Patterning Bioactive Proteins or Peptides on Hydrogel Using Photochemistry for Biological Applications

Summary

In this method, we use photopolymerization and click chemistry techniques to create protein or peptide patterns on the surface of polyethylene glycol (PEG) hydrogels, providing immobilized bioactive signals for the study of cellular responses in vitro.

Abstract

There are many biological stimuli that can influence cell behavior and stem cell differentiation. General cell culture approaches rely on soluble factors within the medium to control cell behavior. However, soluble additions cannot mimic certain signaling motifs, such as matrix-bound growth factors, cell-cell signaling, and spatial biochemical cues, which are common influences on cells. Furthermore, biophysical properties of the matrix, such as substrate stiffness, play important roles in cell fate, which is not easily manipulated using conventional cell culturing practices. In this method, we describe a straightforward protocol to provide patterned bioactive proteins on synthetic polyethylene glycol (PEG) hydrogels using photochemistry. This platform allows for the independent control of substrate stiffness and spatial biochemical cues. These hydrogels can achieve a large range of physiologically relevant stiffness values. Additionally, the surfaces of these hydrogels can be photopatterned with bioactive peptides or proteins via thiol-ene click chemistry reactions. These methods have been optimized to retain protein function after surface immobilization. This is a versatile protocol that can be applied to any protein or peptide of interest to create a variety of patterns. Finally, cells seeded onto the surfaces of these bioactive hydrogels can be monitored over time as they respond to spatially specific signals.

Introduction

There are many stimuli that influence cell behavior. Generally, typical cell culturing techniques rely on soluble factors to elicit cellular responses; however, there are limitations to this approach. These methods are unable to accurately display all signaling motifs commonly found in vivo. Such signaling mechanisms include sequestered growth factors, cell-cell signaling, and spatially-specific biochemical cues.Furthermore, substrate stiffness can play an important role in cell behavior and stem cell differentiation and is not easily manipulated using common cell culturing practices^1,2. Biomaterial a....

Protocol

1. Preparation of Materials for Hydrogel Synthesis

1. Prepare stock solutions of PEGDA, lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), and fibronectin under sterile conditions and based on calculations (Table 1A).
   1. Weigh out and dissolve compounds in phosphate-buffered saline (PBS). Typically, maintain PEGDA working solution concentrations between 50 and 200 mg/mL (5-20% weight/volume). Pipette the PEGDA solution through a 0.22-µm syringe filter for sterilization.
      NOTE: Keep the PEGDA solutions covered with a foil wrapping to protect them from light. Prepare a fresh solution of PEGDA (recommended) for eac....

Results

The protocol to create bioactive patterns on the surface of PEG hydrogels is illustrated in Figure 1. A spreadsheet was developed to calculate the volume and concentration for each stock solution (Table 1A). Proteins to be immobilized onto the surface of the hydrogel are modified with 2-iminothiolane (Figure 1B). This reaction is performed using the volumes from Table 1B. The pre.......

Discussion

This protocol provides a method for creating bioactive protein patterns for biological applications. There are several modifications that can be made to adapt this protocol for different experiments. First, cell attachment requirements will vary for different cell types. If poor cell attachment to the gels is initially observed, increasing the concentration of the ECM protein within the precursor solution is advised. Other ECM proteins can be used instead of fibronectin, including different types of collagen, laminin, or.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
PEG-diacrylate (PEGDA)	Laysan Bio	ACRL-PEG-ACRL-3400	Can also be synthesized or purchased through other venders. Different molecular weights can be used.
Lithium Phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)			Synthesized in lab
Fibronectin	Corning	356008	Other cell attachment proteins can be used, such as laminin, matrigel
Phosphate-buffered saline (PBS)	Sigma	D8537-500ML
Photomask	FineLine Imaging	n/a	Custom prints on transparent sheets with high resolution DPI.
Binder Clips	Various Vendors
Compact UV Light Source (365nm)	UVP	UVP-21	Other UV light sources can be used, calibration of power is required.
2-iminothiolane (Pierce Traut’s Reagent)	Thermo Sci.	26101
Ellman’s Reagent: DTNB; 5,5-dithio-bis(2-nitrobenzoic acid)	Thermo Sci.	22582
human umbilical vein endothelial cells (HUVECs)	Lonza		passage number between 6- 10
EGM-2 Media	Lonza	CC31-56, CC-3162	EGM-2 without growth factors was used in experiments. Full EGM-2 media was used for cell maintainance
0.25% Trypsin EDTA	Life Tech	25200-056
Trypsin Neutralizer	Life Tech	R-002-100
Centrifuge	Various Venders
Hemocytometer	Hausser Sci. Bright-line
Ethylenediaminetetraacetic acid (EDTA)	Sigma Aldrich	E6758
0.22µm filter	Cell Treat	229743
1mL Syringe
Glass Microscope Slides	Fisher Sci.	12-550C
Plastic spacers	Various Venders		0.5mm thickness
70% Ethanol	BICCA	2546.70-1
Bio-shield	Bio-shield	19-150-0010
Bradford Reagent	BIO-RAD
Desalting Resin - Sephadex G-25	GE Healthcare	95016-754
Microspin Columns	Thermo Sci.	PI69725
AR-G2 rehometer	TA Instruments