Novel Hydrogel System Eliminates Subculturing and Improves Retention of Nonsenescent Mesenchymal Stem Cell Populations

Abstract

Aim: To compare the physiological behavior of mesenchymal stem/stromal cells (MSCs) within an expandable tissue-mimetic 3D system relative to in vitro expansion in a traditional 2D system. Methods: Adipose-derived MSCs (ASCs) were continuously cultured for 6 weeks on either 2D culture plastic or in a 3D hydrogel system that eliminated subculturing. ASCs were assessed for senescence, ‘stem-like’ MSC markers, and ability for their secretome to augment a secondary cell population. Results: The 3D hydrogel system resulted in an enhanced retention of more regenerative, nonsenescent ASC populations that exhibited increased expression of ‘stem-like’ MSC surface markers. Conclusion: This study introduces a proof-of-concept design for a novel modular 3D system that can improve in vitro expansion of stem-like cell populations for future regenerative therapies.

Tissue‐mimetic culture enhances mesenchymal stem cell secretome capacity to improve regenerative activity of keratinocytes and fibroblasts in vitro

“…Thus, the porous nature of this system permits mass transport (Figure 1A) and the easy collection of cellular‐derived biologics, such as EVs and proteins, within the secretome (Figures 4 and 5). Therefore, this system was selected because it provided an improved culture environment for ASCs over traditional 2D culture 73 . Additionally, this 3D system enabled more efficient isolation of cellular byproducts (i.e., biologics) relative to non‐porous hydrogel systems, which ultimately lack the capacity for long‐term culture and biologics collection that would be necessary for future clinical therapies.…”

“…Therefore, this system was selected because it provided an improved culture environment for ASCs over traditional 2D culture. 73 Additionally, this 3D system enabled more efficient isolation of cellular byproducts (i.e., biologics) relative to non-porous hydrogel systems, which ultimately lack the capacity for long-term culture and biologics collection that would be necessary for future clinical therapies. For the context of this study, a hydrogel that was mechanically similar to native adipose tissue was generated ($3 kPa; Figure S3).…”

“…For the context of this study, a hydrogel that was mechanically similar to native adipose tissue was generated ($3 kPa; Figure S3). 51,66,[73][74][75] Additionally, the 'bioinert' nature of the hydrogel allowed for the direct observation of the role of the mechanical, dimensional and architectural properties of the 3D system on ASC senescence and phenotype.…”

Mesenchymal Stem Cell Extracellular Vesicles from Tissue-Mimetic System Enhance Epidermal Regeneration via Formation of Migratory Cell Sheets

“…Notably, most current 3D hydrogel systems rely on manipulating densely-packed crosslinked networks in order to modulate the mechanical properties of the hydrogels, which drastically limits the ability for molecules to readily diffuse throughout the system [ 24 , 33 , 34 ]. However, the novel microchannel design of the 3D hydrogel system in this study acts as a “pseudo-vascular” conduit and does not hinder mass transport like traditional poured/molded hydrogel systems, which improved the collection of secreted byproducts, in addition to adequate nutrient exchange and prevention of centralized necrosis within the hydrogel [ 31 ].…”

“…Barriers related to the translatability of cell-based regenerative therapies, such as depleted regenerative capabilities of unhealthy autologous populations and inadequate ex vivo expansion systems, have led to a rising interest in regenerative acellular therapies [ 35 ]. While previous data with MSC-derived acellular products are promising, experiments using MSCs in wound healing to date have mostly utilized standard 2D culture techniques, which are known to induce differentiation and senescence [ 31 , 32 , 36 ]. Thus, traditional 2D culture modalities likely result in a heterogeneous and less regenerative MSC populations, leading to impurities and/or an inconsistent secretive product that subsequently limits the potential clinical benefits of MSC therapies.…”