Protein film formation on cell culture surfaces investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy

Journal Pre-proof Protein film formation on cell culture surfaces investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy Andreas Wargenau, Natalie Fekete, Ariane V. Beland, Gad Sabbatier, Olivia M. Bowden, Mari`eve. D. Boulanger, Corinne A. Hoesli

PII:

S0927-7765(19)30591-0

DOI:

https://doi.org/10.1016/j.colsurfb.2019.110447

Article Number:

110447

Reference:

COLSUB 110447

To appear in:

Colloids and Surfaces B: Biointerfaces

Received Date:

18 February 2019

Revised Date:

15 August 2019

Accepted Date:

18 August 2019

Please cite this article as: Wargenau A, Fekete N, Beland AV, Sabbatier G, Bowden OM, Boulanger MD, Hoesli CA, Protein film formation on cell culture surfaces investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy, Colloids and Surfaces B: Biointerfaces (2019), doi: https://doi.org/10.1016/j.colsurfb.2019.110447

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Protein film formation on cell culture surfaces investigated by quartz crystal microbalance with dissipation monitoring and atomic force microscopy

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Andreas Wargenau‡, Natalie Fekete‡†, Ariane V. Beland, Gad Sabbatier, Olivia M. Bowden,

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Mariève. D. Boulanger, and Corinne A. Hoesli*

address: Saint-Gobain Ceramics & Plastics, Inc., Saint-Gobain Research North-America,

Northborough, MA, USA

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† Present

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‡A.W. and N.F. contributed equally to this work.

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Department of Chemical Engineering, McGill University, Montreal, Quebec H3A 0C5, Canada

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*Corresponding author. E-mail: [email protected]

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Graphical abstract

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Proteins in serum-free medium coated cell culture surfaces within ~2 min Resulting protein films largely consisted of albumin Protein deposition kinetics were comparable for polystyrene and fluoropolymers Protein film topographies were also similar for these surfaces

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   

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Highlights

ABSTRACT

Conventional cell culture surfaces typically consist of polystyrene, with or without surface modifications created through plasma treatment or protein/peptide coating strategies. Other

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polymers such as fluorinated ethylene propylene are increasingly being implemented in the design of closed cell culture vessels, for example to facilitate the production of cells for cancer immunotherapy. Cultured cells are sensitive to culture vessel material changes through different mechanisms including cell-surface interactions, which are in turn dependent on the amount, type, and conformation of proteins adsorbed on the surface. Here, we investigate the protein deposition

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from cell culture medium onto polystyrene and fluoropolymer surfaces using quartz crystal microbalance with dissipation monitoring and atomic force microscopy. Both surfaces showed comparable protein deposition kinetics and resulted in similar mechanical and topographical film properties. At protein concentrations found in typical serum-free media used to culture dendritic cells, two deposition phases can be observed. The protein layers form within the first few minutes of contact with the cell culture medium and likely consist almost exclusively of albumin. It is

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indicated that initial protein film formation will be completed prior to cell settling and initial cell

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contact will be established with the secondary protein layer. The structural properties of the protein film surface will strongly depend on the albumin concentration in the medium, and presumably be

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less affected by the chemical composition of the cell culture surface.

KEYWORDS

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Adsorption, immunotherapy, fluoropolymer, quartz crystal microbalance with dissipation

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monitoring, albumin, insulin, transferrin

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1. Introduction In the process of translating cell therapy products from the bench to the patient's bedside, researchscale cell manufacturing systems need to be adapted to comply with the rules and recommendations issued by the regulatory authorities [1]. Novel 'functionally closed' cell culture systems, such as bag bioreactors and defined serum-free media have been developed. While the traditionally used laboratory-scale T-flasks are made from polystyrene, closed culture bags are

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often produced from fluoropolymers such as fluoroethylene propylene (FEP) or other gas-

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permeable polymers such as polyolefins. These new culture materials are already applied in clinical settings, for example for cancer immunotherapy [2]. In the context of dendritic cell-based

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cancer vaccines, a limited number of studies directly compared the performance of fluoropolymer

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bag systems (e.g., fluorinated ethylene propylene bags) to conventional polystyrene containers [35]. One study reported that monocyte-derived dendritic cell culture in FEP bags markedly reduced

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cell adhesion and altered cell production of critical cell signaling molecules [3]. The interaction of cells with their microenvironment, which is established through matrix-binding

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receptors spanning the cellular membrane, is crucial in determining cell fate both in vivo and in vitro [6]. Cell adhesion to culture substrates or matrix is thus governed by the presence of proteins in the immediate vicinity of the cells. During in vitro cell culture, these proteins are provided via the cell culture medium; proteins rapidly adsorb to the culture surfaces and form a protein surface

