Cell-Sheet Harvesting Based on Smart Surfaces

Apr 24, 2013 - ABSTRACT: A wide range of smart surfaces with novel properties ... like recombinamer onto glass surfaces by means of the well- known an...
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Efficient Cell and Cell-Sheet Harvesting Based on Smart Surfaces Coated with a Multifunctional and Self-Organizing Elastin-Like Recombinamer María Pierna, Mercedes Santos, Francisco J. Arias, Matilde Alonso, and José C. Rodríguez-Cabello* Bioforge Group, University of Valladolid, CIBER-BBN Paseo de Belén 11, 47011 Valladolid, Spain S Supporting Information *

ABSTRACT: A wide range of smart surfaces with novel properties relevant for biomedical applications have been developed recently. Herein we focus on thermoresponsive surfaces that switch between cell-adherent and nonadherent states and their applications for cell harvesting. These smart surfaces are obtained by covalently coupling a tailored elastinlike recombinamer onto glass surfaces by means of the wellknown and widely applied Click Chemistry methodology. The resulting recombinamer-functionalized surfaces have been characterized by means of water contact angle measurements, XPS and TOF-SIMS. A cell-based analysis of these surfaces with human fibroblasts showed a high degree of adhesion to the surface in its adherent state (37 °C), thus, promoting cell viability and proliferation. A temperature decrease triggers reorganization of the recombinamer, thus, markedly increasing the number of nonadherent domains and masking the adherent ones. This process allows a specific and efficient temporal control of cell adhesion and cell detachment. After determination of the properties required for a suitable cell-harvesting system, optimization of the process allows single cells or cell sheets from at least two types of cells (HFF-1 and ADSCs) to be rapidly harvested.

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prove that no trypsin remains in the final product. Additionally, for those cell cultures involving cell differentiation, trypsinization destroys most of the cellular markers that are currently used to determine the resulting cell lineage.3 Recent studies by Okano and co-workers have opened up a new possibility in this field, namely, the use of a smart surface that can switch between a cell-adherent and nonadherent state as a result of a change in temperature.4 In such an approach, poly(N-isopropylacrylamide) (PIPAAm) and its derivatives are grafted onto an appropriate substrate and builds a brush-like structure of the responsive material directly on the surface. In this case, changes in the apparent surface hydrophobicity and, therefore, the physical properties of the surface, seem to govern the mechanism that allows such systems to work. Based on the well-known lower critical solution temperature (LCST) behavior of PIPAAm, the polymer switches between a hydrophobic state at temperatures above its LCST and a hydrophilic state below it, as does the surface on which PIPAAm was grafted. This approach has led to significant progress in this field providing harvesting protocols required are neither complex nor time-consuming and there are no concerns as regards contamination from the reagents used during the harvesting protocol.5 In addition, and more importantly, this approach means that both cells and cell

INTRODUCTION Cell-harvesting systems and technologies are key elements in the development of cell production techniques, especially as regards their subsequent use in human therapies. These systems must be considered as relevant enabling technologies since areas such as tissue engineering and regenerative medicine will not easily become universally applicable without a reliable source of cells for therapeutic purposes. Most cell lineages need to adhere to a substrate to proliferate. As a result, most of the protocols currently used to amplify cell numbers require the use of cell-adherent supports. However, this poses a technical challenge in classical approaches as the cells subsequently need to be harvested from the substrates prior to use. Classical solutions to this problem tend to be based on two different approaches, namely, mechanical and proteolytic enzyme harvesting.1 However, both of these techniques may compromise cell viability and are mainly restricted to flat substrates. Although trypsinization is currently the most widely used proteolytic method, it has some major drawbacks. The most obvious of these is the previously cited cell viability, as excessive exposure of the cultured cells to trypsin activity may damage many different membrane proteins and compromise cell survival.2 Likewise, trypsinization protocols are time-consuming and are strongly dependent on the operator’s expertise. Moreover, the utilization of trypsin, especially in the industrial production of cells for human use, raises the problem of trypsin elimination from the final product. In other words, the manufacturer must be able to conclusively © XXXX American Chemical Society

Received: February 20, 2013 Revised: April 24, 2013

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sheets can now be harvested6 as detachment from the grafted surface is based on changes in the adhesive properties of the support−cell interface rather than alterations to intercellular adhesions.7 However, such systems also exhibit some drawbacks that could hamper the future universal use of this approach. For example, although PIPAAm is an LCST-type polymer, it lacks specific bioactivity, thus, meaning that integrins and other membrane proteins that function as cell anchors to surfaces are not able to directly bind to it. Therefore, at 37 °C, where PIPAAm-grafted surfaces show cell adherence, the mechanism of cell adherence must be indirect and is likely mediated by the adsorption of proteins from the culture medium.8 In practical terms, this results in a lack of effective and total control of the cell-adhesive properties of the surface. Changes in surface adhesiveness may therefore arise as a result of different experimental conditions, such as a change in the culture medium, additions made to it for any purpose, or the use of such surfaces for different time periods during cell culture. Indeed, PIPAAm-grafted surfaces for cell-harvesting purposes are still somewhat of a black box and many relevant aspects of their functioning remain essentially unknown or can currently only be partially explained. One example of this is the dependence of the efficiency of such surfaces on the thickness of the grafted layer, which must be restricted to a narrow range and for which there is currently no convincing explanation.9 In light of this, different approaches based on more direct and controllable properties of the surfaces in terms of cell adherence and switching between the on/off states are desirable to develop a more effective, robust and universal smart platform for cell or cell-sheet harvesting. That is the aim of this work, which concerns the design and testing of a set of bioactive elastin-like recombinamers (ELRs)10 and the creation of functional surfaces by grafting these materials onto glass substrates. Cell adhesiveness is provided by the inclusion of specific cell-adhesion peptides, in the ELR sequence, while the well-known thermal responsiveness is used to trigger a selforganization process that leads to exposure of the bioactive RGD motif to the water interface at physiological temperature to produce a cell adherent surface. In contrast, a temperature decrease triggers a rearrangement of the polymer chain that results in concealment of the cell adhesive motifs beneath an antifouling layer, thereby stimulating cell detachment and establishing a tunable, specific, and controllable system with biomedical applications.

