Sheetal Chowdhury

Human adipose biology is strongly influenced by three-dimensional (3D) architecture, cell–cell interactions, and local oxygen availability maintained over a long-term culture period, features that are not reproduced in traditional two-dimensional (2D) culture systems. To address this gap, we established a long-term human adipose-derived stem cell (hASC) spheroid model using elastin-like polypeptide–polyethyleneimine (ELP-PEI) coating. The ELP-PEI coating facilitated stable spheroid formation and sustained adipogenic differentiation over 56 days. As spheroids enlarged and matured, they exhibited hallmark features of adipocytes, including lipid accumulation, morphological compaction, and transition out of the proliferative state. Glucose uptake increased during maturation and declined as spheroids became larger. This reduction coincided with a marked rise in hypoxia-inducible factor-1α (HIF-1α) expression, indicating the emergence of a hypoxic microenvironment within larger spheroids. Notably, inhibiting HIF-1α restored insulin-stimulated glucose uptake, demonstrating that hypoxia was the primary driver of impaired insulin responsiveness in late-stage spheroids. These findings position ELP-PEI-supported hASC spheroids as a practical and physiologically relevant platform for studying human adipocyte biology, particularly the development and reversibility of hypoxia-associated metabolic dysfunction. This model offers new opportunities for mechanistic studies and for evaluating therapeutic strategies targeting insulin resistance and adipose tissue pathology.

Two-dimensional cell culture systems lack the ability to replicate the complex, three-dimensional (3D) architecture and cellular microenvironments found in vivo. Multicellular spheroids (MCSs) present a promising alternative, with the ability to mimic native cell–cell and cell–matrix interactions and provide 3D architectures similar to in vivo conditions. These factors are critical for various biomedical applications, including cancer research, tissue engineering, and drug discovery and development. Polymeric materials such as hydrogels, solid scaffolds, and ultra-low attachment surfaces serve as versatile platforms for 3D cell culture, offering tailored biochemical and mechanical cues to support cellular organization. This review article focuses on the structure–property relationships of polymeric biomaterials that influence MCS formation, growth, and functionality. Specifically, we highlight their physicochemical properties and their influence on spheroid formation using key natural polymers, including collagen, hyaluronic acid, chitosan, and synthetic polymers like poly(lactic-co-glycolic acid) and poly(N-isopropylacrylamide) as examples. Despite recent advances, several challenges persist, including spheroid loss during media changes, limited viability or function in long-term cultures, and difficulties in scaling for high-throughput applications. Importantly, the development of MCS platforms also supports the 3R principle (Replacement, Reduction, and Refinement) by offering ethical and physiologically relevant alternatives to animal testing. This review emphasizes the need for innovative biomaterials and methodologies to overcome these limitations, ultimately advancing the utility of MCSs in biomedical research.

Three-dimensional (3D) in vitro adipocyte models provide physiologically relevant platforms for studying adipogenesis and obesity-related metabolic dysfunction. However, long-term adipocyte culture is often hindered by limited cell–matrix adhesion and spheroid detachment. Previously, we demonstrated that elastin-like polypeptide (ELP)–polyethyleneimine (PEI) coatings functionalized with a trivalent RGD motif enhanced spheroid retention during frequent media changes. The present study investigates the long-term functional consequences of RGD incorporation over a 28-day culture period. 3T3-L1 preadipocytes were seeded, differentiated, and matured on ELP-PEI or ELP-(RGD)₃-PEI coatings. Spheroid morphology, triglyceride content, expression of PPAR-γ, adiponectin, HIF-1α genes, and insulin-stimulated glucose uptake were assessed. Both coatings supported initial spheroid formation, but only ELP-PEI maintained the 3D architecture and supported adipogenic maturation and insulin responsiveness. ELP-(RGD)₃-PEI promoted early retention but led to spheroid disassembly by mid-culture; notably, by day 28, cells reaggregated into abnormally large spheroids with impaired metabolic function, likely due to continued proliferation. These findings highlight the critical role of extracellular matrix-mediated cell–cell versus cell–substrate interactions in maintaining 3D culture fidelity. While RGD enhances adhesion, it disrupts spheroid integrity and compromises adipogenic and metabolic maturation. Taken together, ELP-PEI coatings offer a more conducive microenvironment for long-term 3D adipocyte culture and hold promise for modeling obesity-associated metabolic dysfunction in vitro.

