Spherical rotary cell seeding system for the production of tissue-engineered small-diameter blood vessels with complex geometry

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Fully bioengineered tissue vessels (TEBVs) have previously been developed for clinical use. Tissue engineering models have also proven to be invaluable tools for disease modeling. In addition, TEBVs with complex geometries are needed to study multifactorial vascular lesions such as intracranial aneurysms. The main goal of the work reported here is to create a fully human clade of small caliber TEBVs. The use of a novel spherical rotating cell seeding system can provide efficient and uniform dynamic cell seeding for viable in vitro tissue engineering models. This report describes the design and manufacture of an innovative 360° random spherical seeding system. The individual seed chambers are housed within the system and secured with polyethylene terephthalate (PETG) Y-brackets. Seeding conditions such as cell concentration, seeding rate and incubation time were optimized by counting the cells attached to the PETG scaffold. This spherical seeding method was compared with other methods such as dynamic and static seeding and clearly showed a uniform distribution of cells on PETG scaffolds. With this easy-to-use spheroid system, fully biobranched TEBV constructs can also be generated by seeding human fibroblasts directly onto custom-made PETG mandrels with complex geometries. The production of small diameter TEBVs with complex geometry and optimized distribution of patient-derived cells could be an innovative approach to model various vascular diseases such as intracranial aneurysms.
In recent years, advances in tissue-engineered vascular grafts have provided promising clinical options for the treatment of vascular disease or have provided alternative in vitro models to study these complex diseases1,2. With model enhancements, it is now possible to create tissue-engineered blood vessels (TEBV) of a patient with a specific genetic background to better understand the pathobiology underlying vascular disease3,4. Various methods for producing TEBV have been developed over the years, each with its own advantages and disadvantages, and can be grouped into three broad categories: (1) vascular channels made from cells seeded on artificial scaffolds, (2) vascular channels made from cells . sheets of vascular catheter engineering and (3) bioprinting5,6. However, one of the challenges of vascular tissue engineering remains the improvement of cell seeding, distribution, and organization for homogeneous integration of cells into tubular structures. Thus, dynamic cell seeding methods have outperformed simpler static methods7,8,9. In addition, in a three-dimensional (3D) environment, uniform monitoring of cell distribution is necessary to promote homogeneous tissue remodeling and avoid competition for nutrients in areas of higher cell density10,11,12,13. The current state of dynamic cell seeding allows for easy production of linear TEBV using roller bottles and perfusion of endothelial cells in tubular structures. However, they are not ideal for the production of three-layer TEBVs with more complex geometries consisting of adventitial, medial, and internal membranes4,14,15,16.
The production of small diameter self-assembling linear vessels seeded with polyethylene terephthalate (PETG) pre-treated with ultraviolet (UV-C) has previously been shown to provide proper cell attachment and optimized extracellular matrix (ECM) secretion/assembly 14 , to produce TEBVs with complex geometries. and improve seeding of cells along the scaffolds, we have developed a rotating system with a random rotational movement that ensures efficient and uniform distribution of cells. We describe here the design and manufacture of an innovative rotary seeding system capable of performing a full 360° rotation and generating fully biologically branched tissue-engineered vascular adventitia (TEBV-A).
Initial features of this new rotary seeding system concept include variable speed 360° rotation, a minimum of 24 hours of operation and a maximum of five TEBVs produced at the same time. The design is simple, clear and easy to use (Fig. 1A). The model consisted of an acrylic sphere in two halves, with spherical rotational motion of two motors on a baseplate so that TEBV could be generated in a seed chamber in the middle of the sphere (Fig. 1B-E). The sowing system is set to 360° random rotation. Rotation was considered random because a constant motor speed was used throughout the seeding time, and sphere shape imperfections caused movement axis changes (Supplementary Video 1). The system also has an adjustable speed from 63 to 135°/min, an operating time of over 24 hours, and a production capacity of five TEBVs (Figure 1F). It can also be placed in a temperature controlled environment during the seeding phase.
carousel system. (A) Computer-aided design (CAD) system using CREO 5.0 software with orientation indicators; (B, C) Photographs of the male and female halves of an acrylic ball with 3D printed closed rings and plates to hold the seed chambers in place; (D) Aluminum base plate with three ball bearings, two motors and an electronic control unit; (E) Five custom-made acrylic seeding chambers; (F) Assembled rotary seeding system. Scale bar = 5 cm.