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layer [7, 8]. The composition of this protein layer will depend on many factors, putatively including the type and quantity of proteins present in the culture medium, the environmental conditions during the adsorption process (e.g., pH and ionic strength of the medium), and the surface properties of the substrate [9]. Moreover, the proteins' conformation inside the layer will depend

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on these factors. Both the composition and the structural properties of the protein layer will determine the availability of cell-receptor binding sites. Water molecules are the first molecules to reach the culture vessel surface after addition of the culture medium [10]. To promote cell adhesion, culture vessel manufacturers alter the surface chemistry of the bare polystyrene by introducing ionizable surface moieties, such as amino or carboxyl groups [11, 12]. In addition to the surface charge, surface free energy, polarity, and

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topography are known to affect protein adsorption and thereby cell adhesion [9, 13]. The co-

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adsorption of multiple proteins present in the medium may also affect the cell adhesive properties of the resulting protein film. For example, when serum-containing media were applied to

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polytetrafluoroethylene surfaces, the presence of albumin in the culture medium significantly

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reduced the co-adsorption of fibronectin on the culture surface, therefore reducing cell attachment [14]. Co-adsorption may involve the displacement of adsorbed proteins by other proteins with

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greater surface affinities (Vroman effect) [15, 16]. These competitive effects are well-known to occur in the presence of serum proteins [17-19]. The dynamics of serum protein adsorption from

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complete serum or individual protein solutions onto a variety of polymeric biomaterials has been thoroughly studied in the context of biomedical device fabrication and implantation [9, 20-22]. Compared to protein adsorption from blood or serum, comparatively few studies have investigated protein deposition dynamics and the architecture of protein layers formed under serum-free cell

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culture conditions. There is an urgent need to study protein adsorption in the context of therapeutic cell manufacturing under current good manufacturing practices (cGMP), where serum-free media are preferred.

Culture media used to manufacture cell therapy products at the clinical scale are cell type- and application-specific formulations. While the exact compositions of ready-to-use media are

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proprietary to the manufacturers, most cell culture media utilize commonly used 'basal media' such as Dulbecco’s Modified Eagle Medium (DMEM) or Roswell Park Memorial Institute (RPMI) 1640 medium as a base for their final products. In addition to specific cell proliferation and/or differentiation-stimulating factors, the majority of mammalian cell culture media are supplemented with albumin, insulin, and transferrin [23]. Albumin is the most abundant protein in human serum and is often added to basal media as a lipid carrier [23]. Insulin and transferrin are part of the

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essential ITS (insulin, transferrin, selenium) supplement routinely added to serum-free media.

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While the hormone insulin plays a central role in the regulation of glucose levels, transferrin is required for the iron transfer into the cells [24]. All three proteins are thus responsible for

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maintaining necessary levels of nutrients and trace elements in cellular homeostasis. Their

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potential effects as cell culture surface proteins, on the other hand, is not well understood. The present study investigates the formation of protein films on polystyrene and

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fluoropolymer-based culture vessels in contact with proteins present in most serum-free media. A clinically-relevant serum-free dendritic cell culture medium was applied as a first step towards

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understanding how protein film composition and structural properties may impact later cell-surface interactions. Quartz crystal microbalance with dissipation monitoring (QCM-D) was used to study the protein deposition kinetics and the mechanical properties of the protein films. Protein film structures were further investigated by atomic force microscopy (AFM). Both characterizations

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were performed on polystyrene and AF1600-coated QCM-D crystals. AF1600 is an amorphous fluoropolymer by DuPont, which was used as a model polymer to mimic the surface properties of fluoropolymer cell culture bags.

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2. Material and methods 2.1. Media and protein solutions Bovine serum albumin (BSA, A9647), holo-transferrin (T0665), and human insulin (I0908) were purchased from Sigma-Aldrich and reconstituted in deionized water or 3 mM HCl (in case of insulin). Antibodies used to detect adsorbed proteins in QCM-D experiments were mouse antihuman albumin (ab24458, Abcam), rabbit anti-human insulin (C27C9, Cell Signaling

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Technology), sheep anti-human transferrin (ab9036, Abcam), and mouse IgG1 isotype control

from CellGenix and Thermo Fisher Scientific, respectively.

