Sample 13
Homo sapiens
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.
ENA first public
2016-06-30
ENA last update
2016-05-25
External Id
SAMEA4008866
INSDC center alias
GenXPro GmbH
INSDC center name
GenXPro GmbH
INSDC first public
2016-06-30T17:01:23Z
INSDC last update
2016-05-25T13:03:14Z
INSDC status
public
Submitter Id
E-MTAB-4747:Sample 13
broker name
ArrayExpress
cell type
HUVEC cell
common name
human
growth condition
HUVEC grown on collagen-coated conventional cell culture plastic
passage number
1
sample name
E-MTAB-4747:Sample 13