Isolation and culture of human ASCs
ASCs were isolated from the subcutaneous fat tissue of four nonsmoking, nondiabetic female donors with an average age of 45 y (32–57 y) and an average body mass index of 24.6 (21.0–26.6) as described previously . The study protocol has been approved by the Research Ethics Committee of National Taiwan University Hospital, and the informed consent has been obtained from each donor of adipose tissue. Small pieces of subcutaneous fat tissue were finely minced and washed using phosphate-buffered saline (PBS; Omics Biotechnology, Taipei, Taiwan), followed by enzymatic digestion using collagenase type I (Gibco, Carlsbad, CA, USA) for 60 min. Pellets were suspended and plated with Dulbecco’s modified Eagle’s medium (DMEM)/F-12 (HyClone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS; HyClone), 1% penicillin–streptomycin (Biological Industries, Kibbutz Beit Haemek, Israel) and 1 ng/mL FGF2 (R&D systems, Minneapolis, MN, USA). The cells were cultured in a humidified atmosphere with 5% CO2 at 37 °C, and the medium was changed every 2–3 day. Upon reaching 90% confluence, the cells were detached using 0.05% trypsin–EDTA (Biological Industries) and re-plated until the third or fourth passage for further experiments. Human ASCs from each donor were pooled to become a single population based on the comparable surface marker expression and differentiation potential demonstrated in each ASC clone. These cells, which were constantly cultured on tissue culture plates, were referred as monolayer ASCs.
Preparation of agarose microwell plates and ASC spheroids
The ASC spheroids were generated as previously described with certain modification . As a non-cell-adhesive substrate, sterile solution of 2% agarose (UniRegion Biotech, Taipei, Taiwan) in PBS was autoclaved and dispensed onto sterile MicroTissues 3D Petri dishes (Merck, Darmstadt, Germany) following the manufacturer’s instruction. After the gelation of agarose molds at room temperature (about 5–7 min), they were gently detached from the petri dishes and immersed in sterile PBS. To equilibrate the petri dishes, they were immersed in a basal medium consisting of DMEM-high glucose (DMEM-HG; HyClone), 10% FBS and 1% penicillin–streptomycin for 15 min before use. Each agarose mold exhibited a structured surface with an array of 256 inverted circular recesses (depth 400 μm, diameter 800 μm). ASCs were seeded onto equilibrated agarose molds at different densities to achieve an average of 2000, 4000 or 8000 cells per microwell. The cells were incubated in the basal medium for 7 days for spontaneous spheroid formation, while the medium was carefully refreshed every 2–3 days.
Fabrication of ASC sheets and spheroid sheets
To create cell sheets, ASCs were seeded in tissue culture plates at a density of 2.5 × 104 cells/cm2 and cultured for 7 days. The culture medium consisted of basal medium and 250 μM A2-P as previously described . The culture medium was refreshed every 2–3 days. While ASC sheets were fabricated, ASC spheroids were also cultured in agarose molds. At day 7, all ASC spheroids from 256 microwells in one agarose mold were carefully transferred onto a prefabricated ASC sheet by inverted placement of the mold onto the ASC sheet. After 3 more days of culture, a composite spheroid sheet was formed, and it could be peeled off from the culture plates for further experiments.
Microscopic images and histology
ASC spheroids consisted of different cell numbers were photographed by an inverted microscope (TS100; Nikon, Tokyo, Japan). The Ferret diameter of the cell spheroids was determined by tracing the periphery of each spheroid using ImageJ. For the electron microscopic study, ASC spheroids consisted of different cell numbers were seeded on µ-slides (ibidi, Fitchburg, WI, USA) overnight. After washing ASC spheroids and ASC sheets thoroughly and fixed in 4% paraformaldehyde for 20 min, they were dehydrated by gradual change of concentrated ethanol, followed by lyophilization. The specimens were subsequently sputter coated with platinum and examined using a scanning electron microscope (JSM-6700F; JOEL, Tokyo, Japan).
A second harmonic generation microscopic system was employed to examine ECM deposition within ASC sheets as previously described . A wavelength-tunable Ti/sapphire laser pumped by a diode-pumped solid-state laser (Spectra Physics, Mountain View, CA, USA) was used as the excitation source. The 880 nm output laser system was guided toward a modified upright microscope (BX51WI; Olympus, Tokyo, Japan). The excitation source was beam expanded and reflected toward the focusing objective by a primary dichroic beamsplitter (Semrock, Rochester, NY, USA). The laser power was set at approximately 70 mW on the tissue section to optimize image quality without laser ablation. The nonlinear autofluorescence signals were spectrally separated by bandpass filters of 434/17 nm and 510/84 nm (Semrock).
As for the histological analysis, ASC spheroids and spheroid sheets were fixed in 4% paraformaldehyde and then paraffin-embedded. Sections were cut perpendicular to the surface of the cell construct with a thickness of 5 µm. Paraffin-embedded sections were deparaffinized, rehydrated and stained with hematoxylin and eosin (H&E; Sigma, St. Louis, MO, USA).
RNA isolation and RNA sequencing analysis
Total RNA from monolayer ASCs, ASC spheroids and ASC sheets was isolated by TRIzol reagent (Invitrogen). RNA quantity and purity were assessed using a microvolume spectrophotometer (Biochrom, Cambridge, UK), and the quality was verified by agarose electrophoresis and a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA) with RNA 6000 LabChip kit (Agilent Technologies). RNA libraries were performed with reagents supplied in Agilent's SureSelect Strand-Specific RNA Library Preparation Kit and sequenced on Illumina NovaSeq6000 platform with 150 paired-end reads. Raw sequences were expected to generate 20M (million reads) per sample according to Illumina’s standard sequencing protocol.
