Lauren Elizabeth Flynn

Cell therapies involving human adipose-derived stromal cells (hASCs) have shown promise for a range of clinical applications due to their ability to stimulate angiogenesis and dampen inflammation via paracrine mechanisms. However, a major barrier to the successful clinical translation of hASC-based therapies is that standard culture methods for expansion on rigid 2D tissue-culture polystyrene under static conditions diminish the pro-regenerative functionality of the cells. To address these limitations, the current project focused on the development of an in vitro bioreactor system for preconditioning hASCs to augment their capacity to stimulate regeneration through paracrine mechanisms. Specifically, the combined effects of decellularized adipose tissue (DAT) coatings, shear-stress stimulation, and varying oxygen tensions on hASC expansion and paracrine factor secretion were assessed. Additional studies were performed to characterize the effects of stimulating hASCs within the rocking bioreactor system using the pro-inflammatory cytokines IFN-gamma and TNF-alpha. Expansion in the bioreactor under all conditions supported hASC growth with no observable morphological differences. However, dynamic culture on DAT coatings enhanced intracellular indoleamine 2,3-dioxygenase (IDO) expression in hASCs cultured under 20% O2. Moreover, culturing under dynamic conditions and/or on DAT coatings significantly increased secretion of the pro-angiogenic factors VEGF, HGF, and angiogenin. When pro-inflammatory cytokine priming was introduced, the expression of all tested paracrine factors was enhanced, particularly the immunomodulatory factors IL-6, IL-8 and MCP-1. Overall, a novel bioreactor system was developed for hASC expansion and preconditioning, demonstrating that the cell microenvironment can be tuned to modulate hASC paracrine factor secretion.

Cellular therapies harnessing the potential of endothelial colony-forming cells (ECFCs) for therapeutic revascularization in patients with critical limb ischemia (CLI) have garnered much interest. However, limited cell persistence following intramuscular injection into ischemic tissues has prompted the development of biomaterials as cell-delivery platforms to support cell survival and enhance therapeutic outcomes. This study explored the use of hydrogels derived from human decellularized adipose tissue (DAT) as a cell-instructive platform for ECFC delivery. In vitro studies confirmed high initial ECFC viability at 1 day following encapsulation in the DAT hydrogels but revealed that cell viability and proliferation were reduced over time in culture as compared to ECFCs cultured on tissue culture polystyrene (TCP). However, the ECFCs cultured within the DAT hydrogels showed similar ECFC cell surface marker expression patterns compared to ECFCs cultured on TCP after 6 days in culture, supporting that their phenotype was maintained. Subsequent testing focused on comparing a low (2.4 × 10⁵ cells) and high (1 × 10⁶ cells) dose of ECFCs delivered in either saline or within DAT hydrogels in a femoral artery ligation-induced CLI (FAL-CLI) model in NOD/SCID mice over 35 days. Bioluminescence imaging results showed that the low dose of ECFCs delivered in DAT hydrogels demonstrated enhanced retention on days 14, 21, and 28 compared to delivery in saline. Despite this finding, there were no observed improvements in hindlimb perfusion between delivery strategies, which may be related to the robust collateral vessel formation observed in this model with a µCT-based angiography method. Overall, this work supports that DAT hydrogels can enhance localized ECFC retention following intramuscular injection in mice with femoral artery ligation but emphasizes that enhanced retention may be insufficient for improving functional vascular regeneration.

