Easy-to-Prepare Coating of Standard Cell Culture Dishes for Cell

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Tissue Engineering and Regenerative Medicine

Easy-to-prepare Coating of Standard Cell Culture Dishes for Cell Sheet Engineering using Aqueous Solutions of Poly(2-n-propyl-oxazoline) Matthias Ryma, Julia Blöhbaum, Raminder Singh, Ana Sancho, Jasmin Matuszak, Iwona Cicha, and Juergen Groll ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b01588 • Publication Date (Web): 13 Feb 2019 Downloaded from http://pubs.acs.org on February 25, 2019

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ACS Biomaterials Science & Engineering

Easy-to-prepare Coating of Standard Cell Culture Dishes for Cell Sheet Engineering using Aqueous Solutions of Poly(2-n-propyl-oxazoline) Matthias Ryma1, Julia Blöhbaum1, Raminder Singh², Ana Sancho1, Jasmin Matuszak², Iwona Cicha², Jürgen Groll1*

1Department

of Functional Materials in Medicine and Dentistry and Bavarian Polymer Institute,

University of Würzburg, Pleicherwall 2, 97070 Würzburg, Germany ²Cardiovascular

Nanomedicine Unit, Section of Experimental Oncology und Nanomedicine

(SEON), Else Kröner-Fresenius-Stiftung-endowed Professorship for Nanomedicine, ENT Department, University Hospital Erlangen, Friedrich-Alexander-Universität Erlangen-Nürnberg, Glückstraße 10a, 91054 Erlangen, Germany *Corresponding author: [email protected]

Keywords: Cell sheet engineering; Fluidic Force Microscopy; Poly(2-propyl-2-oxazoline); Endothelial cells; Fibroblasts; Mesenchymal stem cells.

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Abstract

Cell sheet technology is a well-known method by which cells are grown on thermoswitchable substrates that become non-adhesive upon cooling, such that a complete layer of adherent cells, along with the produced extracellular matrix, detaches as a sheet. To achieve this, polymers that exhibit a lower critical solution temperature (LCST) below physiological temperature in water, commonly poly(N-isopropylacrylamide) (PNIPAM), are covalently grafted or, for blockcopolymers, physisorbed onto substrates in a monomolecular thin film. Consequently, such substrates, and the polymers required for film-formation, can only be prepared in a chemical lab with profound macromolecular expertise.

In this study we present an easy and robust method to coat standard cell culture dishes with aqueous solutions of commercially available poly(2-n-propyl-2-oxazoline) (PnPrOx), a polymer that exhibits LCST behavior. Different standard cell culture dishes were repeatedly coated with 0.1 wt% aqueous solutions of PnPrOx and dried in an oven to create a fully covered and thermoresponsive surface. Using this PnPrOx surface a variety of cell types including endothelial cells, mesenchymal stem cells and fibroblasts, were seeded and cultured until confluency. By decreasing the temperature to 16 °C, viable cell sheets were detached within cell-type dependent time frames and could be harvested for biological analysis. We show that the cytoskeleton rearranges, leading to a more contracted morphology of the cells in the detached cell sheet. The cellular junctions between single cells within the sheet could be detected using immunostainings, indicating that strong and intact intracellular contacts are preserved in the harvested sheets.


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Introduction Cell sheet technology is an established method developed by Okano and co-workers for the creation of multicellular sheets. The method was originally based on culture dishes grafted with a thermoresponsive polymer, poly(N-isopropylacrylamide) (PNIPAM). PNIPAM exhibits a lower critical solution temperature (LCST) in water of 32 °C, meaning that above 32 °C PNIPAM polymer chains are dehydrated, resulting in hydrophobicity of the grafted dish surfaces. Below 32 °C, the polymer reversibly switches to hydrophilic 1. Under standard cell culture conditions, cells are able to adhere and proliferate on the surface and by simply decreasing the temperature, whole cell sheets can be detached 2. Compared to standard cell harvesting methods via trypsinization, cell sheet technology allows non-invasive harvesting, which is important for rapid colonization of cells on biomatrices and for engineering 3D cellular constructs. Until now, many different cell types such as epithelial3-5 and endothelial cells6, cardiomyocytes7-8, or hepatocytes8, have been shown to adhere to PNIPAM, enabling its application in cell sheet technology. Apart from PNIPAM, other polymers also show LCST behavior in water. One example are polymers based on poly(2-oxazolines) (POx). POx have attracted increasing interest because of their tunable thermoresponsive properties and biocompatibility that is based on a structural relation to polypeptides 9-10. In contrast to PNIPAM, POx-based polymers exhibit minimal thermal hysteresis and by exchanging side-chains, copolymerization or functionalization, the LCST of the polymer can be fine-tuned 11. These characteristics have led to the development of many potential applications of POx. For example, it was shown that the conjugation of proteins and small molecule drugs to POx helps to preserve their activity12 and POx micelles have also been used as a drug carrier system 13. Furthermore, by immobilizing poly(2-iso-propyl-2-