A special seed chamber was developed for seeding human fibroblasts on branched PETG scaffolds (Fig. 2). The chamber consists of two polycarbonate halves with a custom Y-notch to fit a 4.8 mm diameter PETG Y-stent (Figure 2A-C). This scaffolding is made up of three separate pieces held together with stainless steel pins so that the various branches can be removed for easy disassembly. The bottom half has a Y-ring that makes the chamber waterproof, and the top half has three small holes that allow cells and media to be added (Figure 2A). The two halves are held together with stainless steel staples. All parts used for the chamber can be sterilized by autoclaving prior to inoculation of human cells using a sterile biofume technique (Figure 2D). Five closed seed chambers must be placed in a rotation system for the spheres to function properly (Figure 2E).
Flowerpots for branches to order. (A) Systematic CAD of the bottom half of the acrylic seed chamber with Y-slots and Y-rings using CREO 5.0 software; (B) CAD of a custom PETG Y-frame with stainless steel positioning pins; (C) CAD of the complete seed chamber closed with fittings and view of the small seed hole screw; (D) photo of all parts and fittings of the seed chamber inside the bio-hood in the sterile field; (E) Photo of the full chamber closed with instrumentation and seeding view of the mouth of the small propeller. Scale bar = 1 cm.
To determine the optimal cell seeding parameters, we first tested different cell concentrations. Three initial cell concentrations, here expressed in millions of cells per milliliter of medium (M/mL), were used to seed the chambers (0.1 M/mL, 0.15 M/mL, and 0.30 M/mL). The cells were allowed to adhere to a 4.8 mm diameter PETG rod at a moderate speed of 90°/min for 22 hours. Then the mandrin seeded with cells were incubated with trypsin for 10 min and the separated cells were counted. A cell concentration of 0.15 M/mL was found to be the optimal cell seeding parameter (0.15 M/mL vs. 0.1 M, P < 0.0010; 0.15 M/mL vs. 0.3 M/mL, P = 0, 3805) (Fig. 3A). There was no significant difference between the conditions in terms of the number of cells in the post-seeding supernatant (FIG. 3A). Then, using this optimal concentration of cells, we wanted to determine the optimal seeding rate, i.e., the optimal speed to set up the spheroidal rotation system to allow the most significant number of cells to adhere to the scaffold. Three different seeding rates were tested (63°/min, 90°/min and 135°/min). Use a moderate seeding rate of 90°/min (63°/min vs 90°/min, P = 0.0321, 90°/min vs 135°/min, P = 0.0119) (Figure 3B). No significant difference was observed in the number of cells in the supernatant after cell seeding (FIG. 3B). Different incubation times (4 hours, 8 hours, 16 hours and 22 hours) were also tested after cell seeding in situ. An incubation time of 22 hours was found to be the optimal setting here, as more cells adhered to the scaffold at any other incubation time tested (P < 0.0001). In addition, there were more cells in the supernatant at 4 hours post-seeding compared to any other time (P < 0.0001), indicating that the cells did not have time to properly attach to the scaffold and remain in the culture medium (Figure 3C). ). , the optimal cell seeding parameters were 0.15 M/ml of cells at 90°/min for 22 h at 37°C.
The number of cells adhering to the scaffold and in the supernatant depends on the cell concentration, seeding rate in the system, and incubation time. (A) Three cell concentrations (M/mL) were evaluated by seeding cells at 90°/min for 22 h. (B) by seeding cells at 0.15 M/mL for 22 h. Three system rates were evaluated (°/ min) (C) Four incubation times (hours) were analyzed by inoculation with 0.15 M/ml at 90°/min. For statistical analysis, a two-way ANOVA was performed with Tukey’s multiple comparison test. Panels (A–C) show the highs and lows of the rectangle and whiskers. n = 4–5/group. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. ns is meaningless.
