organism: Homo sapiens
description: Protocols: Investigations with human umbilical vein endothelial cells (HUVEC) were performed with written informed consent from six paras and following the ethical guidelines. Umbilical cords were collected after birth and stored at 2-8 °C. HUVEC were isolated with pre-warmed (37 °C) GibcoTM dispase solution (3.6-3.8 U/ml; Thermo Fisher Scientific, Darmstadt, Germany, 17105041) within one to four hours after birth. After incubation for one hour, the obtained cell suspension was centrifuged (5 min., 200 g), the supernatant discarded and the cell pellet resuspended in complete endothelial cell growth medium (PromoCell, Heidelberg, Germany, C-22210, Suppl. C-39210) supplemented with 5% heat inactivated fetal bovine serum (FBS; Thermo Fisher Scientific, 10500-064). HUVEC were seeded in an appropriate number and size of culture flasks (Sarstedt AG & Co., Numbrecht, Germany, 83.3911.002) and propagated in complete endothelial cell growth medium supplemented with 5% FBS. To facilitate cell attachment, the flasks have been coated with gelatin type B solution (Sigma-Aldrich, Saint-Louis, MO, USA, G9391) 2% in 0.1 M HCl. At a confluence of 95-100% HUVEC were dissociated with GibcoTM trypsin-EDTA 0.05% (Thermo Fisher Scientific, 25300-054). For Xellulin-based cell culture, Xell-Discs (Xellutec GmbH, Neuried, Germany, XRM-6w-1) have been coated with 0.5 ml Cellovations® rat tail (type I) collagen solution (750 μg/ml in 0.1% acetic acid; PELOBiotech GmbH, Martinsried, Germany, PB-LU-000-0003-100-f), washed two times with DPBS (1x) w/o Ca & Mg (Thermo Fisher Scientific, 14190-094) and then preincubated in complete endothelial cell growth medium supplemented with a total of 20% FBS overnight. Propagated HUVEC passage 1 were seeded at a density of 3.8x104 cells/cm2 per Xell-Disc and cultivated in complete endothelial cell growth medium supplemented with 1% FBS. The culture medium was replaced the next day and then twice a week. After three weeks and six weeks, respectively, HUVEC were detached with collagenase type IV (500 U/ml; Thermo Fisher Scientific, 17104-019). For standard cell culture, propagated HUVEC passage 1 were seeded with a density of 2.2x104 cells/cm2 in 6-well cell culture plates (Techno Plastic Products AG, Trasadingen, Switzerland, 92006) that have been coated with 1 ml Cellovations® rat tail (type I) collagen solution (750 μg/ml 0.1% acetic acid). The HUVEC were fed with complete endothelial cell growth medium supplemented with a total of 1% or 5% FBS. Medium was changed the day after. Confluent monolayers were harvested two days after cell seeding. HUVEC both on collagen-coated Xellulin and on collagen-coated plastic were maintained at 37 °C in a humidified atmosphere with 5% CO2. Native HUVEC were freshly isolated from six umbilical cords and then propagated (passage 0). Ten samples HUVEC passage 1 were cultured on Xellulin and twelve samples HUVEC passage 1 in standard plastic cell culture. Sample nomenclature is given in Supplementary Table 1. Additionally, HUVEC cultivated on Xellulin for one year and for two months, respectively, were detached, propagated on plasticware and then re-seeded on Xellulin. For RNA isolation from native HUVEC, the umbilical vein was washed with DPBS (1x) w/o Ca & Mg and thereafter incubated with 5 to 7 ml RNAprotect cell reagent (Qiagen, Hilden, Germany, 76526) for two minutes. The liquid was transferred to a centrifuge tube. After another washing step with DPBS (1x) w/o Ca & Mg, HUVEC were isolated by incubation with pre-warmed (37 °C) GibcoTM dispase solution (3.6-3.8 U/ml) for one hour. All liquids were collected in one centrifuge tube. The cells were pelleted (5 min., 5,000 g) and the supernatant was discarded. For RNA isolation and purification the RNeasy Mini Kit (Qiagen, 74104) was used according to the manufacturer's protocol. Briefly, the cell pellet was disrupted in lysis buffer. The lysate obtained was applied to QiaShredder columns (Qiagen, 79654), fully homogenized through centrifugation, and precipitated by adding 100% ethanol (Carl Roth GmbH & Co. KG, Karlsruhe, Germany, 5054.5). Afterwards, the elution procedure of the samples was done using RNeasy mini columns. The eluates were stored at -20°C until further processing. For extracting RNA from HUVEC passage 1, the cells were washed with DPBS (1x) w/o Ca & Mg and all liquid was discarded. To guard the cells from changes in their expression pattern, 600 μl RNAprotect cell reagent was added to the cells and incubated for two minutes. Cells were detached with a cell scraper and transferred into a centrifuge tube. For harvesting residual HUVEC, Xellulin and plastic surface, respectively, was washed with an additional ml of DPBS (1x) w/o Ca & Mg and the liquid was transferred to the centrifuge tube used previously. The detached cells were pelleted.
attributes.tag: ENA first public
attributes.value: 2016-06-30
attributes.tag: ENA last update
attributes.value: 2016-05-25
attributes.tag: External Id
attributes.value: SAMEA4008867
attributes.tag: INSDC center alias
attributes.value: GenXPro GmbH
attributes.tag: INSDC center name
attributes.value: GenXPro GmbH
attributes.tag: INSDC first public
attributes.value: 2016-06-30T17:01:23Z
attributes.tag: INSDC last update
attributes.value: 2016-05-25T13:03:14Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-4747:Sample 14
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: HUVEC cell
attributes.tag: common name
attributes.value: human
attributes.tag: growth condition
attributes.value: HUVEC grown on collagen-coated conventional cell culture plastic
attributes.tag: passage number
attributes.value: 1
attributes.tag: sample name
attributes.value: E-MTAB-4747:Sample 14