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oxazoline) (PiPrOx) on surfaces, thermoresponsive nanolayers have been created that allow for non-invasive detachment of fibroblasts 14. Compared to PiPrOx, poly(2-n-propyl-2-oxazoline) (PnPrOx) has not been studied as extensively and has not been utilized in the field of life sciences thus far. Nevertheless, a LCST of 20 °C in 1x Dulbecco’s phosphate buffered saline (PBS)15 makes it a promising candidate for applications in this field. Currently, creating cell sheets for tissue engineering requires either purchasing of commercially available pretreated dishes, or labor- and time-intensive grafting of a thermoresponsive polymer onto cell culture dishes, requiring both pre-defined materials and established methods. However, easy-to-apply coatings have been previously examined by different groups. Schmidt S. et al. created PNIPAM microgels and deposited closely packed films by solvent evaporation, resulting in a thermoresponsive surface for cell sheet engineering 16.

Block copolymers of PNIPAM and poly(butyl methacrylate) (PBMA) have been used to

cover standard cell culture dishes with a thermoresponsive polymer layer by physical deposition 17.

Furthermore, to prevent cell adhesion in defined patterns for precise and long-term control of

neurite outgrowth, POx-based triblock copolymers were used to create easy-to-apply coatings 18. Although these methods share the simplicity of application, the preparation of the relevant thermoresponsive polymers is more complex and requires specialized knowledge. The only exception is an extensive study reported by Nash et al., in which an ethanolic PNIPAM solution was used to create thermoresponsive culture substrates via spin coating as an easy and straightforward preparation method 19. In this study, we aimed to establish an easy-to-apply and especially easy-to-prepare coating procedure purely based on aqueous solutions that can be transferred to standard cell culture plates and utilized in every biological laboratory in possession of standard equipment. We have


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developed a simplified and cheap method to create thermoresponsive polymer layers in cell culture dishes without the use of grafting techniques. By coating the dishes with aqueous solutions of PnPrOx, a thermoresponsive surface for cell growth was prepared. Different cell types were able to adhere and proliferate on PnPrOx in a manner similar to non-treated culture dishes. By reducing the temperature below the LCST of PnPrOx, the polymer became hydrophilic and the detached cell sheets could be harvested for further processing. We showed that the cytoskeleton rearranges, leading to a more contracted morphology of the cells in the detached cell sheet. The cellular junctions between single cells within the sheet could be detected using immunostainings, indicating that strong and intact intracellular contacts are preserved in the harvested sheets. Altogether, we established an easy, user-friendly and low-cost technique for creating thermoresponsive PnPrOx surfaces, applicable for cell sheet technology and tissue engineering purposes, in any cell biology lab. Materials and methods Chemicals All chemicals were purchased from Sigma Aldrich (Munich, Germany) and used as received, unless otherwise stated. Butyronitrile (≥ 99%), ethanolamine (≥ 98%) and zinc acetate-dihydrate (reagent grade) were used for the synthesis of n-propyl-2-oxazoline. The polymer was synthesized using methyl ptoluenesulfonate, benzonitrile (anhydrous, 99%) and piperidine (≥ 99.5%, purified by redistillation). Calcium hydride (92%, coarse powder) was purchased from abcr GmbH&Co. KG (Karlsruhe, Germany). All other solvents like dichloromethane (reagent grade) and diethylether (p.a.) were bought from Fisher Chemical (Waltham, USA).


