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. 2015 May 6;10(5):e0125484.
doi: 10.1371/journal.pone.0125484. eCollection 2015.

Cellular Responses Modulated by FGF-2 Adsorbed on Albumin/Heparin Layer-by-Layer Assemblies

Marta Kumorek  1 , Dana Kubies  1 , Elena Filová  2 , Milan Houska  1 , Naresh Kasoju  1 , Eliška Mázl Chánová  1 , Roman Matějka  3 , Markéta Krýslová  2 , Lucie Bačáková  2 , František Rypáček  1
Affiliations

Cellular Responses Modulated by FGF-2 Adsorbed on Albumin/Heparin Layer-by-Layer Assemblies

Marta Kumorek et al. PLoS One. .

Abstract

In a typical cell culture system, growth factors immobilized on the cell culture surfaces can serve as a reservoir of bio-signaling molecules, without the need to supplement them additionally into the culture medium. In this paper, we report on the fabrication of albumin/heparin (Alb/Hep) assemblies for controlled binding of basic fibroblast growth factor (FGF-2). The surfaces were constructed by layer-by-layer adsorption of polyelectrolytes albumin and heparin and were subsequently stabilized by covalent crosslinking with glutaraldehyde. An analysis of the surface morphology by atomic force microscopy showed that two Alb/Hep bilayers are required to cover the surface of substrate. The formation of the Alb/Hep assemblies was monitored by the surface plasmon resonance (SPR), the infrared multiinternal reflection spectroscopy (FTIR MIRS) and UV/VIS spectroscopy. The adsorption of FGF-2 on the cross-linked Alb/Hep was followed by SPR. The results revealed that FGF-2 binds to the Alb/Hep assembly in a dose and time-dependent manner up to the surface concentration of 120 ng/cm(2). The bioactivity of the adsorbed FGF-2 was assessed in experiments in vitro, using calf pulmonary arterial endothelial cells (CPAE). CPAE cells could attach and proliferate on Alb/Hep surfaces. The adsorbed FGF-2 was bioactive and stimulated both the proliferation and the differentiation of CPAE cells. The improvement was more pronounced at a lower FGF-2 surface concentration (30 ng/cm(2)) than on surfaces with a higher concentration of FGF-2 (120 ng/cm(2)).

