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: