Optimising contraction and alignment of cellular collagen hydrogels to achieve reliable and consistent engineered anisotropic tissue

Abstract

Engineered anisotropic tissue constructs containing aligned cell and extracellular matrix structures are useful as in vitro models and for regenerative medicine. They are of particular interest for nervous system modelling and regeneration, where tracts of aligned neurons and glia are required. The self-alignment of cells and matrix due to tension within tethered collagen gels is a useful tool for generating anisotropic tissues, but requires an optimal balance between cell density, matrix concentration and time to be achieved for each specific cell type. The aim of this study was to develop an assay system based on contraction of free-floating cellular gels in 96-well plates that could be used to investigate cell-matrix interactions and to establish optimal parameters for subsequent self-alignment of cells in tethered gels. Using C6 glioma cells, the relationship between contraction and alignment was established, with 60-80% contraction in the 96-well plate assay corresponding to alignment throughout tethered gels made using the same parameters. The assay system was used to investigate the effect of C6 cell density, collagen concentration and time. It was also used to show that blocking α1 integrin reduced the contraction and self-alignment of these cells, whereas blocking α2 integrin had little effect. The approach was validated by using primary astrocytes in the assay system under culture conditions that modified their ability to contract collagen gels. This detailed investigation describes a robust assay for optimising cellular self-alignment and provides a useful reference framework for future development of self-aligned artificial tissue.

An allogeneic ‘off the shelf’ therapeutic strategy for peripheral nerve tissue engineering using clinical grade human neural stem cells

“…All gels were prepared using 80% v/v Type I bovine dermis collagen (3 mg/ml; Koken, diluted to 2 mg/ml using 1 mM HCl) mixed with 10% v/v 10 × minimum essential medium (Sigma) and neutralised using RAFT Neutralising Solution (Lonza Bioscience) before addition to 10% v/v CTX cell suspension to give a cell density of 2 × 10 6 cells/ml of gel. Gels were allowed to set in tethering moulds at 37 °C for 15 min and then immersed in culture medium and incubated at 37 °C in a humidified incubator with 5% CO 2 /95% air for 24 h, during which time the cells contracted the tethered gels and become aligned 39 (Supplementary Figure 2 ). Using RAFT absorbers (Lonza Bioscience) the aligned gels were stabilised for 15 minutes, a process whereby a biocompatible absorbent material is placed upon the gel and absorbs interstitial fluid to generate a dense robust hydrogel with a 50 fold increase in cell and collagen density.…”

Engineered neural tissue with Schwann cell differentiated human dental pulp stem cells: potential for peripheral nerve repair?

“…Despite the apparent ideal profile of autologous Schwann cells, their isolation is rather invasive and their expansion is difficult. Furthermore, to obtain alignment of cells in constrained hydrogels, cells need to be able to exert a sufficient level of traction forces, a characteristic that is limited in primary glial cell cultures (East et al , ; O'Rourke et al , ). In search of alternative cell sources, mesenchymal stem cells have shown to be promising candidates for regenerative medicine purposes.…”

Anisotropically organized three-dimensional culture platform for reconstruction of a hippocampal neural network

“…For three-dimensional (3D) constructs, several strategies have been introduced to confer anisotropy, which in many cases involve application of strong external forces, such as electric 16 17 or magnetic field 18 19 and mechanical stress 19 20 21 22 23 . Other approaches utilize cellular tension or traction forces 20 24 25 26 27 , which often lead to inhomogeneous alignment. Of note, only a few of such methods that have been devised so far to induce alignment in 3D are compatible with long-term culture of mammalian primary neurons, which are highly sensitive to stress.…”