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(APC labeled, BD). Cell culture media (CellGenix GMP DC and RPMI-1640) were purchased

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Aliquots of protein stock solutions were diluted in phosphate-buffered saline solution (PBS) with

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0.5 M calcium and magnesium or RPMI 1640 medium (Thermo Fisher Scientific) directly before use in QCM-D experiments. The pH of the PBS was adjusted to 7.2 using 1 M HCl. The pH values

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of the RPMI and CellGenix GMP DC media were measured before and after QCM-D experiments and ranged between 7.1 and 7.4. Purchased antibody stocks were diluted in RPMI medium at a

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dilution factor of 1:400. Albumin content in CellGenix GMP DC medium was quantified using a human albumin indirect ELISA kit (Alpco Microalbumin ELISA) according to the manufacturer’s instructions. All antibody and protein solutions as well as PBS and protein-free RPMI media were filtered through a pre-rinsed 0.1 µm Millex VV syringe filter (Millipore) before injection into the

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QCM-D.

2.2. QCM-D crystal preparation

QCM-D crystals with AF1600 and polystyrene coatings were purchased from Biolin Scientific (QSX 331 and QSX 305, respectively) and used without further modifications. Each crystal was

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used only once. No prior cleaning was performed, except that crystals were purged with nitrogen gas to remove dust particles directly before mounting inside the QCM-D flow module. In addition to the polymer-coated QCM-D crystals, silanized and bare silica-coated crystals were used as reference surfaces. Silica-coated crystals (QSX 303, Biolin Scientific) were silanized by vapor deposition at reduced pressure and room temperature using trichloro(1H,1H,2H,2Hperfluorooctyl)silane (Sigma-Aldrich). The silane was allowed to react for 1 h before washing with

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ethanol and drying under a stream of nitrogen. Both modified and unmodified silica-coated crystals

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were purged with nitrogen gas immediately before use. 2.3. QCM-D experiments

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QCM-D experiments were performed with a Q-Sense E1 QCM-D (Biolin Scientific), a Q-Sense

flow module was equilibrated at 22 °C.

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flow module, and a peristaltic pump. The flow rate of the pump was set to 0.1 mL/min and the

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Before injection of the protein solutions, QCM-D crystals were exposed to DI water and proteinfree buffer to minimize the effect of viscosity-induced signal changes. When proteins were

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deposited from CellGenix GMP DC medium, QCM-D signals were equilibrated in the presence of protein-free RPMI medium. For antibody binding tests, CellGenix GMP DC medium was replaced by protein-free RPMI medium after a total of 1.5 h of protein deposition. RPMI medium-dissolved antibodies were then injected in the following order: (1) IgG isotype (control), (2) anti-transferrin,

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(3) anti-insulin, and (4) anti-albumin. Further details on the antibody injection, rinsing, and incubation sequences can be found in the Supporting Information (see Figure S5). All QCM-D experiments were analyzed using the normalized resonance frequency and the energy dissipation factor of the 3rd harmonic (f3 and D3, respectively). Changes in the surface mass density (ms) were calculated by the Sauerbrey equation [25]; i.e.,

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ms = −C × Δf3

(1)

where C is the mass sensitivity constant of the QCM-D crystal (here, C = 17.7 ngs/cm2). Adsorption rate constants (kad) were determined by the ratio of the initial surface mass change rate during the linear portion of the adsorption process (dms/dt) and the mass concentration of the proteins in the medium (Cp): kad = 1/Cp’ × 1/A × dNad/dt = 1/Cp × dms/dt (2)

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where A is the available surface area, Nad is the number of proteins adsorbed onto the surface, and

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Cp’ is the number concentration of proteins in solution.

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2.4. Contact angle measurements

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Contact angles were measured on QCM-D crystal surfaces and 5 mil thick high molecular weight FEP films (CHEMFILM FEP-FS, Saint-Gobain) using an optical goniometer from DataPhysics

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Instruments GmbH (OCA 150). Water droplets of 2 μL volume were deposited at a rate of 0.5 μL/s. Images were captured, and static contact angles were analyzed using the SCA-20 software of the

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manufacturer. The reported values represent the mean ± standard error of a total of 10 measurements, with each measurement representing the average of the left and the right static contact angles.

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X-Ray Photoelectron Spectroscopy

X-ray photoelectron spectroscopy (XPS) was performed on AF1600 and FEP films using a KAlpha instrument from Thermo Scientific. Three survey spectra were obtained from each surface and analyzed with respect to the elemental compositions of the polymers (i.e., carbon, fluorine,

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and oxygen contents). A description of the analytical procedure is provided in the Supplementary Methods section of the Supporting Information. 2.5. AFM imaging Nitrogen-purged QCM-D crystals were mounted under a Dimension FastScan atomic force microscope from Bruker. A triangular-shaped ScanAsyst-Fluid probe with a nominal spring constant of 0.7 N/m and a tip radius of ~ 20 nm was used to image in PeakForce Tapping mode.

