2025-07-08
ENA first public
ERC000011
ENA-CHECKLIST
EMBL
INSDC center name
public
INSDC status
ERS21055634
SRA accession
E-MTAB-14463_3:MB10x02Barcode1
Submitter Id
ArrayExpress
broker name
Primary Human Brain Microvascular Endothelial cells (HBMEC), Primary Human Brain Vascular Pericytes (HBVP), Primary Human Astrocytes (HA), P. falciparum HB3var03
cell_line
Endothelial cells, Primary Pericytes, Astrocytes, P. falciparum HB3var03
cell_type
not collected
collection date
human
common name
Protocols: Primary human brain astrocytes (HA) (ScienCell) and brain vascular pericytes (ScienCell) were added to the 7.5 mg/mL collagen solution in a 7:3 ratio, using a concentration of 7.5x10^5 HA/mL(collagen) : 3.2x10^5 HBVP/mL(collagen). A multi-step process combining soft lithography and injection molding was used to imprint a microfluidic network in the collagen bulk, as previously reported (Zheng et al. 2012; Piatti et al. 2022). Primary human brain microvascular endothelial cells (HBMEC) (CellSystems) were seeded into the microfluidic network at a concentration of 7x10^6 cells/mL under gravity-driven flow until full coverage of the microchannels was achieved. 3D-BBB devices were cultured for 6 days and EGM-2MV medium supplemented with 1% astrocyte and pericyte growth factors (ScienCell) was changed every 12 hours by gravity-driven flow, as previously described (Bernabeu et al. 2019).  P. falciparum-iRBC late-stage parasites at the desired stage of the life cycle (26-34 hpi for trophozoites or 42-48 hpi for schizonts) were purified by magnetic separation (MACS) to above 80% enrichment. After 6 days in culture, 3D-BBB devices were perfused with 150 µL of P. falciparum-iRBC at 5x10^7 iRBC/mL concentration under gravity-driven flow for 30 minutes followed by a 20-minute wash to remove unbound cells. As a control, the same volume and concentration of uninfected erythrocytes was used for perfusion. Devices were then incubated for 6 hours at 37C. After incubation, microvessels were dissociated for scRNAseq. The 3D-BBB models were fabricated, perfused and incubated according to the described conditions. The devices were disassembled and the main microfluidic network was dissected. The collagen piece was dissociated for 6-10 minutes (until complete dissociation) in collagenase diluted in serum-free media (1mg/ml, Sigma-Aldrich C1764). After stopping the collagenase reaction, the cells were trypsinized (0.25% Trypsin-EDTA) for 8 minutes to obtain a single-cell solution. The cells were mechanically dissociated by pipetting with wide-bore tips and washed two times in PBS containing 0.1% bovine serum albumin (BSA). The single-cell solutions were labeled with MULTI-seq barcode oligonucleotides for sample multiplexing as described (McGinnis et al. 2019). Briefly, the cells were resuspended in Cell Prep Buffer (PBS containing 0.1% poly(vinyl alcohol) (PVA) and 1 mM EDTA). A 1:1 mixture of the cholesterol-conjugated Anchor-oligonucleotides (Anchor CMO, synthesized by Integrated DNA Technologies) and Barcode oligonucleotides with a distinct barcode for each sample (final concentration, 0.2 µM) was added and the cells were incubated on ice for 5 min. Next, the same concentration of Co-Anchor CMO (synthesized by Integrated DNA Technologies) was added and incubated for another 5 min, followed by three washes with PBS containing 1% BSA. The cells in each sample were counted after washing and were combined so that the multiplexed suspension contained the same numbers of cells from each sample. The combined sample was filtered through a 35 µm cell strainer and counted again before 10x barcoding. After obtaining the samples from the 10x Chromium Controller the samples were processed and the libraries were constructed with Chromium Next GEM Single Cell 3' - Reagent Kits v3.1 (10x Genomics) and single-indexed for sequencing according to the manufacturer's protocol.
description
not collected
geographic location (country and/or sea)
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
Homo sapiens
scientific_name
6 hours
time
MB10x02Barcode1
title
P. falciparum-infected red blood cells (Trophozoite or Schizont stage)
treatment
5x10^7 P. falciparum-infected RBC/mL
treatment concentration