Journal: bioRxiv

Article Title: Surface potential as a strong early osteogenic trigger via mechanotransduction and calcium accumulation

doi: 10.64898/2026.04.26.720950

Figure Lengend Snippet: Colorimetric staining and quantification of ALP activity in hBM-MSCs cultured for seven days on collagen type I-coated (a) and fibronectin-coated (b) substrates. Results are expressed as mean ± SD (n=3). Data were analyzed assuming a Gaussian (normal) distribution and equal variances across groups; statistical differences were assessed using one-way ANOVA. Only p-values <0.1 are shown. Scale bar represents 200 µm.

Article Snippet: hBM-MSCs (Cytion, 300665) were seeded at an approximate density of 5.000 cells/cm 2 on the different surfaces in basal medium (DMEM; 11995-065, Gibco), 10% fetal bovine serum (FBS; 11560636, Gibco), 1% Glutamax (13462629, Gibco) and 1% antibiotics (11548876, Gibco), and after 24 hours the culture medium was replaced with osteogenic medium (Basal medium supplemented with 0,28 mM ascorbic acid (A4544, Sigma-Aldrich), 10 mM β-glycerol-2-phosphate (G9422, Sigma-Aldrich), and 10 nM dexamethasone (D4902, Sigma-Aldrich)).

Techniques: Staining, Activity Assay, Cell Culture

Journal: bioRxiv

Article Title: Surface potential as a strong early osteogenic trigger via mechanotransduction and calcium accumulation

doi: 10.64898/2026.04.26.720950

Figure Lengend Snippet: Fluorescence images of hBM-MSCs cultured on β-PVDF films coated with either collagen type I (a) or fibronectin (b) and corresponding morphological features. Nuclei are stained in blue, vinculin in green and F-actin in red. Results are expressed as mean ± SD (n=3). Only p-values <0.1 are shown. Scale bars represent 200 µm.

Article Snippet: hBM-MSCs (Cytion, 300665) were seeded at an approximate density of 5.000 cells/cm 2 on the different surfaces in basal medium (DMEM; 11995-065, Gibco), 10% fetal bovine serum (FBS; 11560636, Gibco), 1% Glutamax (13462629, Gibco) and 1% antibiotics (11548876, Gibco), and after 24 hours the culture medium was replaced with osteogenic medium (Basal medium supplemented with 0,28 mM ascorbic acid (A4544, Sigma-Aldrich), 10 mM β-glycerol-2-phosphate (G9422, Sigma-Aldrich), and 10 nM dexamethasone (D4902, Sigma-Aldrich)).

Techniques: Fluorescence, Cell Culture, Staining

Journal: bioRxiv

Article Title: Surface potential as a strong early osteogenic trigger via mechanotransduction and calcium accumulation

doi: 10.64898/2026.04.26.720950

Figure Lengend Snippet: MYPT1 phosphorylation in hBM-MSCs cultured on collagen type I-coated (a) and fibronectin-coated (b) β-PVDF films of varying surface potential. Results are expressed as mean ± SD (n=3). Only p-values <0.1 are shown.

Article Snippet: hBM-MSCs (Cytion, 300665) were seeded at an approximate density of 5.000 cells/cm 2 on the different surfaces in basal medium (DMEM; 11995-065, Gibco), 10% fetal bovine serum (FBS; 11560636, Gibco), 1% Glutamax (13462629, Gibco) and 1% antibiotics (11548876, Gibco), and after 24 hours the culture medium was replaced with osteogenic medium (Basal medium supplemented with 0,28 mM ascorbic acid (A4544, Sigma-Aldrich), 10 mM β-glycerol-2-phosphate (G9422, Sigma-Aldrich), and 10 nM dexamethasone (D4902, Sigma-Aldrich)).

Techniques: Phospho-proteomics, Cell Culture

Journal: bioRxiv

Article Title: Surface potential as a strong early osteogenic trigger via mechanotransduction and calcium accumulation

doi: 10.64898/2026.04.26.720950

Figure Lengend Snippet: Fluorescence images of hBM-MSCs cultured on β-PVDF films coated with either collagen type I (a) or fibronectin (b) immunostained against YAP and counterstained with Hoechst 33342, and the corresponding quantifications (right panels). Scale bars represent 200 µm. Results are expressed as mean ± SD (n=3). Only p-values <0.1 are shown.

