Cell culture
Primary cultured NHDFs, neonatal hASCs and HUVECs were purchased from Lonza (Walkersville, MD). Each cell type was derived from single donor. NHDFs were cultured in Dulbecco’s modified Eagle medium (DMEM, Wako Pure Chemical, Osaka, Japan) containing 10% fetal bovine serum (FBS, SAFC Biosciences; Lenexa, KS) under 5% CO2 at 37 °C as described in our previous study4. For cultivation of HUVECs, Endothelial Growth Medium-2 (Lonza; Walkersville, MD) was used as described previously3. For cultivation of hASCs, Mesenchymal Stem Cell Growth Medium 2 (Takara Bio Inc., Kusatsu, Japan) was used. Transwell inserts with porous polyester bottom (pore size: 0.4 µm) for 24-well culture plate were purchased from CORNING Inc. (New York, NY).
Animals
Female nude mice (BALB/cAJcl-nu) at 6 weeks of age were purchased from CLEA Japan, Inc. (Tokyo, Japan). All the animal experiments in this study were approved by the Animal Research Committee at Hirosaki University and were conducted according to the Guidelines for Animal Experimentation, Hirosaki University. Mice were maintained under controlled light (12-h light : dark cycle) and temperature (21 °C) conditions. The study was carried out in accordance with the ARRIVE guidelines.
Construction of 3D artificial human vascular network tissue
Using a cell accumulation method by layer-by-layer cell coating technique, we constructed multi layered 3D tissue as previously described8,9,10,60. Briefly, cultured cells (NHDFs and hASCs within passage eight or HUVECs within passage six) were collected by trypsinization. The cells were suspended in 0.04 mg/mL bovine plasma-derived fibronectin [FN, Sigma-Aldrich (St. Louis, MO)] in 50 mM Tris–HCl buffer at pH 7.4, and incubated for 1 min with gentle rotation. Afterward, the cells were treated with 0.04 mg/mL of porcine skin gelatin [G, Sigma-Aldrich (St. Louis, MO)] in 50 mM Tris–HCl buffer. Nine steps of alternate FN and G treatment resulted in about 10 nm-thick coating of ECM nano-film on each cell type8,9.
The coated cells which were suspended in DMEM with 10% FBS were seeded on Transwell inserts. The inserts were set in 24-well cell culture plate supplemented with the same medium. To construct the vascular tissue, four bottom layers of NHDFs or hASCs, a single layer of HUVECs, and four top layers of NHDFs or hASCs were overlaid daily, and the tissues were continuously cultured in DMEM containing 10% FBS at 37 °C with 5% CO2 for 4 days. Alternatively, NHDFs or hASCs and HUVECs were mixed in the cell number ratio of 8:1, seeded on Transwell inserts, and cultured in DMEM containing 10% FBS at 37 °C with 5% CO2 for 5 days. In order to confirm the formation of the vascular network, the whole of fabricated 3D tissue was immunostained for human CD31 or human CD34 according to the method described below.
In this study, the artificial vascular tissues constructed by NHDFs/HUVECs and hASCs/HUVECs are termed FbVT and ASCVT, respectively. At least 10 times of ASCVT construction were performed for the analysis of tissue and transplantation in this study.
Cryopreserved cell accumulation method (CP-CAM)
ECM nano-film-coated cells that were prepared as described in the former cell accumulation method above were cryopreserved using several cell freezing media as shown in Fig. S3a. After the coating, the cells were collected by centrifugation at 1000 rpm for 5 min, 15 °C, then resuspended in a cell freezing medium (CultureSure Freezing Medium; Fujifilm Wako, Osaka, Japan), termed as CSFM in this study, at the concentration of 5 × 105–8 × 106 cells/ml and placed on ice. The other four cryopreservation media: conventional cell freezing medium [DMEM containing 10% FBS and 10% dimethyl sulfoxide (DMSO)], CELLBANKER 1 (Takara Bio Inc.), STEMCELLBANKER (Takara Bio Inc.), and our originally designed cell freezing medium [DMEM containing 5 mg/ml of recombinant human albumin expressed in plants (Fujifilm Wako, Osaka, Japan) and 10% DMSO], termed as DMSO-albumin FM, were also examined. The cell suspensions were aliquoted in the cryovials, kept at − 80 °C overnight, and then stocked in liquid nitrogen.