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Table 1. Five Different Building Blocks (A−E) and Their Amino Acid Composition with a Given Intrinsic Functionality (Top), and the Schematic Cartoon of the Elastin-Like Recombinamers Used in this Work (Bottom) Composed by the Association of Previously Described Building Blocks

* The graphics shown are not intended to accurately reflect relative molecular sizes and are not drawn to scale. (BB: Building block; AA: amino acids).

MATERIALS AND METHODS

1. ELR Biosynthesis and Characterization. The recombinamers used in this work and their detailed amino acid sequence are shown in Table 1. The gene-polymerization, cloning, and molecular-biology techniques, ELR biosynthesis in Escherichia coli expression systems, and purification protocols have been described in detail previously.11,12 The molecular and physical characterization of these recombinamers was routinely carried out by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), mass spectroscopy (MS), aminoacid composition determination, nuclear magnetic resonance (NMR) spectroscopy, and differential scanning calorimetry (DSC). 2. Preparation of ELR-N3. ELRs were chemically modified by transformation of the ε-amine group present in the lateral chain of the lysine residue into an azide group. For this purpose, a substitution reaction was carried out using triflyl azide as nucleophilic reagent. Triflyl azide was freshly prepared in situ prior to each reaction as described previously.13 The transformation was corroborated by MALDI-TOF, infrared spectroscopy (FITR-ATR), amino-acid composition determination, and DSC in PBS at pH 7.

3. Synthesis of Alkynyl-Glass. Round glass coverslips (diameter: 12 mm; Thermo Fisher) were used as the substrate for polymer grafting. These slips were activated with piranha solution (3H2O2/ 7H2SO4) and then thoroughly rinsed with deionized water and ethanol.14 An aminosilylated layer was then formed on the activated surface by treatment with (3-aminopropyl)silanol, which was generated in situ by hydrolysis of (3-aminopropyl)trimethoxysilane (APTS) in an ethanol/water/silane (9:1:0.5, w/w) mixture and subsequent dehydration by curing at 110 °C for 1 h. The resulting amine groups were treated with 2% pentynoic anhydride15 and triethylamine (TEA; Sigma Aldrich) in tetrahydrofuran (THF; Sigma Aldrich). The surfaces were then rinsed abundantly with THF (3 × 5 min sonicated) and ethanol (1 × 5 min sonicated). 4. Synthesis of ELR Surfaces by Click Chemistry. The alkynylglass coverslips were immersed in an aqueous modified-polymer solution (5 mg/mL) in the presence of CuSO4 (0.45 mg/mL) and sodium ascorbate (0.95 mg/mL) and incubated at 4 °C for 2 h and then at room temperature for 3 h. After that time, the substrates were washed with a large volume of cold deionized water. B

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5. Surface Characterizations. The surfaces obtained after each modification step and the final biofunctionalized surfaces were evaluated by X-ray photoemission spectroscopy (XPS) using a Physical Electronics (PHI) 5500 spectrometer equipped with a monochromatic X-ray source (Al Kα line of 1486.6 eV energy and 350 W), time-of-flight secondary ion mass spectrometry (TOF-SIMS; ION-TOF, Munster, Germany), at a pressure of 5 × 10−9 mbar using 25 keV Bi3+2 primary ions, and contact angle measurements determined by a sessile drop method using a Data Physics OCA20 system instrument equipped with an adapted CCD video camera and a peltier (Linkam PE94) to control the temperature within ±0.1 °C . 6. Cell Manipulation. Human primary fibroblasts (HFF-1; ATCC, U.S.A.) were maintained in DMEM with GlutaMAX-I (Invitrogen), supplemented with 15% fetal bovine serum (FBS; Gibco) and penicillin/streptomycin (100 μg/mL; Gibco), at 37 °C in a humidified atmosphere compromising 5% CO2 in air. The medium was changed every three days during the experiment. Cell passages 4−7 were used in all experiments. Human adipose-derived stem cells (ADSCs; Invitrogen, U.S.A.) were maintained in MesenPro RS Medium (Invitrogen) supplemented with MesenPro RS Growth Supplement (Invitrogen) and gentamicin/amphotericin B (10 mg/mL, 0.25 μg/ mL; GIBCO), at 37 °C in a humidified atmosphere compromising 5% CO2 in air. The medium was changed every three days during the experiment. Cell passages 2−4 were used in all experiments. 7. Cell Adhesion and Cell Detachment. HFF-1 cells were harvested by treatment with 0.01% EDTA and 0.25% trypsin in PBS solution and suspended in DMEM with GlutaMAX supplemented with penicillin/streptomycin (100 μg/mL; serum-free medium). Cells were seeded at a density of 2 × 104 cells/mL on the various surfaces studied and cultured at 37 °C in an incubator under an atmosphere comprising 5% CO2 in air. The different functionalized and glass surfaces were sterilized by UV irradiation for 30 min prior to seeding, then placed in a 24-well polystyrene tissue culture plate previously incubated overnight with BSA 0.1% in PBS and washed with phosphate buffered saline (PBS; pH 7.2). The morphology of the culture cells was photographed periodically (at 0.5, 4, 8, and 24 h postseeding) under a phase-contrast microscope (NIKON Eclipse Ti) equipped with a digital camera system (Digital sight DS-2MBWc). At 24 h (0 min) post-seeding, the cell-containing surfaces were cooled to 4 °C. After incubation for a further 60 min (25 h in total), 120 min (26 h), and 180 min (27 h), the cell morphology was observed as described above. Cells were counted randomly in multiple areas of the microphotographs for the different surfaces. Rounded, bright and refractive cells were considered to be detached cells and spread, dark cells were considered to be well adhered.16,17 The number of adhered cells and the percentage of adhered and nonadhered cells with respect to the total number of cells were calculated as the mean (n = 12) and standard deviation. 8. Cell Viability Assays. After low-temperature treatment, the cells remaining on the different surfaces were evaluated and quantified using the LIVE/DEADViability/Cytoxicity Assay Kit (Molecular probes). The live cells correspond to green cells and the red ones to dead cells. The percentage of live cells to total number of cells was calculated as the mean (n = 12) and standard deviation. Detached cells were transferred to a new 24-well polystyrene tissue culture plate and the viability and proliferation evaluated over 14 days by measuring the fluorimetric reduction of Alamar Blue (AbDSerotec) at regular time intervals. The same number of fresh human fibroblasts cultured in the same substrate was used as a control. Cell viability data were expressed as the mean (n = 12) and standard deviation. 9. Cell Analysis by Flow Cytometry. HFF-1 cells were cultured as described previously, whereas ADSCs cells were grown in MesenPRO RS with Growth Supplement supplemented with gentamicin/amphotericin B (10 mg/mL, 0.25 μg/mL). Cells were seeded at a density of 2 × 104 cells/mL on surfaces and cultured at 37 °C in an incubator under an atmosphere of 5% CO2. At 24 h postseeding, both types of cells were incubated at 10 °C for 15 min and then warmed again to 37 °C for 5 min. The supernatant was then removed and the dissociated cells evaluated and quantified using the