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A droplet of deionized water was deposited onto the surface of an AF1600 or polystyrene-coated

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QCM-D crystal and a 2 µm × 2 µm square surface area was scanned immediately. Subsequently, the water droplet was removed carefully using a pipette without touching the polymer surface, and

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a drop of CellGenix GMP DC culture medium was placed onto the polymer surface. Following an

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incubation period of 10 min, the same surface area was scanned again. The initial scan locations were recognized based on topographical imperfections found on both polymer coatings. These

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imperfections were excluded from the analysis.

Images from the height sensor were used to analyze surface roughness using the NanoScope

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Analysis software (Bruker). Prior to roughness analysis, the inbuilt ‘Flatten’ function was applied to all images. This function acts as a filter to remove low frequency noise and tilt features from the image through polynomial fitting of all unmasked scan lines. After applying this filter, the nano-scale surface roughness was characterized by calculating the root mean square roughness

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(Rq value) for each image.

2.6. Direct ELISA to detect adsorbed proteins

ELISA experiments were conducted on high protein-binding capacity coated 96-well plates (Nunc Maxisorp, Invitrogen), on untreated polystyrene 96-well plates (Nunc Edge 2.0, Invitrogen), on

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aminoalkysilane glass slides (Electron Microscopy Sciences), and on custom well plates to study protein adsorption to FEP films and AF1600-coated QCM-D crystals. A detailed description of the custom well plate fabrication as well as direct ELISA experiments is provided in the Supporting Information (see Supplementary Methods and Figure S1). Briefly, test surfaces were exposed for 1 h to protein solutions at room temperature, washed, blocked and then incubated with diluted horseradish peroxidase (HRP)-conjugated anti-insulin or anti-albumin antibodies. After washing,

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surfaces were reacted with the HRP substrate 3,3',5,5' tetramethylbenzidine (TMB), followed by

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stopping the reaction with 0.16 M sulfuric acid and detection of TMB cleavage products by measuring the absorbance at 450 nm using a Bio-Rad Benchmark Plus microplate

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spectrophotometer.

3. Results and Discussion

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3.1. Surface deposition of individual cell culture proteins To compare the adsorption kinetics of the individual cell culture proteins, we deposited albumin,

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transferrin, and insulin on polystyrene and AF1600-coated QCM-D crystals. The proteins were injected in PBS buffer at concentrations of 10 g/mL and 1 g/mL. The results are summarized in

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Figure 1.

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Figure 1. Deposition of insulin, albumin, and transferrin onto AF1600 and polystyrene-coated QCM-D crystals. Proteins were deposited from PBS buffer using mass concentrations of 10 µg/mL (A, B) and 1 µg/mL (C). Vertical arrows indicate injection points. Figure 1A shows the QCM-D responses upon protein deposition on AF1600. The adsorption of

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the proteins to the polymer surface resulted in a sharp frequency drop within the first 2 min of the deposition time. In the cases of albumin and insulin, the resonance frequency decreased to

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~ -10 Hz, while an almost two-fold greater shift was observed for transferrin. The energy

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dissipation, on the other hand, did not change significantly. In fact, none of the observed energy dissipation shifts exceeded a value of 0.5 × 10-6, indicating the formation of rigid, monomolecular

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protein layers [26]. After the initial deposition phases, albumin and transferrin continued to deposit at notably slower deposition rates, whereas no secondary deposition phase was observed for insulin.

Deposition on polystyrene resulted in a qualitatively similar adsorption behaviour of the proteins

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(Figure 1A). Similar to the fluoropolymer, albumin and transferrin adsorbed within two steps, while insulin stopped depositing after the initial adsorption step. A quantitative comparison reveals that the deposition of albumin and transferrin resulted in somewhat more pronounced resonance frequency and energy dissipation shifts on polystyrene as compared to AF1600. These differences could be due to a greater water content inside the protein films [27] and may be explained by the lower hydrophobicity of the polystyrene surface (see below). Page 12 of 28

Because of the comparatively small changes in energy dissipation (i.e., ΔD3/Δf3 < 10-7 s), we can use the Sauerbrey equation to calculate the surface mass densities of the protein films after the initial protein adsorption. For instance, the adsorption of the smallest protein insulin onto the fluoropolymer surface resulted in a final surface mass density of 1.7 mg/m2. Assuming a mass density of the hydrated protein film between 1 kg/L and 1.5 kg/L [28], this value would correspond to an effective thickness between 1.1 nm and 1.7 nm. As a reference, the approximate Stokes radius

