Journal: Advanced healthcare materials

Article Title: Precision Culture Scaling to Establish High-Throughput Vasculogenesis Models.

doi: 10.1002/adhm.202400388

Figure Lengend Snippet: Figure 1. a) Schematic summary of precision culture scaling (PCS-X) to customize high-throughput 3D tissue and disease models. PCS-X comprises design of experiments (DOE) methods to systematically adjust the composition and automated and parallelized preparation of cultures, the collection and analysis of quantitative readouts, and multiple linear regression (MLR) modeling of the data to identify individual and interactive effects of the investigated parameters to instruct further adjustment. Exemplifying the approach, high-throughput 3D vasculogenesis models were developed from hydrogel culture-based protocols using human umbilical vein endothelial cells or human retinal microvascular endothelial cells in mono- and cocultures with mesenchymal stromal cells or retinal microvascular pericytes, respectively. The hydrogels were made of multi-armed poly(ethylene glycol) and the sulfated glycosaminoglycan heparin (starPEG-sGAG hydrogels), functionalized with covalently bound cell-adhesive RGDSP peptides (to sGAG) and sGAG-complexed growth factors. b) Parallelized hydrogel culture fabrication: The stock solutions, containing the starPEG-peptide conjugate and the sGAG (heparin-maleimide)/RGDSP peptide/growth factor/cell mixture, respectively, were automatically transferred and mixed in a 96-well plate with V-shaped wells, then transferred into a low-volume 384-well plate. c) Image analysis of vasculogenesis was performed by a dedicated 3D image analysis routine (exemplary shown image displays a HUVEC monoculture stained for F-Actin): Confocal input images were filtered (Gaussian filter) to minimize over-quantification of subcellular features, a 3D rendition of these structures was computed, masked, and subjected to a filament algorithm generating skeletonized trajectories of cellular structures. Scale bar 200 μm. d) DOE methods were used to assess effects of three crucial culturing parameters on vasculogenesis of hydrogel-embedded endothelial cells in balanced experimental designs (central composite designs). The experimental data were analyzed by MLR, generating robust models to predict endothelial cell vasculogenesis across the parameter space studied.

Article Snippet: Human telomerase reverse transcriptase (hTert) immortalized human retinal microvascular cells (HRMVECs) and hTert immortalized GFPexpressing human retinal microvascular pericytes (HRMVPs), as well as non-fluorescent hTert immortalized HRMVPs (Angio-Proteomie, US), were cultured on tissue culture flasks pre-coated with quick coating solution (Angio-Proteomie, US) in endothelial growth medium and pericyte growth medium (Angio-Proteomie, US) at 37 °C and 5% CO2 in a humidified incubator, respectively.

Techniques: High Throughput Screening Assay, Adhesive, Staining

Journal: Advanced healthcare materials

Article Title: Precision Culture Scaling to Establish High-Throughput Vasculogenesis Models.

doi: 10.1002/adhm.202400388

Figure Lengend Snippet: Figure 5. Effects of vasculogenesis inhibitors on interactions between HRMVECs and HRMVPs. a) 3D image analysis routine to quantify cell-cell contacts (Imaris, Oxford Instruments). 1) The relevant channels are filtered (Gaussian), 2) a channel-specific surface algorithm is performed, 3) the Imaris Xtension ‘surface surface contact area’ is applied for creating a surface at the junction of both cell types. 4) Close-up of contacts (yellow) between HRMVECs (CellTracker Orange, green) and HRMVPs (GFP, orange) (scale bar 1–3. 200 μm and 4. 50 μm). Total filament lengths for b) HRMVECs and c) cocultured HRMVPs. d) Percentage of all HRMVECs surfaces in contact with HRMVPs. e) Total number of contacts. f) Total surface area of contacts. Data presented as violin plots with horizontal lines indicating quartiles 1–3 (n = 4–6). Asterisks indicate multiplicity adjusted P values of one-way ANOVA with post hoc Tukey test, comparing inhibitor treatments to respective vehicle controls; *P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001.