LIVE/DEADViability/Cytoxicity Assay Kit (Molecular probes) with a flow cytometer (FC-500 Beckman Coulter).

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RESULTS AND DISCUSSION 1. ELR Biosynthesis and Characterization. Five different recombinamers have been designed, produced, and studied in order to achieve a functional substrate and test the working hypothesis. All five have been designed and constructed, at the genetic level, using a modular approach involving the combination of five different binding blocks (blocks A−E), each of which confers a given intrinsic functionality. The composition of these five building blocks can be seen in Table 1. Block A is an N-terminal block containing a repeat of three lysines, thus meaning that it contains four free amino groups (three from the lysine ε-amino groups and the fourth from the terminal α-amino group). This block is used as a chain-end grafting domain. Building block B is a tandem repeat of the well-known elastin pentapeptide (VPGVG) and is responsible for both the thermal sensitivity, due to the well-known LCST behavior of poly(VPGVG), and the antifouling nature.18 Building block C is a bioactive domain. The central area of this domain is made up of the RGD loop of human fibronectin, (AVTGRGDSPASS), which contains the well-known integrinmediated cell adhesion tripeptide RGD.19,20 The whole RGD loop found in the natural protein, rather than just the bioactive tripeptide, was included in this design to mimic the occurrence of this bioactive motif in natural proteins because it is known that the whole loop adopts a folded structure that helps the RGD motif to be more accessible for cell interactions.21−23 This central domain is flanked by two (VPGIG)10 wings in building block C. VPGIG is similar to VPGVG, but the second L-valine of the latter has been replaced by L-isoleucine, which is slightly more hydrophobic but maintains the LCST behavior and antifouling properties.18 This building block is therefore markedly amphiphilic as the RGD loop is clearly hydrophilic and contains several amino acids whose side chains are hydrophilic or even charged at physiological pH, whereas the VPGIG wings are somewhat apolar and show LCST behavior. This will form the basis of self-organization processes, as discussed later. Building block D is similar to C but with one significant difference: the central RGD peptide has been scrambled to RDG.24 Finally, building block E is again a modification of block C where the L-isoleucines present in the third and eighth (VPGIG) pentapeptides of the two flanking domains of this block have been substituted by L-lysine. Block E therefore contains four reactive amino groups that can be used for click grafting. Five different recombinamers, the composition and molecular arrangements of which can be seen in Table 1, were designed by combining the previous building blocks. Recombinamer 3K-RGD was conceived as the functional recombinamer, whereas the other four were used as controls to test the working hypothesis as described below. The molecular architecture of 3K-RGD was designed to ensure that it would be in the folded state at 37 °C. Due to the amphiphilic nature of this recombinamer, the hydrophilic central domain is expected to be exposed to the surface water interface in this folded state. Furthermore, as this domain bears the cell adhesion peptide, the surface onto which the recombinamer is grafted will show specific cell adherence. 3K-RGD was also designed to unfold as a consequence of a decrease in temperature. In this state, the antifouling domain, which is opposite to the grafted side chain, will be oriented toward the C

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Table 2. Summary of MALDI-TOF Characterization Data for the Nonmodified and Modified ELRs (*), DSC Data To Evaluate Transition Temperatures and Their Increase, and Amino-Acid Analysis To Determine the Number of Lysine Residues in Each Polymer Chaina

a

polymer

3K-RGD

3K-RDG

3K-V84

N-RGD

RGD+24K

MW (Da) MW(Da)* Tt (°C) Tt (°C)* ΔT (°C) No. Lys No. Lys*

31225 ± 7.6 31262 ± 6.8 24.2 22.8 1.4 2.9 1.6

31250 ± 8.5 31291 ± 9.6 23.5 21.8 1.4 2.9 1.6

35085 ± 3.1 35185 ± 5.5 29.5 27.7 1.7 2.8 1.5

31388 ± 2.8 31375 ± 9.9 23.9 24.1 −0.2 0.0 0.0

60542 ± 9.8 60929 ± 9.0 32.2 22.2 10 23.9 2.3

MW, molecular weight; Tt, transition temperature; No. Lys, number of lysine residues.

Scheme 1. Synthesis of ELR Biofunctionalized Surfacesa

a (A) Chemical modification of the glass surface to create an alkyne layer. (B) Polymer modification by chemical transformation of lysine amine groups into azide groups. (C) Click reaction between both substrates to obtain a covalent grafting.

surface−aqueous interface, thereby hiding the RGD motifs from that interface. As the surface has now shifted from a celladherent to a nonadherent substrate, the cells previously cultured on that surface will detach and become ready for harvesting. The first control recombinamer is 3K-RDG, which is essentially the same as 3K-RGD but with the integrinmediated, cell-adherent RGD motif replaced by its scrambled version (RDG), which shows no cell adherence.25 The second control is 3K-V84 (Table 1), which is essentially a graftable poly(VPGVG). This material should show a temperaturemediated transition and should therefore change the physical properties of any surface to which it is grafted, mainly the apparent hydrophilicity. This control was designed to be used to test whether simple changes in surface hydrophobicity are sufficient to govern a functional surface for cell harvesting. The third control is N-RGD (Table 1), which is the same recombinamer as 3K-RGD but lacking the grafting domain. This material was therefore used to test whether the presence of the grafting domain is essential or whether the adsorbed

recombinamer is sufficient. The fourth control was RGD+24K (Table 1). This material contains lysine residues distributed along the recombinamer chain, thus meaning that it is not grafted only at one end but at many different points along the chain. This material was used test the suitability of a recombinamer that must be grafted from one end according to the working hypothesis and the molecular design of the functional recombinamer 3K-RGD. SDS-PAGE, MALDI-TOF, NMR, and amino acid analyses were performed to verify the effectiveness of the production and purification methods for all the six recombinamers used in this work. SDS-PAGE of the purified polymers confirmed their purity and monodisperse nature, and MALDI-TOF corroborated their molecular weight within the bounds of experimental error (Table 2). Likewise, the measured amino acid contents fitted well with the theoretical composition. DSC was used to calculate the transition temperature (Tt) for all the polymers shown in Table 2. As all values obtained were in the range D