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Ultroxa® poly(2-n-propyl-2-oxazoline) (Mn 10,000, PDI ≤1.2) was used as a commercial reference to our synthesized polymer. Instrumentation for chemical characterization 1H

NMR spectra were recorded on a Bruker Fourier 300 at 300 MHz. Deuterated chloroform

(CDCl3) and deuterated acetonitrile (CD3CN) spectra were recorded with nondeuterated solvent signals as internal reference. Gel permeation chromatography measurements were performed on an OmniSEC Resolve & Reveal system purchased from Malvern (Herrenberg, Germany). The system is equipped with an RI-, a viscosity- and a RALS/LALS-detector. A precolumn (Dguard, organic guard column 10x4.6 mm) and two separating columns (D2000 + D3000, 300x7.8 mm) are installed in series. The solvent is dimethylformamide (DMF) with 1 g/L lithium bromide and solvent flow is 1.0 mL/min. For triple calibration, a Polymethylmethacrylate standard (Malvern) was used. Turbidimetry measurements were made by observing the count rate over a temperature range between 15 °C to 25 °C (1 °C steps) on a Zetasizer Nano-ZSP, Malvern, with a HeNe Laser at 633 nm. The LCST was determined to be at the temperature point where the count rate increased drastically. For these measurements, 1 wt% and 0.1 wt% PnPrOx in ultrapure water and 1x Dulbecco’s phosphate buffered saline (PBS) (Thermo Fischer Scientific, Waltham, USA) buffer were used. Synthesis of PnPrOx The monomer was synthesized as described in literature 20. To a round-bottom flask, 3.970 g (0.018 mol, 0.025 eq.) zinc acetate dehydrate, 50.0 g (0.723 mol, 1 eq.) butyronitrile and 53.029


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g (0.868 mol, 1.2 eq.) ethanolamine were added. The solution was heated to 130 °C under reflux and stirred for 24 h. After the reaction, dichloromethane was added, and the monomer solution was washed two times with water and one time with brine. Dichloromethane was evaporated and the monomer was distilled to dryness over calcium hydride under reduced pressure. 1H

NMR (300 MHz, CDCl3): δ/ppm = 4.10-4.16 (t, 2H, NCH2CH2O), 3.70-3.76 (t, 2H,

NCH2CH2O), 2.14-2.19 (t, 2H, CCH2CH2CH3), 1.51-1.64 (sext., 2H, CCH2CH2CH3), 0.86-0.91 (t, 3H, CCH2CH2CH3). The polymer of 2-n-propyl-2-oxazoline was synthesized via ring-opening polymerization using methyl p-toluenesulfonate (MeTos) as the initiator and benzonitrile as the solvent, as described in literature 21. The initiator was weighed and dried in a flame-dried round-bottom schlenk flask. The solvent benzonitrile and the dried monomer were added under argon. The monomer concentration was 3.1 M and the ratio [I]/[M] was 1:400. The polymerization was carried out for 41 h at 100 °C in an oil bath. The terminating agent piperidine (3 eq. in respect to initiator) was added under argon and the solution was stirred overnight. The solvent was evaporated and the polymer redissolved in chloroform and precipitated three times into ice-cold diethylether. In the following, the polymer was dialyzed for three days in regenerated cellulose membranes (MWCO: 3.5 kDa, Carl Roth, Karlsruhe Germany) against ultrapure water and afterwards freeze-dried. 1H

NMR (300 MHz, CD3CN): δ/ppm = 3.40-4.33 (m, 4H, NCH2CH2), 2.96-2.98 (m, 2H,

CH3N), 2.88 (s, 1H, CH3N), 2.24-2.36 (m, 2H, CCH2CH2CH3), 1.55-1.62 (m, 2H, CCH2CH2CH3), 0.89-0.94 (t, 3 H, CCH2CH2CH3). Coating of cell culture dishes with PnPrOx


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PnPrOx was dissolved at 4 °C in ultrapure water to a final concentration of 0.1 wt%. Wells of PS-treated (polystyrene) standard cell culture dishes (Thermo Fischer Scientific) were fully covered with PnPrOx-solution (Table 1). Evaporation of water was performed in an oven at 60 °C. After the initial drying process, the coating could be seen by eye as a thin film. Nevertheless, the coating in the central region was noticeably thinner after the drying, due to capillary effects. Therefore, another drop of PnPrOx-solution was added to the center and placed again in the oven for the complete evaporation of water. As a result, the entire surface was covered with a layer of PnPrOx (Figure 1).

