SAM-based cell transfer to photopatterned hydrogels for microengineering vascular-like structures - PubMed (original) (raw)

SAM-based cell transfer to photopatterned hydrogels for microengineering vascular-like structures

Nasser Sadr et al. Biomaterials. 2011 Oct.

Abstract

A major challenge in tissue engineering is to reproduce the native 3D microvascular architecture fundamental for in vivo functions. Current approaches still lack a network of perfusable vessels with native 3D structural organization. Here we present a new method combining self-assembled monolayer (SAM)-based cell transfer and gelatin methacrylate hydrogel photopatterning techniques for microengineering vascular structures. Human umbilical vein cell (HUVEC) transfer from oligopeptide SAM-coated surfaces to the hydrogel revealed two SAM desorption mechanisms: photoinduced and electrochemically triggered. The former, occurs concomitantly to hydrogel photocrosslinking, and resulted in efficient (>97%) monolayer transfer. The latter, prompted by additional potential application, preserved cell morphology and maintained high transfer efficiency of VE-cadherin positive monolayers over longer culture periods. This approach was also applied to transfer HUVECs to 3D geometrically defined vascular-like structures in hydrogels, which were then maintained in perfusion culture for 15 days. As a step toward more complex constructs, a cell-laden hydrogel layer was photopatterned around the endothelialized channel to mimic the vascular smooth muscle structure of distal arterioles. This study shows that the coupling of the SAM-based cell transfer and hydrogel photocrosslinking could potentially open up new avenues in engineering more complex, vascularized tissue constructs for regenerative medicine and tissue engineering applications.

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Figures

Fig. 1

Cell transfer and vascular construct generation schematic. (A) The oligopeptide CGGGKEKEKEKGRGDSP was chemically adsorbed onto a gold surface and seeded with HUVECs. SAM desorption from the surface resulted in cell detachment. (B) HUVECs seeded on gold substrates modified with oligopeptide based-SAM were transferred to the hydrogel after GelMA prepolymer photocrosslinking with or without electrical potential. (C) Micrometric gold rods enveloped with cells were positioned in culture chambers that were then filled with GelMA. After photocrosslinking, an electrical potential was applied and the endothelial cells were transferred. Following rod removal, the device was connected to a microsyringe pump and cultured under perfusion. (D) Double-layer cell microvascular structures were generated by dip-coating the gold rods enveloped with HUVECs in a GelMA solution containing 3T3 cells. The double-layered rods were then encapsulated in the hydrogel, cells were transferred by electrical potential application to the gel and the rod was removed.

Fig. 2

Effect of PI&UV on SAM and HUVEC adhesion. (A) Resonant frequency of bare gold surface (I) decreased with oligopeptide adsorption over time (II, 10 min; III, 180 min), and increased after PI&UV exposure, due to mass desorption (V). (B) XPS surface analyses show decreased Oxygen, Nitrogen and Carbon peaks after PI&UV exposure. (C) Percentage of adherent HUVECs on modified substrates upon exposure to PI&UV (Pep+ PI&UV) show almost 30% cell detachment as compared to non-modified substrates (Pep− PI&UV) or modified substrates exposed to PBS (Pep+ PBS). Representative phase contrast images show that HUVECs adhering on modified substrates (D) acquire a rounded morphology after PI&UV (E). The cells maintained a spread morphology on modified substrates when exposed to PBS (F) or on non-modified substrates when exposed to PI&UV (G). PI&UV exposure induce loss of fraction of the adsorbed SAM leading to partial cell detachment. (Error bars: ± SD; statistically significant difference from Pep+ PBS # and Pep− PI&UV *)