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23.5−32.2 °C, all the recombinamers would be folded at 37 °C and unfolded at temperatures below 20 °C. 2. Surface Biofunctionalization and Characterization. The three-step approach employed to produce the biofunctionalized glass surfaces is shown in Scheme 1. Briefly, the ELR-N3 polymers (indicated with an asterisk in Scheme 1B) were first prepared by chemical transformation of the ε-amine group present in the lateral chain of the lysine residue and the amine terminal residue into an azide group by treatment with triflyl azide (TfN3).13,26 The success of this reaction was confirmed by MALDI-TOF, FTIR-ATR, amino acid composition determination, and DSC. Mass analysis showed molecular weight differences between the nonmodified and modified polymers (Table 2) as each amine group transformation from the lysine residue increases the molecular weight by 26 Da. This results, for example, in a difference of 46 Da between the experimental molecular weights of polymers 1 and 1* due to substitution, on average, of two of the amine groups present in the lysine residues for an azide group. As can be seen from Table 2, the remaining recombinamers also presented a similar difference, thus indicating the efficacy of the reaction. The only exception to this was the N-RGD* recombinamer, where only the Nterminal amine group of the chain polymer is available for azide transformation. The FTIR-ATR spectra of the modified polymers displayed a band at 2100 cm−1, which is characteristic of an azide group (Figure S1, Supporting Information); this signal was absent in the IR spectra of the precursor polymers. The presence of this band was most marked for the recombinamer RGD+24K* due to the high number of lysine residues (24 K) along its chain. The success and efficiency of this reaction were further corroborated by amino-acid analysis, which showed a decrease in the number of lysine residues as a consequence of their transformation into azide derivatives (Table 2). The content of the remaining amino acids remained constant. Recombinamer 3K-RGD* showed a decrease from three lysine residues to 1.6, thus, indicating an average of 46% lysine transformation. The most appreciable change was observed for RGD+24K* due to the presence of 24 lysines per polymer chain. Thus, this polymer showed a reduction in the number of lysine amino acids from 24 to 2.3 (95.8% transformation into azide groups). The modified polymers were also studied by DSC in PBS at pH 7 to investigate how the transition temperature was modified by transformation of the amine groups. As the azide group is much more hydrophobic than the initial amine group, the Tt of the polymers should decrease after transformation. As a result, those recombinamers with a higher number of Lys residues will show a higher Tt shift. This is the case for RGD+24K*, whereas the shift for the four 3K* recombinamers is very small and that for N-RGD* negligible. Commercial glass coverslips chemically modified to create an alkyne-functionalized layer (Scheme 1A) were used as the substrate for grafting the different recombinamers. Glass was chosen as substrate as it is a common and inexpensive surface and, in addition, it also enables microscopic observation in view of the optical properties to be used for biomedical studies. The modified recombinamers were covalently coupled to these alkynyl-functionalized glass surfaces by a Cu(I)-catalyzed cycloaddition reaction between the azide groups of the recombinamers and the alkyne groups on the glass surface (Scheme 1C). This reaction forms part of the click chemistry

techniques developed by Sharpless and co-workers27 and provided the formation of 1,2,3-triazoles in water as solvent. Each modification step from glass coverslips to biofunctionalized surface was characterized by XPS, TOF-SIMS, and contact-angle measurements. Table 3 shows the XPS summary Table 3. XPS Results for Glass, Activated Glass, and Amine-, Alkyne-, and Polymer-Functionalized Surfacesa peak (eV)

C 1s 284

O 1s 529

N 1s 398

Si 2p 101

glass ACT glass amine alkyne 3K-RGD* 3K-RDG* 3K-V84* N-RGD* RGD+24K*

12.10 9.80 33.96 48.24 59.15 69.99 64.24 52.75 54.61

41.81 64.40 39.52 25.13 23.19 15.29 18.18 24.87 21.62

0.23 0.00 2.06 8.15 11.16 9.65 9.10 8.21 13.50

29.70 25.80 24.46 18.18 4.21 5.18 6.59 16.17 9.19

a

The atomic concentrations for the C 1s, O 1s, N 1s, and Si 2p components present on the different surfaces are shown.

spectra for glass, activated glass, amine, alkyne and polymerfunctionalized surfaces. Activation of the surface and the attachment of hydroxyl groups to it was confirmed by the large increase in the oxygen ratio (64.4%) with respect to the starting glass surface (41.81%). After activation, the glass was treated with aminosiloxane to functionalize the surface with amino groups. XPS analyses after this step showed the presence of an N 1s signal (2.1%). An increase in the C 1s ratio and a decrease in the O 1s ratio were also observed, thus, suggesting coating of the surface with aminopropylsiloxane groups. High-resolution XPS spectroscopy showed that 98.09% of the N 1s atoms introduced onto the surface were amine groups (399.5 eV). Likewise, the high-resolution C 1s spectra showed the presence of two carbons in different oxidation states corresponding to C−C (284.9 eV) and C−N (286.2 eV) peaks (Table S1, Supporting Information). The low-resolution XPS survey spectra of the alkyne-functionalized surfaces revealed an increase in the C 1s and N 1s components due to the introduction of pentenyl groups and surface-bound amides. Similarly, a decrease in the organic environmental contaminant oxygen was also deduced from the slight decrease in the O 1s signal caused by the thicker coating resulting from the longer chain group on the surface. The silicon ratio also decreased slightly for the same reason. A new C 1s signal appeared at 287.8 eV in the high-resolution XPS spectrum and the signal for the amine group (399.5 eV) decreased, thus, confirming the presence of an amide group (Table S1, Supporting Information). ELR grafting resulted in several changes in the atomic composition of the resulting surfaces, especially an increase in the C 1s component, a maintenance or slight decrease in the O 1s and N 1s components, and a significant decrease in the Si 2p component. These changes suggest a thicker coating in comparison with the previous amine- and alkyne-functionalized surfaces (Table 3), thereby confirming the success of the click reaction at the surface and the corresponding formation of a covalently bonded recombinamer layer. The proteinaceous character of the ELR studied herein explains the maintenance or slight increase of the N 1s and C 1s percentages (these are the main constituents of proteins). Moreover, the recombinamer coating resulted in a strong decrease in the Si 2p signal, thus, confirming that the surfaces E

