Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Fabricating Microfluidic Co-Cultures of Immortalized Cell Lines Uncovers Robust Design Principles for the Simultaneous Formation of Patterned, Vascularized, and Stem Cell-Derived Adipose Tissue.

doi: 10.1002/smll.202501834

Figure Lengend Snippet: Figure 1. GFP-hAECs and hAD-MSCs spontaneously form robust and phenotypical 2D microvascular networks with a morphological dependence on cell seeding ratio. A) Fluorescence images of GFP-hAEC microvascular networks formed after 7-days culture in complete vascular cell media, with GFP- hAEC:hAD-MSC seeding ratios of 2:1 (i), 1:1 (ii), 1:2 (iii), 1:5 (iv) and 1:10 (v, scale bars = 1 mm). B) Quantitative analysis of vessel network morphology at day-7, detailing the number of network junctions (i), vessel end points (ii), total vessel length (iii) and total vessel area (iv) within a field of view 3.67 × 3.67 mm (n = 3 biological replicas, mean ± standard deviation). Results were compared via an ordinary one-way ANOVA followed by a Tukey multiple comparisons test (𝛼= 0.05). p > 0.05 (ns), p ≤0.05 (*), p ≤0.01 (**), p ≤0.001 (***) and p ≤0.0001 (****). C) Time lapse imaging of GFP-hAECs in 7-day vasculogenesis co-culture with a seeding ratio of 1:5 (scale bars = 1 mm). D) Quantitative analysis of every tenth time-lapse image (47 of 478 total images) over the entire 7-day culture period with graphs depicting the number of network junctions (i), vessel end points (ii), total vessel length (iii) and total vessel area (iv) within a field of view 3.67 × 3.67 mm (n = 3 biological replicas, mean ± standard deviation). E) Immunocytochemical staining panel of a 1:2 seeding ratio sample after 7-days culture detailing the nuclear counter stain DAPI (i), endogenous GFP expression (ii), and detection of CD31 (iii), 𝛼SMA (iv), CD90 (v, false colored cyan) and F-actin (vi, false colored yellow, scale bars = 500 μm). Images i–iv, v and vi represent three independent replicate cultures. At times, the 2:1 and 1:1 seeding ratio conditions detached from culture surfaces. The minimum time directly before which a culture commenced detachment (2:1, 22-h; 1:1, 82-h; depicted as dotted lines in 1.B and D) was used for the analysis of that condition and therefore was not statistically compared to the cultures which remained attached for the culture duration. EC: endothelial cell, MSC: mesenchymal stem cell, DAPI: 4′,6-diamidino-2-phenylindole, GFP: green fluorescent protein, CD31: cluster of differentiation 31, 𝛼SMA: alpha smooth muscle actin, CD90: cluster of differentiation 90, F-actin: filamentous actin.

Article Snippet: Cell Culture and Imaging: This project was supported by The University of Queensland’s Human Research Ethics Committee through approval 2021/HE002698: “Tissue culturemodels for in vitro optimization of engineered biomaterial scaffolds and bioprocess techniques”, as well as The University of Queensland’s Institutional Biosafety Committee through approval IBC/602E/ChemEng/2023: “Risk group 2 immortalized cell line activities.” Cell Culture and Imaging—Routine Cell Line Maintenance: Human telomerase reverse transcriptase (hTERT) immortalized adipose-derived mesenchymal stem cells (hAD-MSCs; ASC52telo, SCRC-4000, American Type Culture Collection ATCC) were routinely maintained as per manufacturer’s instructions in MSC Basal Medium (#500-040) supplemented with Mesenchymal Stem Cell Growth Kit for Adipose and Umbilical-derived MSCs – Low Serum (#500-030), herein referred to as “complete MSC media”, and subcultured at a confluence of ≈80%. hTERT immortalized green fluorescent protein-expressing human aortic endothelial cells (GFP-hAECs; TeloHAEC-GFP, CRL-4054, ATCC) were routinely maintained as per manufacturers instruction in Vascular Cell Basal Medium (#100-0303) supplemented with Endothelial Cell Growth Kit – VEGF (#100-041) and 33 μM phenol red (#999-001), herein referred to as “complete vascular cell media”, and subcultured at a confluence of ≈80%.