Figure 1: formula of poly(2-n-propyl-oxazoline) (A) and coating procedure of a standard cell culture dish with aqueous 0.1 wt% PnPrOx solution. The culture dish is filled with aqueous 0.1 wt% PnPrOx solution until the whole well is covered and dried in the oven at 60 °C (B). During the drying process, water evaporates from the inside of the well to the outside (B). After evaporation of the residual water, the middle of the well is less coated with polymer than the outside. To counteract the uneven coating, Tthe middle of the well is again covered with aqueous 0.1 wt% PnPrOx solution (C). Since the solution does not touch the walls of the well, the water evaporates from the outside to the inside covering the previously less coated part of the well.

Contact angle measurement


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Contact angle measurement was performed with the Contact Angle System OCA (DataPhysics Instruments, Filderstadt, Germany) and the corresponding software SCA20. The drop volume was 3 µL and the contact angle was calculated via Laplace-Young Fitting. Cell culture Human microvascular endothelial cell line isolated from human dermal microvascular endothelium (HMEC-1) was acquired from ATCC. The cells were cultivated in MCDB 131 media (Thermo Fischer Scientific) with additional 10 ng/mL Epidermal Growth Factor (EGF), 1 µg/mL hydrocortisone, 10 mM glutamine, fetal calf serum (FCS) (Thermo Fischer Scientific) to a final concentration of 10% and Pen/Strep to a final concentration of 1%. Primary human mesenchymal stem cells (hMSCs) from trabecular bone were isolated via plastic adherence from the femoral heads of patients undergoing total hip arthroplasty 22. Cells were cultured in DMEM F´12 media (Thermo Fischer Scientific,) with additional FCS, 1% PenStrep (Thermo Fischer Scientific) and 50 µg/mL L-ascorbic-acid-2-phosphate (Sigma-Aldrich,) in 175 cm2 cell culture flasks (Greiner Bio-One). Undifferentiated hMSCs in passage 2 were used for the experiments. All experiments were approved by the Local Ethics Committee of the University of Wuerzburg and each donor gave the informed consent. Primary human umbilical vein endothelial cells (HUVECs) were isolated from freshly collected umbilical cords by a standard technique23 and grown in endothelial cell growth medium (Promo Cell) with endothelial cell growth supplement containing 5% FCS, 4 μL/mL heparin, 10 ng/mL epidermal growth factor and 1 μg/mL hydrocortisone, at humidified 7.5% CO2 atmosphere. The use of human material was approved by the Institutional Ethical Committee on Human Research at the University Hospital Erlangen (ethical review number 85_14 B) and all


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donors have given an Informed Consent according to the ethical guidelines. In all experiments, HUVECs at passage 1-2 were used. Commercially available normal human dermal fibroblasts (PromoCell, Heidelberg, Germany) were cultured in DMEM supplemented with 10% (v/v) FCS and 1% (v/v) antibiotic-antimycotic, at 37 °C with a controlled atmosphere of 5% CO2 and 95% relative humidity. Murine fibroblast cell line (L929) was acquired from ATCC. L929 cells were cultivated in DMEM (Dulbecco’s Modified Eagle medium) (Thermo Fischer Scientific) with additional HEPES Buffer, FCS to a final concentration of 10% and Pen/Strep to a final concentration of 1%. Passaging of the cells was executed via 5 minutes of incubation with PBS/EDTA (0.45M EDTA) and afterwards trypsinization with 0.05% Trypsin-EDTA 1X (Thermo Fischer Scientific), unless otherwise stated. Cells were cultivated in 75 cm2 and 175 cm² cell culture flasks (Greiner Bio-One, Kremsmuenster, Germany). Cytocompatibility L929 cells (40,000 cells/cm²) were seeded in PnPrOx-coated and uncoated wells of standard PS-treated 96-well-plates. Cell viability was examined via CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Germany) by quantification of the presence of ATP, indicating cell viability, for 24, 48 and 72 hours. Additionally, L929 cells (25,000 cells/cm²) were seeded in wells of 24-well-plates. Proliferation was examined via cell counting for 24, 48 and 72 hours.