The new spherical seeding method was compared with dynamic and static seeding methods. First, the distribution of cells on PETG scaffolds after seeding can be visualized with Rhodaniel blue staining (Figure 4A). When using the spheroidal technique, a surprisingly uniform distribution of cells on the mandrel was observed compared to other seeding methods (Fig. 4A and Supplementary Fig. 1). In addition, there were no statistically significant differences between spheroid seeding and other methods after proper counting of cells adhering to the scaffold after a 22-hour seeding period (Fig. 4B). Notably, TEBV-A generated using the described sphere system was statistically superior to TEBV-A generated using other seeding methods after a 42 day post-inoculation period in culture (P < 0.05 and P < 0.0001 ) (picture 1). 4C,D), suggesting that uniform distribution may be important to obtain thicker TEBV-A.
Cell distribution and viability after seeding and mature TEBV-A obtained by various seeding methods. (A) Photograph of Rhodaniel blue stained cells on PETG scaffolds after seeding. Scale bar = 1 cm; (B) The number of cells adhered to the scaffold after a 22 hour seeding period; (C) TEBV-A tissue thickness (µm) measured after a 42 day maturation period; (D) Hematoxylin and eosin (H&E) stained histological sections collected from mature TEBV-A obtained using various inoculation techniques (spherical, dynamic and static). Scale bar = 100 µm; (E, F) analysis of live/dead cells collected after seeding of TEBV-A analyzed by flow cytometry; (G, H) Analysis of live/dead mature cells harvested by TEBV-A. (B,C) Boxes and whiskers with highs and lows. For statistical analysis, the Kruskal-Wallis test and Dunn’s multiple comparison test were performed. n = 4–24. (F, H) Stacked columns with standard deviation. For statistical analysis, a two-way ANOVA was performed with Tukey’s multiple comparison test. n = 5. *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001. ns is meaningless.
Another important aspect to consider and compare different seeding methods is cell viability. Therefore, flow cytometry was used to quantify cell viability after 22 hours of seeding (post-seeding) and a 42-day maturation period. Significantly higher post-inoculation energy was found when using a spherical plating system compared to other plating methods, both dynamic (P < 0.001) and static (P < 0.001) (Fig. 4E, F and Supplementary Fig. 2A). Despite the rather low level, no difference in cell viability was observed after a maturation period of 42 days post-plating with any of the seeding methods tested (Fig. 4G, H and Supplementary Fig. 2B). The lower cell viability measured in mature TEBV-A can simply be explained by the 30 min enzymatic digestion required to harvest cells from the ECM for flow cytometry analysis; this step was not required for the later seeding stages. Overall, more uniform cell distribution, thicker TEBV-A, and increased cell viability were measured using the developed spherical rotation system compared to other dynamic and static seeding methods.
Fully bifurcated TEBV-A derived from human fibroblasts was generated using predetermined optimal cell seeding parameters. After a 22 hour cell seeding incubation period, the seeding chamber was removed from the system. The seeded scaffolds were then placed in culture dishes and kept in culture for 42 days. Measure tissue thickness with a laser micrometer on each strand (Figure 5A). For all three branches of TEBV, no statistically significant differences were found, indicating a uniform distribution of cells over the inoculated mandrel (Fig. 5A, B). In order to obtain TEBVs with complex geometries such as Y-shaped scaffolds, it is indeed important to design a cell seeding system that ensures uniform distribution of cells along the scaffold, especially at branching/junction sites. All branched TEBV-A macroscopically showed evenly distributed cells at key junction sites (Fig. 5C,D). In addition, no statistically significant differences were found in tissue thickness measurements across histological cross sections of branches and junctions (Fig. 5E,F) (Fig. 5G).
