MB10x01Barcode
Homo sapiens
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). To obtain the P. falciparum egress media, P. falciparum-iRBC at 42-48 hours post-infection (hpi) were purified by gelaspan (40 mg/mL) and egress was inhibited for 5 hours using the reversible PKG inhibitor C2 (kindly donated by Michael Blackman, The Francis Crick Institute). After drug removal, parasites were resuspended in EGM-2MV media supplemented with 1% astrocyte and pericyte growth factors (ScienCell), gassed at a 5% O2 concentration and left in the incubator overnight on a shaker (50 rpm) to facilitate parasite egress. The concentration was adjusted at 5x10^7 ruptured P. falciparum-iRBC/mL before the suspension was centrifuged at 1000 rpm, aliquoted and flash frozen in liquid nitrogen. The same protocol was applied to uninfected erythrocytes to be used as a negative control medium. After 6 days in culture, 3D-BBB devices were perfused with 150 µL of P. falciparum-iRBC egress media or RBC-media by gravity-driven flow for 30 minutes, followed by a 20-minute wash. Control and P. falciparum stimulated microvessels were incubated for 24 hours at 37C. Reservoirs were refilled once after 12 hours. 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.
ENA first public
2025-07-08
INSDC center name
EMBL
INSDC status
public
Submitter Id
E-MTAB-14463_3:MB10x01Barcode
broker name
ArrayExpress
cell_line
Primary Human Brain Microvascular Endothelial cells (HBMEC), Primary Human Brain Vascular Pericytes (HBVP), Primary Human Astrocytes (HA), P. falciparum HB3var03
cell_type
Endothelial cells, Primary Pericytes, Astrocytes, P. falciparum HB3var03
collection date
not collected
common name
human
geographic location (country and/or sea)
not collected
isolate
not applicable
sample name
E-MTAB-14463_3:MB10x01Barcode
scientific_name
Homo sapiens
time
24 hours (re-application after 12 hours)
treatment
P.falciparum-infected red blood cell egress media
treatment concentration
Egress media from 50x10^6 P. falciparum-infected RBC/mL