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Temperature dependent detachment of cell sheets After the cells reached confluency the medium was exchanged with 16 °C cold medium and the culture dish was also kept at 16 °C for 30-90 minutes, depending on the cell type. Layers of fibroblasts and hMSCs detached after 30 minutes spontaneously from the outside inwards, while endothelial cell layers detached after 90 minutes. If the monolayer did not detach by itself, simple shaking and soft agitation with a pipette improved the detachment. Immunofluorescent Analysis Cell sheets were fixed with 4% phosphate buffered formaldehyde and permeabilised with 0.2% Triton X-100 (Sigma-Aldrich) in PBS. Samples were then stained with rhodamine phalloidin (PromoKine, Heidelberg, Germany), which selectively binds F-actin. To specifically stain intercellular junctions in fibroblast cell sheets, samples were blocked with 1% bovine serum albumin (BSA) in PBS, followed by immunohistochemical staining with antiN-cadherin antibody (Santa Cruz, Heidelberg, Germany) diluted 1:100 in PBS containing 1% BSA. Cell sheets were incubated with primary antibody overnight and then washed three times with PBS. Afterwards, the samples were incubated for 45 min at room temperature with the appropriate secondary antibody (Alexa-488-conjugated anti-rabbit antibody, 1:500 in PBS, Molecular Probes, Thermo Fisher Scientific). The samples were subsequently stained with Hoechst 33342 (Life Technologies GmbH, Darmstadt, Germany) to visualize the nuclei of cells. Images were obtained via fluorescence microscopy (Zeiss AxioObserver) at different magnifications. Cell-substrate adhesion forces


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Cell-substrate adhesion forces were measured by detachment of individual cells using a FlexFPM (Nanosurf GmbH, Germany), which combines Atomic Force Microscopy (AFM) and FluidFM® technology (Cytosurge AG, Switzerland). Flex-FPM was mounted on an inverted microscope Axio Observer Z1 (Carl Zeiss, Germany) and a stage of 100 µm retraction range. Tipless cantilevers of 200 µm in length and 36 µm in width, with an aperture of 8 µm and nominal spring constant of 2 N/m were used (FluidFM Micropipette, Cytosurge AG, Switzerland). For the force spectroscopy, a set point of 40 nN, approach speed of 5 µm/s, pause of 3 seconds, retraction speed of 3 µm/s and underpressure of 700 mbar were used. Due to the weak adhesiveness of L929 at low temperature, the set point in this condition was set at 15 nN and the underpressure at 400 mbar. HMEC-1 and L929 were seeded on Thermanox plastic coverslips (Thermo Scientific) previously coated with PnPrOx at a density of 15,000 – 40,000 cells/cm2, to exclude the forces derived from cell-cell junctions, as explained previously 24. Cells were kept in culture between 1 and 4 days prior to the force measurements. For the experiments in L929 at temperatures above the LCST samples were kept out of the incubator no longer than 20 minutes or were placed on a hotplate at 37 °C during the waiting time between measurements. For the experiments below LCST, cells were cooled down to 14 °C for 30 minutes and then proceeded with the force measurements. Compared to 16 °C in other experiments, 14 °C was used to increase the timeframe for measurements, because no external cooling system was available for the Flex-FPM device. A temperature increase above the LCST of PnPrOx would lead to precipitation of the polymer. Cells were kept cool between measurements. Data curves were then extracted in SPIP 6.2.0 software (Image Metrology, Denkmark) to automatically calculate the forces proportionally to the cantilever deflection. The maximum deflection peak of the cantilever indicates the maximum detachment force, which is the parameter used here as the


