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Figure 1. 

Schematic overview of the biofabrication strategy for mature adipocyte (AC) bioprinting. (a) Conventional extrusion-based bioprinting of gellan gum-based bioinks containing mature adipocytes lacks dimensional stability due to the softness and buoyancy of the bioink. (b) An adapted workflow employing a yield-stress agarose-based support bath with divalent ions enables stabilized extrusion, resulting in improved printing accuracy demonstrated by the fabrication of defined grid structures and more complex three-dimensional dome-shaped constructs.

Figure 2. 

Support bath preparation and rheological characteristics of agarose support baths and gellan-based bioinks for printing mature adipocytes. (a) Preparation of agarose support bath by mixing agarose with PBS⁺, autoclaving it, and stirring it overnight. (b) Viscosity of different support bath compositions (50%, 75%, and 100% PBS⁺) at 10 °C. (c) Viscosity of different acellular GG solutions (10% and 20% PBS+ in stock solution) at 20 °C. (d) Inversion test of different acellular GG solutions (10%, 20%, and 30% PBS+ in stock solution) at room temperature (20 °C). Black arrows indicate the GG solution level after the Falcon tubes were inverted. Each analysis was performed in triplicate. Mean values represent the mean ± standard deviation.

Figure 3. 

Bioprinting characteristics and printing protocol. (a) Macroscopic images of grid structures printed without (−) and with (+) support bath; red markings and numbers highlight the analyzed parameters for printing fidelity. Scale bar: 10 mm. (b) Analysis of (1) construct length, (2) pore width, and (3) filament width of support-bath printed grid structures. Design parameters for each measure are indicated above each graph, with a construct length of 20 mm, a pore width of 5 mm, and a filament width of 1.2 mm. (c) Macroscopic pictures of dome-shaped constructs in side and top view with CAD model and support-bath printed and recovered construct. Scale bar: 10 mm. (d) Overview of the printer setup using the BioX printer and a Petri dish as printing vessel. (e) Listed bioprinting windows for printing AC constructs. Mean values represent the mean ± SD of six independent experiments for accuracy measurements of printed grid structures.

Figure 4. 

Viability of mature adipocytes in the printed dome-shaped constructs. (a) Live/dead staining of printed dome-shaped constructs on days 1 and 3 post-printing, living cells in green, dead cells in red, and cell nuclei in blue. Scale bar: 100 µm. (b) Normalized LDH release of printed dome-shaped constructs on days 1 and 3 post-printing, normalized to 1% Triton X-100 control (set to 100%). Data are represented as the mean ± standard deviation, with single values shown as dots from five independent experiments with five different biological donors. ** p ≤ 0.01.

Figure 5. 

Morphology and function of mature adipocytes in the printed dome-shaped construct. (a) Intracellular lipid (BODIPY) staining of printed dome-shaped constructs on days 1 and 3, lipids in green, cell nuclei in blue. Scale bar: 50 µm. (b) Basal (−) and isoproterenol-stimulated (10 µM) (+) glycerol release of dome-shaped AC constructs on days 1 and 3 post-printing. (c) Lipid vacuole (perilipin A) staining of printed dome-shaped constructs with two different magnifications (20x and 40x), lipid vacuoles in yellow, nuclei in blue. Scale bars: 50 µm and 20 µm. Mean values are shown as mean ± standard deviation, with individual values as dots from five independent experiments with five different biological donors. ** p ≤ 0.01, *** p ≤ 0.001.