Stem Cell

Bioengineering of vascularized porcine flaps using perfusion-recellularization – Scientific Reports

Time Stamp: March 30, 2024 8:00 PM
Source Node: 566245

Animal use

Yorkshire pigs (30–40 kg; age approximately 12 weeks old) were used for all decellularization and recellularization experiments. All studies were approved by the Institutional Animal Care and Use Committee (IACUC) of the University Health Network and Toronto General Hospital Research Institute. Humane care was provided to all animals in accordance to the “Principles of Laboratory Animal Care” defined by the National Society for Medical Research and the “Guide for the Care of Laboratory Animals” issued by the National Institutes of Health. Reporting of use of experimental animals in this study followed recommendations specified by the ARRIVE guidelines.

Surgical procurement of porcine omentum and tensor fascia lata flap

Pigs were fasted for 12 h prior to surgery. Sedation was achieved with ketamine (20 mg/kg IM), atropine (0.04 mg/kg IM) and midazolam (0.3 mg/kg IM). Anesthesia was induced by inhalation of 5% isoflurane through a mask at a flow rate of 22 to 44 mL/kg/min to facilitate peripheral line insertion and intubation. Anesthesia was maintained with isoflurane (0.5 to 2%). Pigs were intubated with an appropriate endotracheal tube (7–8 mm) and ventilated to a tidal volume of 8 mL/kg, positive end-expiratory pressure of 5 cm H₂O, FiO₂ of 0.5 and respiratory rate of 14 breaths per minute. Pigs were prepped and draped in the usual sterile fashion prior to flap procurement. Surgical procedure for porcine omentum and TFL flaps procurement were as previously described³⁰. Briefly, the omental flap was procured by midline laparotomy and the left gastroepiploic artery and vein was used as the dominant vascular conduit. The right gastroepiploic vessels were ligated to prevent perfusion flow-through.

The TFL flap was procured with pigs in the lateral decubitus position. The main vascular pedicle was defined by the ascending branch of the lateral circumflex femoral artery and veins. The overlying skin island was removed to produce a pure fascial flap. Following flap detachment, the vascular pedicle was cannulated with 20–22 G Angiocath (Becton Dickenson) under direct vision and flushed with 20 U/mL heparin sodium (LEO Pharma, Denmark) in 0.9% normal saline and transported under sterile conditions to the lab.

Perfusion decellularization of porcine vascularized flaps

Porcine flaps were perfusion-decellularized using low-concentration SDS followed by DNase (Sigma Aldrich) reconstituted to a concentration of 10 mg/mL, as previously described³⁰. Cannulated flaps were each connected to a perfusion system to allow antegrade perfusion via the arterial inlet at 2 ml/min, in which solutions: 0.05% SDS followed by 0.1 mg/mL deoxyribonuclease (DNase) were perfused through the flap vasculature with 1 × phosphate buffered saline (PBS) perfusion in between to remove residual detergent. Flaps were sterilized in 0.1% paracetic acid (PAA) / 4% ethanol (EtOH) (Sigma Aldrich) and then washed in 1 × PBS prior to recellularization. As described previously³⁰, omental and TFL flaps were perfused with SDS for 2 and 3 days, respectively. Following SDS perfusion, flaps were washed with PBS for 24 h and then perfused with DNase for 2 h, PBS for again for 24 h, and finally PAA/EtOH for 3 h. With the exception of DNase, each step included an exchange of the submersion fluid to match the given perfusate. For the DNase step, flaps were submerged in fresh PBS.

In vitro culture and expansion of HUVECs and MSCs

Commercially available HUVECs (American Type Culture Collection/ATCC, USA) were cultured in EGM-2 (Lonza, Switzerland) supplemented with SingleQuots (Lonza) of Growth Supplements including: FBS 2%, hEGF, hydrocortisone, Gentamicin/Amphotericin-B, VEGF, hFGF-B, R3-IGF-1, ascorbic acid, and heparin (concentrations proprietary). Commercially obtained human bone-marrow derived MSCs (Promocell, Germany) were cultured in MSCGM (Promocell) containing proprietary media supplement and 5% FBS. HMSCs and HUVECs between passage 4 and 6 were used for recellularization. Both cell types were verified for correct functional and phenotype expression. HUVECs expressed CD31/VE-Cadherin using flow cytometry and were functionally capable to undergo angiogenesis. MSCs were CD90/73/44 positive and CD34/45/11b negative using flow cytometry and capable of undergoing trilineage differentiation (Supplementary Fig. 1). These findings were consistent with the minimal criteria to define MSCs according to the International Society for Cellular Therapy Criteria⁴⁷.

