Cerebellar Cells Self-Assemble into Functional Organoids on Synthetic, Chemically Crosslinked ECM-Mimicking Peptide Hydrogels

Abstract

Hydrogel-supported neural cell cultures are more in vivo-relevant compared to monolayers formed on glass or plastic substrates. However, there is a lack of synthetic microenvironment available for obtaining standardized and easily reproducible cultures characterized by tissue-mimicking cell composition, cell–cell interactions, and functional networks. Synthetic peptides representing the biological properties of the extracellular matrix (ECM) proteins have been reported to promote the adhesion-driven differentiation and functional maturation of neural cells. Thus, such peptides can serve as building blocks for engineering a standardized, all-synthetic environment. In this study, we have compared the effect of two chemically crosslinked hydrogel compositions on primary cerebellar cells: collagen-like peptide (CLP), and CLP with an integrin-binding motif arginine-glycine-aspartate (CLP-RGD), both conjugated to polyethylene glycol molecular templates (PEG-CLP and PEG-CLP-RGD, respectively) and fabricated as self-supporting membranes. Both compositions promoted a spontaneous organization of primary cerebellar cells into tissue-like clusters with fast-rising Ca2+ signals in soma, reflecting action potential generation. Notably, neurons on PEG-CLP-RGD had more neurites and better synaptic efficiency compared to PEG-CLP. For comparison, poly-L-lysine-coated glass and plastic surfaces did not induce formation of such spontaneously active networks. Additionally, contrary to the hydrogel membranes, glass substrates functionalized with PEG-CLP and PEG-CLP-RGD did not sufficiently support cell attachment and, subsequently, did not promote functional cluster formation. These results indicate that not only chemical composition but also the hydrogel structure and viscoelasticity are essential for bioactive signaling. The synthetic strategy based on ECM-mimicking, multifunctional blocks in registry with chemical crosslinking for obtaining tissue-like mechanical properties is promising for the development of fast and well standardized functional in vitro neural models and new regenerative therapies.

Investigation of Cancer Cell Migration and Proliferation on Synthetic Extracellular Matrix Peptide Hydrogels

“…The 1 H NMR spectra of CLP was similar to earlier published spectra (Islam et al, 2016). The CLP-RGD spectra had an additional arginine peak at δ 3.10 ppm and serine peak at δ 3.90 ppm (marked by blue arrows in Figure 1) from RGD motif (Balion et al, 2020). The CLP-IKVAV had an apparent valine peak at δ 0.75 ppm, isoleucine peak at δ 0.82 ppm, and alanine peak at δ 1.22 ppm (marked by green arrows in Figure 1) from additional IKVAV motif.…”

“…In this study we have investigated the interaction between synthetic PEG-CLP hydrogels, alone and with conjugated cell adhesion peptides RGD or IKVAV, and three cancerous cell lines: human glioblastoma HROG36, rat glioma C6, and human melanoma A375. The background for this research were previous studies on PEG-CLP hydrogels as human corneal implants (Islam et al, 2016) and research performed on primary cerebellar cells grown on PEG-CLP and PEG-CLP-RGD (Balion et al, 2020). In the above-mentioned research, the material was characterized for mechanical properties, circular dichroism spectra, and surface topography (Islam et al, 2016;Balion et al, 2020).…”

“…The background for this research were previous studies on PEG-CLP hydrogels as human corneal implants (Islam et al, 2016) and research performed on primary cerebellar cells grown on PEG-CLP and PEG-CLP-RGD (Balion et al, 2020). In the above-mentioned research, the material was characterized for mechanical properties, circular dichroism spectra, and surface topography (Islam et al, 2016;Balion et al, 2020). It was found that the values of storage modulus G' modulus of the materials were between 0.02 and 0.07 MPa, and the values of elastic (Young) E modulus were between 0.06 and 0.180 MPa, respectively.…”

“…A nanometer-scale structure of hydrogel surfaces evaluated by atomic force microscopy (AFM) revealed relatively smooth and densely packed hydrogel surfaces with the surface roughness values measured as root mean square (RMS) between 2.7 ± 1.6 and 4.6 ± 2.8 nm. The data indicated the elastomechanical properties of the material is within the range of soft biological tissue (McKee et al, 2011), and it has the bioactive capacity to organize primary cerebellar cells to functional interconnecting organoids (Balion et al, 2020). Therefore, the idea of the study was to test whether PEG-CLP-based hydrogels might serve as an ECM mimetic to cancerous cell lines.…”

“…In this study, we examine ECM-mimicking hydrogels made of self-assembling collagen-like peptide attached to eight-armed polyethylene glycol (PEG-CLP; Islam et al, 2016) functionalized with fibronectin active site motif RGD (PEG-CLP-RGD) and laminin motif IKVAV (PEG-CLP-IKVAV) as matrices for cancer in vitro modeling. Previously, the hydrogels have promoted the attachment and self-assembly of primary neuronal-glial cells from developing rat cerebellum to functional organoids (Balion et al, 2020). In this study, the matrices were tested with two cancerous cell lines of astrocytic origin: rat glioma cells C6 and human glioblastoma HROG36.…”