Article Snippet: hBM-MSCs (Cytion, 300665) were seeded at an approximate density of 5.000 cells/cm 2 on the different surfaces in basal medium (DMEM; 11995-065, Gibco), 10% fetal bovine serum (FBS; 11560636, Gibco), 1% Glutamax (13462629, Gibco) and 1% antibiotics (11548876, Gibco), and after 24 hours the culture medium was replaced with osteogenic medium (Basal medium supplemented with 0,28 mM ascorbic acid (A4544, Sigma-Aldrich), 10 mM β-glycerol-2-phosphate (G9422, Sigma-Aldrich), and 10 nM dexamethasone (D4902, Sigma-Aldrich)).

Techniques: Fluorescence, Cell Culture

Journal: bioRxiv

Article Title: Surface potential as a strong early osteogenic trigger via mechanotransduction and calcium accumulation

doi: 10.64898/2026.04.26.720950

Figure Lengend Snippet: Volcano plots with the −log 10 (p-value) plotted against their respective log 2 (fold change) of genes differentially expressed in hBM-MSCs, Venn diagrams and histogram plots showing genes that are down- or upregulated (p<0.05) in hBM-MSCs cultured on the indicated surfaces for 24 hours (a) or four days (b). Circle area in a and b is proportional to the number of genes. GSEA of the cells cultured on the different surfaces for 24 hours (c) and 4 days (d).

Article Snippet: hBM-MSCs (Cytion, 300665) were seeded at an approximate density of 5.000 cells/cm 2 on the different surfaces in basal medium (DMEM; 11995-065, Gibco), 10% fetal bovine serum (FBS; 11560636, Gibco), 1% Glutamax (13462629, Gibco) and 1% antibiotics (11548876, Gibco), and after 24 hours the culture medium was replaced with osteogenic medium (Basal medium supplemented with 0,28 mM ascorbic acid (A4544, Sigma-Aldrich), 10 mM β-glycerol-2-phosphate (G9422, Sigma-Aldrich), and 10 nM dexamethasone (D4902, Sigma-Aldrich)).

Techniques: Cell Culture

Journal: bioRxiv

Article Title: Surface potential as a strong early osteogenic trigger via mechanotransduction and calcium accumulation

doi: 10.64898/2026.04.26.720950

Figure Lengend Snippet: (a) Immunostaining of hBM-MSCs cultured on the different β-PVDF surfaces with glutaraldehyde-crosslinked collagen type I coating and corresponding morphologic analysis (b). MYTP1 phosphorylation (c) and YAP translocation (d and e) in hBM-MSCs cultured on the substrates. Only p-values <0.1 are shown. Scale bars represent 200 µm. Results are expressed as mean ± SD (n=3).

Article Snippet: hBM-MSCs (Cytion, 300665) were seeded at an approximate density of 5.000 cells/cm 2 on the different surfaces in basal medium (DMEM; 11995-065, Gibco), 10% fetal bovine serum (FBS; 11560636, Gibco), 1% Glutamax (13462629, Gibco) and 1% antibiotics (11548876, Gibco), and after 24 hours the culture medium was replaced with osteogenic medium (Basal medium supplemented with 0,28 mM ascorbic acid (A4544, Sigma-Aldrich), 10 mM β-glycerol-2-phosphate (G9422, Sigma-Aldrich), and 10 nM dexamethasone (D4902, Sigma-Aldrich)).

Techniques: Immunostaining, Cell Culture, Phospho-proteomics, Translocation Assay

Journal: bioRxiv

Article Title: Surface potential as a strong early osteogenic trigger via mechanotransduction and calcium accumulation

doi: 10.64898/2026.04.26.720950

Figure Lengend Snippet: Immunofluorescent images (a) and corresponding morphologic analysis (b) of hBM-MSCs treated with the ROCK inhibitor Y-27632 for 24 hours. YAP immunolocalization (c) and translocation (d) in treated cells. Only p-values <0.1 are shown. Scale bars represent 300 µm. Results are expressed as mean ± SD (n=4).

