Introduction: Cells cultured in flat, stiff, 2D environments results in cultures with irregular cell morphology and unnatural cell-cell interactions[1]. This can lead to physiological inaccuracies that can be extremely problematic for pre-clinical drug screening. Likewise, animal models can also be limited in their ability to mimic human physiology[2]. Both these scenarios can have considerable detrimental impacts on the progression of new drug candidates from pre-clinical trials to clinical trials, and can be particularly evident when modelling complex disease states such as those found in the central nervous system[3].
By exploring the use of induced pluripotent stem cells (iPSCs) and emulsion templated porous polymer scaffolds, this project aims at creating a 3D neural ‘organoid’ cell culture that can be used to improve the accuracy of current pre-clinical neurological drug screening techniques. The work presented here describes a novel method for the preparation of a culture system for the induction of iPSCs to neural cells. This work particularly focusses on developing a coating method for porous thiol-ene polyHIPE[4] scaffolds with poly-L-ornithine (PLO)/laminin, which are necessary for cell adhesion and to facilitate neural differentiation[5]. Both scaffold and coating were stained and then imaged using confocal microscopy to investigate the appearance and morphology of different coating concentrations, and to validate the coating technique.
Materials and Methods: Preparation: 80% porous polymer scaffolds were prepared with a trimethylolpropane tris(3-mercaptopropionate) and trimethylolpropane triacrylate ‘click’ reaction using the method previously described by Caldwell et al[4].
Coating and Staining: All incubation steps were carried using a Low Speed Orbital Shaker at 60 rpm for 5 minutes at room temperature unless specified otherwise. Naked scaffolds were stained using a solution of Nile Red in methanol and incubated for 10 minutes. Scaffolds were then washed with phosphate buffered saline (PBS). Scaffolds were incubated with 0.01% PLO for 2 hours. Scaffolds were then coated in varying laminin solutions (0.01-0.33 g/L) in PBS, and incubated for 2 hours. Laminin coatings were then stained using Alexa Fluor 488.
Results and Discussion: The concentrations of laminin chosen were based on well-established 2D coating protocols of 1-2 μg/cm2[6]. This equates to a concentration of 0.08 g/L for the porous polymer scaffold. Concentrations below this gave poor laminin surface coverage resulting in large red areas of exposed scaffold when analysed under fluorescence. Concentrations greater than the theoretically optimal 0.08 g/L interestingly did not reveal a complete surface coverage, revealing aggregates rather than a uniform surface distribution. It was hypothesised that high concentrations may lead to voids filling with protein, but this was not the case. The 0.08 g/L laminin concentration did in fact give an even film of laminin covering the majority of the scaffold (Figure 1), and importantly not filling voids, which could prevent cell intrusion.
Figure 1. Scaffold stained with Nile Red (red) and coated with 0.08 g/L laminin (green).
Conclusion: Porous polymer scaffolds were coated in PLO/laminin by immersion and using an orbital shaker for dispersion. A laminin concentration of 0.08 g/L was found to give an ideal, evenly dispersed surface coverage for the culture and neural induction of iPS cells.
Monash Micro Imaging, Monash University, Victoria, Australia; This work was supported by the Australian Post Graduate Award funded through Monash University
References:
[1] Carletti, E., A. Motta, and C. Migliaresi, Scaffolds for Tissue Engineering and 3D Cell Culture, in 3D Cell Culture, J.W. Haycock, Editor. 2011, Humana Press. p. 17-39
[2] Mak, I.W.Y., N. Evaniew, and M. Ghert, Lost in translation: animal models and clinical trials in cancer treatment. American Journal of Translational Research, 2014. 6(2): p. 114-118
[3] Xu, X.-h. and Z. Zhong, Disease modeling and drug screening for neurological diseases using human induced pluripotent stem cells. Acta Pharmacol Sin, 2013. 34(6): p. 755-764
[4] Caldwell, S., et al., Degradable emulsion-templated scaffolds for tissue engineering from thiol-ene photopolymerisation. Soft Matter, 2012. 8(40): p. 10344-10351
[5] Flanagan, L.A., et al., Regulation of Human Neural Precursor Cells by Laminin and Integrins. Journal of neuroscience research, 2006. 83(5): p. 845-856
[6] Kleinman, H.K., et al., Use of extracellular matrix components for cell culture. Analytical Biochemistry, 1987. 166(1): p. 1-13