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Fig 1
Fig 1. Schematic illustration of the formation of albumin/heparin assembly with adsorbed FGF-2.
Step 1: eLbL assembly of (Alb/Hep)2 in a citrate buffer (pH 4); step 2: cross-linking of (Alb/Hep)2 assembly with glutaraldehyde and pH reversal to PBS buffer (pH 7.4); step 3: adsorption of FGF-2 on the cross-linked (Alb/Hep)2.
Fig 2
Fig 2. The adsorption of albumin (Alb) and heparin (Hep) layers and cross-linking with glutaraldehyde on a gold surface observed by SPR.
The Alb and Hep was adsorbed from 1 mg/mL solutions in a citrate buffer (CB, pH 4); phosphate buffer saline (PBS, pH 7.4), glutaraldehyde (1% GA in CB). Arrows indicate replacement of the solutions.
Fig 3
Fig 3. The adsorption of albumin (Alb) and heparin (Hep) layers and cross-linking with glutaraldehyde on a PS-coated ZnSe observed in situ by FTIR MIRS.
The Alb (■) and Hep (●) was adsorbed from 1 mg/mL solutions in a citrate buffer (CB, pH 4); phosphate buffer saline (PBS, pH 7.4), glutaraldehyde (1% GA in CB).
Fig 4
Fig 4. UV/Vis absorption spectra of AlbFITC/Hep films assembled on a silanized quartz slide with increasing bilayer numbers (a—e).
The AlbFITC and Hep was adsorbed from 1 mg/mL solutions in a citrate buffer (CB, pH 4; a—e). The (AlbFITC/Hep)5 assembly cross-linked with 1% glutaraldehyde (1% GA in CB; f) and transferred to phosphate buffer saline (PBS, pH 7.4; g).
Fig 5
Fig 5. Adsorption of FGF-2 and albumin on (Alb/Hep)2 film.
(A) SPR sensogram and (B) time dependent mass adsorption of (1) 0.1% albumin, (2) 500 ng/mL FGF-2 and (3) 1000 ng/mL FGF-2 to (Alb/Hep)2. The FGF-2 solutions contained 0.1% albumin.
Fig 6
Fig 6. AFM topography images.
(A) TCPS, (B) (Alb/Hep)2 assembly, (C) (Alb/Hep)2 with FGF-2ads (30 ng/cm2), (D) (Alb/Hep)2 with FGF-2ads (120 ng/cm2) in PBS. The root mean squared surface roughness (RRMS) is depicted in each image. Image size: 1 × 1 μm, Z-scale: 25 nm.
Fig 7
Fig 7. Images of living CPAE cells on TCPS (A) and (Alb/Hep)2 (B).
The cells were cultivated in 5% FBS media. Images taken on day 3 after seeding. Obj. ×10, scale bar = 200 μm.
Fig 8
Fig 8. The effect of the amount of FGF-2sol added into the cultivation media on the spreading area 24 h after seeding (A), doubling time between day one and three (B), doubling time between day three and six (C), and the intensity of immunofluorescence staining of von Willebrand factor (D) of CPAE cells cultivated on (Alb/Hep)2 surfaces and the control TCPS.
The amount of FGF-2sol added into the media was 0, 0.1, 1, 10 and 100 ng/mL. The cells were cultivated in 5% FBS media. The fluorescence intensity (D) is normalized per cell. The data is expressed as mean ± S.E.M (A) and median ± quartiles (B-D). Statistically significant differences (p < 0.05) are depicted above the bars in comparison with 0 ng/mL (*), 0.1 ng/mL (&), 1 ng/mL (**), 10 ng/mL (***), 100 ng/mL (#), and TCPS ($).
Fig 9
Fig 9. Density of CPAE cells on day 1 (A), 3 (B) and 7 (C), doubling time between day 1 and 3 (D) and between 3 and 7 (E), growth curves (F), cell spreading area on day 1 (G) and BrdU labelling index on day 3 (H) on different surfaces.
(1) tissue culture polystyrene (TCPS); (2) TCPS with 10 ng/mL of FGF-2sol in media (TCPS_FGF-2sol); (3) (Alb/Hep)2; (4) (Alb/Hep)2 with 10 ng/mL of FGF-2sol in media ((Alb/Hep)2FGF-2sol); (5) (Alb/Hep)2 with FGF-2ads adsorbed (30 ng/cm2, (Alb/Hep)2FGF-2adsLow); (6) (Alb/Hep)2 with FGF-2ads adsorbed (120 ng/cm2, (Alb/Hep)2FGF-2adsHigh). The cells were cultivated in 5% FBS media. The data is expressed as mean ± S.E.M (A-C, F-H) and median ± quartiles (D, E); Statistically significant differences (p < 0.05) between surfaces (denoted in numbers) are depicted above bars.
Fig 10
Fig 10. Immunofluorescence staining of VE-cadherin (A,B) and von Willebrand factor (C,D) in CPAE.
Cells cultivated on (Alb/Hep)2 (A,C) and on (Alb/Hep)2 with the adsorbed FGF-2ads (30 ng/cm2; (Alb/Hep)2FGF-2adsLow; B,D). The cells are counterstained with Hoechst 33342. Obj. ×20 (A,B), obj. ×10 (C,D), scale bar = 200 μm.
Fig 11
Fig 11. Intensity of immunofluorescence staining for vinculin (on day 3, A), VE-cadherin (on day 7, B) and von Willebrand factor (on day 7, C) of CPAE cells cultured on different surfaces.
(1) tissue culture polystyrene (TCPS); (2) TCPS with 10 ng/mL of FGF-2sol in media (TCPS_FGF-2sol); (3) (Alb/Hep)2; (4) (Alb/Hep)2 with 10 ng/mL of FGF-2sol in media ((Alb/Hep)2FGF-2sol); (5) (Alb/Hep)2 with adsorbed FGF-2ads (30 ng/cm2, (Alb/Hep)2FGF-2adsLow); (6) (Alb/Hep)2 with adsorbed FGF-2ads (120 ng/cm2, (Alb/Hep)2FGF-2adsHigh). Cells were cultured in 5% FBS media. Fluorescence intensity is normalized per cell. The data is expressed as mean ± S.E.M. Statistically significant differences (p value <0.05) between surfaces (denoted in numbers) are depicted above bars.
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Grants and funding

The study was funded by the Ministry of Education, Youth and Sports, Czech Republic (grant number EE2.3.30.0029), by the Grant Agency of the Czech Republic (grant number P108/11/1857 and grant number P108/12/P624), and by the European Regional Development Fund (Project BIOCEV: Biotechnology and Biomedicine Centre of the Academy of Sciences and Charles University, grant number CZ.1.05/1.1.00/02.0109). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.