Direct photo-patterning on anthracene containing polymer for guiding stem cell adhesion
© The Author(s). 2016
Received: 8 June 2016
Accepted: 26 July 2016
Published: 3 August 2016
Various micropatterned surfaces capable of guiding the selective adhesion of biomolecules such as proteins and cells are of great interests in biosensor, diagnostics, drug screening, and tissue engineering. In this study, we described a simple photo-patterning method to prepare micro-patterned films for stem cell patterning using anthracene containing polymers (PMAn). This micro patterned polymer film was prepared by the facile photo-reaction of anthracene units in polymer backbone structure.
The UV irradiation of PMAn through a photomask resulted in the quenching of fluorescent intensity as well as the changes in surface wettability from hydrophobic to hydrophilic surface. As a result, UV exposed regions of PMAn film show lower fluorescent intensity as well as higher proliferation rate of mesenchymal stem cells (MSCs) than unexposed region of PMAn film. Furthermore, the selective MSC attachment was clearly observed in the UV exposed regions of PMAn film.
We developed a simple cell patterning method with a fluorescent, biocompatible, and patternable polymer film containing anthracene units. This method provides a facile stem cell patterning method and could be extended to various patterning of biomaterials without labor-intensive preparation and no pre-treatment for complex interactions of cell-microenvironment.
KeywordsAnthracene Fluorescent polymer Photolithography Polymer pattern film Photoreaction Human mesenchymal stem Cell patterning
Micropatterned surfaces that can selectively interact with biomaterials such as proteins and cells are of great interest in genomics, diagnostics, drug screening, and tissue engineering, as these “micro-scale patterned surfaces” enable us to define the adhesion of single cells or cell groups [1–3]. Beyond the control of cell adhesion, these surfaces are particularly valuable for designing and developing cell culture systems which reflect better the complexity of cell-microenvironment interactions in order to regulate cell phenotype and cell fate [4–6].
A number of micropatterning approaches have been extensively exploited including microcontact printing, photoresist lithography, microfluidics, and self-assembled monolayers (SAMs) [7–11]. Also, a number of laboratories, including our own, have been performing surface engineering with poly (ethylene glycol) (PEG) photolithography to control cell-surface interactions, originating from anti-fouling effects of PEG hydrogel [12–14]. However, SAM method with alkylthiols requires labor intensive preparation and also is only applicable to gold and glass substrate. Also, the major limitations of PEG hydrogel micropatterned surfaces is the instability of PEG adhesion on substrates. The stability of PEG hydrogel micropatterns is influenced by not only the molecular weight and concentration of PEG but also the treatment of silane as a coupling layer. In general, thiol-silane anchored PEG hydrogel are durable for up to 3 days but this hydrogel started being detached after 4 days, in spite of the anchoring of hydrogel microstructures with silane coupling agents . Thus, simple but solid methods for cell micropatterns still remains a significant challenge.
The patterning of functional polymers with conductive and fluorescent properties has recently garnered considerable attention for sensor, bioengineering, display and electrical circuits because of their specific opto-electronic properties [16–19]. Recently, we reported direct photopatterning approaches for functional polymers to avoid decomposition of the functional group in polymer structure during the patterning step [20–22]. Leveraging simple and direct photo-patterning process, we have developed a highly fluorescent and biocompatible p-phenylene vinylene (PPV) polymer pattern film that allows stem cell micropatterns . This enables us to easily detect the location of cells or other biomaterials without the labeling cells because the pattern is fluorescent.
For the application of direct photo-patterning, it is important to prepare a patternable fluorescent polymer. Anthracnee moleuclue has been frequently utilized to fabriciate photo-responsive biocompatible drug delivery systems due to the its ability of photo-truggered reaction upon UV exposure [24, 25]. In this study, we report a photo patternable anthracene containing fluorescent polymer (PMAn) for the selective patterning of mesenchymal stem cells (MSCs). We utilized micro polymer (PMAn) patterns formed by direct photo-reaction of anthracene units for guiding stem cell adhesion.
