Self-assembling nanofibre scaffolds for 3D cell cultures

Conventionally, researchers who want to study cellular function have grown cells on two-dimensional culture dishes. This normally involves dissecting out from an animal the tissue containing the cells being studied. The tissue is pulled through a series of pipettes of decreasing diameter; this disaggregates it, resulting in a suspension of single cells. The suspension is then ‘plated’ onto a culture dish coated with laminin, poly-D-lysine, or a similar substance, which acts as a substrate to which the cells can adhere. A medium containing growth factors and other nutrients is then added to the culture dish. As the cells grow and divide, they absorb the nutrients in the culture medium, so it is removed and replaced with fresh medium periodically during the time over which the culture is kept.

Cells cultured in this way normally form a single layer covering the surface of the culture dish. These 2D cultures enable researchers to investigate certain properties of cells. For example, the formation of synapses can be observed in cultured nerve cells, or RNA transcripts can be isolated from the cells to investigate the effects of reagents on gene expression in the cells. 2D cultures do, however, have their limitations. In situ, all cells are found within a three-dimensional micro-environment – they are surrounded by an extra-cellular matrix which is synthesized and secreted by the cells. They are in contact with other cells and are exposed to diverse, diffusible chemical signals which reach the cells through nano-sized pores in the matrix.

“The time has come to move on from two-dimensional dishes to culture systems that better represent the natural context of cells in tissues and organs,” says Shuguang Zhang, associate director of Massachusetts Institute of Technology’s Center for Biomedical Engineering, who together with collaborators from Milan, Italy, has developed a three-dimensional cell culture system.

The system is based on 18 short peptides containing of up to amino acid residues. When a solution of containing a number of these peptides is added to a culture dish, the protein fragments self-assemble into a three-dimensional nanofibre scaffold upon addition of the culture medium. Zhang and his colleagues cultured neural stem cells using this 3D system. Incorporated into the amino acid sequences of the peptides are motifs from cell adhesion molecules, from proteins which induce differentiation, and from proteins which promote neurite outgrowth. The cultured stem cells therefore adhered to the scaffold, and began dividing and differentiating, by synthesizing proteins characteristic of either nerve cells or glia.

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Confocal microscope image of neural stem cells cultured for 21 days on a 3D scaffold. Cells are labelled green, cell nuclei blue.

In the scaffold assembled from one peptide mixture, most of the cultured cells differentiated into neurons; another combination of peptides promoted mainly astrocyte differentiation; and in a scaffold consisting of yet another peptide mixture, most of the cells remained in an undifferentiated state. The 3D culture system resembles the micro-environment found in the living body more than any previous cell culture system, because the dimensions of the scaffold are similar to those of the extracellular matrix which surrounds cells in their natural environment. Furthermore, the scaffold enhances the survival of the cltured cells, and growth factors do not need to be added to the culture medium.

Zhang and his colleagues at the Laboratory for Molecular Self-Assembly discovered that self-assembling peptides could form stable membrane-like structures in 1993. Since then, members of the lab have shown that the peptides support neurite outgrowth and promote neuronal differentiation. Earlier this year, it was demonstrated that the peptides could partially restore vision in hamsters blinded by the severing of axonal pathways in the visual cortex; a solution of peptides injected directly into the hamsters’ visual cortex self-assembled into a 3D scaffold which promoted the regeneration of the severed axons. Self-assembling peptide scaffolds are also highly effective in stopping bleeding when applied to wounds in the brain, spinal cord, cardiovascular system, skin or liver.

Because they consist purely of amino acids, the peptide solutions can be transplanted into the body without side effects, unlike 3D scaffolds , such as Matrigel, which are made from synthetic biopolymers. The potential applications of the 3D scaffolds are diverse, because of the versatility obtained by different combinations of the peptides. Undoubdtedly, new petides containing different structural motifs will be designed and added to the current repertoire. Because the peptides are synthesized chemically, this can be done quickly and cheaply, and will soon result in scaffolds that can be used to study gene expression, cell migration and cell-to-cell signalling. The scaffolds may also be used as templates for the engineering of tissues and organs.

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Reference:

Gelain F., et al (2006). Designer Self-Assembling Peptide Nanofiber Scaffolds for Adult Mouse Neural Stem Cell 3-Dimensional Cultures. PLoS ONE 1 (1): e119. doi:10.1371/journal.pone.0000119

One thought on “Self-assembling nanofibre scaffolds for 3D cell cultures

  1. Great post – this has a lot of potential for changing the future of treatment of bleeds. I did my Masters thesis in Nanotechnology and I believe and know the potential of nanoscience to change our lives.
    Happy New Year Neurophilosopher!!

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