SAMN32028212
External Id
Koltzov Institute of Developmental Biology of the Russian Academy of Sciences
INSDC center name
2022-12-04T00:00:00Z
INSDC first public
2022-12-04T15:20:02.217Z
INSDC last update
SRS15958444
INSDC secondary accession
live
INSDC status
Human
NCBI submission model
Human.1.0
NCBI submission package
SRS15958444
SRA accession
IDB RAS
biomaterial_provider
K7
cell_line
2DF-protocol
cell_subtype
differentiating induced macrophages
cell_type
day 19 of differentiating
dev_stage
Homo sapiens
http://purl.obolibrary.org/obo/NCBITaxon_9606
organism
Human sample from Homo sapiens
title
The generation of iMacs using the 2DF protocol (2DF-iMacs) was performed as it was previously described by [Takata et al., 2017], with some modifications. Briefly, iPSCs were expanded on MEFs and depleted from MEFs by culturing them on Matrigel-coated tissue culture plates in mTeSRTM1 medium for 2-3 days. MEF-depleted iPSC colonies were collected using collagenase IV and transferred to new Matrigel-treated tissue culture 6-well plates at 1:10-1:12 ratio (day -6). The cells were then cultured in StemPro-34 medium that was supplemented with mixtures of factors stimulating mesoderm/hemogenic endothelium (day -6 to day 0) and hematopoietic/myeloid differentiation (day 0 to day +10). The medium was changed every other day. On day +10, StemProTM-34 was replaced by supplemented RPMI-1640 medium containing M-CSF (50 ng/ml). The medium was changed every 3 days. When the first 2DF-iMacs appeared in the cultures (which usually occurred around day +19), they were collected and used for phenotypic and functional analysis or RNA isolation.The remaining cultures were re-stimulated with supplemented X-VIVO 15 medium containing IL-3 (25 ng/ml) and M-CSF (50 ng/ml). Seven days later, monocyte-like precursors of 2DF-iMacs appeared in the cultures; they were collected, transferred to new 12-well or 6-well plates and cultured in supplemented RPMI-1640 medium containing M-CSF (50 ng/ml) for 7 days to obtain a new portion of 2DF-iMacs. The rounds of iMac generation were repeated multiple times while cell generation lasted (usually for more than 90 days). Culture conditions were hypoxic from day -6 to day +2 and normoxic starting day +2.
treatment