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indicator of cell-substrate adhesion force 24. Data sets did not follow a gaussian distribution according to the Shapiro-Wilk test; therefore the statistical significance of the data was evaluated by the non-parametric Mann-Whitney U test. Results Synthesis and characterization of nPrOx and PnPrOx Synthesis of the monomer 2-n-propyl-2-oxazoline was successfully performed according to Seeliger et al.25 as confirmed by 1H NMR (Figure S1). After the synthesis, the monomer was distilled to dryness. Cationic ring-opening polymerization, with methyl p-toluenesulfonate as initiator and benzonitrile as solvent to enable the polymerization at higher temperatures, and thus a higher reaction rate, was used to prepare poly(2-propyl-2-oxazoline). 1H NMR spectroscopy of the polymer before dialysis revealed ~397 repeating units (Figure S2). However, the distinct signal of the methyl group of the initiator was no longer visible in the NMR after dialysis. Gel permeation chromatography (GPC) was performed using dimethylformamide (DMF) as solvent and molecular weights were calculated via triple calibration using a PMMA standard (Figure S4). The Mn of the polymer was calculated to be 41,720 g/mol; which indicates ~370 repeating units. This was in accordance with the result of the NMR measurement and the theoretical degree of polymerization (Pn = 400). Despite a slight fronting, the elugram showed a monodisperse polymer with a dispersity of 1.3. With an increasing molecular weight, the polymerization reaction of 2-alkyl-2-oxazoline suffers an increase in side reactions, which results in dispersities exceeding 1.2, the limit for living polymerizations. Presumably, this increase in side reactions might be the reason for the slight fronting observed in the elugram 26. The turbidimetry


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measurements showed a LCST of 21 °C for PnPrOx in ultrapure water, which decreased to 19 °C in PBS. PnPrOx coating PnPrOx was dissolved in water to a final concentration of 0.1 wt%. Using lower concentrations of PnPrOx in water led to difficulties with cell detachment (data not shown). Therefore, 0.1 wt% was determined as the minimum effective concentration. Different culture dishes were filled with the solution until the whole well was covered (using volumes indicated in Table 1). After drying, another layer of coating was applied in order to completely cover the bottom of the well.

Table 1: Volumes of aqueous PnPrOx solution used to coat cell culture dishes of different sizes Volume dish

per 48-Well-Dish

24-Well-Dish

12-Well-Dish

6-Well-Dish

1. Coating

100 µL

200 µL

400 µL

800 µL

2. Coating

100 µL

100 µL

100 µL

200 µL

Thermoresponsive capabilities of the dried surface coating were examined via contact angle measurement (Table 2) on standard cell culture dishes. Contact angle measurement of PnPrOxcoating at 37 °C revealed a hydrophobic surface with a contact angle of 65°, whereas the measurements at 16 °C resulted in a decreased contact angle of 39°, indicating a more hydrophilic surface. The contact angle of the uncoated culture dish was also hydrophobic at both temperatures.


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Table 2: Contact angles of cell culture dishes coated with PnPrOx at 37 °C and 16 °C. Uncoated dishes were used as control surfaces (n=3). 37 °C

16 °C

PnPrOx-coated dish

64.6° ± 5.9°

39.9° ± 3.2°

Uncoated dish

73.1° ± 1.1°

69.5° ± 1.2°

Furthermore, 6-Well-Plates were coated with 10 mg of PnPrOx and incubated in 2 mL dH2O for 72 hours at 37 °C. After decreasing the temperature to 16 °C, dissolved PnPrOx was collected past 30, 60, 90 and 120 minutes and subsequently freeze-dried and weighed (Figure 2). Longer incubation in water at 16 °C led to increased amount of dissolved PnPrOx with most of the polymer being dissolved after 120 minutes.


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Figure 2: 6-Well plates were coated with 10 mg PnPrOx and incubated for 72 hours at 37°C. The weight of PnPrOx in solution was determined after decreasing the temperature to 16°C for 30, 60, 90 and 120 minutes. Error bars refer to standard deviation with 3 separate samples.