Article Snippet: Human telomerase reverse transcriptase (hTert) immortalized human retinal microvascular cells (HRMVECs) and hTert immortalized GFPexpressing human retinal microvascular pericytes (HRMVPs), as well as non-fluorescent hTert immortalized HRMVPs (Angio-Proteomie, US), were cultured on tissue culture flasks pre-coated with quick coating solution (Angio-Proteomie, US) in endothelial growth medium and pericyte growth medium (Angio-Proteomie, US) at 37 °C and 5% CO2 in a humidified incubator, respectively.

Techniques:

Journal: Cell Death & Disease

Article Title: Cinacalcet-mediated activation of the CaMKKβ-LKB1-AMPK pathway attenuates diabetic nephropathy in db/db mice by modulation of apoptosis and autophagy

doi: 10.1038/s41419-018-0324-4

Figure Lengend Snippet: a The changes of [Ca 2+ ]i in HGECs exposed to cinacalcet and high-glucose media. To determine whether the addition of cinacalcet might modulate [Ca 2+ ]i in HGECs, FURA-2AM-loaded HGECs were stimulated using different concentrations (15, 100 nM) of cinacalcet in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) media. The area under curve (AUC) was estimated from the baseline of normalized data (at the point of injection) to a fluorescence level and between time points of injection (0 min) and 10 min. The peak of the curve was measured as highest value of the curve. The peak amplitude and AUC of [Ca 2+ ]i were significantly increased by cinacalcet in dose-dependent manners in both LG and HG media. In Fig. 1a, the arrow denotes the administration of cinacalcet (15 and 100 nM, respectively) ( n = 6 independent experiments in each experiments). * p < 0.05; ** p < 0.01 compared with LG and HG. b The changes of intracellular signaling in HGECs exposed to cinacalcet and high-glucose media. Representative immunofluorescent ( n = 6 independent experiments in each experiments) and western blot analyses ( n = 4 independent experiments in each experiments) of CaSR, CaMKKα/β, phospho-Ser 428 LKB1, and phospho-Thr 172 AMPK in the cultured HGECs in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) conditions with or without cinacalcet treatment (15 nM) and the quantitative analyses of the results are shown. * P < 0.05; ** P < 0.01 and # P < 0.001 compared with other groups

Article Snippet: Human glomerular endothelial cells (HGECs) were purchased from Anigio-Proteomie (Boston, MA) and subcultured in endo-growth media (Angio-Proteomie, Boston, MA).

Techniques: Injection, Fluorescence, Western Blot, Cell Culture

Journal: Cell Death & Disease

Article Title: Cinacalcet-mediated activation of the CaMKKβ-LKB1-AMPK pathway attenuates diabetic nephropathy in db/db mice by modulation of apoptosis and autophagy

doi: 10.1038/s41419-018-0324-4

Figure Lengend Snippet: The effect of cinacalcet on intracellular signaling for AMPK-eNOS oxidative stress and apoptosis in the HGECs cultured in low-glucose (LG; 5 mmol/l D-glucose) or high-glucose (HG; 30 mmol/l D-glucose) conditions with or without cinacalcet treatment (1, 5, 15 nM) ( a–d ). Representative Western blot analyses and quantitative analyses of total AMPK, phosphor-Thr 172 AMPK, total eNOS, phospho-Ser 1177 eNOS ( a , * P < 0.05 and ** P < 0.01 compared with LG control), SOD1 and SOD2 ( b , * P < 0.05 compared with other groups), dihydroethidium expression (as an oxidative stress marker; c , * P < 0.05 and # P < 0.001 compared with other groups), Bcl-2, Bax, and TUNEL-positive HGECs ( e , * P < 0.05 and ** P < 0.01 compared with other groups), and β-actin levels in the cultured HGECs and their quantitative analyses of the results are shown ( n = 4 independent experiments in each experiments). d The effect of BAPTA-AM (25 μM) on cinacalcet-indueced in the HGECs cultured in low-glucose or high-glucose (HG; 30 mmol/l D-glucose) with or without cinacalcet treatment (15 nM). Representative Western blot analyses and quantitative analyses of CaMKKβ, phospho-LKB1, and total LKB1 ( n = 4 independent experiments in each experiments). * P < 0.05 and ** P < 0.01 compared with LG control. f The changes of intracellular signaling related to autophagy in HGECs exposed to cinacalcet and high-glucose media. Representative Western blot analyses and quantitative analyses of beclin-1, LC3-II/LC3-I ratio, and β-actin levels in the cultured HGECs and their quantitative analyses of the results are shown ( n = 4 independent experiments in each experiments). Representative immunofluorescent analyses of LC3 punctae in HGECs and the quantitative analyses of the results are shown ( n = 6 independent experiments in each experiments). ** P < 0.01 compared with other groups