Methylene bridged anthracene polymer (PMAn) was synthesized via Friedel-Crafts alkylation reaction [26, 27]. Weight average molecular weight (M w) of the resultant polymer by gel permeation chromatography (GPC) was 6700 with M w /M n of 1.7. α-Minimum Essential Medium (α-MEM), Fetal Bovine Serum (FBS), antibiotics (Penicillin/Streptomycin), Phosphate-Buffered Saline (PBS, pH 7.4), Trypsin/EDTA (0.05 %), and Tryphan blue (0.4 %) were purchased from Gibco (Invitrogen, USA). Albumin (5 %) was purchased from Green cross corporation (Korea). Bone Marrow (BM)-derived Mesenchymal Stem Cells (MSCs), on patient compliance, were used for this study. A frozen stock of MSCs was provided by Cell Therapy Center, Severance hospital (from University of Yonsei, Seoul, Korea) at passage 3. Other chemicals and solvents were purchased from Aldrich.
1H spectrum was determined on a Bruker ARX-300 spectrometer. The average molecular weight of the polymer was characterized by a gel permeation chromatography (GPC) (model: Waters R-401 ALC/GPC) with THF as an eluent and polystyrene standard for calibration. Fluorescence spectra were obtained with a luminescence spectrometer (PerkinElmer, Model LS55) under excitation at 370 nm. The polymer films were illuminated with a UV lamp (Rolence Enterprise, Inc., Taiwan, power: 13.05 mW/cm2), model POWERARC UV 100. The surface wettability was investigated by a water (DI) drop contact angle measurement using Contact Angle Meter-CAM 101 model (KSV Instruments Ltd, FINLAND). AFM analysis was carried out in room temperature with a Dimension 3100 SPM equipped with Nanoscope IVa devised by Digital Instruments from Santa Barbara, CA. The fluorescent patterns such as were imaged under Olympus-BX51 fluorescence microscope with WB – dichroic mirror DM500, excitation filter BP450-480 and barrier filter BA515. The optical MSCs patterns were obtained from Olympus inverted research microscope model IX71. To detect cell patterns more in detail, MSCs were observed with field emission-scanning electron microscope (HITACHI S-800, Tokyo, Japan) and the picture was taken by scanning microscope image analysis system (ESCAN-4000, Bummi Universe, Tokyo, Japan)
The preparation of PMAn film and PMAn patterned substrates
PMAn films having average thickness of 185 nm were prepared by spin coating of chloroform solution of polymers (1 wt %) at 1200 rpm for 15 s and then dried under room temperature for solvent removal. These pristine PMAn films, without pattern, were used for the spectroscopic measurements, contact angle measurement, and the proliferation assay of MSCs. For fluorescent pattern formation, the PMAn films were illuminated with a high-intensity UV lamp (13.05 mW/cm2) through a photomask. These PMAn patterns were used for the AFM and MSCs pattern.
The analyses of PMAn surface with contact angle and AFM measurements
Surface wettability of the PMAn films through photo-reaction was investigated by water (DI) drop contact angle measurement. PMAn films were illuminated by high intensity UV source for 1, 3, 5, 10 mins. For surface morphology experiments, the polymer films on silicon wafer were illuminated by a high-intensity UV source for 15 min through a 10 μm line pattered photomask. AFM analyses were carried out at room temperature with a Dimension 3100 SPM equipped with Nanoscope IVa devised by Digital Instruments from Santa Barbara, CA. The AFM tip was oscillated at its resonance frequency (75 kHz). Next, the tip was lifted with fixed distance above the sample surface and scanned at that constant height with a voltage applied.
Cell culture and proliferation assay on PMAn film substrates
MSCs were thawed, placed at a density of about 10,000 cells/cm2 in 15 mL medium (α-MEM supplemented with 10 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin) in a 75 cm flask (Nunc, Denmark), at 37 °C in 5 % humidified CO2. Medium was replaced with fresh α-MEM with 10 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin every 3 or 4 days and cells were grown to 90–95 % of confluence over about 3–7 days. Once the cells reached confluence, they were detached using 0.05 % Trypsin/EDTA and replaced for further expansion. The MSCs in this study were between passage of 4 and 7.