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of insulin is 1.1 nm [29, 30], and a monolayer of densest packed spheres (i.e., hexagonal packing)

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of 1.1 nm radius exhibits an effective film thickness (volume equivalent) of ~ 1.3 nm. Interestingly, the initial adsorption of albumin, which has a Stokes radius of 3.5 nm, resulted in very similar

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frequency shifts as insulin, indicating a similar mean film thickness after the first two minutes of

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albumin deposition. An albumin film thickness below 4 nm suggests side-on orientation with possible denaturation of the proteins at the surface [27].

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To evaluate potential differences in the initial adsorption rates of the cell culture proteins more accurately, we deposited proteins at a mass concentration of 1 g/mL. Figure 1C compares the

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initial slopes in the resonance frequencies upon protein deposition on AF1600 and polystyrene. The corresponding adsorption rates constants are listed in Table 1. Most notably, insulin and albumin showed almost identical mass deposition patterns and comparable adsorption rate constants. Transferrin, in contrast, showed an almost two-fold larger adsorption rate constant for

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AF1600 and a 2.5 to 3 times larger adsorption rate constant for polystyrene. However, transferrin also resulted in a significantly larger surface mass density after the initial protein adsorption (approx. 2-fold on average), suggesting that protein films developed within comparable time scales.

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Table 1. Adsorption rate constants for the individual cell culture proteins kad = 1/Cp × dms/dt [m/s] * Insulin

albumin

transferrin

1.6 × 10-6

1.5 × 10-6

2.7 × 10-6

Polystyrene 1.3 × 10-6

1.1 × 10-6

3.3 × 10-6

AF1600

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*Surface mass change rates were calculated from the initial frequency changes depicted in Figure 1C

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Considering typical total protein concentrations in serum-free cell culture media (> 0.1 mg/mL), the observed adsorption rate constants suggest an immediate protein film formation once the

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culture medium is injected into the culture container. Furthermore, the initial surface layer will

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almost exclusively consist of albumin. Culture media typically contain ~ 2 magnitudes of order higher albumin concentrations as compared to insulin and transferrin [31-34]. Since the adsorption

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rate constants of all three proteins are of the same order of magnitude, the initial albumin layer will likely be formed without significant competition by the other two proteins. Insulin and

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transferrin, however, could add to the surface layer after the initial adsorption step. For example, even at 200-fold lower concentration, adsorbed insulin was detected on polycarbonate membranes (with hydrophilic surface treatment) and was found to be enriched compared to concentrations in solution [35].

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3.2. Protein deposition from cell culture media

To study the effect of competitive protein deposition, we exposed differently coated QCM-D crystals to CellGenix GMP DC culture medium. The medium contains all of the investigated proteins, with albumin being the most abundant supplement. The albumin concentration in CellGenix GMP DC medium was determined to be 3.9 mg/mL. Insulin and transferrin were confirmed by the medium producers to be > 100-fold less concentrated than albumin in the medium Page 14 of 28

formulation (personal communication). In addition to the three proteins, the medium contains various salts, sugars, amino acids, vitamins and buffer components. However, none of these constituents showed comparable surface adsorption when tested in the absence of the cell culture

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proteins (data not shown).

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Figure 2. CellGenix GMP DC protein deposition on fluoropolymer, polystyrene, and silica-coated QCM-D surfaces. Proteins were deposited on AF1600, polystyrene (A-C), silica, and perfluorooctylsilane modified silica (D-E). Diagrams A, B, D, and E show changes in the resonance frequency and energy dissipation at different time scales. Diagrams C and F show the corresponding ΔD/Δf-ratios as a function of frequency shift. Dashed lines indicate the different deposition phases: (i) initial protein deposition, (i-ii) intermittent phase, (ii) secondary deposition phase. Vertical arrows indicate injection points.

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Figure 2A depicts the results of the protein deposition onto AF1600 and polystyrene. In agreement with the protein deposition from PBS, both surfaces resulted in comparable resonance frequency and energy dissipation shifts. However, the deposition kinetics differed significantly from those observed for the lower protein concentrations. As opposed to the adsorption behaviour of albumin, the resonance frequency decreased to ~ -50 Hz and the energy dissipation increased to ~ 5×10-6;

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i.e., ~ 10 times the dissipation shifts shown in Figure 1. Moreover, the QCM-D signals didn’t change uniformly, but within two distinct steps (see deposition data at magnified time scale in Figure 2B). On both surfaces, the resonance frequency passed through an inflection point ~ 0.5 min after the proteins entered the QCM-D chamber. At these points, the resonance frequency and energy dissipation values were ~ 15 Hz and ~ 0.5×10-6, respectively, which is consistent with the QCM-D signals for the initial albumin layers formed during the albumin deposition from PBS.