Article Snippet: Human glomerular endothelial cells (HGECs) were purchased from Anigio-Proteomie (Boston, MA) and subcultured in endo-growth media (Angio-Proteomie, Boston, MA).

Techniques: Cell Culture, Western Blot, Control, Expressing, Marker, TUNEL Assay

Journal: Cell Death & Disease

Article Title: Cinacalcet-mediated activation of the CaMKKβ-LKB1-AMPK pathway attenuates diabetic nephropathy in db/db mice by modulation of apoptosis and autophagy

doi: 10.1038/s41419-018-0324-4

Figure Lengend Snippet: Immunoblot for CaMKKβ, LKB1, phospho-AMPK, SIRT1 and phospho-Ser 1177 eNOS in AMPKα1 siRNA, AMPKα2 siRNA, or SIRT1 siRNA knock-down HGECs in a high-glucose environment with cinacalcet treatment ( a and b ). The cultured HGECs were transfected with a final concentration of 50 nM CaMKKβ and LKB1, α1 and α2-AMPK, SIRT1 siRNAs for 24-h by transfection reagent and treated with cinacalcet (15 nM) in high-glucose media. Representative Western blot analyses of CaMKKβ and phospho-Ser 428 LKB1 ( a ), as well as phospho-Thr 172 AMPK, total AMPK, SIRT1, phospho-Ser 1177 eNOS ( b ) and β-actin levels and the quantitative analyses of the results are also shown ( a and b , respectively) ( n = 4 independent experiments in each experiments).* P < 0.05, ** P < 0.01 compared with control siRNA with HG

Article Snippet: Human glomerular endothelial cells (HGECs) were purchased from Anigio-Proteomie (Boston, MA) and subcultured in endo-growth media (Angio-Proteomie, Boston, MA).

Techniques: Western Blot, Knockdown, Cell Culture, Transfection, Concentration Assay, Control

Journal: Cell reports

Article Title: Cancer-associated fibroblasts maintain critical pancreatic cancer cell lipid homeostasis in the tumor microenvironment

doi: 10.1016/j.celrep.2024.114972

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human pancreatic microvascular endothelial cells were cultured in Endothelial Cell Basal Medium (iXCells, MD-0010B) cultured for 48 h under 21% O 2 or 0.5% O 2 .

Techniques: Recombinant, Western Blot, Transfection, Reverse Transcription, Flow Cytometry, Bicinchoninic Acid Protein Assay, Plasmid Preparation, ROS Assay, Mass Spectrometry, shRNA, Control, Software, Microscopy, Real-time Polymerase Chain Reaction

Journal: Cancers

Article Title: CAFs and TGF-β Signaling Activation by Mast Cells Contribute to Resistance to Gemcitabine/Nabpaclitaxel in Pancreatic Cancer

doi: 10.3390/cancers11030330

Figure Lengend Snippet: CM-HMC-1 and CM-CAF did not alter GEM/NAB-dependent inhibition of tumor angiogenesis but reduced the efficacy on tumor invasion. ( a ) Capillary morphogenesis. Both CM-HMC-1 and CM-CAF did not alter angiogenesis as demonstrated by microvascular formation and did not reduce the antiangiogenic potential of GEM/NAB. Pictures represent three different experiments. ( b ) Invasion assay. Both CM-HMC-1 and CM-CAF altered tumor invasion, by inducing the decrese and the increase of MIA PaCa-2 invasion, respectively. Both conditioned media reduced the efficacy of GEM/NAB-dependent inhibion of tumor invasion as reported in the histogram plot showing the recovery of tumor invasion on CM-HMC-1/CM-CAF + GEM/NAB treated cells. The pictures represent three different experiments.