Cytocompatibility The casted PnPrOx coating was tested by determining proliferation and viability to ensure cytocompatibility. Compared to uncoated PS-treated wells, cell growth on PnPrOx-coated dishes was slower in the beginning. This effect was observed for all tested cell types. However, cells proliferated over time, reaching numbers comparable to the PS-control within 72 h, indicating comparable cytocompatibility. Cell proliferation tested on L929 cells seeded on PS and PnPrOx is shown in Fig 3A. Furthermore, the results obtained with CellTiter-Glo® Luminescent Cell Viability Assay confirmed equal performance in terms of viability and metabolic activity of cells grown on PnPrOx compared with standard PS control dishes (Figure 3B).


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Figure 3: Cell proliferation (A) and viability (B) results of L929 cells; n=3. L929 cells were cultivated on standard PS culture dishes or PnPrOx-coated dishes. Cell viability was estimated using CellTiter-Glo® Luminescent Cell Viability Assay. Error bars refer to standard deviation of 3 independent samples with 5 technical replicates each.

Temperature-dependent detachment of cell sheets To confirm the temperature-dependent detachment of cell sheets, different types of cells were seeded on dishes pre-coated with 0.1 wt% aqueous solution of PnPrOx and grown until 100% confluency. Subsequently, the temperature was decreased to 16 °C and cells grown on the PnPrOx coating were incubated for 30-90 minutes, depending on the cell type. After the specified incubation time, most cell sheets began to detach spontaneously starting from the periphery of the well. If cell-monolayers did not detach by themselves, slow shaking or soft agitation of medium on the sides of the well led to faster detachment of the whole monolayer. No


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differences in detachment were seen by varying the size of the culture dish (Figure 4). In order to corroborate that detachment of cell sheets is caused by alterations in the underlaying polymer and not due to the state of cells, cells seeded on uncoated culture dishes were brought to confluency and incubated at 16 °C for 30-90 minutes. After testing every cell type, no cell sheet detachment occurred on uncoated dishes.

Figure 4: HMEC-1 cell sheets. (A) Macroscopic images of spontaneously detached and contracted cell sheet of HMEC-1 in a PnPrOx-coated well of a 12-well-plate after 60 minutes incubation at 16 °C; (B) Spontaneously detached hMSC cell sheet in a PnPrOx-coated 30 mm Petri dish after 30 minutes incubation at 16 °C; (C), and (D): Representative microscopic images of spontaneously detaching cell sheet of HMEC-1 after 60 min incubation at 16 °C in a well precoated with PnPrOx; (E) Control HMEC-1 cells grown on uncoated dish. Incubating for 60 minutes did not lead to spontaneous cell sheet detachment

We first tested the system with microvascular endothelial cell line HMEC-127. HMEC-1 were seeded at a density of 150,000 cells/cm² and cultivated for 48 hours, before exchanging the medium and cooling the dish down to 16 °C for 60 minutes. After this cooling step monolayers already started to detach, going from the periphery of the coated wells towards the central region (Figure 4). The detached monolayer shrunk to around 10% of its initial size but could be further


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used for tissue engineering purposes. By transferring to another uncoated well and incubating for 1 hour at 37 °C, the monolayer reattached again. Monolayers grown in uncoated control dishes did not detach during this time period. To validate this method in different cell types, the thermosensitive coating was tested using primary hMSCs, isolated from bone tissue of the femoral head. The hMSCs were seeded at a density of 100,000 cells/cm² and cultivated until a confluent monolayer was obtained. The temperature was then reduced to 16 °C, and after less than 30 minutes the monolayer detached spontaneously.

Figure 5: Phalloidin-staining of primary HUVEC monolayer cultured in (A) a standard cell culture dish (attached cells); (B) Phalloidin staining of HUVEC monolayer grown on PnPrOx after detachment. F-actin, red; nuclei, green.