All cells were maintained in 150 cm² dishes until reaching 90% confluency (resulting in approximately 50,000 cells/cm²). Cells were detached from culture vessels with 0.25% trypsin–EDTA solution (Gibco) prior to recellularization. Cell media was replaced every other day, and the cultures were maintained in a humidified 95% air/5% CO₂ incubator at 37 °C.

Perfusion recellularization bioreactor and culture

A closed-system bioreactor was set up in an incubator for recellularization within the flap scaffold matrix. We used a modified airtight snap-lid container, previously used for decellularization with a closed-circuit L/S-16 (Masterflex, Fisher Scientific) silicone tubing. The end of the tubing external to the tissue chamber was fitted with a female Luer thread-style panel (Cole-Parmer), which connected to a 3-stop tubing compatible with peristaltic pump (Ismatec, Cole-Parmer) tubing cassette as previously used for perfusion-decellularization. The opposite end of tubing was reconnected to the second port from the tissue chamber to allow closed-loop circulation of medium from tissue chamber into the flap via the arterial cannula at a flow rate of 2 mL/min. Just proximal to the tissue chamber, silicone tubing was connected to a three-way stopcock (Baxter, USA). The chamber was filled with 200 mL of EGM-2 media, which was primed through the tubing to remove air bubbles. Decellularized flaps were perfused with EGM-2 at 2 mL/min in conventional cell culture incubator at standard conditions (95% air/5% CO₂) overnight before cell seeding to equilibrate flaps with culture medium.

Flap vascular seeding of HUVEC and MSC co-culture

Cell seeding was performed as follows: HUVECs and human bone-marrow derived MSCs were lifted from tissue culture plastic with 0.25% trypsin and centrifuged at 500×g for 5 min. The resultant cell pellet was resuspended in 10 mL media, strained with 75 µm pore mesh, and counted via automated hemocytometer (Vi-Cell XR, Beckman Coulter). A total of 8 × 10⁷ cells, divided equally with 4 × 10⁷ HUVEC co-cultured with 4 × 10⁷ MSCs, were used for recellularization of each scaffold. A combined cell suspension of the two cells were slowly manually injected into the vascular arterial inlet through a three-way stopcock. Following the introduction of cells, flaps were placed in a standard cell culture incubator for 2 h of static culture to allow cell attachment. Afterwards, perfusion-culture was initiated with the peristaltic pump (Ismatec, Cole-Parmer) running at 2 mL/min for 6 days. Media passed through the flap was recovered back into the reservoir using a separate pump channel that drained the bioreactor at an equal rate to the perfusion, allowing for recycling and reuse. Media was exchanged every other day for fresh EGM-2. A total of 750 mL of culture medium was used over 6 days for each flap.

Histology and immunohistochemistry

Native, decellularized, and recellularized tissues were biopsied near the distal margin of the flap, fixed in 10% formalin (Fisher Scientific), embedded in paraffin, and sliced into 5 µm sections on microtome (Leica Biosystems). Slides of the paraffin-embedded samples were processed for histological and IHC staining. Histologic staining was performed on xylene-deparaffinized slides with the following stains: H&E (Sigma Aldrich), Masson’s Trichrome (American MasterTech Scientific), and Verhoeff Van Gieson Elastin Stain (Abcam).

