Simple Polyacrylamide-based Multiwell Stiffness Assay for the Study of Stiffness-dependent Cell Responses

Abstract

Currently, most of the in vitro cell research is performed on rigid tissue culture polystyrene (~1 GPa), while most cells in the body are attached to a matrix that is elastic and much softer (0.1 -100 kPa). Since such stiffness mismatch greatly affects cell responses, there is a strong interest in developing hydrogel materials that span a wide range of stiffness to serve as cell substrates. Polyacrylamide gels, which are inexpensive and cover the stiffness range of all soft tissues in the body, are the hydrogel of choice for many research groups. However, polyacrylamide gel preparation is lengthy, tedious, and only suitable for small batches. Here, we describe an assay which by utilizing a permanent flexible plastic film as a structural support for the gels, enables the preparation of polyacrylamide gels in a multiwell plate format. The technique is faster, more efficient, and less costly than current methods and permits the preparation of gels of custom sizes not otherwise available. As it doesn't require any specialized equipment, the method could be easily adopted by any research laboratory and would be particularly useful in research focused on understanding stiffness-dependent cell responses. Video LinkThe video component of this article can be found at http://www.jove.com/video/52643/ 12 Several promising high-throughput hydrogel technologies have been developed, including PEG-based microarrays, 13 microfluidic devices for the production of agarose hydrogel microbeads, 14 or micro and nano-rods where stiffness is modulated by the diameter and height of the microrods. 15 However, the technologies to prepare such substrates are sophisticated and available to limited number of laboratories. Much research involving stiffness modulated cell responses utilizes polyacrylamide (PA) gels which are not only inexpensive and simple to implement, but also exhibit a physiologically relevant range of Young's modulus, namely 0.3 -300 kPa. [16][17][18][19][20][21][22] However, existing methods to fabricate PA gels for cell culture are labor intensive and consequently prepared in small batches. Some of the difficulties associated with the preparation of PA gels as cell substrates stem from the requirement that the gels have to be prepared: 1) in the absence of oxygen to allow complete polymerization, 2) with a flat and smooth surface to permit uniform cell attachment and spreading, and 3) permanently affixed to the bottom of the cell culture dish to prevent floating.Several groups have attempted to produce PA gels for cell culture in large batches. Semler et al. prepared thick sheets of PA gels which were then "cut" with a hole punch and placed into 96-well plates. 23 However, this method is limited to stiffer gels, i.e., > 1 kPa in Young's modulus, because softer gels are "sticky", difficult to cut, and easily damaged. Mih et al. developed a more sophisticated technique which allows the gels to be directly polymerized in a glass-bottom multiwell plate. 6 This was achieved by pouring the gel solutions into functionalized gla...

A straightforward cell culture insert model to incorporate biochemical and biophysical stromal properties into transplacental transport studies

“…Importantly, these mechanical stiffness ranges cannot be achieved with numerous hydrogel formulations, several of which, like type I collagen gels, are an order of magnitude softer than healthy placenta (28). However, PAA gels can be reliably created from 0.2-300 kPa by altering the ratio of acrylamide to bis-acrylamide to PBS of the gel solution (20, 21, 29). Mechanistically, PAA hydrogels have been used to study the ability of trophoblasts to fuse on different stiffnesses.…”

Large polyacrylamide hydrogels for large-batch cell culture and mechanobiological studies

“…The polymerized hydrogel is thus physically or covalently attached to a rigid substrate at its bottom surface, and the hydrophobic slide is removed to expose the cell-contacting surface. However, this approach as well as other modified methods [13a, 14a, 15] is associated with difficulties, especially for cell culture in large batches. For example, the glass-sandwich method is technically demanding requiring several chemical reactions that take multiple experimental steps.…”

Large Polyacrylamide Hydrogels for Large‐Batch Cell Culture and Mechanobiological Studies

“…The polymerized hydrogel is thus physically or covalently attached to a rigid substrate at its bottom surface, and the hydrophobic slide is removed to expose the cell‐contacting surface. However, this approach as well as other modified methods [ 13a,14a,15 ] is associated with difficulties, especially for cell culture in large batches. For example, the glass‐sandwich method is technically demanding requiring several chemical reactions that take multiple experimental steps.…”