In order to construct artificial tissue, the cryopreserved cells were thawed in the warm water at 37 °C for 2 min, resuspended in DMEM without the serum, and centrifuged at 1000 rpm for 5 min, 15 °C as illustrated in Fig. S3b. Then, the cells were washed with DMEM containing 10% fetal bovine serum and resuspended in the same medium with appropriate cell concentrations to fabricate the artificial tissue by the former cell accumulation method.
Verification of the viable recovery rate after thawing of frozen cells in CP-CAM
The frozen cells (ECM nano-film-coated NHDFs, hASCs, and HUVECs) in a cryovial (1 ml) were thawed and the total cell number was counted. Then, the number was compared with that before freezing, and represent as % recovered rate;
$$Recovery, rate, (%) = [cell, number, after, thawing/cell, number, before, freezing] times 100.$$
The recovery rate of NHDFs with or without ECM nano-film coating and freezing by conventional medium was also assessed to verify the effectiveness of CP-CAM.
Analysis of the angiogenesis-related factors profile
After cultivation of NHDFs and hASCs with suitable media, two-dimensional (2D) and 3D cultures were performed. For the 2D culture, 8 × 105 of NHDFs or hASCs without ECM nano-film were seeded onto 3 cm-culture dishes with 2.3 ml of DMEM and incubated for 24 h under 5% CO2 at 37 °C. For the 3D culture, 8 × 105 of NHDFs or hASCs with ECM nano-film were seeded on Transwell inserts and incubated with 2.3 ml of DMEM for 24 h under 5% CO2 at 37 °C. Then, the culture supernatants were collected from 2D/3D cultures (NHDFs or hASCs) and used to analyze the angiogenesis-related factors profile. The expression of 55 factors was analyzed using Proteome Profiler Human Angiogenesis Array Kit (R&D Systems) according to the manufacturer’s protocol. The quantitative analysis of detected factors was performed by using FIJI software (https://imagej.net/Fiji). The duplicated blots were obtained per factor, and their averaged scores were calculated.
Transplantation of 3D artificial human vascular network tissue
Transplantation of the artificial vascular tissue was performed according to the method described in the previous studies3,4. Briefly, FbVT or ASCVT were collected from the Transwell inserts by cutting the bottom polyester membranes and kept in DMEM at 37 °C until transplantation into the subcutaneous tissue of nude mice. Under anesthesia, the dorsal skin of the mice was cut to a length of 1.5 cm, and the artificial tissues were then inserted subcutaneously, facing the artificial tissue up to the skin tissue. The wound was then immediately closed using silk sutures. For immunosuppression, cyclosporin (Neoral, Novartis, Rueil-Malmaison, France) was added to drinking water (120 mg/L), 1 week before and all along the engraftment period as previously reported61. After 2 weeks and 4 weeks of transplantation, the mice were euthanized, then the dorsal skin including the graft was collected for histological examination.
EGFP labeling of hASCs and HUVECs by lentiviral transfection
For the fluorescence labeling of hASCs and HUVECs, GFP‐bearing CS‐CDF‐CG‐PRE lentiviral plasmid was used59. The experimental procedures were approved by the Genetically Modified Organisms Safety Committee of Hirosaki University (registration number: 16S001-1) and Tokyo Medical and Dental University (registration number: G2019-026C). To generate viral particles, the 293FT cells were co‐transfected with the pCMV‐VSV‐G‐RSV‐Rev expression plasmid and pCAG‐HIVgp packaging plasmid using Lipofectamine 2000 (11668019; Thermo Fisher Scientific). The lentiviral particles in the supernatants were collected 48 h after transfection, and infected to 5.0 × 104 hASCs or HUVECs per well in 12‐well tissue culture plates.
