Generation of 3D Soluble Signal Gradients in Cell‐Laden Hydrogels Using Passive Diffusion

Abstract

Soluble signal gradients play an important role in organ patterning, cell migration, and differentiation. Currently, signal gradients in 2D cell culture are realized using microfluidics and here cells are exposed to high and nonphysiological shear stress. Tissue morphogenesis (organogenesis) however occurs in 3D and therefore there is a need for simple and practical systems to impose gradients to cells dispersed in 3D matrix. Herein, a 3D gradient generator based on passive diffusion elements that recapitulates interstitial flow and is capable of imposing predictable gradients over long length scales (6 mm) lasting up to 48 h to cells dispersed in a hydrogel environment is reported. Using recombinant human WNT3A (rhWNT3A), the spatiotemporal activation of the canonical WNT pathway in human epithelial kidney cells and human mesenchymal stems cells expressing a green fluorescence protein reporter on a transcription factor/lymphoid enhancer‐binding factor (TCF/LEF) promoter is demonstrated. By refining computation models based on experimental findings, the diffusion coefficient of rhWNT3A in presence of human cells in 3D is determined. Furthermore, the formation of rhBMP4 gradients is visualized using immunohistochemistry by staining for phospho‐SMAD1/5, the downstream targets of the bone morphogenetic protein (BMP) pathway. The simplicity of the gradient generator is expected to spur its adoption in studying developmental biology paradigms in vitro.

Incorporation of Cross-Linked Gelatin Microparticles To Enhance Cell Attachment and Chondrogenesis in Carboxylated Agarose Bioinks for Cartilage Engineering

“… 37 Moreover, glutaraldehyde cross-linked gelatin drug delivery devices have been successfully explored in investigating signaling pathways in human marrow-derived mesenchymal stem cells without any adverse outcomes on cell viability and function. 38 , 39 In order to ensure minimal glutaraldehyde in the GMPs, GMPs were thoroughly rinsed until no residual glutaraldehyde was undetectable ( Figure S3 ). Additionally, as demonstrated in Figure S2 , this rinsing procedure enabled NIH 3T3 cells to adhere to the surface of cross-linked CA-GelA hydrogels and proliferate, indicating that the nondetectable residual glutaraldehyde level in the culture media was noncytotoxic.…”

Modulation of Short-Term Delivery of Proteins from Hydrogels

“…Based on our earlier work on the development of gelatinbased matrix delivery for the release of soluble signals relevant in endochondral ossification, 8,21 here we blended type-B gelatin with CA in various ratios and prepared devices by vapor-phase glutaraldehyde cross-linking as described earlier 21 and compared the release behavior of BSA, LF and rhFGF2. The release profiles are presented in Figure 4.…”

“…3 However, much of these efforts have focused on developing sustained drug delivery systems (DDS) with long-term release over weeks, 4 months, 5 and years. 6 While such long-term DDS are very relevant for the treatment of chronic diseases or cancer and the administration of contraceptives, 7 however, for modulating cellular function, for example in studying signal cascades 8 or inducing tissue morphogenesis, local delivery of molecules in a controlled fashion over a shorter duration is desirable. Many developmental processes such as vertebrate limb bud development, e.g.…”

Stochastic Nanoscale Biophysical Cues as a Basis for the Induction of Glioblastoma‐Like Transcriptional Programs in Astrocytes

“…To elucidate the underlying molecular mechanisms that might lead to astrocyte aggregation, further studies could examine outcomes between surfaces that present deterministic roughness (pillars, lines) in the same length scales as the R q values investigated in this study. Since inflammatory processes and biochemical changes associated with TBI are not captured by the stochastic nanoroughness model, future studies should include temporal presentation of soluble cues using delivery systems [ 117 ]. Also, a time course analysis of changes to astrocyte phenotype within spheroids using single cell omics would further shed light on the extent to which the astrocytes adopt GBM‐like transcription traits and when a GBM‐like phenotype might emerge.…”