Techniques: Fluorescence, Standard Deviation, Imaging, Co-Culture Assay, Staining, Expressing

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Fabricating Microfluidic Co-Cultures of Immortalized Cell Lines Uncovers Robust Design Principles for the Simultaneous Formation of Patterned, Vascularized, and Stem Cell-Derived Adipose Tissue.

doi: 10.1002/smll.202501834

Figure Lengend Snippet: Figure 2. GFP-hAECs and hAD-MSCs spontaneously form robust and phenotypical 3D hydrogel-embedded microvascular networks with a morphological dependence on cell seeding ratio. A. Fluorescence images of GFP-hAECs as microvascular networks formed after 7-days fibrin hydrogel-embedded microfluidic device culture in complete vascular cell media with GFP-hAEC:hAD-MSC seeding ratios of 5:1 (i), 2:1 (ii), 1:1 (iii) and 1:2 (iv, scale bars = 300 μm). B) Quantitative analysis of vessel network morphology detailing the number of network junctions (i), vessel end points (ii), total vessel length (iii) and total vessel area (iv) within a 1.33 × 1.33 mm field of view (n = 3 biological replicas, mean ± standard deviation). Results were compared via an ordinary one-way ANOVA followed by a Tukey multiple comparisons test (𝛼= 0.05). p > 0.05 (ns), p ≤0.05 (*), p ≤0.01 (**), p ≤0.001 (***) and p ≤0.0001 (****). C) Time lapse imaging of GFP-hAECs in 7-day vasculogenesis co-culture with a seeding ratio of 1:1 after 0 (i), 24 (ii), 49 (iii) and 153 h (iv) post-seeding (scale bars = 300 μm). D) Quantitative analysis of every tenth time-lapse image (28 of 274 images total) of the entire 7-day culture period, with graphs depicting the number of network junctions (i), vessel end points (ii) and total vessel length (iii) within a 1.33 × 1.33 mm field of view (mean ± standard deviation). E) Immunocytochemical characterization of a 1:1 seeding ratio sample after 7-days culture, detailing the nuclear counter stain DAPI (i), endogenous GFP expression (ii), and detection of CD31 (iii), 𝛼SMA (iv), CD90 (v) and F-actin (vi, scale bars = 100 μm). F) DAPI nuclear counter stain, CD31 and F-actin sample cross sections demonstrating vessel patency (scale bar = 20 μm). Images (E.i, ii, vi and F), (E.iii and iv) and v represent three independent replicate cultures. EC: endothelial cell, MSC: mesenchymal stem cell, DAPI: 4′,6-diamidino-2-phenylindole, GFP: green fluorescent protein, CD31: cluster of differentiation 31, 𝛼SMA: alpha smooth muscle actin, CD90: cluster of differentiation 90, F-actin: filamentous actin.

Article Snippet: Cell Culture and Imaging: This project was supported by The University of Queensland’s Human Research Ethics Committee through approval 2021/HE002698: “Tissue culturemodels for in vitro optimization of engineered biomaterial scaffolds and bioprocess techniques”, as well as The University of Queensland’s Institutional Biosafety Committee through approval IBC/602E/ChemEng/2023: “Risk group 2 immortalized cell line activities.” Cell Culture and Imaging—Routine Cell Line Maintenance: Human telomerase reverse transcriptase (hTERT) immortalized adipose-derived mesenchymal stem cells (hAD-MSCs; ASC52telo, SCRC-4000, American Type Culture Collection ATCC) were routinely maintained as per manufacturer’s instructions in MSC Basal Medium (#500-040) supplemented with Mesenchymal Stem Cell Growth Kit for Adipose and Umbilical-derived MSCs – Low Serum (#500-030), herein referred to as “complete MSC media”, and subcultured at a confluence of ≈80%. hTERT immortalized green fluorescent protein-expressing human aortic endothelial cells (GFP-hAECs; TeloHAEC-GFP, CRL-4054, ATCC) were routinely maintained as per manufacturers instruction in Vascular Cell Basal Medium (#100-0303) supplemented with Endothelial Cell Growth Kit – VEGF (#100-041) and 33 μM phenol red (#999-001), herein referred to as “complete vascular cell media”, and subcultured at a confluence of ≈80%.