For IHC, heat induced antigen retrieval was done with citrate buffer (pH 6.0; Thermo Fisher Scientific) in a 95 °C autoclave for 10 min. Endogenous peroxidases were blocked with a peroxide block (Cardinal Health), and nonspecific binding was blocked with Dako Serum-Free Protein-Block (Agilent). Sections were incubated with the primary antibodies at 4 °C overnight with dilutions as follows: rabbit polyclonal anti-Collagen IV (Abcam, ab6586, 1:300), rabbit polyclonal anti-Fibronectin (Abcam, ab23751; 1:400); and rabbit polyclonal anti-Laminin (Abcam, ab11575, 1:400) and anti-CD31 (Abcam, ab28364, 1:50) at 4 °C overnight. Slides were washed three times in PBS with 0.1% Tween and goat anti rabbit IgG HRP-conjugated secondary antibody (ImmPRESS Peroxidase Polymer Reagent, Vector Laboratories) was applied for 30 min. Slides were again washed thrice in PBS-Tween and then diaminobenzidine solution (Vector Laboratories) applied for 10 min. Slides were counterstained with hematoxylin. After staining, all slides were dehydrated in ethanol to xylene exchange, mounted and imaged on Aperio CS2 Slide Scanner (Leica Biosystems).

Immunofluorescence staining was performed using paraffin embedded sections cut to 5 μm thickness and deparaffinized using xylene and rehydrated in serial dilutions of ethanol. Tissue sections in were incubated in antigen retrieval buffer (10 mM citrate buffer, pH 6.0) at 95 °C for 10 min in an autoclave. Tissue sections were then blocked with 5% blocking serum (goat serum) in 1% bovine serum albumin (BSA) before adding primary antibody. Slides were then incubated with primary antibodies for VE-Cadherin (Abcam, ab33168, 1:100) and vimentin (Abcam, ab92547, dilution 1:200) diluted in 1% BSA at 4 °C overnight. After washing three times with PBS-Tween, slides were then incubated for 1 h at RT in the secondary antibody goat anti-rabbit IgG conjugated with AlexaFluor 647 (Thermo Fisher Scientific, 1:500). Finally, slides were washed three times with PBS-Tween in the dark and counterstained with DAPI (Abcam; 1:5000). Negative controls were used by replacing the primary antibody with the corresponding isotype (IgG) of the primary antibody. Images were taken on a Leica SP8 confocal microscope with LAS X software (Leica Biosystems) installed.

GAG quantification

Tissue pieces (~ 30–40 mg) were obtained by punch biopsy tool and dried in 60 °C oven overnight. Dried tissue pieces were digested in papain solution at 65 °C for 18 h. Corresponding native flap tissues were dried and digested in parallel as controls. Papain (Sigma Aldrich, ≥ 16 units/mg protein) 15–30 mg/mL stock was solubilized to working concentration of 0.1 mg/ml in 0.1 M phosphate buffer (pH 6.0), with 5 mM cysteine hydrochloride (Sigma Aldrich), and 5 mM EDTA (Sigma Aldrich). The lysates were used for detection of sulfated glycosaminoglycan (sGAG) and DNA content. The Blyscan Sulfated GAG Assay kit (Biocolor) was used to measure sGAG according to manufacturer’s instruction. Briefly, tissue specimen lysates were mixed with Blyscan Dye Reagent to bind the GAG for 1 h at room temperature. The GAG-dye complex was then collected by centrifugation at 10,000×g. After the supernatant was removed and the tube drained, Dissociation Reagent was added and 100 μl of analyte solution was transferred to a 96-well plate. Absorbance against the background control was obtained at a wavelength of 656 nm with a SpectraMax spectrophotometer (Molecular Devices). GAG amount was interpolated from a standard curve (0–5 µg) using a known GAG standard provided in the kit. Final GAG content was standardized to the total dry tissue mass (mg) used for assay.

DNA quantification

For DNA content quantitation, the tissue lysate following papain digestion (above) was used. The Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen) was used to measure DNA content according to manufacturer’s instruction. Fluorescence reading (excitation: 485 nm and emission: 528 nm) was taken on a plate reader (Cytation 5, Biotek), and the absolute amount of DNA (ng) was quantified against a lambda DNA standard curve (0–1000 ng) provided by the manufacturer; final DNA content was standardized to total dry tissue mass (mg) used for assay.

Statistical analysis

All statistical analysis was performed using GraphPad Prism, version 9.0 (GraphPad, Inc.). Statistical analyses was conducted with multiple unpaired t test with a significance level of p < 0.05. Values are presented as mean, with S.D. unless stated otherwise.