Meniscus tears are one of the most common musculoskeletal injuries, but treatment options remain limited. Recognizing that the extracellular matrix (ECM) provides cell-instructive cues that can direct cell differentiation, the current study focused on the development and characterization of bioscaffolds derived from decellularized meniscus with a view towards future tissue-engineering applications. The first phase of research validated a new decellularization protocol for porcine meniscus that avoided the use of anionic detergents. The inner and outer regions of the meniscus were processed separately, and cell extraction, collagen content, and glycosaminoglycan (GAG) content were characterized in comparison to a published protocol also involving treatment with the non-ionic detergent Triton X-100 and enzymatic digestion with DNase and RNase. Our new protocol effectively removed cellular content while preserving key ECM constituents including glycosaminoglycans (GAGs) in both the inner and outer meniscus regions. Next, the cell-instructive effects of the decellularized ECM generated with our new protocol were probed by encapsulating human adipose-derived stromal cells (ASCs) within alginate beads incorporating pepsin-digested decellularized inner meniscus (DIM) or decellularized outer meniscus (DOM). Viability staining confirmed that the encapsulated ASCs remained highly viable over 28 days in culture in proliferation or chondrogenic differentiation media. Compared to alginate alone, the viable cell density within the alginate beads at 28 days was significantly higher in the beads incorporating DIM under both media conditions, as well as in the beads incorporating DOM in proliferation medium. In addition, immunofluorescence staining supported that the incorporation of the pepsin-digested ECM within the alginate enhanced fibrochondrogenic ECM marker expression in the samples cultured in chondrogenic differentiation medium. Overall, these studies provide new insight supporting that decellularized meniscus ECM can be harnessed to direct cell phenotype and function towards the goal of developing tissue-specific bioinks for tissue-engineering applications.

Meniscus tears are one of the most common musculoskeletal injuries, but treatment options remain limited. The meniscus can be divided into distinct inner and outer regions based on differences in the tissue structure and composition. Recognizing that the extracellular matrix (ECM) provides cell-instructive cues that can direct cell differentiation, the current study investigated the effects of incorporating region-specific ECM derived from decellularized meniscus on the viability, growth, and lineage-specific differentiation of human adipose-derived stromal cells (ASCs) encapsulated in alginate beads. The first phase of research focused on validating a novel decellularization protocol for porcine meniscus, which demonstrated the effective removal of cellular content while preserving key ECM constituents including glycosaminoglycans (GAGs) in both the inner and outer meniscus regions. Subsequently, the decellularized inner meniscus (DIM) and decellularized outer meniscus (DOM) were digested with pepsin and combined with a propriety alginate formulation to enable rapid cell encapsulation under mild conditions. Viability staining confirmed that encapsulated human ASCs remained highly viable over 28 days in culture in proliferation or chondrogenic differentiation media. Under both media conditions, the ASC density was significantly higher in the alginate beads incorporating DIM or DOM as compared to alginate alone controls at 28 days. In addition, gene expression analysis and immunofluorescence staining supported that the incorporation of the pepsin-digested ECM within the alginate enhanced fibrochondrogenic marker expression in the samples cultured in chondrogenic differentiation medium. Qualitatively, more intense staining for collagen types I and II were observed within the beads incorporating DOM as compared to DIM, supporting that the formulations had varying effects. Overall, these studies provide new insight supporting that region-specific meniscus ECM can be harnessed to direct cell phenotype and function towards the goal of developing tissue-specific bioinks for tissue-engineering applications.

Cellular therapies involving the co-delivery of cells with complementary pro-regenerative functionality hold promise as a strategy to promote soft tissue regeneration. In particular, the co-delivery of adipose-derived stromal cells (ASCs) and endothelial colony-forming cells (ECFCs) has shown promise for regenerating stable blood vessels in vivo. The current study developed novel “cell-assembled” scaffolds for co-delivering human ASCs and ECFCs within a supportive decellularized adipose tissue (DAT) matrix, with the objective of enhancing their localized retention and augmenting their capacity to stimulate adipose tissue regeneration. Human ASCs and ECFCs were seeded separately onto human-derived DAT microcarriers under cell-type specific conditions. The cell-seeded microcarriers were then combined and cultured for 8 days under conditions that promoted matrix remodeling to fuse the microcarriers into 3D engineered tissues containing ASCs+ECFCs, ASCs alone, or ECFCs alone. Co-culture with ECFCs within the scaffolds was shown to modulate ASC pro-angiogenic gene expression, with some ECFCs forming tubule-like structures in vitro in both the ASC+ECFC and ECFC alone groups. In vivo bioluminescence imaging using a dual luciferase reporter system showed that co-delivery with ASCs enhanced ECFC retention following subcutaneous implantation in athymic nu/nu mice, but co-delivery did not alter the localized retention of viable ASCs. Interestingly, while immunofluorescence staining for CD31 and microcomputed tomography angiography indicated that vascular regeneration was similar in the cell-assembled scaffolds containing ASC+ECFCs, ASCs alone, and ECFCs alone, histological staining revealed that extensive regions of the ECFC alone scaffolds had remodelled into adipose tissue at 29 days post-implantation.