Macroscopic and microscopic characteristics of branched TEBV-A obtained using the developed rotary seeding system. (A) Photograph of the Y-geometry of TEBV-A on a branched PETG scaffold, (B) Tissue thickness of the three branches (I, II, III) of TEBV-A in µm. n=4, n=4; (C) Close-up of TEBV-A connections on scaffolds (D) and cut from scaffolds; (E) Branches and (F) connections of TEBV-A samples cut from scaffolds, H&E staining at ; (G) Tissue thickness of tissue sections of the TEBV-A branch and junction in µm. n = 12. The arrows show the connections. For statistical analysis, the Kruskal-Wallis test and Dunn’s multiple comparison test were performed in panel (B), and the Welch t-test was performed in panel (G). Panels (B, G) show a scatterplot with bars and standard deviations. Scale bar = 100 µm. ns is meaningless.
In this paper, we have successfully designed and constructed a rotating spheroid cell seeding system capable of producing biologically small TEBVs of complex geometry composed entirely of human cells. Cell seeding parameters have been optimized to improve cell distribution and adhesion throughout the scaffold, even at critical branching/junction points of the designed TEBV in a ‘Y’ shape for proof of concept. Compared to other dynamic and static seeding methods, our new spherical seeding method provides a more even distribution of cells on the generated TEBV, demonstrates higher cell viability after seeding, and produces thicker tissues. The described system consists of a custom made and easily sterilizable seed chamber containing a UV-C treated PETG support designed to be installed in the seed chamber. Notably, the seed chamber can be easily changed to different geometries/dimensions to accommodate different vessel shapes, sizes and bifurcation angles.
During the initial chamber seeding, a cell concentration of 0.15 M/mL combined with random rotation at 90°/min in all directions (x, y, and z) for 22 h is the optimal seeding setting to favor cells along adhesion and distribution. PETG frame. After a 22 hour incubation period in the seeding chamber, seeded cell scaffolds were removed from the chamber and cultured in culture medium for 42 days to stimulate production, assembly, and maturation of TEBV ECM. The dynamics of interaction between cells and solid scaffolds follows the Langmuir model, whose three assumptions apply to cell culture: (i) cells cannot attach to form more than a monolayer; (ii) cells can attach to all scaffold surfaces; (iii) Cells can attach to the scaffold independently of already attached cells until a monolayer is reached. UV treatment of PETG is known to promote cell adhesion by changing the carbonyl groups of the plastic, which in turn increases the hydrophilicity of the material14,19,20,21. According to Langmuir’s theory, the rotational motion of the dynamic seeding system described promotes cell-scaffold interaction across the entire surface of the treated plastic rod, resulting in better cell adhesion and distribution7,8,22. In fact, the random 360° movement of this new system allows the cells to collide with all parts of the complex geometric scaffolds during seeding. This is indicated by uniform tissue thickness measured along the branches of the Y-shaped TEBV. Langmuir’s third hypothesis suggests that once a monolayer is reached, cells cease to attach independently to the scaffold and begin to interact with other attached cells. An increase in cell-to-cell interactions over time may have contributed to the separation of cells from the scaffold and explained why the same or fewer cells were recovered from higher cell density PETG, effectively reaching a plateau 17, 21.
The average speed of the system is considered optimal for seeding cells at 90°/min. Although they were all tested at relatively low speeds, the systems had to be fast enough to keep the cells suspended in the medium for the entire seeding period, but slow enough so that the cells could properly interact and attach to the plastic. A longer 22 hour seeding period was also required to increase cell attachment. Prolonged sluggishness has also been shown to promote better cell attachment in other models23,24. Cell seeding parameters such as speed, cell density, and time depend on the cell type (cell-scaffold interaction, cell adhesion, matrix production capacity) and therefore need to be optimized for each tissue engineering model and cell type to be seeded9,25 , 26, 27, 28. Using the Stokes law in fluid mechanics, V = (g(ρp − ρf)d2)/18µ, we calculated that the sedimentation rate (V) of the tested fibroblast population in the seed chamber was estimated to be 2.5 µm s using the following constants /s: (density (g: 9.81 m/s2), cell density (ρp: 1050 kg/m3), density of Dulbecco’s Modified Eagle’s Medium (DMEM) (ρf: 1007 kg/m3), fibroblast diameter ( d: 10 µm) and DMEM viscosity (μ: 0.00093 Pa s) 29, 30. Despite the low rate of fibroblast settling combined with the random rotational movement of the spheroids for 22 hours, the cells were able to rotate on the scaffold in the seed chamber. complex geometry 30 , 31 .