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Even though the initial protein deposition from CellGenix GMP DC medium was too rapid for

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quantitative analysis of the adsorption kinetics, the two-step deposition behaviour provides further evidence for the formation of an initial albumin layer before the bulk of the protein mass is

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deposited. The initial adsorption step likely involved spreading of the globular protein along the

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polymer surface [36-38], allowing for the formation of a secondary protein layer [39]. The secondary layer could be established through protein adsorption to spatially confined surface areas

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and/or form on top of the initial protein layer involving long-range surface interactions [40, 41]. Since we observed very similar absorption characteristics on both polymers, it is likely that the

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adsorption mechanism is linked to the surface hydrophobicity of the different materials [42]. In general, higher protein binding energy is observed on non-polar, hydrophobic surfaces as compared to polar surfaces [9]. Tight binding of albumin has been observed on fluorinated surfaces, and albumin retention during washes was correlated with surface free energy [43]. Water

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contact angles of the AF1600 and polystyrene surfaces were determined to be 104 ± 4°, and 87 ± 2°, respectively. These values are above the critical water contact angle of ~ 60 reported to mark the transition between weaker and stronger protein surface interactions [44]. Despite the structural differences between AF1600 and FEP, which impact the stability of the surface [45], the water contact angle measured on FEP films (107 ± 3°) was not significantly

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different from the contact angle on AF1600-coated QCM-D crystals. This observation coincides with the presence of similar amounts of fluorine in both polymers as detected by XPS, suggesting comparable amounts of surface exposed CF2 and CF3 moieties (see Figure S2 in the Supporting Information). As shown in Figure S3 in the Supporting Information, direct ELISA experiments also showed no significant differences between FEP and AF1600 when quantifying surface-bound albumin or insulin epitopes available for antibody binding. This observation was contrary to other

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control surfaces used in these experiments (silanized glass as well as untreated and MaxiSorp

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treated polystyrene).

The effect of surface hydrophobicity was further investigated by comparing the protein deposition

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from CellGenix GMP DC medium between a bare and a fluorosilanized silica surface (contact

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angles of 40° ± 4° and 98° ± 2°, respectively). The results of the experiments are shown in Figure 2D and E. Interestingly, protein deposition onto the fluorosilane resembled the two-step deposition

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behaviour of the two polymer surfaces. The hydrophilic silica, in contrast, resulted in a more uniform protein adsorption. Although a less pronounced inflection point could be observed for the

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hydrophilic surface, the adsorption phase change occurred at a significantly smaller frequency shift as compared to the hydrophobic surface (~ -30 Hz). In addition, the protein adsorption on the hydrophilic surface resulted in a notably greater energy dissipation shift, indicating the formation of a softer, more hydrated protein film.

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The effect of surface hydrophobicity becomes further apparent when analysing ΔD/Δf-ratios as a function of the corresponding frequency shifts (Figures 2C and G). The more hydrophobic surfaces showed smaller negative ΔD/Δf-ratios during the initial mass deposition, suggesting a more rigid initial protein layer. In the case of AF1600 and polystyrene, initial negative ΔD/Δf-ratios even decreased before a rapid increase could be observed starting at resonance frequencies of ~ -10 Hz.

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Protein adsorption on silica, on the other hand, resulted in a more moderate change of the ΔD/Δfratio. However, final values were comparable to those obtained for the hydrophobic surfaces. The time-independent adsorption patterns suggest a fundamental impact of surface hydrophobicity on protein film formation from culture medium. Our results indicate that the mechanical properties of the initial albumin layer are governed by strong hydrophobic interactions, and that these interactions are responsible for a more pronounced surface denaturation of the globular protein

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[36-38]. While the mechanical properties of the initial protein layer appear to be independent of

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the bulk protein concentration (c.f., Figure 1 and 2), the ΔD/Δf-changes obtained during the secondary deposition phase suggest a concentration-dependent mechanism. Negative ΔD/Δf values

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calculated for the albumin deposition at 10 µg/mL (Figure 1) increased less rapidly than indicated

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in Figure 2C and did not exceed a value of 0.4×10-7 s. Additional control experiments in which AF1600-coated QCM-D crystals were exposed to albumin-spiked RPMI media confirmed the