EGFP-labeled hASCs were used for visualization of their perivascular localization in the artificial vascular tissue. The NHDFs, HUVECs, and EGFP-labeled hASCs were coated with ECM nano-film and seeded in the ratio of 7:1:1. At 4 days after the seeding, the tissues were fixed, embedded and immunostained for human CD34 and EGFP. These tissues were also subcutaneously transplanted to nude mice, and engrafted tissues at 2 weeks after transplantation were fixed and immunostained according to the method described below.
EGFP-labeled HUVECs were used for visualization of engrafted artificial human vascular structures in nude mice. The ASCVTs were constructed by using EGFP-labeled HUVECs and subcutaneously transplanted as described above. The engrafted tissues at 4 weeks after transplantation were fixed and immunostained for EGFP.
Light and fluorescence microscopy
The fabricated artificial vascular tissues in the Transwell inserts were fixed with 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for overnight at 4 °C and collected from the Transwell inserts by cutting the bottom polyester membranes. In order to achieve whole-mount immunostaining, the tissues were treated with 0.3% Triton X-100 in 0.1 M phosphate buffer at 4 °C for 3 days. Then, the tissues were stained according to the method described in our previous studies3,4,10. The specimens were observed using a confocal microscope Nikon C2 (Nikon, Tokyo, Japan). For the histological analysis, the fixed artificial tissues embedded in paraffin, and then 5 µm thick serial tissue sections were prepared. The engrafted tissues collected from mouse dorsal skin were also fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) and embedded in paraffin. Masson–Goldner staining was performed using the conventional method. After deparaffinization, the sections were treated with acid Fuchsin solution, phosphomolybdic acid-Orange G solution, and Light Green solution, respectively. Afterward, the sections were mounted using cover slips before observation. Immunohistochemistry was performed as follows. The deparaffinized sections were boiled twice in 10 mM citric acid (pH 6.0) using a microwave oven at 500 W for 5 min each for antigen retrieval. Then, the sections were treated with blocking solution [3% normal goat serum (Wako) in 0.1 M phosphate buffer (pH 7.4) containing 0.05% Tween 20] for 1 h at room temperature, and incubated with the primary antibodies overnight at 4 °C. For dark field microscopy, the sections were visualized by incubation with fluorescence-labeled secondary antibodies, goat anti-mouse IgG conjugated with Alexa-Fluor 594 or goat anti-rabbit IgG conjugated with Alexa-Fluor 488. The specimens were observed using a light microscope BX-50 (Olympus, Tokyo, Japan) or a fluorescence microscope BZ-X700 (Keyence, Tokyo, Japan).
Transmission electron microscopy
The fixative containing 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) were used to fix the artificial vascular tissues and engrafted tissues at 4 °C. After that, the tissues were cut into 1 mm × 1 mm in size and post-fixed with 1% osmium tetroxide in 0.1 M phosphate buffer. Subsequently, the tissues were dehydrated and embedded in Epon 812 (Nisshin EM, Tokyo, Japan). Ultra-thin sections with 70 nm-thickness were prepared using an ultramicrotome (REICHERT ULTRACUT S, Leica, Wetzlar, Germany). After staining with 4% uranyl acetate and lead stain solution (Sigma Aldrich, St. Louis, MO), the tissues were observed under transmission electron microscope (JEM-1200, JEOL, Tokyo, Japan).
Quantitative analysis of image data
Areas of human CD34-positive vascular network in immunofluorescent photographs were extracted by using Photoshop software (Adobe, San Jose, CA) and their total length, branches and areas were quantified by using Image J software (https://imagej.nih.gov/ij/) and Angiogenesis Analyzer (http://image.bio.methods.free.fr/ImageJ/?Angiogenesis-Analyzer-for-ImageJ&lang=en). Three-dimensional analysis of vascular network was also performed by using FIJI software (https://imagej.net/Fiji).
Ki67-positive areas in immunofluorescent photographs were extracted by using Photoshop software and quantified by using Image J software.
Statistical analysis
The statistical comparisons between two independent groups of the data were performed by t tests. The normal distribution in all sample data was confirmed by the degree of skewness and kurtosis. Then, the equality of variances in two groups of the data was validated by F test. For the data with equal variances or unequal variances, Student’s t test or Welche’s t test were used, respectively. P-values of less than 0.05 were considered statistically significant.
