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Tissue Engineering

Tissue Engineering - Poster Session D

Poster BB14 - A Method for Manufacturing Reproducible Scaffold-Free Adipose Spheroids

Friday, October 25, 2024
3:30 PM - 4:30 PM EST
Location: Exhibit Hall E, F & G

Introduction:  With more than 2 in 5 adults in the US currently battling obesity, ongoing research strives to unravel the intricacies of adipose tissue and its role in metabolic syndromes[1], autoimmune diseases[2], cardiovascular diseases[3], and cancers[4]. Therefore, it is critical to develop in vitro models that not only allow the study of how healthy adipocytes behave but also shed light on how dysfunctional cells may contribute to these pathologies.
Conventional monolayer models of adipogenic differentiation, while convenient, have significant limitations. Cells cultured in monolayers exhibit decreased efficiency in adipogenic differentiation, resulting in a heterogeneous population. Only a minority express the classic mature adipocyte phenotype, characterized by large, unilocular, lipid-rich droplets with substantially increased production of hormones such as adiponectin, along with decreased pro-inflammatory cytokine production [5]. Over the years, several 3D models of adipose tissue have been developed, progressively increasing in complexity. Protocols utilizing various scaffolding techniques, such as mechanically tunable 3D environments[6] and microfluidics chips[7] have been created. While these methods are fascinating, they require specialized resources, expertise, and/or technologies that limit their applicability to a broader group of biomedical researchers.
We have distilled our methods for generating adipose spheroids into two formats that meet a wide range of experimental goals: a 96-well spheroid plate format and agarose micromold format. Both methods are relatively simple allowing their adoption across many biomedical research labs. We compared the two methods for experimental design and use them to highlight the differences between monolayer and spheroid culture and the importance of characterizing donor variability.

Materials and

Methods:  Adipogenic differentiation of adMSCs in monolayer or 96-well spheroid plates: adMSCs were plated in their prospective plate formats to achieve confluency or form spheroids. Cells were cultured within Preadipocyte Differentiation Medium (PDM-2) (Lonza) for the first week and changed to DMEM with insulin the following week to mature.
Adipogenic differentiation of adMSCs in agarose micromold format: A 4% w/v solution of agarose was heated to 80℃ to achieve molten state. Once solubilized, the solution was transferred to either a Microtissues© 35-well micromold cast, or in-house printed 10-well micromold cast for 2 minutes until gelled. Gels were transferred to a 24-well plate and acclimated within PDM-2 for 1 hour, media was aspirated, and cells were seeded within the 7x5 array at various concentrations to achieve ~1k, 2k, 5k, 10k, or 17.5k cells/spheroid. Once formed, the remaining media was added and cells were differentiated as previously described.
Adipokine production analysis: Media was collected at the end of the two week differentiation for adipokine analysis via LegendPlex Adipokine Panel (BioLegend) according to manufacturer’s protocols.
Lipid staining and imaging: Cells cultured within monolayer were gently washed 2x with PBS -/-, fixed for 30 minutes, and stained for 30 minutes with Hoechst 33342 and BODIPY 493/502 within PBS followed by fluorescent microscopy. Cells cultured within spheroid format followed the same procedure, with increased fixing and staining times of 1hr followed by inverted fluorescent confocal microscopy.

Results, Conclusions, and Discussions: Study 1: Adipogenic differentiation in 2D monolayer and 3D spheroid culture formats. Cells differentiated within a 3D spheroid format exhibited a substantial increase in adiponectin signaling in comparison to that produced by cells differentiated in monolayer (Fig. 1A). Pro-inflammatory cytokine production decreased across the board from 2D to 3D culture. Average concentrations of MCP-1, IL-8, and IL-6 decreased from 2D to 3D culture (Fig. 1B-D). Cells differentiated within 3D also illustrated greater lipid droplet production compared to their 2D counterpart (Fig. 1E,F).

Study 2: Donor-specific adipogenic variability within 3D culture format. After the 14-day spheroid differentiation, Donors 2302 and 2414 had increased average lipid droplet counts compared to Donors 2334, 2220, and 11738 (Fig. 2B). When analyzing adiponectin, donors 2414 and 11738 had 11x increased production compared to donors 2334, 2220 and 2302 (Fig. 2C). Donor 2334 had higher pro-inflammatory cytokine production across the board compared to the remaining four (Fig. 2D-F).

Study 3: Spheroid production and function via agarose micromold or spheroid plate. Spheroids formed within agarose micromolds have a greater size variability compared to those formed within spheroid plates (Fig. 3C). For the spheroids formed within a 10- or 35-well micromold, increasing the number of cells per spheroids leads to a drastic decrease in adiponectin production, while increasing the overall number of spheroids leads to a further decrease (Fig. 3D).

These findings demonstrate the efficacy of using simple, scaffold-free techniques for adipogenic differentiation without the need for specialized scaffolding, machinery, or materials expertise. Both methods require minimal intervention after initial seeding, reducing user error and enhancing experimental reproducibility. Additionally, they provide full access to the spheroids, facilitating scale-up and experimental flexibility. We used these techniques to compare how 3D adipogenic differentiation leads to increased differentiation efficiency and decreased pro-inflammatory cytokine production compared to 2D as seen in prior work[5]. This is more in line with the low levels of inflammation expected within adipose tissue. We then directly compare spheroids from the methods showing the benefits and drawbacks of each. Finally, we use the 96-well plate method to reveal donor variability.

Acknowledgements (Optional):  This work was supported from NIGMS R01GM145626 (ES, JA), NIH P42 ES013661 (J.A. and A.K.), and a Diabetes Action Research and Education Grant (J.A.). Adipose was supplied by the Tissue Procurement Core at the University of Iowa which is supported by an award from NIH (NCI award number P30CA086862) and the University of Iowa Carver College of Medicine.