Hcmec

An undifferentiated human iPSC line (Thermo Fisher Scientific) was routinely maintained in complete Essential 8 (E8) cell culture medium (Thermo Fisher Scientific) on 6-well plates coated with 1 : 100 growth factor–reduced Matrigel (R&D Systems). The cells were passaged every 3–4 days using 0.5 mM EDTA in D-PBS (Thermo Fisher Scientific) and re-plated in E8 medium supplemented with 2 μM of the ROCK inhibitor Thiazovivin (Stratech Scientific) for the first 24 h following passaging. Human cardiac microvascular endothelial cells (hCMEC; PromoCell) were grown in PromoCell Cell Growth Medium and passaged using PromoCell DetachKit. The media was changed every two to three days. Human cardiac fibroblasts (hCFib; kindly provided by Prof. Terracciano) were cultured in DMEM high glucose (Thermo Fisher Scientific) supplement with 10% (v/v) FBS (Thermo Fisher Scientific) and dissociated using 0.25% trypsin (Sigma-Aldrich).

The primary human cardiac microvascular endothelial cells (HCMEC) were obtained from PromoCell (Heidelberg, Germany) and cultured according to the manufacturer’s protocol in endothelial cell growth medium MV. Medium was changed every 2–3 days while subculturing was performed at 70–90% confluence using the detach kit from PromoCell.
The polymer films disks of 14 mm diameter were generated by punching. Disinfection was achieved by incubation in 70% ethanol followed by washing with sterile water and equilibration in medium. Material disks were placed into the wells of a 24 well PS plate and fixed with Teflon rings. Cell-culture treated polystyrene (TCPS) plates were used as controls for ideal cell growth. Cells were seeded on top of the disks (5⋅10⁴ cells per disk) and grown for 24 h under standard cell culture conditions. Staining was done using the Live/Dead Cell Staining Kit II (PromoKine, Heidelberg, Germany) followed by fluorescence microscopy using the IX 51 microscope. At least 2 independent samples were visualized per material and at least 5 images of different areas per sample were recorded and counted.

Primary ventricular HCMEC (PromoCell, Heidelberg, Germany C-12286), 51-year-old male (Lot #470Z011.7), 63-year-old nondiabetic (Lot no. 447Z026.3), or 63-year-old type 2 diabetic (Lot no. 451Z015.1) Caucasian males were cultured in PromoCell microvascular media MV (C-22020) or MV2 (C-22022), supplemented with their corresponding supplement mixes (C-39225 or C-39226, respectively) and 0.1% penicillin/streptomycin (PS). Cells were kept in a humidified incubator at 37 °C and 5% CO₂ and used for experiments between passages 2 and 8.
Mouse cardiac microvascular endothelial cells (MCMEC) (Cedarlane, Burlington, ON, Canada CLU510) were cultured according to the provider’s instructions in DMEM with 10 mM, 10 mmol/L HEPES, 1% PS, and 5% fetal bovine serum (FBS).
THP-1 monocytes (ATCC, Manassas, VA, United States TIB-202) were maintained in RPMI medium with 10% FBS, 0.05 mM 2-mercaptoethanol, and 1% PS.

Microvessels were fabricated in collagen and hylauronan (HA) composite hydrogels polymerized inside polydimethylsiloxane (PDMS)-based microfluidic devices fabricated using soft lithography^{43 (link)}. p20–p23 human cerebral microvascular endothelial cells (hCMEC/D3) and p7–p15 GFP-labeled human brain vascular pericytes (hBVP) (Neuromics) were seeded in the hydrogels at densities of 2 M/mL and 0.4 M/mL, respectively. These ratios were obtained from a previous study that used co-cultures of human blood outgrowth endothelial cells and human pericytes to form microvasculature networks^{13 (link)}. hCMEC/D3 were cultured in Endothelial Cell Basal Medium (PromoCell) supplemented with 5 µg/mL ascorbic acid (Sigma), 1 ng/mL hBFGF (Sigma), 1/100 chemically defined lipid concentrate (Thermo Fisher), 5% fetal bovine serum (VWR Life Science), 10 mM HEPES (Quality Biological), 1.4 µM hydrocortisone (Sigma), and 1% penicillin-streptomycin (Corning). hBVP were cultured in DMEM (Corning) supplemented with 10% FBS, 1% penicillin-steptomycin, and 1X MEM Amino Acid Solution (Thermo Fisher). Final hydrogel formulation concentrations consisted of 3 mg/mL HA (Sigma), 5 mg/mL collagen type I (MP Biomedical), and 0.85–1 mg/mL Matrigel (Corning). These reagents were combined with 0.1 M sodium hydroxide (NaOH) and 10x phosphate buffer solution (PBS) to facilitate polymerization and maintain physiological pH.