Micro-structured Materials and Mechanical Cues in 3D Collagen Gels

Abstract

Collagen gels provide a versatile and widely used substrate for three-dimensional (3D) cell culture. Here we describe how cell-seeded Type-I collagen gels can be adapted to provide powerful 3D models to support a wide range of research applications where cell/substrate alignment, density, stiffness/compliance, and strain are critical factors. In their fully hydrated form, rectangular collagen gels can be tethered such that endogenous forces generated as resident cells attach to and remodel the fibrillar collagen network can align the substrate in a controllable, predictable, and quantifiable manner. By removing water from collagen gels (plastic compression), their density increases towards that of body tissues, facilitating the engineering of a range of biomimetic constructs with controllable mechanical properties. This dense collagen can be used in combination with other components to achieve a range of functional properties from controlled perfusion, or tensile/compressive strength to new micro-structures. Detailed methodology is provided for the assembly of a range of 3D collagen materials including tethered aligned hydrogels and plastic compressed constructs. A range of techniques for analysing cell behaviour within these models, including microscopy and molecular analyses are described. These systems therefore provide a highly controllable mechanical and chemical micro-environment for investigating a wide range of cellular responses.

Hormonal Regulation of Epithelial Organization in a Three-Dimensional Breast Tissue Culture Model

“…22 Gels were rinsed with phosphate-buffered saline (PBS) and transferred to 2 mL of a 0.125% collagenase type 3 (from Clostridium histolyticum) (Worthington) solution in the DMEM/ F12. Gels were mechanically disrupted by gently pipetting up and down and the suspension was incubated at 37°C for 25 min.…”

Monitoring Neuron and Astrocyte Interactions with a 3D Cell Culture System

“…The interface mould has a stainless steel divider that separates sections of the chamber during casting, and can be removed to enable integration during setting [ 17 ]. The alignment mould contains nylon mesh structures at each end to provide tethering points to facilitate cellular self-alignment [ 19 ] 1. Standard glass slides and cover slips can be combined with various imaging chambers (e.g., CoverWell™) for microscopy of 3D cultures after removal from moulds, or cultures can be imaged in situ if cast within plates or moulds with appropriate transparency.…”

“…Neurons can either be added to the surface of the aligned construct or mixed with the astrocytes prior to gelling, and the resulting growth of neurites will be guided by the aligned astrocyte environment [ 15 ]. To achieve cellular self-alignment, set the gels within rectangular moulds that have attachment points for tethering the collagen at opposite ends, as in Fig. 1 [ 19 ] ( see Note 3 ).…”

“…It is important to ensure proper integration of collagen with the tethering bars prior to setting, and to use an appropriate density of cells to ensure suffi cient tension develops within the gels to align the cells [ 19 ]. If the astrocytes need to be seeded at a density which is too low for suffi cient tension to develop, then stimulating them with TGFβ1 (10 ng/ml for 48 h) or admixing a more contractile cell type (e.g., 5 % fi broblasts) will facilitate contraction and alignment [ 15 ].…”

“…For example cells can be lysed in situ for mRNA extraction or Western blotting, or cells can be collected from gels following collagenase incubation [ 19 ]. Cellular collagen gels can also be embedded and sectioned in the same way as tissues for…”