Article Snippet: HMC-1 human mast cell line-1 cells were kindly provided by Prof. L. Macchia, University of Bari, CAF cells were purchased from Vitro Biopharma, and MVEC human dermal microvascular endothelial cells were purchased from Lonza, Switzerland.

Techniques: Inhibition, Invasion Assay

Journal: ACS biomaterials science & engineering

Article Title: Vascular Network Formation by Human Microvascular Endothelial Cells in Modular Fibrin Microtissues

doi: 10.1021/acsbiomaterials.6b00274

Figure Lengend Snippet: Endothelial sprouting in bulk fibrin hydrogels. A) Fluorescent images of embedded MVEC stained red with UEA-1 in gels made with different MVEC:FB ratios and cultured in different medium volumes. B) Quantitation of the average sprout length in bulk gels (n=3). C) Quantitation of the number of sprouts >100 µm in length in bulk gels (n=3). Error bars represent standard deviation of the mean. * indicates statistical significance (*p<0.05, **p<0.01 and ***p<0.001).

Article Snippet: Human neonatal foreskin microvascular endothelial cells (MVEC) and normal human lung fibroblasts (FB, passage 9–12) were obtained from a commercial vendor (Lonza Inc., Walkersville, MD).

Techniques: Staining, Cell Culture, Quantitation Assay, Standard Deviation

Journal: ACS biomaterials science & engineering

Article Title: Vascular Network Formation by Human Microvascular Endothelial Cells in Modular Fibrin Microtissues

doi: 10.1021/acsbiomaterials.6b00274

Figure Lengend Snippet: Influence of cell ratios and culture media volume on total sprouts.

Article Snippet: Human neonatal foreskin microvascular endothelial cells (MVEC) and normal human lung fibroblasts (FB, passage 9–12) were obtained from a commercial vendor (Lonza Inc., Walkersville, MD).

Techniques:

Journal: ACS biomaterials science & engineering

Article Title: Vascular Network Formation by Human Microvascular Endothelial Cells in Modular Fibrin Microtissues

doi: 10.1021/acsbiomaterials.6b00274

Figure Lengend Snippet: Influence of cell ratios and culture media volume on average sprout length.

Article Snippet: Human neonatal foreskin microvascular endothelial cells (MVEC) and normal human lung fibroblasts (FB, passage 9–12) were obtained from a commercial vendor (Lonza Inc., Walkersville, MD).

Techniques:

Journal: ACS biomaterials science & engineering

Article Title: Vascular Network Formation by Human Microvascular Endothelial Cells in Modular Fibrin Microtissues

doi: 10.1021/acsbiomaterials.6b00274

Figure Lengend Snippet: Characterization of modular microtissues. A) Single MVEC:FB microtissues (1:1 and 1:3) under phase contrast showing microtissue morphology and embedded cells immediately after microbead fabrication. B) Single MVEC:FB microtissues (1:1 and 1:3) under fluorescence showing cell viability (green = live cells, red = dead cells). C) Quantitation of cell viability for a population of microtissues. D) Quantitation of total cells per unit volume for a population of microtissues. E) Total DNA in microtissues as a function of time. Best viewed in color. Error bars represent standard deviation of the mean. * indicates statistical significance (*p<0.05).

Article Snippet: Human neonatal foreskin microvascular endothelial cells (MVEC) and normal human lung fibroblasts (FB, passage 9–12) were obtained from a commercial vendor (Lonza Inc., Walkersville, MD).