Using a brush motor to randomly rotate a sphere 360° at low speed has some limitations as it has reached its maximum power. The servo motor upgrade provides more precise rotation, while eliminating the risk of motor stall due to overheating, high resistance and voltage drop. This rotary cell seeding system uses a potentiometer to adjust the voltage to the motor and determine the rotation speed. The addition of a microcontroller system and an electronic display will allow for better voltage regulation and more accurate rotation speed during cell seeding.
Compared to other seeding methods, the developed spherical seeding system method achieved uniform distribution of cells on the PETG scaffolds and produced denser mature TEBV-A after 42 days of cell culture, regardless of the number of cells that had originally attached to the scaffold at the time of seeding. Then, a more even distribution of cells can promote tissue maturation, thereby improving extracellular matrix secretion and assembly32,33,34. Disease modeling has been shown to require cell and cell-ECM interaction in tissue engineering to better represent crosstalk between cells, tissue components and microenvironments relevant to complex human pathologies11,35,36.
Although complete TEBV models cover adventitia (fibroblasts), media (smooth muscle cells), and intima (endothelial cells), the state of the art for branched TEBV models is rapidly changing from branched vessel prostheses to clinical branched vessel stents and branched collagen tubes with endothelial cells and 3D printed branched vascular grafts4,37,38,39. To the best of our knowledge, we are the first research group to create a small-diameter branched-channel TEBV outer membrane model without exogenous materials. Of particular interest was the fact that critical connections remained intact and did not break after dismantling the scaffolding. This innovative system makes it possible to produce tissue of sufficient thickness for manipulation, observation and research using only a single layer of fibroblast sheet, although it cannot yet be perfused. Multiple sequential seeding of cells may be the next appropriate step to fabricate thicker multilamellar TEBVs.
The production of small-diameter, complex-geometry TEBVs obtained from patients could be an innovative approach to modeling various vascular diseases. For example, the engineered Y-shaped TEBV will be a unique in vitro model for studying intracranial aneurysms (IAs), which primarily occur at the bifurcation of the circle of Willis in affected individuals 41, 42 . AI is a cerebrovascular disorder in which weakness of the cerebrovascular walls leads to focal ballooning43. AIs show 50% mortality and 30-50% morbidity among survivors of their rupture44. Artificial but physiological blood flow can be created using Y-shaped TEBVs cultured in bioreactors, which is of particular interest in the study of IA and hemodynamics. Since the genetic components of IA are still not well understood, multilamellar and branching TEPs in patients with fully vascularized intima, media, and adventitia are also of interest for studying the pathogenic and pathophysiological mechanisms associated with IA.
In conclusion, we present an innovative rotary cell seeding system that is easy to use, washable and sterilizable and can produce five TEBVs with a small diameter tubular outer membrane with complex geometries. In general, the seeding system and chambers have been thought out and adapted for ease of use. Therefore, given that uniform distribution of cells across scaffolds during cell seeding is critical for 3D culture and tissue engineering, the described system will have a significant impact on future research.