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Table 4. TOF-SIMS Results for Glass and Amine-, Alkyne-, and Polymer-Functionalized Surfacesa glass amine alkyne 3K-RGD* 3K-RDG* 3K-V84* N-RGD* RGD+24K* a

30 (CH4N+)

45 (CH3NO+)

68 (C4H6N+)

70 (C4H8N+)

72 (C4H10N+)

86 (C5H12N+)

0.99 64.07 88.99 72.60 61.69 100.00 70.72 71.71

2.84 63.75 94.31 8.75 8.98 28.46 10.21 4.62

2.03 3.96 12.07 36.61 29.52 55.95 38.65 28.48

1.35 0.52 1.46 100.00 100.00 63.83 100.00 100.00

1.75 0.97 1.11 92.84 97.75 98.01 93.86 72.41

0.20 1.43 2.43 40.16 30.54 5.75 35.05 65.22

The intensity of the most representative ions present on the surfaces is shown.

Table 5. Contact Angle (CA) Measurements at 37 and 10 °C for Glass, Active Glass, and Amine-, Alkyne-, and PolymerFunctionalized Surfaces and the Variations Produced by the Temperature Change (n = 9)

had been almost completely covered. The only recombinamer to show a dissimilar behavior was N-RGD*, for which a modest decrease in this component (Si 2p 16.17%) similar to that for the alkyne-coated surface (18.18%) was observed. The values for the remaining components were also similar to those for the alkyne-coated surface, thereby probably indicating that the absence of the grafting domain in this recombinamer results in incomplete coating of the modified glass substrate. TOF-SIMS analysis, which is commonly used to complement XPS, showed that the amine- and alkyne-coated surfaces provide similar spectra, which clearly differ from that for the nonfunctionalized glass surface (Table 4), with a clear signal from the coating at m/z 30 (CH4N+) and 45 (CH3NO+). The CH4N+ signal is more intense for the alkyne-functionalized surface than for its amine-functionalized counterpart, whereas the CH3NO+ signal is present in a similar proportion for both surfaces, thus, confirming the organic nature of the coating layer. In contrast, TOF-SIMS analysis (Table 4) of the polymer-functionalized surfaces allowed us to infer the presence of a protein coating due to the appearance of species that have previously been identified as originating from amino acid residues, such as glycine (Gly, CH4N+, 30), alanine (Ala, C2H6N+, 68), proline (Pro, C4H8N+, 70), valine (Val, C4H10N+, 72), and leucine or isoleucine (Leu/Ile C5H12N+, 86).28 The recombinamers 3K-RGD*, 3K-RDG*, and N-RGD* showed similar ion percentages due to their similar amino-acid composition, whereas 3K-V84* displayed higher values for the valine residue peak (C4H10N+, 72, 98.01) and lower values for the isoleucine amino acid peak (C5H12N+, 86, 5.75) as a result of its particular composition. On the other hand, RGD +24K* showed a higher value for the isoleucine residue peak (C5H12N+, 86, 65.22) as this polymer contains more than twice as much of this amino acid in its chain as the other recombinamers containing a VPGVG repeat sequence. Taken together, these results suggest an adequate surface coating with the different ELRs studied herein, thereby corroborating the XPS analysis. Additional fluorescence studies (Figure S2, Supporting Information) corroborated the stability of the covalent immobilization in comparison with adsorbed biopolymer coatings, and AFM measurements (Table S2, Supporting Information) verified the thickness (∼16.7 nm) and roughness (∼9 nm) homogeneity of the polymer-grafted surfaces. Water contact angle measurements showed hydrophobicity changes on the surface during the different surface-functionalization steps and as a result of the change in polymer conformation as the temperature was varied. Thus, Table 5 shows how the contact angle decreased from 48.1 ± 0.5° to 27.3 ± 1.1° when the surfaces were first activated as the presence of hydroxyl groups increases their hydrophilicity.

surface glass ACT glass amine alkyne 3K-RGD* 3K-RDG* 3K-V84* N-RGD* RGD+24K*

CA 37 °C 48.1 27.3 70.9 64.6 75.8 74.9 64.8 64.1 65.1

± ± ± ± ± ± ± ± ±

0.5° 1.1° 1.7° 0.3° 0.7° 0.7° 1.1° 1.5° 0.6°

CA 10 °C

Δ

± ± ± ± ± ± ± ± ±

−0.4° 0.6° 0.5° −0.2° 10.3° 9.0° 5.1° 3.8° −1.8°

48.5 26.7 70.4 64.8 65.5 65.9 59.7 60.3 67.1

0.6° 0.8° 0.7° 1.1° 0.9° 1.0 2.1° 0.5° 0.7°

However, these values increased to 70.9 ± 1.7° during the second surface-functionalization step, thus indicating the presence of an aminosilylated layer, which is more hydrophobic than its hydroxyl-coated precursor. As the alkyne layer is less hydrophobic, the surface contact angle decreased again to 64.6 ± 0.3°, thus, indicating the presence of a more hydrophilic coating that probably results from the amide group conformation at the alkynyl-functionalized surface. After verification of the polymer coating efficacy using the previous analytical techniques, the contact angle measurements allowed us to determine whether the biofunctionalized surfaces maintained their thermal responsive behavior after grafting of the ELRs onto them. Thus, contact angle measurements were performed at 37 and 10 °C, in other words at temperatures above and below the corresponding T t for all the recombinamers studied. The resulting contact angles showed no changes with temperature (10 and 37 °C) for the glass surfaces functionalized with amine and/or alkynyl groups, whereas a different behavior was found for glass surfaces with ELRs covalently bonded to them. As expected, the contact angles for surfaces biofunctionalized with recombinamers 3KRGD* and 3K-RDG* were similar in light of their essentially identical structure. The contact angle was higher at 37 °C than at 10 °C in both cases, with a difference of about 10° between them. The contact angles for glass surfaces functionalized with the recombinamers 3K-V84* and N-RGD* showed a similar behavior, with higher values at 37 °C than at 10 °C, although smaller increases were observed in these cases. The lowest shift in contact angle was found for N-RGD* (increase of 3.8°). Furthermore, the contact angle at 37 °C was similar to that obtained for the alkynyl-functionalized surface despite presenting a similar amino acid composition to recombinamers 3KRGD* and 3K-RDG*. It therefore appears that the absence of the grafting domain in N-RGD* leads to a decreased amount of F

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recombinamer on the surface. The contribution to the hydrophobicity, and its dependence on the temperature of the substrate beneath the recombinamer layer, is most evident in this particular case. The general behavior of the recombinamer-coated surfaces can be explained by assuming that the transition and the resulting conformational changes in the recombinamer, which evolves from a hydrophobic state at 37 °C, where the polymer is folded, to a hydrophilic state at 10 °C, where the recombinamer is extended, take place on these surfaces. The surfaces functionalized with recombinamers 3K-RGD* and 3KRDG* showed the most efficient changes following the temperature decrease. Additionally, 3K-RGD* surfaces showed fully reversible wettability changes under repeated cooling and heating cycles (Figure S3, Supporting Information), thus, suggesting a clear reversibility and reproducibility of the temperature responsiveness of these surfaces. The kinetic data showed that the conformation change driven by the change in temperature is completed within seconds after heating and within less than one minute after cooling according to the data obtained in solution ELR,30 thus, indicating that the grafted 3KRGD* shows a similar kinetic folding and unfolding to other model ELRs in solution. Indeed, these changes are similar to those found in PIPAAm-based systems.29 Finally, the surfaces functionalized with recombinamer RGD+24K*, which contains 24 internal lysine residues, showed a negligible change in contact angle. The extensive grafting at different points along the polymer chain most likely hampers conformational change in the recombinamer, thus, masking any changes in the apparent hydrophilicity of the surface. 3. Cell Adhesion to Functionalized Surfaces. Generally speaking, cell adhesion to surfaces can be considered to take place via a two-step process. The first step is considered to be “passive adhesion”, which is characterized by the adsorption of proteins from the medium onto the substrate. This adsorption is controlled by complex combinations of physicochemical interactions. The second stage is best termed “active adhesion” as it requires the participation of cellular metabolic process. Attached cells are well-known to change their shape and expend metabolic energy in order to stabilize the interface between their membrane and the underlying biomaterial.4 “Active adhesion” is mainly mediated by integrin receptors, which bind to adhesive motifs present in the ECM proteins.8 The bioactive recombinamers used in this work include one such adhesive motif (RGD) in their chains. As a result, surfaces coated with a recombinamer containing the RGD sequence can interact directly with cells via “active adhesion” without needing to pass through a prior passive adhesion step. In an initial set of experiments, recombinamer-functionalized surfaces were examined by means of cell analysis with human fibroblasts to study cell adhesion at different times over a 24 h period. Fibroblast cells were used due to their extraordinary capacity to adhere, which makes them a good candidate for testing a cell-harvesting system, as they do not readily detach. The number of cells adhered to each surface at pre-established time intervals can be seen in Figure 1. The percentage of adhered and nonadhered cells with respect to total cells was calculated relative to the unmodified glass surface, which was used as control and considered to represent 100% adhesion.31 Although cell adhesion and cell morphology are two distinct biological phenomena, both parameters can be directly related. Thus, the cellular response to an adherent surface is usually manifested by an increase in cell spreading, the activation of

Figure 1. Cell adhesion and detachment from ELR-functionalized surfaces at different times before (h) and after (min) a temperature change. Untreated glass surfaces were used as control (n = 12).

survival signaling pathways, and the activation of focal assembly.20,32 Integrin-mediated cell adhesion to extracellular matrix ligands, such as the RGD-related domains, is defined by the well-known focal adhesion complex formation33 by which rounded, bright and refractive cells change to flat, dark cells. Therefore, rounded, bright, and refractive cells were considered to be detached cells and flat, dark cells were considered to be well-adhered cells.16,17 The quantitative analysis (Figure 1) showed that those recombinamers bearing an RGD motif showed the highest adhesion level at 30 min, thus, confirming that the presence of this adhesive sequence is the main factor controlling early cell adhesion.20 No significant differences (p > 0.05; Table S3, Supporting Information) were found between the glass control and recombinamers 3K-RGD* and RGD+24K* at this time point. In contrast, the recombinamer N-RGD*, which also contains an RGD sequence in its amino acid chain, displayed significant differences (p < 0.001) when compared with the three previous surfaces. This could be due to a defective NRGD* polymer coating, which would allow the cells to adhere to the modified glass, thereby avoiding the need to form a specific interaction. Our findings (Figure 1) also indicated that the cell behavior over the bioactive RGD and grafted polymers (3K-RGD* and RDG+24K*) at 24 h remained similar to that for the control glass surface, with no significant differences being observed (p > 0.05). The number of adhered cells and their morphology (Figure 2) were similar for all three surfaces, with a high degree of adhesion in a serum-free medium. Although the differences between RGD and non-RGD surfaces became more noticeable at this time, the differences between RGD surfaces were minimal, with recombinamer N-RGD* presenting a similar adhesion level as 3K-RGD* and RGD+24K*, where no significant differences were found. As suggested above, the behavior of this recombinamer may result from an incomplete RGD polymer coating and a consequent decrease in the density of the RGD motifs on the surface. A single amine group may therefore be insufficient to graft the recombinamer and create a fully functional coating. Those surfaces lacking a functional RGD motif presented approximately half the number of adhered cells than the previous surfaces, although there were G

