Directed cell growth on protein-functionalized hydrogel surfaces

Abstract

Biochemical surface modification has been used to direct cell attachment and growth on a biocompatible gel surface. Acrylamide-based hydrogels were photo-polymerized in the presence of an acroyl-streptavidin monomer to create planar, functionalized surfaces capable of binding biotin-labelled proteins. Soft protein lithography (microcontact printing) of proteins was used to transfer the biotinylated extracellular matrix proteins, fibronectin and laminin, and the laminin peptide biotin-IKVAV, onto modified surfaces. As a biological assay, we plated LRM55 astroglioma and primary rat hippocampal neurons on patterned hydrogels. We found both cell types to selectively adhere to areas patterned with biotin-conjugated proteins. Fluorescence and bright-field modes of microscopy were used to assess cell attachment and cell morphology on modified surfaces. LRM55 cells were found to attach to protein-stamped regions of the hydrogel only. Neurons exhibited significant neurite extension after 72h in vitro, and remained viable on protein-stamped areas for more than 4 weeks. Patterned neurons developed functionally active synapses, as measured by uptake of the dye FM1-43FX. Results from this study suggest that hydrogel surfaces can be patterned with multiple proteins to direct cell growth and attachment.

Fig. 1

Patterned hydrogel surfaces. (A) A streptavidin-acrylamide hydrogel surface was microcontact printed with the biotinylated extracellular matrix proteins laminin and fibronectin. PDMS stamps were used to transfer proteins in a pattern corresponding to 10 μm-wide lines with 90 μm-gap spacing. Proteins were detected using primary antibodies to laminin and fibronectin, and secondary antibodies for laminin (Alexa 488 secondary antibody, green) and fibronectin (Texas Red secondary antibody, red). Scale bar = 40 μm. (B) The laminin peptide biotin-IKVAV was stamped onto a hydrogel using a PDMS stamp grid pattern consisting of orthogonal 2 μm-wide lines intersecting at 15 μm diameter nodes, with a repeat spacing of 50 μm. The IKVAV epitope was visualized using an anti-laminin primary antibody and Alexa 488-conjugated secondary antiboidy (green). Imaging revealed that the peptide localized to stamped regions only. Scale bar = 50 μm. (C) Examples of non-specific protein deposition that can occur following stamping of hydrogel surfaces. Panel 1 (left). Protein diffusion across hydrogel surface during μCP. The result is areas of protein-containing buffer flowing across the hydrogel surface. Panel 2 (right). Areas of hydrogel are devoid of stamped biomolecules due to the presence of a defect (bubble) in the PDMS stamp. Bubbles in the stamp are due to incomplete de-gassing of the elastomer mixture prior to curing. Defect-containing stamps produce areas not in close contact with the hydrogel surface. Scale bar = 50 μm.

Fig. 1

Patterned hydrogel surfaces. (A) A streptavidin-acrylamide hydrogel surface was microcontact printed with the biotinylated extracellular matrix proteins laminin and fibronectin. PDMS stamps were used to transfer proteins in a pattern corresponding to 10 μm-wide lines with 90 μm-gap spacing. Proteins were detected using primary antibodies to laminin and fibronectin, and secondary antibodies for laminin (Alexa 488 secondary antibody, green) and fibronectin (Texas Red secondary antibody, red). Scale bar = 40 μm. (B) The laminin peptide biotin-IKVAV was stamped onto a hydrogel using a PDMS stamp grid pattern consisting of orthogonal 2 μm-wide lines intersecting at 15 μm diameter nodes, with a repeat spacing of 50 μm. The IKVAV epitope was visualized using an anti-laminin primary antibody and Alexa 488-conjugated secondary antiboidy (green). Imaging revealed that the peptide localized to stamped regions only. Scale bar = 50 μm. (C) Examples of non-specific protein deposition that can occur following stamping of hydrogel surfaces. Panel 1 (left). Protein diffusion across hydrogel surface during μCP. The result is areas of protein-containing buffer flowing across the hydrogel surface. Panel 2 (right). Areas of hydrogel are devoid of stamped biomolecules due to the presence of a defect (bubble) in the PDMS stamp. Bubbles in the stamp are due to incomplete de-gassing of the elastomer mixture prior to curing. Defect-containing stamps produce areas not in close contact with the hydrogel surface. Scale bar = 50 μm.