CREO 5.0 software (PTC, Boston, Massachusetts, USA) was used to create a CAD system for the seeding system prior to construction (Figure 1A). The spheroid was made from two halves of a 10 inch diameter hollow acrylic sphere (California Quality Plastics, Ontario, CA, USA) (Figure 1B,C). Two 3D printed closed loops made from 1.75 mm PETG filament (Filaments.ca, Mississauga, Ontario, Canada) printed on an H800 3D printer (Afinia, Chanhassen, MN, USA) and Silastic™ medical adhesive . Bond to Hemisphere Superior elastomer (DuPont, Wilmington, NC, USA). The closed rings were secured with stainless steel hex screws (McMaster-Carr, Chicago, IL, USA) screwed into a PETG male ring and locked into a PETG female ring. The 3D printing plates were conceived and printed as described earlier to protect the seed chamber. The plates are attached to the spheres with 3D printed supports that are glued to the center of the hemisphere with Silastic™. To keep the seed chamber in place as the system rotates, the pressure is increased by a corrosion resistant stainless steel spring (McMaster-Carr). The base plate was fabricated from 6061-T6 aluminum alloy (Acier Picard, Levis, QC, Canada) using a 3-axis milling machine (Fryer Machine Systems, Patterson, NY, USA) (Figure 1d). Two DC collector motors 12 V, 10 rpm (RobotShop, Mirabel, QC, Canada), placed perpendicular to each other, provided the rotational movement of the system. Two motor mounts and three aluminum ball bearing mounts were custom made and attached to the board. Corrosion-resistant stainless steel balls are placed in support bearings to ensure proper ball rotation and provide extra support (McMaster-Carr). The electronic components that provide the current and control the speed are housed inside a 3D printed PETG box. The motors were connected to separate 3-position switches, 25 kΩ potentiometers, linear regulators (Digi-Key, Thief River Falls, Min, USA) and 5V medical AC/DC power supplies (Newark, Mississauga, Ontario, Canada) . Standard 125V outlet. All photos and videos were taken with an iPhone 12 mini camera (Apple, Cupertino, CA, USA).
The CREO 5.0 software (PTC) was also used to create the CAD for the seed chamber (Fig. 2A-C). The chamber consists of two polycarbonate halves (Groupe PolyAlto, Quebec, QC, CA). Cut out Y-shaped recesses in both halves using a 3-axis milling machine (Fryer Machine Systems). The bottom half is surrounded by a 3/32 wide Y-shaped high temperature soft silicone O-ring (McMaster-Carr) to seal the two halves of the seed chamber to each other (Figure 2D). The top half consists of three holes, used as priming ports, sealed with a heat-resistant soft silicone O-ring, 3/64 wide, and a 18-8 stainless steel cone end set screw, M4 × 0.7 mm thread, 4 mm long (McMaster -Carr). ). Y-shaped removable braces were custom-made from 4.8 mm diameter PETG rods (McMaster-Carr) using a 5-axis milling machine (Fryer Machine Systems, Patterson, NY, USA) and secured with 18–8 pins stainless steel 1/. 32″ diameter, 1/4″ length (McMaster-Carr). This scaffolding is placed in the center of the boarding chamber, inside a Y-notch, and the two halves are connected together with 316 stainless steel hex head screws, thread M4 x 0.7mm, length 16mm, screw size M4. Inner diameter 4300 mm, outer diameter 8 mm. General Purpose 316 Stainless Steel Washers and Extra Corrosion Resistant 316 Stainless Steel Hex Nuts, M4 x 0.7mm Thread (McMaster-Carr) (Figure 2E). All photos taken with iPhone 12 (Apple) mini camera.