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maintained their adhesion and cell morphology (Figure 2), with detachment levels of 16.92 and 11.36%, respectively, during the 3 h, low-temperature treatment. The differences between these two surfaces were not significant (p > 0.05). Neither of these surfaces undergoes a physical transition, as supported by the contact angle measurements (Table 4). RGD+24K* contains lysine amino acids along the polymer chain, thus resulting in extensive grafting in a nonbrush layer that hinders its folding and therefore the change in surface properties necessary for cell detachment. These surfaces show a high adhesion capacity due to the presence of the RGD sequence but are unable to undergo temperature-driven cell detachment. The N-RGD*grafted substrates were also unsuitable, mainly due to their limited cell adhesion, which hardly varied after a change in temperature (34.90%). In light of the low number of ELRs absorbed on the glass substrate, the main attachment mechanism for cells in this case would be nonspecific adhesion to the modified glass. As such, a decrease in temperature would have little effect on its properties, thereby preventing detachment. In contrast, it is interesting to note that the few cells that were able to adhere to 3K-RDG* and 3K-V84* could be efficiently detached (83.90 and 76.49%, respectively; Figure 2). This suggests that, as indicated by the contact angle measurements, the thermal behavior of these ELRs is responsible for detachment, although the very limited adhesion capacity of these cells probably facilitates this process. The response of human fibroblasts over 3K-RGD* surfaces was completely different to that for the surfaces described above during the low-temperature treatment, with a cell detachment percentage of 73.41%. The fact that recombinamer 3K-RGD* bears a lys-rich chain end means that it grafts in the form of a brush, thus allowing conformational changes in the chain as a function of temperature, as seen from the contact angle measurements. As a result, the polymer architecture and this covalent coupling onto modified glass induce hydrophobic changes that modify the surface functionality by exposing or hiding cell-adhesion motifs. Above Tt, cells can adhere to RGD motifs as they are much more hydrophilic than the rest of the chain and, as explained before, they tend to present at the water interface. However, below Tt, the recombinamer chains unfold, thus, shifting to their apparent hydrophilic state and resulting in the hydration and spread of the molecule. This process hides the cell-adhesion domains beneath an antifouling layer of (VPGVG)n, thus resulting in the detachment of cells from the surface. This behavior is further enhanced by the total absence of polydispersity in the molecular weight of those recombinamers, which means that all individual molecules are virtually identical and behave essentially the same. In this state the cells are able to maintain their cell−cell and cell−own extracellular matrix interactions but are unable to remain attached to the surface. 5. Cell Viability and Proliferation Assays. The viability of the cells remaining on the surfaces after low-temperature treatment was evaluated by fluorescence techniques and found to be greater than 95% in all cases (Table S4, Supporting Information), thus, demonstrating the biocompatibility and suitability of the ELRs in each of the different surfaces. The viability and proliferation capacity of the fibroblasts detached from the 3K-RGD* surfaces were also analyzed. Thus, the harvested fibroblasts were transferred to a conventional 24well tissue culture polystyrene plate and evaluated over 14 days by measuring the fluorimetric reduction of resazurin at regular time intervals. This analysis revealed a continual increase in

Figure 2. Cell-adhesion and -detachment phase-contrast images of the same surface location after incubation at 37 °C for 24 h (0 min in the figure) and after low temperature treatment for 60, 120, and 180 min. (A) 3K-RGD*; (B) 3K-RDG*; (C) RGD+24K*; (D) N-RGD*.

significant differences between them (p < 0.001). In contrast, the RDG surfaces displayed very few adhered cells due to the presence of this scrambled, nonadherent sequence. The 3KV84* surfaces also exhibited a very limited adhesion, which is coherent with the antifouling and nonadherent properties of (VPGVG) polypeptides.18 These quantitative results highlight the importance of the presence and availability of an RGD domain for inducing cell adhesion. Indeed, with the exception of the untreated glass control surface, which showed the expected high unspecific adhesion, fibroblasts were unable to efficiently adhere to surfaces lacking an RGD sequence even after incubation at 37 °C for 24 h. Consequently, adhesion in these RGD-containing systems must be an active step that involves both the specific adhesion sequences and cellular metabolic processes. This is remarkable because it implies that the functioning of these systems is not based on a change in the physical properties of the surface as is the case with PIPAAm systems. The most similar surface to the PIPAAm systems reported previously tested here is the 3K-V84* surface, which markedly fails to promote cell adherence, behaving in the folded state as a predominantly nonadherent surface. Additionally, some PNIPPAm studies have concluded that the covalent immobilization of cell adhesive peptides, such as RGD, on PNIPPAm surfaces promotes initial adhesion in a serum-free medium. Nevertheless, the present system provide the advantage of included the RGD motif into the polymer chain, thereby avoiding the need to coimmobilize it.17,30 These differences in cell adhesion were more clearly demonstrated by phase-contrast micrographs (Figure 2). The marked decrease in cell adhesion on those surfaces lacking bioactivity (3K-RDG* and 3K-V84*), together with the somewhat higher adhesion for N-RGD* surfaces and the much higher adhesion to 3K-RGD* and RGD+24K* surfaces, which was similar to that found for the positive control (untreated glass), should be noted. 4. Cell Detachment from Functionalized Surfaces. To determine whether the adhered cells could readily be detached from the temperature-responsive, polymer-coated surfaces, a low-temperature treatment was performed after incubation at 37 °C for 24 h. Both the control glass and RGD+24K* surfaces H

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Figure 3. Analysis of cells detached using the two-step procedure. (A) Cell adhesion (24 h at 37 °C) and detachment from 3K-RGDV* (circle), glass surface (triangle) and RGD+24K (square) functionalized surfaces at different times (n = 12). (B, C) Flow cytometry images showing the complexity of fibroblast cells detached from the 3K-RGD* surface before trypsin treatment (B) and after 5 min of incubation with trypsin (C). (D) Percentage of detached cells with respect to total number of seeded cells obtained using the two-step procedure for all three surfaces. Two different types of cells were evaluated: ADSCs (in white) and HFF-1 (in streaks; n = 3; p < 0.05). The dead cell percentages with respect to detached cells are shown in black.