Fig. 1

Patterned hydrogel surfaces. (A) A streptavidin-acrylamide hydrogel surface was microcontact printed with the biotinylated extracellular matrix proteins laminin and fibronectin. PDMS stamps were used to transfer proteins in a pattern corresponding to 10 μm-wide lines with 90 μm-gap spacing. Proteins were detected using primary antibodies to laminin and fibronectin, and secondary antibodies for laminin (Alexa 488 secondary antibody, green) and fibronectin (Texas Red secondary antibody, red). Scale bar = 40 μm. (B) The laminin peptide biotin-IKVAV was stamped onto a hydrogel using a PDMS stamp grid pattern consisting of orthogonal 2 μm-wide lines intersecting at 15 μm diameter nodes, with a repeat spacing of 50 μm. The IKVAV epitope was visualized using an anti-laminin primary antibody and Alexa 488-conjugated secondary antiboidy (green). Imaging revealed that the peptide localized to stamped regions only. Scale bar = 50 μm. (C) Examples of non-specific protein deposition that can occur following stamping of hydrogel surfaces. Panel 1 (left). Protein diffusion across hydrogel surface during μCP. The result is areas of protein-containing buffer flowing across the hydrogel surface. Panel 2 (right). Areas of hydrogel are devoid of stamped biomolecules due to the presence of a defect (bubble) in the PDMS stamp. Bubbles in the stamp are due to incomplete de-gassing of the elastomer mixture prior to curing. Defect-containing stamps produce areas not in close contact with the hydrogel surface. Scale bar = 50 μm.

Fig. 2

Cell attachment to hydrogel surfaces (A) LRM55 astroglioma cells plated onto PAA/PEGDA hydrogel surfaces. Non-patterned hydrogel surfaces do no allow cell attachment. However, cells still have the ability to attach to cell-permissive (protein-stamped) hydrogel surfaces. Cells were stained using Alexa 568-conjugated phalloidin (cytoskeleton, red) and Hoescht 33342 (nuclei, blue). Scale bar = 50 μm. (B) Unattached cells from (A) were plated onto poly-L-lysine coated glass coverslips. Cells were able to successfully attach to glass coverslips. Scale bar = 50 μm.

Fig. 2

Cell attachment to hydrogel surfaces (A) LRM55 astroglioma cells plated onto PAA/PEGDA hydrogel surfaces. Non-patterned hydrogel surfaces do no allow cell attachment. However, cells still have the ability to attach to cell-permissive (protein-stamped) hydrogel surfaces. Cells were stained using Alexa 568-conjugated phalloidin (cytoskeleton, red) and Hoescht 33342 (nuclei, blue). Scale bar = 50 μm. (B) Unattached cells from (A) were plated onto poly-L-lysine coated glass coverslips. Cells were able to successfully attach to glass coverslips. Scale bar = 50 μm.

Fig. 3

Cell attachment to protein-functionalized hydrogel surfaces. (A) At 24 hr, rat astroglioma LRM55 cells were seen to attach and extend on regions stamped with either the laminin (left to right) or the fibronectin (top to bottom) proteins. Cells were stained using Alexa 568-conjugated phalloidin (cytoskeleton, red) and Hoescht 33342 (nuclei, blue). Scale bar = 25 μm. (B) Patterned LRM55 cells were shown to incorporate BrdU, denoting that cells were actively synthesizing DNA prior to cell division. BrdU-positive cells are indicated by arrows. Scale bar = 25 μm. (C) At 4 weeks in vitro cells were found to maintain compliance with the stamped patterning of the hydrogel. Cells were observed to extend along both the laminin and the fibronectin stampings, or they spread where the two proteins intersected (arrow), indicating that μCP proteins retain long-term functionality. Scale bar = 50 μm.

Fig. 3

Cell attachment to protein-functionalized hydrogel surfaces. (A) At 24 hr, rat astroglioma LRM55 cells were seen to attach and extend on regions stamped with either the laminin (left to right) or the fibronectin (top to bottom) proteins. Cells were stained using Alexa 568-conjugated phalloidin (cytoskeleton, red) and Hoescht 33342 (nuclei, blue). Scale bar = 25 μm. (B) Patterned LRM55 cells were shown to incorporate BrdU, denoting that cells were actively synthesizing DNA prior to cell division. BrdU-positive cells are indicated by arrows. Scale bar = 25 μm. (C) At 4 weeks in vitro cells were found to maintain compliance with the stamped patterning of the hydrogel. Cells were observed to extend along both the laminin and the fibronectin stampings, or they spread where the two proteins intersected (arrow), indicating that μCP proteins retain long-term functionality. Scale bar = 50 μm.