The generated sequences went through a filtering process to obtain qualified reads. Low-quality reads containing adapters or ploy-N from the raw reads were trimmed or removed according to the quality score. The filtered reads were aligned to the reference genomes using Bowtie2 (version 184.108.40.206). Qualified reads after filtering low-quality data were analyzed using RSEM (RNA-Seq by expectation–maximization) for gene expression estimation. The gene expression level was calculated as TPM (transcript per million). For differential expression analysis, edgeR v3.5 was employed to perform statistical analyses of gene expression profiles. The reference genome and gene annotations were retrieved from Ensembl database. The raw sequencing data were submitted to the NCBI Sequence Read Archive with BioProject ID PRJNA742860.
Functional enrichment analysis
After filtering out the low expressed genes which were less than 10 RPKM expression level in samples, differential expression genes were determined with 2× fold change compared with monolayer group (whether is upregulated or downregulated). The molecular relationships were generated using the core analysis showing significant (p < 0.05) association by Ingenuity Pathway Analysis software (IPA; QIAGEN, Redwood City, CA, USA). Canonical pathway analysis found by core analysis in IPA is given with a p value.
Quantitative polymerase chain reaction (qPCR)
Total RNA was extracted using RNeasy Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. After the RNA was isolated, complementary DNA (cDNA) was synthesized from the RNA using High-Capacity cDNA Reverse Transcription Kits (Applied Biosystems, Foster City, CA, USA). Real-time qPCR was performed with iQ SYBR Green Supermix (Bio-Rad, Hercules, CA, USA) using CFX Connect Real-Time PCR Detection System (Bio-Rad). The expression level was analyzed and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) for each cDNA sample. The relative quantity (RQ) of gene expression was calculated by relative quantification based on threshold cycle number. The sequences of the gene-specific primers are shown in Additional file 1: Table S1.
ASC spheroid labeling and immunofluorescence
ASCs labeled with PKH26 (Sigma) were subjected to spheroid formation process. After seeded on ASC sheet for 4 days, the spheroid sheet was fixed in 4% paraformaldehyde and treated with anti-CD31 (Abcam, Cambridge, MA, USA) overnight at 4 °C. After rinsing for three times, the secondary antibody was applied, followed by counterstaining with 4′,6-diamidino-2-phenylindole (DAPI; Sigma). Spheroid sheets were analyzed by a fluorescent microscope (Leica DMI 6000), and negative controls without primary antibodies were also prepared to rule out nonspecific labeling.
The angiogenic potential of ASC-conditioned medium
Monolayer ASCs, ASC sheets and spheroid sheets were washed with PBS and supplied with serum-free DMEM-HG. The conditioned medium was collected after 48 h as conditioned medium. The concentration of HGF, VEGF and FGF2 in the conditioned medium was determined using relevant ELISA kits (R&D Systems). Data were expressed as the secreted factor per 104 cells at the time of harvest.
Human umbilical vein endothelial cells (HUVECs) were seeded on μ-slides (Ibidi) coated with Matrigel (Corning, Corning, NY, USA) at a density of 8 × 103 cells/well, and they were cultured in a mixture of endothelial basal medium (EBM; PromoCell, Heidelberg, Germany) and conditioned medium from experimental groups of monolayer ASC, ASC sheet and spheroid sheet with a volume ratio of 4:1. Tube formation assay was performed as previously described . A mixture of EBM and DMEM-HG with a volume ratio of 4:1 served as a negative control, while endothelial growth medium 2 (EGM2, PromoCell) was used as a positive control. At 6 h, formation of tubelike structures was visualized by a phase-contrast microscope, and the images were analyzed using ImageJ.
In ovo angiogenesis assay
We employed the chick embryo chorioallantoic membrane (CAM) model as an in ovo assay for angiogenesis evaluation. Briefly, fertilized chicken eggs were incubated at 37 °C with 60% humidity. On day 3, a circular window was made on the upper side of the egg to evaluate the embryo viability. On day 7, ASC sheets or spheroid sheets were placed onto the CAM through the open window. The opening window in the shell was sealed with Tegaderm (3M, St. Paul, MN, USA) to prevent dehydration and contamination, and the eggs were placed into the incubator at 37 °C for 3 more days. On day 10, the embryos were infused with 4% paraformaldehyde and placed at − 80 °C overnight. ASC sheets or spheroid sheets with adjacent CAM tissues were removed and transferred to 6-well plates containing 4% paraformaldehyde. Untreated CAM tissues were used as controls. The CAM specimens were photographed, and the blood vessels were quantified by measuring the capillary area and counting the capillary branch points using ImageJ. Moreover, the immunohistochemical staining of the CAM sections was performed using anti-CD31 antibody as described previously . The CAM sections were photographed under a microscope, and the ratio of CD31-positive area was quantified using ImageJ.
All measurements are presented as means ± standard deviation. Statistical significance was evaluated using one-way ANOVA followed by the Tukey’s post hoc test. When Tukey’s test was used, each group was compared to all other groups in the experiment. All statistical analyses were performed using GraphPad Prism 7 (La Jolla, CA, USA), and statistically significant values were defined as p < 0.05.