HdE-CYST-Matrigel-d5-HIO-1
Homo sapiens
Protocols: Human derived epithelial organoids (HdEs) or transplanted human derived epithelial organoids (tHdEs)were collected as pools from individual wells of a 24-well plate and seperated manually from the culture matrix prior to RNA extraction. Generation of HdEs and tHdEs from HIOs and tHIOs was performed using previously described methods to isolate the epithelium (Finkbeiner et al., 2015b; Hill et al., 2017; Tsai et al., 2018). In summary, HIOs and tHIOs were incubated in dispase (07923; STEMCELL Technologies) for 30 minutes on ice. Following incubation, dispase was removed and replaced with 100% fetal bovine serum for 15 minutes on ice. To mechanically separate the epithelium from mesenchyme, a volume of advanced Dulbecco's modified Eagle medium/F12 (12634010; Gibco) equal to the initial volume of fetal bovine serum was added to the tissue before vigorously pipetting the mixture several times. Epithelial fragments then settled to the bottom where they were collected manually on a stereoscope by pipet. The epithelium was washed with ice-cold advanced Dulbecco's modified Eagle medium/F12 and allowed to settle to the bottom of a 1.5-mL Eppendorf tube. The media was then withdrawn from the loose tissue pellet and replaced with Matrigel on ice. The Matrigel containing the isolated epithelium was gently mixed to evenly suspend the cells before being pipetted into individual 50-μL droplets in a 24-well plate. The plate containing the droplets was incubated at 37°C for 15 minutes to allow the Matrigel to solidify before adding LWRN growth media containing Thiazovivin (2.5 μmol/L), SB431542 (100 nmol/L), CHIR99021 (4 μmol/L), and Y27632 (10 μmol/L). After 24 hours, the media was replaced with LWRN growth media containing TZV (2.5 μmol/L), SB431542 (100 nmol/L), and CHIR99021 (4 μmol/L). After 3 days, cultures were maintained with LWRN growth media replaced every other day. Prior to transplantation, human intestinal organoids (HIOs) were mechanically dissociated from either alginate or Matrigel. HIOs were implanted under the kidney capsules of immunocompromised NOD-scid IL2Rg-null (NSG) mice (Jackson Laboratory strain no. 0005557) as previously described (Finkbeiner et al., 2015b). In summary, mice were anaesthetized using 2% isoflurane. A left-flank incision was used to expose the kidney after shaving and sterilization with isopropyl alcohol. HIOs cultured in alginate and Matrigel were surgically implanted beneath mouse kidney capsules using forceps. Mice were euthanized for retrieval of transplanted HIOs (tHIOs) after 10 weeks. RNA from each sample was isolated using MagMAX-96 Total RNA (AM1830; Applied Biosystems) RNA isolation kits and used as input for library generation with Takara SMARTer Stranded Total RNA Sample Prep Kit (634876; Takara Bio USA).
ENA-FIRST-PUBLIC
2019-06-27T04:02:30Z
ENA-LAST-UPDATE
2018-07-17T16:15:25Z
External Id
SAMEA4795436
INSDC center name
University of Michigan Medical School: Department of Internal Medicine, Division of Gastroenterology
INSDC first public
2019-06-27T04:02:30Z
INSDC last update
2018-07-17T16:15:25Z
INSDC status
public
Submitter Id
E-MTAB-7000:HdE-CYST-Matrigel-d5-HIO-1
broker name
ArrayExpress
cell line
H9
cell type
intestinal epithelial cell
common name
human
developmental stage
embryo
genotype
wild type genotype
growth condition
organoid culture in matrigel
progenitor cell type
embryonic stem cell
sample name
E-MTAB-7000:HdE-CYST-Matrigel-d5-HIO-1
scientific_name
Homo sapiens