Fig. 3

Cell transfer from gold substrate to hydrogel. Cells were transferred from non-modified substrates with electrical potential (Pep− El+), modified substrates without electrical potential (Pep+ El−), and modified substrate with electrical potential (Pep+ El+). Representative phase contrast images of the same substrate before (A–C) and after (D–E) transfer show negligible number of cells still adherent on peptide coated gold after transfer as opposed to the high number of cells on non-modified substrates. The corresponding fluorescent images (GFP/EthD-1) of the hydrogels after transfer display few rounded cells for the Pep− El+ (G) and numerous viable spread HUVECs for both Pep+ El− (H) and Pep+ El+ (I). (J) Mean percentage of cell transferred to hydrogel confirms that peptide modification enables complete HUVEC transfer as compared to only 20% of cells for Pep− El+. (K) Mean cell viability after transfer similarly illustrates that both modified substrates promoted transfer with high cell viability as opposed to non-modified ones (less than 50% viable cells). Gold surface coating with oligopeptide SAM enables HUVEC transfer to GelMA hydrogels with high efficiency and viability independent of electrochemical SAM desorption. (Scale bars: 100 μm; error bars: ± SD; statistically significant difference from Pep+ El− # and Pep+ El+ *)

Fig. 4

Cell morphology and proliferation upon transfer. (A) Mean cell area after transfer shows that Pep− El+ cells occupy significantly smaller area than Pep+ El− and Pep+ El+, the latter being statistically more spread among the modified substrates. (B) Cell area distribution shows that cells transferred from the modified substrates using potential (Pep+ El+) maintained a similar morphology whereas those transferred without potential (Pep+ El−) tended to be less spread than on gold substrates. (C) Similarly, a higher percentage (Mean ± SD) of cells (E) presented a spread morphology at 12 h on Pep+ El+ hydrogels compared to cells on Pep+ El− (D). However, electrochemical oligopeptide cleavage did not show significant effect on the percentage (Mean ± SD) of viable cells (C) and on proliferation (F) after transfer. While electrochemical SAM desorption has an effect on the cell morphology immediately after transfer, no differences are evidenced on longer cultures both conditions maintaining proliferative cell population. (Scale bars: 50 μm; error bars: ± SD; statistically significant difference from Pep+ El− # and Pep+ El+ *)

Fig. 5

HUVEC monolayer transfer. Representative phase contrast images of hydrogels after transfer from modified substrates seeded at high density and cultured for 16 h (HD-16h) (A,B) or at low density and cultured for 72 h (LD-72h) (C,D) transferred either without (A,C) or with (B,D) potential. (E) Mean percentage of cells transferred to hydrogel decreases upon longer culture, maintaining however high transfer efficiency (over 80%) when SAM was electrochemically desorbed (Pep+ El+). (F–M) Representative confocal images of gold and hydrogel samples show intact VE-Cad junctions while Cx43 is mostly internalized upon transfer. Both processes were not affected by transfer method and culture period on gold. Although endothelial monolayers can be transferred both with and without electrical potential, the longer culture requires electrochemical oligopeptide cleavage for efficient transfer. (Scale bars: 100 μm; error bars: ± SD; statistically significant difference between culture period * and between transfer protocol #)

Fig. 6

Fabrication of microvascular-like structures. (A) HUVECs were cultured on gold rods for three days to create a confluent monolayer. (B) After GelMA photocrosslinking, electrical potential application and rod removal, HUVECs were transferred uniformly on the hydrogel channel surface. (C) The constructs, cultured under perfusion, maintained a continuous endothelial monolayer lining the surface of the channel (D) mimicking the 3D endothelial cell organization in microvasculature (E). (F) Over 5 days of culture, the constructs maintained a stable shape and a compact cell layer on the channel surface. (G) After 15 days of culture, samples stained with DAPI and cross-sectioned displayed a hollow channel structure that was maintained over long term culture. (H) GelMA encapsulated PKH26-stained 3T3 cells photopatterned on HUVEC monolayer seeded on gold rods. (I) Confocal image showing cross-section of hydrogel channel covered with 3T3 cells, (J) lined with HUVEC monolayer. (K) Magnified merged confocal image shows both 3T3 (PKH26) and HUVEC (GFP) layer patterned in close proximity (inset shows the entire channel). (All scale bars: 100 μm, except F: 200 μm).

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SAM-based cell transfer to photopatterned hydrogels for microengineering vascular-like structures - PubMed (original) (raw)