The detached cells (both HFF1 and ADSC) were harvested and analyzed by flow cytometry after vital staining (Figure 3). The initial images show the cell complexity, with a heterogeneous population containing a wide variety of sizes and shapes (Figure 3A) due to the fact that cell−cell and cell− ECM interactions remain intact after detachment, with only the interaction between the cells and the material surface being affected. In order to accurately quantify the number and vitality of the harvested cells, it was necessary to disaggregate the harvested cells and provide individual cells suitable for cytometric analysis. Under these conditions, the image (Figure 3B) shows dissociated and homogeneous cell populations. Figure 3C shows the recovery ratio for both cell types and the percentage of dead cells (in black) for all three surfaces. The recovery ratio was equivalent for both cell types, with no significant differences (p > 0.05), and the viability percentage was also similar to that obtained in previous experiments. However, the surface coated with 3K-RGD* again showed a higher cell recovery (95.07% and 91.10%) in comparison with that obtained for glass and RGD+24K* surfaces (range: 8.15− 11.54%). This new harvesting procedure therefore represents an effective, specific and controllable method that allows cell−cell and cell−ECM interactions to be maintained and can be used to harvest different types of cells in a fast and efficient manner without affecting cell viability.

metabolic activity with time (Figure S5, Supporting Information), with no significant differences (p < 0.05) in the proliferative tendency of these cells compared with the fresh recently trypsinized cells used as control. The fibroblasts therefore preserve their viability and proliferation capacity after thermal treatment. 6. Optimization of the Cell-Harvesting Method with HFF-1 and ADSCs. To optimize and reduce the time needed for efficient cell detachment from these new substrates, a new cooling stage that takes into account certain properties recently described regarding the cell biology of cell detachment must be introduced. It has previously been demonstrated that reduced temperatures provide the conditions for a shift in the surface properties of this kind of cell-harvesting substrate. However, such temperatures also slow cell metabolism,4 which has a negative influence on the kinetics of cell detachment as this process is accompanied by a change in cell shape that is known to consume internal energy and therefore must have a metabolic component. The detachment protocol was therefore redesigned to take this effect into account by including an incubation at reduced temperature (10 °C in this case) for just 15 min followed by a temperature increase to 37 °C for a period of 5 min. This latter step was introduced to activate the metabolic steps required by the cells for active detachment and subsequent shape and cytoskeleton remodeling. The new twostep procedure was assayed using untreated glass as control and test surfaces coated with 3K-RGD* and RGD+24K*, the best adherent surfaces in our previous studies. In this case both fibroblasts and human adipose-derived stem cells (ADSCs) were studied. Visual tracking of the surfaces showed rapid fibroblast detachment during the second step in the procedure (37 °C for 5 min) for those surfaces functionalized with the polymer 3K-RGD* (see video link in WEOs) and insignificant detachment and changes in cell morphology for the other two surfaces. The detachment process seemed to be cooperative, with the best results being obtained at higher cell densities, and was also successful for cellular sheets, as can be seen qualitatively in the WEOs provided.

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CONCLUSION We have successfully demonstrated an efficient approach for functionalizing surfaces to make them suitable for temperaturedriven cell harvesting. This approach involved grafting a multifunctional ELR with a dedicated molecular architecture onto the previously prepared surface. The functionalized surfaces obtained were characterized by XPS, TOF-SIMS, fluorescent analysis, AFM, and contact angle measurements to check the coating for each of the different surfaces tested, thereby demonstrating that the desired intrinsic smart behavior was maintained by the final surfaces. This thermoresponsive character allows molecular conformational changes and I

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ACKNOWLEDGMENTS We acknowledge financial support from the EU through the European regional development fund (ERDF), from the MICINN (Projects MAT 2007-66275-C02-01, MAT 200914195-C03-03, MAT-2010-15982, MAT2010-15310, ACI20090890, and PRI-PIBAR-2011-1403), the JCyL (Projects VA030A08, VA049A11, VA152A12, and VA155A12), the CIBER-BBN, and the JCyL and the Instituto de Salud Carlos III under the “Network Center of Regenerative Medicine and Cellular Therapy of Castilla and León”.

subsequent rearrangement of the molecule to provide either a cell-adherent or a cell-repellent surface. The different controls used in this work provide a strong body of evidence that this behavior relies on a direct interaction between the cell adhesion domains present in the molecule and the cell surface, with other factors, such as changes in surface hydrophilicity, having little or no influence. Only recombinamer 3K-RGD*-derived surfaces proved to be fully functional for efficient cell harvesting, with the remainder proving unsuitable either because they did not release the adhered cells, were unable to induce cell adhesion, or both. Moreover, the efficiency of obtaining single cells and cell sheets was demonstrated with different cell types in an easy, fast and reliable manner. In summary, the creation of an efficient, ELR-based, cellharvesting system requires, first of all, on the basis of the cell adhesion results, an intrinsic cell-adhesive functionality provided by the inclusion of cell adhesive domains, such as RGD in this case, and, second, as deduced by the detachment results, the appropriate positioning of this functionality along the polymer chain to link the conformational changes and selfassembly process that take place during the temperature-driven LCST behavior of these recombinamers to a cell-adhesive/ repellent behavior in a synergetic manner. Other factors, including an efficient grafting capacity, which must be enhanced and favored by an increase in the number of reactive side chains at one of the recombinamer chain ends, have also been found to be relevant. In addition, the identification of the mechanism governing this system and the evidence that this mechanism relies on direct interactions between the cell-adhesion domains of the recombinamers and the cells themselves by “active adhesion” opens the way to the development of much more sophisticated systems that cannot be achieved using other alternative cell-harvesting technologies, either alone or in combination with other available techniques such as microcontact printing and so on. These may include selective cell adhesion by exploiting the wide panoply of cell-adhesion domains described in the literature, cell shortening, using coculturing techniques involving the simultaneous culture of different cell types, in this case with the possibility of cell organization and confinement, and, finally, selective cell harvesting.

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REFERENCES

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ASSOCIATED CONTENT

* Supporting Information S

FTIR-ATR study of recombinamers 3K-RGD, 3K-RGD*, RGD +24K, and RGD+24*, high-resolution XPS data for amine- and alkyne-functionalized surfaces, fluorescence studies over the surfaces to corroborate the stability of the covalent grafted, pictures of the cell-adhesion and -detachment experiments over all different biofunctionalized surfaces, statistical analysis of cell adhesion and detachment from ELR-functionalized surfaces at different times and viability, and proliferation capacity of the fibroblasts remaining and detached from the 3K-RGD* surfaces. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest. J

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K

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