Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

Abstract

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

A Tumor‐on‐a‐Chip System with Bioprinted Blood and Lymphatic Vessel Pair

“…More recently, tumor cells have been seeded within 3D scaffolds or matrices, and similar to an in vivo tumor, they freely extend [8] , polarize [9] , migrate, and cluster [10,11] within the matrix. Moreover, these 3D tumor models display advantages in reestablishing cell functions [12] , signaling pathways [13,14] , and drugs responses [15] as compared to traditional 2D cultivation models, hence becoming more favorable in screening of pharmaceutical compounds [16][17][18] . For example, we and colleagues have reported an approach to generate a glioblastoma microenvironment featuring a co-culture of macrophages and glioblastoma cells in a mini-brain model, where it was shown that the glioblastoma cells were able to actively recruit the macrophages and then prime them to glioma-associated macrophages (GAMs), similar to the in vivoscenario [19] .…”

Development of a 3D Co-Culture System as a Cancer Model Using a Self-Assembling Peptide Scaffold

“…For cell encapsulation, we modified a protocol described in the previous work [22]. The commercial peptide RAD16-I (1% in water) was prepared at a final concentration of 0.3% ( w / v ) in 10% sucrose and sonicated for 20 min at room temperature.…”

Designer Self‐Assembling Peptide Hydrogels to Engineer 3D Cell Microenvironments for Cell Constructs Formation and Precise Oncology Remodeling in Ovarian Cancer

“…Most recently, Carlos and co‐workers perform the cancer cell behavior assays in designer RADA16‐I peptide hydrogel. [ 169 ] An easy and reliable methodology is established to decipher cancer cell behaviors and explore tumor progression in 3D context, which is also widely applicable to any type of cells, including any type of functional cell, embryonic or adult stem cells, or eventually, dysfunctional cells isolated from biopsies. As an instance, 3D cell cocultures in RADA16‐I peptide hydrogel maintain the expanded human articular chondrocytes with good viability during 4 weeks period.…”

“…[ 103 ] Moreover, peptide backbone functionalized by relevantly biological recognition and signal motifs can regulate the physiological cell behaviors in all hydrogel volume. [ 169,171 ] In comparison with other synthetic nanofiber hydrogels to incorporate nanofibrous architectures with spatially controllable biochemical features, [ 172 ] synthetic designer peptide hydrogels are ideal reductionist mimics of the ECMs for cell behavior studies, which may create the proper topography, biophysical and biochemical cell microenvironments with physiologically relevant elasticity and viscoelasticity to direct cell behaviors. [ 17,109 ]…”