To mimic the important features of progressing adiposity, in vitro adipose cell culture models must allow gradual intracellular fat accumulation in the three-dimensional (3D) arrangement of adipose-derived stem cells (ASCs) over a long-term culture period. Previously, elastin-like polypeptide (ELP) and polyethyleneimine (PEI) have been used to culture human adipose-derived stem cells (hASCs) as 3D spheroids and to differentiate them to adipocytes over a relatively long culture period of up to 5 weeks. In this study, to further enhance the spheroid adhesion properties, ELP was fused with Arginine–Glycine–Aspartic Acid (RGD) residues, known for their role as cell-attachment sites. This study aimed to assess whether the addition of RGD to the C-or N-terminus of ELP would impact the spheroid-forming ability of ELP-PEI coatings. ELP-RGD conjugates were produced using genetically modified Escherichia coli to express ELP-(RGD)₃ and (RGD)₃-ELP, followed by chemical conjugation with PEI. SDS gel electrophoresis, FTIR spectroscopy, and turbidimetry analyses revealed that ELP was conjugated with RGD without much alteration in the molecular weight, functional groups present, and transition temperature of ELP. The addition of RGD to ELP also did not affect the chemical conjugation capacity of ELP to PEI. We observed that the ELP-PEI coating formed slightly larger spheroids (61.8 ± 3.2 µm) compared to the ELP-(RGD)₃-PEI and (RGD)₃-ELP-PEI coatings (56.6 ± 3.0 and 53.4 ± 2.4 µm, respectively). Despite the size difference, ELP-(RGD)₃-PEI coatings exhibited superior spheroid retention during media changes, with minimal spheroid loss. DNA assay results confirmed a significant decrease in the DNA concentration (p < 0.05) after the 20 media changes for spheroids cultured on the ELP-PEI coating, indicating spheroid loss. However, there was no significant difference in DNA concentration before and after 20 media changes for spheroids cultured on the ELP-(RGD)₃-PEI and (RGD)₃-ELP-PEI coatings (p > 0.05). These findings suggest that RGD incorporation does not hinder the initial spheroid formation ability of the ELP-PEI coating and enhances spheroid retention under dynamic culture conditions.

Titanium has a long history of clinical use, but the naturally forming oxide is not ideal for bacterial resistance. Anodization processes can modify the crystallinity, surface topography, and surface chemistry of titanium oxides. Anatase, rutile, and mixed phase oxides are known to exhibit photocatalytic activity (PCA)-driven bacterial resistance under UVA irradiation. Silver additions are reported to enhance PCA and reduce bacterial attachment. This study investigated the effects of silver-doping additions to three established anodization processes. Silver doping showed no significant influence on oxide crystallinity, surface topography, or surface wettability. Oxides from a sulfuric acid anodization process exhibited significantly enhanced PCA after silver doping, but silver-doped oxides produced from phosphoric-acid-containing electrolytes did not. Staphylococcus aureus attachment was also assessed under dark and UVA-irradiated conditions on each oxide. Each oxide exhibited a photocatalytic antimicrobial effect as indicated by significantly decreased bacterial attachment under UVA irradiation compared to dark conditions. However, only the phosphorus-doped mixed anatase and rutile phase oxide exhibited an additional significant reduction in bacteria attachment under UVA irradiation as a result of silver doping. The antimicrobial success of this oxide was attributed to the combination of the mixed phase oxide and higher silver-doping uptake levels.

Clinical and basic science applications using adipose-derived stem cells (ADSCs) are gaining popularity. The current adipose tissue harvesting procedures introduce nonphysiological conditions, which may affect the overall performance of the isolated ADSCs. In this study, we elucidate the differences between ADSCs isolated from adipose tissues harvested within the first 5 min of the initial surgical incision (well-vascularized, nonpremedicated condition) versus those isolated from adipose tissues subjected to medications and deprived of blood supply during elective free flap procedures (ischemic condition). ADSCs isolated from well-vascularized and ischemic tissues positively immunostained for several standard stem cell markers. Interestingly, the percent change in the CD36 expression for ADSCs isolated from ischemic versus well-vascularized tissue was significantly lower in males than females (p < 0.05). Upon differentiation and maturation to adipocytes, spheroids formed using ADSCs isolated from ischemic adipose tissue had lower triglyceride content compared to those formed using ADSCs isolated from the well-vascularized tissue (p < 0.05). These results indicate that ADSCs isolated from ischemic tissue either fail to uptake fatty acids or fail to efficiently convert those fatty acids into triglycerides. Therefore, more robust ADSCs suitable to establish in vitro adipose tissue models can be obtained by harvesting well-vascularized and nonpremedicated adipose tissues.

Crystalline titanium oxides have shown photocatalytic activity (PCA) and the formation of antibacterial reactive oxygen species (ROS) when stimulated with UV light. Polyaniline (PANI) is a conductive polymer that has shown antibacterial effects. Previously, titanium oxides have been PANI-doped using a multi-step approach. In the present study, we compared PANI-doped specimens produced with a two-step method (ACV), to PANI-doped specimens produced by a novel single-step direct anodization (AAn) method, and a control group of anodized un-doped specimens. The surface morphology, oxide crystallinity, surface elemental composition, surface roughness, surface wettability, oxide adhesion, corrosion resistance, PCA, and ROS generation of each oxide group were evaluated. All groups exhibited mixed anatase and rutile phase oxides. The AAn group revealed less anatase and rutile, but more PANI-surface coverage. The AAn group exhibited significantly increased PCA after 60 min of direct UVA illumination compared to the ACV group, despite containing lower amounts of anatase and rutile. The ACV and AAn groups showed significant increases in ROS production after 4 h UVA illumination while the control group showed similar ROS production. These findings suggested that PANI doping using the novel direct anodization technique significantly improved PCA even for oxides containing less crystallinity. The S. aureus attachment response to each oxide group was also compared under UVA pre-illumination, UVA direct illumination, and no illumination (dark) lighting conditions. Although no significant differences were shown in the bacterial response, both PANI-doped groups exhibited less average bacterial attachment compared to the control group. The response of MC3T3-E1 pre-osteoblast cells to each oxide group was evaluated using MTT and live/dead assays, and no evidence of cytotoxicity was found. Since many, if not most, titanium implant devices are routinely anodized as a part of the manufacturing processes, these study findings are applicable to a wide variety of implant applications.