The generation of a tissue-specific intestinal hydrogel derived from the native intestine has the potential to support and promote the growth of intestinal organoids. In this study, we aimed to develop hydrogels derived exclusively from intestinal extracellular matrix (ECM) or composites comprised of intestinal ECM combined with alginate that allow for greater tuning of the hydrogel properties. A novel mouse intestinal decellularization protocol was developed and the ECM characterized. Our analyses demonstrate that our protocol effectively removed cellular and nuclear content while preserving key ECM components including collagens, glycosaminoglycans, fibronectin and laminin. When the decellularized small intestine (DSI) was used to generate hydrogels, the resulting ECM showed bioactivity as demonstrated by metabolic and pro-proliferative effects on NIH 3T3 murine fibroblasts. Importantly, our novel DSI hydrogels also supported murine intestinal and colonic organoid growth similar to Matrigel® controls. These studies demonstrate that murine tissue-specific DSI hydrogels can provide a supportive environment for the culture of intestinal and colonic organoids in vitro.

Cell therapies using human mesenchymal stromal cells (MSCs) are promising for a wide variety of clinical applications. However, broad‐scale clinical translation is limited by conventional culture methods for MSC expansion within 2D tissue‐culture flasks. MSC expansion on ECM‐derived microcarriers within stirred bioreactor systems offers a promising approach to support MSC growth. Previously, our team established methods for fabricating ECM‐derived microcarriers from a variety of decellularized tissue sources using electrospraying techniques. However, these microcarriers are relatively large and have a broad size distribution, which may limit their utility. Smaller and more uniform microcarriers may be favorable for MSC growth within bioreactors and have greater potential to serve as a minimally invasive injectable cell delivery platform. To address these limitations, the current project focused on the development of a new microfluidic‐based approach enabling both uniform and small microcarrier production. Using a novel, modified 3D T‐junction design, we successfully generated microcarriers using human decellularized adipose tissue (DAT) as the ECM source. Our new cost‐effective device produced microbeads that were small and monodisperse, at a range of flow rate combinations and with high production rates. Photo‐crosslinking using rose bengal allowed for the generation of microcarriers that retained their shape and could withstand rehydration, with a mean diameter of 196 ± 47 μm. Following methods optimization and microcarrier characterization, in vitro studies confirmed that the new microcarriers supported human adipose‐derived stromal cell (hASC) attachment and growth, as well as ECM production, across 14 days within spinner flask bioreactors. Overall, this study demonstrates the feasibility of using our novel, cost‐effective, and reusable microfluidics device to generate cell‐supportive microcarriers comprised exclusively of ECM that show promise as an MSC expansion platform.

Cell therapies using human mesenchymal stromal cells (MSCs) are promising for a wide variety of clinical applications. However, broad-scale clinical translation is limited by conventional culture methods for MSC expansion within 2D tissue-culture flasks. MSC expansion on ECM-derived microcarriers within stirred bioreactor systems offers a promising approach to support MSC growth. Previously, our team established methods for fabricating ECM-derived microcarriers from a variety of decellularized tissue sources using electrospraying techniques. However, these microcarriers are relatively large and have a broad size distribution, which may limit their utility. Smaller and more uniform microcarriers may be favorable for MSC growth within bioreactors and have greater potential to serve as a minimally invasive injectable cell delivery platform. To address these limitations, the current project focused on the development of a new microfluidic-based approach enabling both uniform and small microcarrier production. Using a novel, modified 3D T-junction design, we successfully generated microcarriers using human decellularized adipose tissue (DAT) as the ECM source. Our new cost-effective device produced microbeads that were small and monodisperse, at a range of flow rate combinations and with high production rates. Photo-crosslinking using rose bengal allowed for the generation of microcarriers that were stable following rehydration, with a mean diameter of 196 ± 47 µm. Following methods optimization and microcarrier characterization, in vitro studies confirmed that the new microcarriers supported human adipose-derived stromal cell (hASC) attachment and growth, as well as ECM production, across 14 days within spinner flask bioreactors. Overall, this study demonstrates the feasibility of using our novel, cost-effective, and reusable microfluidics device to generate cell-supportive microcarriers comprised exclusively of ECM in a size range that could be injected via a small gauge needle and were stable in long-term culture.