Fig. 3

Cell attachment to protein-functionalized hydrogel surfaces. (A) At 24 hr, rat astroglioma LRM55 cells were seen to attach and extend on regions stamped with either the laminin (left to right) or the fibronectin (top to bottom) proteins. Cells were stained using Alexa 568-conjugated phalloidin (cytoskeleton, red) and Hoescht 33342 (nuclei, blue). Scale bar = 25 μm. (B) Patterned LRM55 cells were shown to incorporate BrdU, denoting that cells were actively synthesizing DNA prior to cell division. BrdU-positive cells are indicated by arrows. Scale bar = 25 μm. (C) At 4 weeks in vitro cells were found to maintain compliance with the stamped patterning of the hydrogel. Cells were observed to extend along both the laminin and the fibronectin stampings, or they spread where the two proteins intersected (arrow), indicating that μCP proteins retain long-term functionality. Scale bar = 50 μm.

Fig. 4

Neurons on hydrogel peptide-stamped hydrogel surfaces. (A–C) Neurons at 24 hr, 48 hr and 10 days in culture. At 24 hr, growth-cone lamellipodia were extensive and extended. After 48 hr in culture, cell somata were predominantly located at the nodes of the grid (15 μm-diameter discs at the intersections of 2 μm-wide orthogonal lines). By 10 days in vitro neurons had formed into neuronal networks. Cells were stained for neuron-specific βIII-tubulin (red) and nuclei were stained with Hoescht 33342 (blue). Scale bar = 50 μm. (D) Hydrogel patterned neuronal network at 4 weeks in vitro. Arrows indicate where neuronal processes have deviated from the patterned areas. Pattern fidelity was maintained over a 1cm² area of the hydrogel. Neurons were stained with βIII-tubulin (red). Scale bar = 100 μm. (E) Neurons at 10 days in vitro were analyzed for the presence of active synapses, using the FM1-43FX dye. Neurons were depolarized in the presence of FM1-43FX, rinsed extensively to remove excess dye, and stimulated for 2 min in a high K⁺ solution. Following stimulation and washing, cells were fixed with 4% paraformaldehyde. Active synapses as revealed by the FM1-43FX dye are labelled in green (arrows). Neurons were imaged using an Olympus widefield upright microscope with ImagePro software (MediaCybernetics, Silverspring, MD). Scale bar = 50 μm. (F) Depolarization-dependent FM1-43FX labelling. Neurons from Figure 4E were subject to a second round of depolarization prior to fixation. FM-143FX synapses (green) were co-labelled with antibodies to post-synaptic density protein 95 (PSD95; red). Putative synapses (yellow; arrows) were identified by the co-localization of both FM 1-43FX and PSD95. Scale bar = 10 μm.

Fig. 4

Neurons on hydrogel peptide-stamped hydrogel surfaces. (A–C) Neurons at 24 hr, 48 hr and 10 days in culture. At 24 hr, growth-cone lamellipodia were extensive and extended. After 48 hr in culture, cell somata were predominantly located at the nodes of the grid (15 μm-diameter discs at the intersections of 2 μm-wide orthogonal lines). By 10 days in vitro neurons had formed into neuronal networks. Cells were stained for neuron-specific βIII-tubulin (red) and nuclei were stained with Hoescht 33342 (blue). Scale bar = 50 μm. (D) Hydrogel patterned neuronal network at 4 weeks in vitro. Arrows indicate where neuronal processes have deviated from the patterned areas. Pattern fidelity was maintained over a 1cm² area of the hydrogel. Neurons were stained with βIII-tubulin (red). Scale bar = 100 μm. (E) Neurons at 10 days in vitro were analyzed for the presence of active synapses, using the FM1-43FX dye. Neurons were depolarized in the presence of FM1-43FX, rinsed extensively to remove excess dye, and stimulated for 2 min in a high K⁺ solution. Following stimulation and washing, cells were fixed with 4% paraformaldehyde. Active synapses as revealed by the FM1-43FX dye are labelled in green (arrows). Neurons were imaged using an Olympus widefield upright microscope with ImagePro software (MediaCybernetics, Silverspring, MD). Scale bar = 50 μm. (F) Depolarization-dependent FM1-43FX labelling. Neurons from Figure 4E were subject to a second round of depolarization prior to fixation. FM-143FX synapses (green) were co-labelled with antibodies to post-synaptic density protein 95 (PSD95; red). Putative synapses (yellow; arrows) were identified by the co-localization of both FM 1-43FX and PSD95. Scale bar = 10 μm.