For further testing, primary HUVECs were seeded at a density of 150,000 cells/cm² and cultivated on PnPrOx coated wells. Phalloidin staining was performed to visualize intracellular F-actin. As shown in Figure 5A, attached HUVECs spread cell bodies and formed a monolayer with typical cobblestone morphology. In the cells grown on control dishes F-actin was uniformly distributed. By contrast, in the detached cell sheet, HUVEC monolayers showed a more


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contracted cell morphology and F-actin was localized at the edges of the cell. This localization of F-actin indicates its rearrangement to cortical regions and to cell-cell junctions linking neighboring cells upon detachment from focal adhesions (Figure 5B). To test whether this technique is also suitable for non-monolayer forming cells we used primary fibroblasts, which form multilayered cell structures on cell-compatible materials. Fibroblasts were seeded at a density of 100,000 cells/cm² and cultivated for 2 weeks to obtain thick multilayers. Following detachment at 16 °C for 30 minutes, dense cell sheets were obtained (Figure 6 A-B). Immunohistochemical staining demonstrated the strong expression of N-Cadherin, an intercellular junction protein in the detached sheets, localized mainly to the cortical cell region. By contrast, the N-cadherin distribution was more uniform in the cytoplasm of attached cells grown on uncoated dishes (Figure 6 C-D).


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Figure 6: Multilayered fibroblast sheets. (A) Macroscopic overview, (B) Microscopic image of detached cell sheet at x10 magnification. (C) N-Cadherin expression in fibroblasts cultured in a standard cell culture dish. (D) N-Cadherin expression in detached fibroblast cell sheet. N-cadherin, green; nuclei, red.

This protocol to create cell sheets was further used with the commercially available PnPrOx by Ultroxa®, which differs from the synthesized polymer in terms of molecular weight. The synthesized PnPrOx has a molecular weight of about 50 kDa, while the molecular weight is 10kDa for the commercially available alternative. After seeding and cultivating with HMEC-1 and subsequent temperature decrease, cell sheets were also obtained. However, concentrations had to be increased from 0.1 wt% to 0.2 wt% (Figure 7 C-D). When using only 0.1% aqueous PnPrOx the cell sheet detached from the edge but did not continue to detach fully (Figure 7 AB).


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Figure 7: Cell sheets of HMEC-1 created with surface coating of Ultroxa® PnPrOx. (A) Macroscopic overview of non-detaching HMEC-1 cell sheet seeded on 0,1 wt% Ultroxa® PnPrOx, (B) Microscopic image of non-detached cell sheet at x10 magnification. (C) Macroscopic overview of detached HMEC-1 cell sheet seeded on 0,2 wt% Ultroxa® PnPrOx, (B) Microscopic image of detached cell sheet at x10 magnification.

Reduction in cell-substrate adhesion forces Going deeper in the study of the detachment of cells, the adhesion forces between cells and the underlying PnPrOx were measured at temperatures above and below the LCST. Cell-substrate adhesion forces of HMEC-1 were measured on individual cells, specifically cells not in direct contact with other cells, in order to quantify the forces between the cell and the substrate only


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and to exclude the intercellular forces. The measurements were accomplished using Fluidic Force Microscopy (FluidFM®) as explained previously 24. The samples were cooled to 14 °C for 30 minutes right before starting with the force measurements below LCST. After this time, some HMEC-1 cells were completely detached from the substrate, others were still attached and not presenting noticeable alterations yet, and many cells were attached but showed altered phenotype that was more contracted and with a smaller projected area. These cells that were still attached but showed alterations due to changes in the polymer were chosen for the force measurements. By contrast, at room temperature HMEC-1 cells showed no alterations in phenotype compared to culture conditions (Figure S5 A-B). Results show a statistically significant decrease of cellsubstrate adhesion forces at temperatures below LCST compared to the forces registered above LCST for HMEC-1 cells (Figure 6). In addition to the highly cohesive endothelial cells, we confirmed the same tendency on non-cohesive cells, by measuring the cell-substrate adhesion forces of L929 fibroblasts above and below LCST (Figure S5 C-D). In this case, most cells showed a round phenotype after cooling for 30 minutes and were selected for force measurements. L929 also showed a statistically significant decrease in cell-substrate adhesion forces upon reduction of the temperature but appeared to be more sensitive to temperature changes than the endothelial cells. Therefore, they were measured no longer than 20 minutes upon removal from the incubator or, alternatively, were kept at 37 °C in the waiting times between measurements (Figure 8).


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Figure 8: Box plot representing the adhesion forces of individual HMEC-1 and L929 cells onto PnPrOx at temperatures above and below LCST; 15 cells per condition were measured. *** p

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