Article Snippet: hBM-MSCs (Cytion, 300665) were seeded at an approximate density of 5.000 cells/cm 2 on the different surfaces in basal medium (DMEM; 11995-065, Gibco), 10% fetal bovine serum (FBS; 11560636, Gibco), 1% Glutamax (13462629, Gibco) and 1% antibiotics (11548876, Gibco), and after 24 hours the culture medium was replaced with osteogenic medium (Basal medium supplemented with 0,28 mM ascorbic acid (A4544, Sigma-Aldrich), 10 mM β-glycerol-2-phosphate (G9422, Sigma-Aldrich), and 10 nM dexamethasone (D4902, Sigma-Aldrich)).

Techniques: Translocation Assay

Journal: Journal of orthopaedic research : official publication of the Orthopaedic Research Society

Article Title: Expression of neural and neurotrophic markers in nucleus pulposus cells isolated from degenerated intervertebral disc.

doi: 10.1002/jor.22098

Figure Lengend Snippet: Figure 2. Mesenchymal Profile of Human NPs-IVD: (A) Immunophenotypic profile, by citofluorimetric analysis, of nucleus pulposus from 14 human degenerated intervertebral discs. (B) NPs-IVD show mesenchymal properties under chondrogenic, adipogenic, and osteogenic differentiation.

Article Snippet: Human Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN) was used to induce adipogenic, chondrogenic, and osteogenic differentiation.

Techniques:

Journal: Molecular Therapy. Nucleic Acids

Article Title: Ratio of miRNA-29 to miRNA-199 expression coordinates mesenchymal stem cell repair of bleomycin-induced pulmonary injury

doi: 10.1016/j.omtn.2025.102461

Figure Lengend Snippet: Bleomycin-induced lung injury, which is reduced after adipose-derived mesenchymal stem cell instillation (A) Representative μCT transverse and coronal lung sections acquired from aged (22-month-old) male C57BL/6 mice at baseline (left) and 7 days following intratracheal bleomycin (BLM, 2.0 U/kg) administration (right) demonstrating increased lung density and loss of airspaces. (B) Saline treatment did not result in evidence of lung injury on μCT scan at baseline (left) or 7 days post-instillation (right). Histological sections of lung tissue collected at day 21 post-BLM were stained with Masson’s trichrome as described in . (C and D) Representative photomicrographs (20× and 40× magnifications) of lung sections from saline-treated control mice (C) and BLM-treated mice (D). (E) Infusion of adipose-derived mesenchymal stem cells (ASCs) 12 days post-BLM instillation resulted in reduced severity of pulmonary fibrosis (PF). (F) Degree of PF on histological sections was measured by semi-quantitative Ashcroft score as described in . BLM-induced lung injury resulted in increased Ashcroft score compared to saline controls. Infusion with ASCs 12 days post-BLM injury resulted in decreased Ashcroft score. (G) Intratracheal BLM instillation increased lung collagen content as measured by hydroxyproline assays as described in . Mice treated with ASCs on day 12 post-BLM had decreased lung collagen content compared to BLM-only controls. Each data point represents an individual biological replicate (mouse); n = 6–10 mice/group. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001. (H) Ratio of pAKT to AKT protein expression in lung tissue of mice was quantified by western blot analysis at day 21 post-BLM sacrifice. Aged C57BL/6 mice treated with intratracheal BLM demonstrated increased pAKT/AKT protein expression compared to saline-treated controls. Lungs from mice treated with intravenous infusion of ASCs 12 days post-BLM-induced injury demonstrated decreased expression of pAKT/AKT compared to BLM-only group. Inset shows a representative western blot and β-actin loading control. Data are graphed as individual biological replicates ( n = 6–8 mice/group); ∗ p < 0.05.

Article Snippet: For mesenchymal differentiation potential, the Mouse Mesenchymal Stem Cell Functional Identification Kit (R&D Systems, Minneapolis, MN) was used according to the manufacturer’s instructions.

Techniques: Derivative Assay, Saline, Staining, Control, Expressing, Western Blot

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