Techniques: Fluorescence, Standard Deviation, Imaging, Co-Culture Assay, Staining, Expressing

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Fabricating Microfluidic Co-Cultures of Immortalized Cell Lines Uncovers Robust Design Principles for the Simultaneous Formation of Patterned, Vascularized, and Stem Cell-Derived Adipose Tissue.

doi: 10.1002/smll.202501834

Figure Lengend Snippet: Figure 4. GFP-hAEC and hAD-MSC gradient co-culture supports the co-formation of microvascular networks and differentiation with vascular network- enhanced adipogenesis. A) Color bright field microscope images of 1:1 seeding ratio microfluidic device cultures after 17- (i) and 31-days (ii) gradient culture and green fluorescence after 31-days culture (iii, scale bars = 500 μm). High magnification monochromatic bright field image of the culture compartment (iv, scale bar = 50 μm). B) Color bright field microscope images of 0:1 seeding ratio microfluidic device cultures after 17- (i) and 31-days (ii) gradient culture and green fluorescence after 31-days culture (iii, scale bars = 500 μm). High magnification monochromatic brightfield image of the culture compartment (iv, scale bar = 50 μm). C) LipidTOX staining of 1:1 seeding ratio cultures at day-31 at low magnification (i, scale bar = 50 μm) and high magnification with F-actin (green) and GFP (ii, scale bar = 30 μm). D) Quantitative analysis of lipid coverage across the culture compartment at days-17 and -31 comparing seeding ratios of 0:1, 1:2, 1:1, 2:1 (n = 3 biological replicas, mean ± standard deviation). Results were compared via an ordinary two-way ANOVA followed by a Tukey multiple comparisons test (𝛼= 0.05). E) Immunocytochemical detection of 1:1 co-cultures at day-17 for PPARG with images depicting the nuclear counter stain DAPI (i), PPARG (ii) and GFP-hAECs (iii, scale bars = 500 μm). F) Percentage lipid coverage localized to each third of the culture compartment defined as vasculogenesis, middle and adipogenesis (inset). Seeding ratios of 0:1, 1:2, 1:1 and 2:1 were investigated at days-17 and -31 of culture (n = 3 biological replicas, mean ± standard deviation). Results were compared via an ordinary one-way ANOVA followed by a Tukey multiple comparisons test (𝛼= 0.05). p > 0.05 (ns), p ≤0.05 (*), p ≤0.01 (**), p ≤0.001 (***) and p ≤0.0001 (****). G) Lipid coverage was similarly quantified in 370 μm increments across the entire length of the culture compartment at day-7 and -31 of culture and plotted for seeding ratios of 0:1, 1:2, 1:1 and 2:1 (n = 3 biological replicas, mean). EC: endothelial cell, MSC: mesenchymal stem cell, D: day, F-actin: filamentous actin, DAPI: 4′,6-diamidino-2-phenylindole, PPARG: peroxisome proliferator-activated receptor gamma, GFP: green fluorescent protein.

Article Snippet: Cell Culture and Imaging: This project was supported by The University of Queensland’s Human Research Ethics Committee through approval 2021/HE002698: “Tissue culturemodels for in vitro optimization of engineered biomaterial scaffolds and bioprocess techniques”, as well as The University of Queensland’s Institutional Biosafety Committee through approval IBC/602E/ChemEng/2023: “Risk group 2 immortalized cell line activities.” Cell Culture and Imaging—Routine Cell Line Maintenance: Human telomerase reverse transcriptase (hTERT) immortalized adipose-derived mesenchymal stem cells (hAD-MSCs; ASC52telo, SCRC-4000, American Type Culture Collection ATCC) were routinely maintained as per manufacturer’s instructions in MSC Basal Medium (#500-040) supplemented with Mesenchymal Stem Cell Growth Kit for Adipose and Umbilical-derived MSCs – Low Serum (#500-030), herein referred to as “complete MSC media”, and subcultured at a confluence of ≈80%. hTERT immortalized green fluorescent protein-expressing human aortic endothelial cells (GFP-hAECs; TeloHAEC-GFP, CRL-4054, ATCC) were routinely maintained as per manufacturers instruction in Vascular Cell Basal Medium (#100-0303) supplemented with Endothelial Cell Growth Kit – VEGF (#100-041) and 33 μM phenol red (#999-001), herein referred to as “complete vascular cell media”, and subcultured at a confluence of ≈80%.

