The cells were harvested from near-confluent cultures by brief exposure to a solution containing 0.25% w/v trypsin-1 mmol/L ethylenediamine tetraacetic acid-4Na solution with phenol red (FUJIFILM Wako Pure Chemical, Ltd., Osaka, Japan). Trypsinization was stopped using RPMI-1640 medium supplemented with 10% FBS. Cells were concentrated by centrifugation at 300 × g for 5 min at 23°C.
Growth protocol
Adherent SNP cells (2D-SNP cells) were maintained as adherent monolayer cultures in Roswell Park Memorial Institute (RPMI)-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Subsequently, these cells were supplemented with 10% heat-inactivated fetal bovine serum (FBS; Nichirei Biosciences Inc., Tokyo, Japan) and antibiotics (5 mg/mL penicillin, 5 mg/mL streptomycin, and 10 mg/mL neomycin; Invitrogen; Thermo Fisher Scientific, Inc.) in an atmosphere containing 5% CO2 at 37°C. We observed non-adherent SNP cells in the growth medium. SNP spheroids (3D-SNP cells) were established by selecting the non-adherent SNP cells and culturing them without the addition of basement membrane components and ECM-like biomaterials.
Extracted molecule
total RNA
Extraction protocol
RNA samples were extracted from cells using miRNesy mini kit (Qiagen).
Label
Cy5
Label protocol
Extracted total RNA was labeled with Cy5 using the 3D-Gene miRNA labeling kit (Toray, Kamakura, Japan)
Hybridization protocol
Hybridized for 16 h at 32 C with rotary shake (250 rpm). Hybridization buffer and washing protocol was followed by the protocol supplied by TORAY Industries, Inc..
Scan protocol
3D-Gene Scanner ((Toray Industries Inc., Tokyo, Japan) was used for scanning. Images were quantified using Extraction(Toray Industries Inc., Tokyo, Japan).
Description
All samples were analyzed using an Agilent 2100 Bioanalyzer, and the 18S and 28S ribosomal RNA peaks were identified. The RNA integrities of the 2D- and 3D-SNP cells were 9.8 and 9.7, respectively.
Data processing
The raw data of each spot was normalized by substitution with a mean intensity of the background signal determined by all blank spots’ signal intensities of 95% confidence intervals.