To investigate the proliferation of MSCs on PMAn film substrates, two PMAn films were prepared on glass substrates by spin coating. One of the films was exposed to UV source for 5 min and the other film was not exposed to UV. The prepared PMAn film substrates were washed with α-MEM supplemented with 10 % FBS, 100 U/mL penicillin and 100 ug/mL streptomycin. Cells (from passage 4) were harvested by treating a solution of 0.05 % Trypsin/EDTA for 3–5 min at 37 °C, placed at 2,000 cells/cm2 in unexposed PMAn substrate and UV-exposed PMAn substrate. Cells (from passage 4) were harvested also on a tissue culture polystyrene as a reference experiment. Each well was dispensed with 3 mL α-MEM medium and changed once after 2 days. Cultures were maintained for 5 days and 7 days and then harvested for cell counting, respectively. This proliferation assay was examined at three different times. The growth rates for MSCs on the sample after 5 days and 7 days of culture were determined by counting the number of cells with a hemacytometer after tryphan blue staining in a counting chamber.
Cell attachment to micro-patterned PMAn substrate
For cell patterning studies, the PMAn pattern on a glass substrate (1.6 cm × 2.8 cm) was prepared by exposing the pristine PMAn film on UV light for 5 min. The micro-patterned substrates were placed in six well plate (Nunc, Denmark) containing culture medium, α-MEM supplemented with 10 % FBS, 100 U/mL penicillin and 100 μg/mL streptomycin. MSCs were detached from the cell culture substrates by trypsinization. The cells at a concentration of 4,000 cells/cm2 were seeded on a patterned substrate in six well plate (Nunc, Denmark) and maintained under culture condition for 2 days at 37 °C in 5 % humidified CO2.
Results and discussion
In order to understand the difference in the photo-oxidation and photo-dimerization of PMAn in an atmosphere of air or argon (Ar), the decrease in emission intensity was examined by irradiating the PMAn film at various exposure doses (Fig. 2b).
When UV light was exposed to PMAn film surface in an atmosphere of Ar, the relative emission intensity at 535 nm decreased from 1.0 to 0.58 (42 %). However, in the presence of oxygen, relative emission intensity at 535 nm reduced drastically from 1.0 to 0.15 (85 %). The relative decrease in emission at 535 nm in an atmosphere of Ar was smaller than that in air. This seems to be because the photo-dimerization of the anthracene units in the polymer is the only photochemical reaction in the Ar condition, while the photo-oxidation as well as photo-dimerization took place concomitantly in an atmosphere of air. This result indicated that both photo-oxidation and photo-dimerization led to fluorescence quenching in air.
We have shown simple cell patterning method that doesn’t require labor-intensive preparation for cell patterning. The UV exposure to PMAn film leads to fluorescent quenching as well as the change in surface wettability into hydrophilic surface due to direct photo-reactions of anthracene units. In contrast to UV unexposed regions of PMAn film (PMAn-UV), UV exposed regions of PMAn film (PMAn + UV) showed high proliferation rate and selective attachment of MSCs. We envision that this simple cell pattern method may be valuable for designing cellular platform for drug screening, diagnostics, and cellular/tissue engineering.
AFM, atomic force microscopy; Ar, argon; FBS, Fetal Bovine Serum; GPC, gel permeation chromatography; MSCs, mesenchymal stem cells; PBS, Phosphate-Buffered Saline; PEG, poly (ethylene glycol); PMAn, anthracene containing polymers; PMAn + UV, UV exposed PMAn film; PMAn-UV, UV unexposed PMAn film; PPV, p-phenylene vinylene; SAMs, self-assembled monolayers; TCPS, tissue culture polystyrene; α-MEM, α-Minimum Essential Medium;
This work was supported by the National Research Foundation of Korea (NRT) grant funded by the Korea government (MSIP) and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic Korea.
This work was supported by the National Research Foundation of Korea (NRT) grant funded by the Korea government (MSIP) (No. 2015R1C1A1A01054258) and a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic Korea (No. HI15C0942).
Availability of data and materials
There was no available data and supporting materials.
JY, HOK, and EK designed and drafted the manuscript. JY and JSH carried out all of experiments. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Ethics approval and consent to participate
All primary human cell experiments were reviewed and approved by a board of Severance Hospital (University of Yonsei, Seoul, Korea).
Consent for publication
Authors consent for publication.
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