Techniques: Co-Culture Assay, Microscopy, Staining, Standard Deviation

Journal: Small (Weinheim an der Bergstrasse, Germany)

Article Title: Fabricating Microfluidic Co-Cultures of Immortalized Cell Lines Uncovers Robust Design Principles for the Simultaneous Formation of Patterned, Vascularized, and Stem Cell-Derived Adipose Tissue.

doi: 10.1002/smll.202501834

Figure Lengend Snippet: Figure 5. Proven inducers of lipolysis do not inhibit lipid formation in GFP-hAEC and hAD-MSC gradient co-cultures. A) A schematic representing the interactome of vascular PTEN-mediated lipolysis proposed by Monelli et al,[4] the PTEN-inhibiting effect of VO-OHpic and lipolysis-enhancing effect of spermidine. Created with BioRender.com. B) Monochromatic brightfield images of day-31 1:1 seeding ratio cultures without inhibiting supplements (i) with 50 nM VO-OHpic (ii) and 1 μM spermidine (iii); and 0:1 seeding ratio cultures without inhibiting supplements (iv, scale bars = 500 μm). C) Quantitative analysis of lipid coverage across the culture compartment after 31-days gradient culture comparing seeding ratios of 0:1 and 1:1 (n = 3 biological replicas for spermidine and for VO-OHpic, n = 2 control replicates, mean ± standard deviation). 𝛽-AR: beta-adrenaline receptor, PI3K: phosphoinositide 3-kinase. PIP2: phosphatidylinositol 4,5-bisphosphate, PIP3: phosphatidylinositol (3,4,5)-trisphosphate, mTOR: mammalian target of rapamycin, PTEN: phosphatase and tensin homolog, VO-OHpic: hydroxyl(oxo)vanadium 3-hydroxypiridine-2-carboxylic acid, EC: endothelial cell, MSC: mesenchymal stem cell.

Article Snippet: Cell Culture and Imaging: This project was supported by The University of Queensland’s Human Research Ethics Committee through approval 2021/HE002698: “Tissue culturemodels for in vitro optimization of engineered biomaterial scaffolds and bioprocess techniques”, as well as The University of Queensland’s Institutional Biosafety Committee through approval IBC/602E/ChemEng/2023: “Risk group 2 immortalized cell line activities.” Cell Culture and Imaging—Routine Cell Line Maintenance: Human telomerase reverse transcriptase (hTERT) immortalized adipose-derived mesenchymal stem cells (hAD-MSCs; ASC52telo, SCRC-4000, American Type Culture Collection ATCC) were routinely maintained as per manufacturer’s instructions in MSC Basal Medium (#500-040) supplemented with Mesenchymal Stem Cell Growth Kit for Adipose and Umbilical-derived MSCs – Low Serum (#500-030), herein referred to as “complete MSC media”, and subcultured at a confluence of ≈80%. hTERT immortalized green fluorescent protein-expressing human aortic endothelial cells (GFP-hAECs; TeloHAEC-GFP, CRL-4054, ATCC) were routinely maintained as per manufacturers instruction in Vascular Cell Basal Medium (#100-0303) supplemented with Endothelial Cell Growth Kit – VEGF (#100-041) and 33 μM phenol red (#999-001), herein referred to as “complete vascular cell media”, and subcultured at a confluence of ≈80%.

Techniques: Control, Standard Deviation