bulkRNAseq of immortalized HUVEC cells isolated from MVNs in devices on day 15, 8 days after flow
For each timepoint analyzed, the fibrin hydrogel containing the MVNs was cut out of the devices and placed in 5 mL of 100 g mL-1 Liberase (Millipore Sigma), 100 g mL-1 DNase I (Sigma Aldrich), and 9.2 g mL-1 Elastase (Thermo Fischer Scientific) in DMEM (Thermo Fischer Scientific) for 75 minutes at 37oC with intermittent agitation. Cells from 3-4 devices were pooled for each replicate. Dead cells were removed using the Dead Cell Removal Kit and an OctoMACS Separator (Miltenyi Biotec), cells were incubated with CD31 MicroBeads (CD31 MicroBead Kit, Miltenyi Biotec) and HUVECs and FBs were sorted using the OctoMACS Separator (Miltenyi Biotec). The sorted cells were lysed with TRIzol (Thermo Fischer) and stored at -80oC. Once the cell lysates from all timepoints were collected, the lysates were thawed, and 0.2 mL of chloroform (Millipore Sigma) per 1 mL of TRIzol reagent was added, the samples were centrifuged for 15 minutes at 12,000 x g at 4oC, and the aqueous phase was collected. The samples were then prepared using the Preparation of Cell Pellets protocol for RNA extraction using the chemagic 360 (Revvity).
SUB15245623
D24-152081