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assumption of a concentration-dependent adsorption mechanism. Spiking the medium with increasing concentrations of albumin (0.5 mg/mL, 2 mg/mL, and 8 mg/mL) resulted in increasing

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dissipation and decreasing frequency shifts (see Figure S4 in the Supporting Information). Furthermore, only one continuous adsorption phase could be observed at the two lower protein concentrations (0.5 mg/mL and 2 mg/mL), while deposition at the highest concentration (8 mg/mL) resulted in a similar adsorption pattern and ΔD/Δf-ratios as observed during protein

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deposition from CellGenix GMP DC medium, although absolute changes in resonance frequency and energy dissipation were less pronounced. To test for the presence of insulin and transferrin at the protein film surface, we injected antiinsulin and anti-transferrin antibodies into the QCM-D chamber after the formation of the protein film. However, none of the two antibodies led to detectable changes in the resonance frequency

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(see Figure S5 in the Supporting Information). By contrast, injection of anti-albumin antibodies resulted in a frequency shift of -18 Hz, suggesting a tightly packed antibody monolayer with an effective molecular area of ~ 80 nm2 (note that the viscosity radius of a 150 kDa antibody of 5–6 nm [46, 47] suggests a cross-sectional area of ~ 100 nm2). Even though it cannot be ruled out that small amounts of transferrin and insulin were present at the film surface, the observation of a high anti-albumin density confirms the predominance of albumin proteins, consistent with the

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significantly larger albumin concentration in the CellGenix GMP DC medium.

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of ro -p re lP ur na Jo Figure 3. Topography of AF1600 and polystyrene-coated QCM-D surfaces before and after protein deposition from cell culture medium. The polymer surfaces were first analyzed in water (A and C), and subsequently in the presence of CellGenix GMP DC medium (B and D). The graphs underneath the AFM images represent cross-sections obtained at the indicated dashed lines.

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3.3. AFM characterization. In addition to the QCM-D deposition experiments, protein film formation was further analyzed from topographic AFM images (Figure 3). Prior to protein adsorption, polystyrene and AF1600 crystal coatings were found to show comparable topographies with Rq values of 0.42 nm and 0.58 nm, respectively (Figure 3A and C). When investigating surface areas greater than 2 µm × 2 µm, regions with greater heterogeneity in surface topography were observed, as may be expected for

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spin-coated QCM-D crystals (see Figure S6 in the Supporting Information). However, similar

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adhesion strengths measured between the tip and the surface suggests a uniform chemical composition of the polymer. Based on the size of the adsorbed protein features (radii < 10 nm), it

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is unlikely that the larger topographic features altered protein adsorption locally.

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Topographic AFM images obtained after protein deposition are shown in Figure 3B and D. Vertical cross-sections of the films on both types of polymers revealed the presence of

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heterogeneously distributed proteins. Both surfaces show distinct peaks ranging between 3 nm and 6 nm in height. These features could correspond to loosely bound albumin and/or albumin patches

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forming isolated protrusions of the secondary protein layer. The effect of the protein deposition is also reflected in the change in surface roughness. Calculated Rq values increased from 0.42 nm to 0.88 nm and from 0.58 nm to 1.69 nm for polystyrene and AF1600, respectively. Note that the dense and thin initial protein layer as characterized by QCM-D is not expected to change the

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surface topography to such extent.

It is possible that the secondary layer formed on top of the primary protein layer with little or no direct contact with the polymer. On the other hand, the primary layer may have also undergone surface re-arrangements during the secondary adsorption step [44]. Such a mechanism could create restricted binding sites for the depositing proteins and potentially result in a more heterogenous

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surface structure of the protein film. In this scenario, the height peaks observed in AFM could

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represent albumin molecules with end-on conformation [19].

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Figure 4. Proposed mechanism of protein adsorption onto untreated polystyrene and FEP surfaces prior to cell adhesion. A first layer of albumin adsorbs onto the hydrophobic surface, likely leading to protein conformation changes to create a 1-2 nm protein layer. After this initial deposition, a secondary layer of albumin deposits either on top or through the formation of new binding sites via primary layer rearrangement, or both mechanisms may occur simultaneously. 3.4. Proposed protein adsorption mechanism in the context of serum-free cell culture The scheme in Figure 4 summarizes the mechanisms of protein adsorption onto non-polar cell

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culture surfaces emerging from this study. When hydrophobic culture containers, such as polystyrene flasks or fluoropolymer bags are exposed to culture media, a protein film will be formed within the order of time required for cell settling (approx. 0.2–0.4 mm/min with 2 to 4 mm typical liquid height). A protein film will therefore be present on the surface prior to cell attachment, but the film may evolve as cells settle onto the surface. The protein film will most likely consist of at least two mechanically distinct layers. The first layer, which has an initial Page 22 of 28

thickness between 1 nm and 2 nm, presumably consists of albumin of side-on orientation with some extent of conformational changes [27, 48, 49]. This initial layer potentially changes its structure during the second adsorption step. The second protein layer is suggested to exhibit a similar protein composition as the initial protein layer. However, this protein layer is significantly more viscose (i.e., more hydrated), and therefore proteins involved are more likely to retain their ability to interact with specific cell surface receptors. While the chemical composition of the

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underlying polymer may affect the conformation of the first layer (Figure S3), it does does not

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appear to impact protein deposition kinetics (Figure 1 and 2), film mechanical properties (Figure 2), or topography (Figure 3), which were mainly correlated with substrate hydrophobicity. Since

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cell adhesion occurs concurrently with protein deposition, it stands to reason that proteins released

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by the cells will further affect the structure of the evolving protein film. Factors such as the time at which medium is introduced into the culture container or the time required for cell settling may

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therefore introduce variability in cell adhesion processes and hence product performance. Conditioning the cell culture surface sufficiently in advance would ensure that protein layer

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formation reaches a steady state before cells are able to interact with the proteins, which could improve the reproducibility of cell manufacturing processes. 4. Conclusions

In this study, no clear difference concerning adsorption kinetics, mechanical film properties, and

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surface structure could be observed between polystyrene and fluoropolymer substrates. Even though direct ELISA measurements suggested differences in the protein conformation of the initial protein layer, these differences are not reflected in the overall mass, mechanical properties, or topography of the final protein film formed. It may be concluded that the chemical surface properties of the culture container materials will have no significant influence on the structural

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protein film properties if protein adsorption is governed by hydrophobic surface interactions as is the case for untreated polystyrene or FEP. Differences in the culture performance of polystyrene flasks (without surface modifications) and fluoropolymer bags observed by others may thus likely be attributed to different material factors, such as the material’s rigidity, gas permeability, or the formation of toxic organic compounds during cell culture [3, 50]. Fluoropolymer-based culture surfaces show much higher elasticity and provide higher gas exchange rates compared to ‘classic’

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culture vessels made of polystyrene. The rigidity of culture substrates is understood to directly

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impart fate-decision cues to adherent cells. In conjunction with surface roughness and spatial arrangements, this constitutes one of the major biomechanical factors to be considered when

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selecting the appropriate culture system [51-54]. Differences in gas exchange rates and capacity

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to retain water vapor may affect pH levels and the concentration of other medium components, thereby altering cell attachment and metabolism. As our understanding of the highly complex

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interactions of cells with their culture microenvironment advances, so will our ability to design

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tailored culture systems that can drive cell performance in vitro.

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5. Supporting Information The Supporting Information file contains supplementary ELISA and XPS methods as well as the following supplementary figures: Figure S1 (Fabrication of custom well plates), Figure S2 (XPS survey spectra of FEP and AF1600 and calculated relative amounts of fluorine, carbon, and oxygen), Figure S3 (Albumin and insulin direct ELISA after protein adsorption on test surfaces), Figure S4 (Albumin deposition at varying mass concentrations), Figure S5 (Antibody binding to

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CellGenix GMP DC protein films on polystyrene and AF1600), and Figure S6 (Topography,

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rigidity modulus, and adhesion strength mapping of 10 m × 10 m of polystyrene-coated QCM-

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D crystals).

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ACKNOWLEDGEMENTS

We thank Dr. Katie Campbell, Dr. Rachel Pytel and Natasha Boghosian (Saint-Gobain Research

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North America) for useful discussions. We thank Patricia Moraille (Agente de recherche, Laboratoire de caractérisation des matériaux, Département de chimie, Université de Montréal) for

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providing technical expertise on atomic force microscopy measurements. We thank Lisa Danielczack and Omar Bashth for protein quantification. This work was supported by the following research networks: the Quebec Cell, Tissue and Gene Therapy Network –ThéCell (a thematic network supported by the Fonds de recherche du Québec–Santé); PROTÉO (supported

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by the “Fonds de recherche sur la nature et les technologies du Québec”); the Québec Center for Advanced Materials (QCAM, supported by the “Fonds de recherche sur la nature et les technologies du Québec”). This research was undertaken, in part, thanks to funding from the Canada Research Chairs program.

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