Fig. 4

Neurons on hydrogel peptide-stamped hydrogel surfaces. (A–C) Neurons at 24 hr, 48 hr and 10 days in culture. At 24 hr, growth-cone lamellipodia were extensive and extended. After 48 hr in culture, cell somata were predominantly located at the nodes of the grid (15 μm-diameter discs at the intersections of 2 μm-wide orthogonal lines). By 10 days in vitro neurons had formed into neuronal networks. Cells were stained for neuron-specific βIII-tubulin (red) and nuclei were stained with Hoescht 33342 (blue). Scale bar = 50 μm. (D) Hydrogel patterned neuronal network at 4 weeks in vitro. Arrows indicate where neuronal processes have deviated from the patterned areas. Pattern fidelity was maintained over a 1cm² area of the hydrogel. Neurons were stained with βIII-tubulin (red). Scale bar = 100 μm. (E) Neurons at 10 days in vitro were analyzed for the presence of active synapses, using the FM1-43FX dye. Neurons were depolarized in the presence of FM1-43FX, rinsed extensively to remove excess dye, and stimulated for 2 min in a high K⁺ solution. Following stimulation and washing, cells were fixed with 4% paraformaldehyde. Active synapses as revealed by the FM1-43FX dye are labelled in green (arrows). Neurons were imaged using an Olympus widefield upright microscope with ImagePro software (MediaCybernetics, Silverspring, MD). Scale bar = 50 μm. (F) Depolarization-dependent FM1-43FX labelling. Neurons from Figure 4E were subject to a second round of depolarization prior to fixation. FM-143FX synapses (green) were co-labelled with antibodies to post-synaptic density protein 95 (PSD95; red). Putative synapses (yellow; arrows) were identified by the co-localization of both FM 1-43FX and PSD95. Scale bar = 10 μm.

Fig. 4

Neurons on hydrogel peptide-stamped hydrogel surfaces. (A–C) Neurons at 24 hr, 48 hr and 10 days in culture. At 24 hr, growth-cone lamellipodia were extensive and extended. After 48 hr in culture, cell somata were predominantly located at the nodes of the grid (15 μm-diameter discs at the intersections of 2 μm-wide orthogonal lines). By 10 days in vitro neurons had formed into neuronal networks. Cells were stained for neuron-specific βIII-tubulin (red) and nuclei were stained with Hoescht 33342 (blue). Scale bar = 50 μm. (D) Hydrogel patterned neuronal network at 4 weeks in vitro. Arrows indicate where neuronal processes have deviated from the patterned areas. Pattern fidelity was maintained over a 1cm² area of the hydrogel. Neurons were stained with βIII-tubulin (red). Scale bar = 100 μm. (E) Neurons at 10 days in vitro were analyzed for the presence of active synapses, using the FM1-43FX dye. Neurons were depolarized in the presence of FM1-43FX, rinsed extensively to remove excess dye, and stimulated for 2 min in a high K⁺ solution. Following stimulation and washing, cells were fixed with 4% paraformaldehyde. Active synapses as revealed by the FM1-43FX dye are labelled in green (arrows). Neurons were imaged using an Olympus widefield upright microscope with ImagePro software (MediaCybernetics, Silverspring, MD). Scale bar = 50 μm. (F) Depolarization-dependent FM1-43FX labelling. Neurons from Figure 4E were subject to a second round of depolarization prior to fixation. FM-143FX synapses (green) were co-labelled with antibodies to post-synaptic density protein 95 (PSD95; red). Putative synapses (yellow; arrows) were identified by the co-localization of both FM 1-43FX and PSD95. Scale bar = 10 μm.