Techniques: Fluorescence, Quantitation Assay, Standard Deviation

Journal: ACS biomaterials science & engineering

Article Title: Vascular Network Formation by Human Microvascular Endothelial Cells in Modular Fibrin Microtissues

doi: 10.1021/acsbiomaterials.6b00274

Figure Lengend Snippet: Flow cytometry analysis of MVEC and FB cell population in fibrin co-cultures over time. A,B) Mono-culture of MVEC and FB. C- H) MVEC and FB cell population in co-cultures over time. Error bars represent standard deviation of the mean. * indicates statistical significance (*p<0.01).

Article Snippet: Human neonatal foreskin microvascular endothelial cells (MVEC) and normal human lung fibroblasts (FB, passage 9–12) were obtained from a commercial vendor (Lonza Inc., Walkersville, MD).

Techniques: Flow Cytometry, Standard Deviation

Journal: ACS biomaterials science & engineering

Article Title: Vascular Network Formation by Human Microvascular Endothelial Cells in Modular Fibrin Microtissues

doi: 10.1021/acsbiomaterials.6b00274

Figure Lengend Snippet: Sprouting of neovessels from modular microtissues. A) Fluorescence images (green = fibrin, red = MVEC) at day 7 and 14 with different MVEC:FB ratios. B) Quantification of vessel area normalized to microtissue area. C) Box plot of vessel diameter. The solid center line in the box plot represents the median, the dotted center line in the box represents the mean, and the lower and upper boundaries of the box represent the 25th and 75th percentiles, respectively. Whiskers (error bars) above and below the box indicate the 90th and 10th percentiles. Large dots represent outliers.

Article Snippet: Human neonatal foreskin microvascular endothelial cells (MVEC) and normal human lung fibroblasts (FB, passage 9–12) were obtained from a commercial vendor (Lonza Inc., Walkersville, MD).

Techniques: Fluorescence

Journal: ACS biomaterials science & engineering

Article Title: Vascular Network Formation by Human Microvascular Endothelial Cells in Modular Fibrin Microtissues

doi: 10.1021/acsbiomaterials.6b00274

Figure Lengend Snippet: Vessel lumen diameter at day 7 and day 14 in embedded microtissue cultures

Article Snippet: Human neonatal foreskin microvascular endothelial cells (MVEC) and normal human lung fibroblasts (FB, passage 9–12) were obtained from a commercial vendor (Lonza Inc., Walkersville, MD).

Techniques:

Journal: ACS biomaterials science & engineering

Article Title: Vascular Network Formation by Human Microvascular Endothelial Cells in Modular Fibrin Microtissues

doi: 10.1021/acsbiomaterials.6b00274

Figure Lengend Snippet: Inosculation of MVEC neovessels. A) MVEC (red) networks (i) sprouted from microtissues (green) and formed branches (ii) with hollow lumens (iii, iv). B) Serial histological sections showed inosculation of adjacent MVEC vessels.

Article Snippet: Human neonatal foreskin microvascular endothelial cells (MVEC) and normal human lung fibroblasts (FB, passage 9–12) were obtained from a commercial vendor (Lonza Inc., Walkersville, MD).

Techniques:

Journal: ACS biomaterials science & engineering

Article Title: Vascular Network Formation by Human Microvascular Endothelial Cells in Modular Fibrin Microtissues

doi: 10.1021/acsbiomaterials.6b00274

Figure Lengend Snippet: Microtissues cultured in suspension aggregated to form larger tissue structures. A, B) By day 7, neovessels were evident in tissue masses. C, D) By day 14, networks of vessels had formed in tissue masses made with 1:3 MVEC:FB microtissues, but were less evident in those made at 1:1. E) Fluorescence staining and F) PECAM/CD-31 IHC confirmed that vessels within tissue structures were created by MVEC.

Article Snippet: Human neonatal foreskin microvascular endothelial cells (MVEC) and normal human lung fibroblasts (FB, passage 9–12) were obtained from a commercial vendor (Lonza Inc., Walkersville, MD).

Techniques: Cell Culture, Suspension, Fluorescence, Staining