Human dermal fibroblasts were isolated as described previously and cultured in DMEM containing 10% fetal bovine serum (FBS; VWR, Radnor, PA, USA), 100 IU/mL penicillin G, and 25 µg/mL (Invitrogen, Burlington, ON ). , Canada) to a culture of gentamicin (Sigma-Aldrich, Kawasaki, OL, Japan). The use of human cells was approved by the CHU de Quebec Ethical Research Committee (protocol numbers: 1115C and 1115D) and people were recruited voluntarily after informed consent. Cells were seeded with 10 ml of DMEM medium into chambers containing 4.8 mm diameter PETG rods treated with UV-C (McMaster-Carr). The seed chambers were placed in a rotary seeding system and maintained at 37° C. for subsequent experiments. UV-C treatment was performed as previously described for 30 minutes per side (90° rotation) followed by coating with 0.2% gelatin (Fisher Scientific, Waltham, MA, USA)14. To determine the optimal cell concentration to use during the initial inoculation, three conditions (0.10, 0.15, and 0.30 M/mL) were tested and added to the cell chamber, which was then placed in a rotating system at a moderate speed. for 22 hours. Three different rotation speeds were also tested to better optimize cell seeding parameters. The chambers were seeded with fibroblasts (0.15 M/ml) and placed in rotating spheres for 22 hours at rotation speeds of 63°, 90° or 135°/min, which were considered the minimum, average and maximum achievable speed determined by the sphere. Various incubation periods after cell seeding were also tested. The inoculation chamber was inoculated with fibroblasts (0.15 M/ml) placed in spheroids at a rotation speed of 90°/min for 4, 8, 16, and 22 hours.
At the end of each incubation time, the supernatant was removed and centrifuged at 300 g for 10 minutes. The remaining unattached and centrifuged cells present in the supernatant were resuspended in 1 ml of DMEM medium and counted using a cell counter (Beckman Coulter, Pasadena, CA, USA). Cell seeded PETG rods or Y scaffolds were first incubated with 0.05% trypsin (Fisher Scientific)/0.01% EDTA (Teknisciences, Terrebonne, QC, Canada) for 10 min to separate cells and then centrifuged at 300× g for 10 min. 10 minutes. The recovered cells were resuspended in 1 ml of culture medium and then counted using a Coulter cell counter (Beckman Coulter).
Human dermal fibroblasts were cultured in DMEM containing 10% FBS and a mixture of antibiotics. Inoculation of 0.15 M/ml of cells into a special seed chamber through the seed hole. Inject a total of 10 ml of DMEM into the inoculum chamber containing a Y-shaped holder made of 4.8 mm diameter PETG rods (McMaster-Carr). PETG scaffolds were pretreated with UV-C and then coated with 0.2% gelatin (Fisher Scientific, Waltham, MA, USA) as previously described 14 . To inoculate the spheroids, place the inoculation chamber in a 37°C room at 90°/min for 22 hours. For dynamic seeding, place the seed chamber on an orbital shaker (BlotBoy, Benchmark Scientific, Sayreville, NJ, USA) in an incubator at 37°C, 8% CO2 for 22 hours. For static inoculation, place the culture chamber directly in an incubator at 37°C, 8% CO2 for a 22-hour inoculation period. The seeded scaffolds were then removed and placed in 500 cm2 cell culture plates containing DMEM supplemented with 50 µg/mL ascorbic acid (Sigma) for 42 days in an incubator at 37°C, 8% CO2. Change media every 2-3 days by rotating the holder 180° for each media change each day.
Macroscopic images of cell-seeded PETG Y-scaffolds were obtained using an iPhone 12 mini camera (Apple). Tissue thickness on three different branches of the Y-frame was measured using a high-precision laser micrometer (Keyence, Mississauga, ON, Canada). For statistical analysis, four different measurements were used on each branch of four different Y-shaped TEBVs. TEBV was fixed overnight in 3.7% formalin (ChapTec, Montreal, Quebec, Canada). Macroscopic photographs of TEBV biopsy compounds were also taken prior to histological analysis. Fixed 10 µm TEBV transverse sections were then stained with hematoxylin and eosin (H&E) as previously described36. Microscopic images were acquired and measured under bright field conditions using an upright microscope (AxioImager.M2; Carl Zeiss Microscopy, Jena, TH, Germany). For distribution analysis, the seeded matrices were fixed in 3.7% formalin for 30 min, then stained with rhodanium blue for 15 min, washed and allowed to dry before taking macroscopic photographs of the scaffolds from all sides.
After a 22 hour inoculation period, supernatants were removed and centrifuged at 300 g for 10 minutes for each inoculation method. Seeded PETG rods were incubated with 0.05% trypsin (Fisher Scientific)/0.01% EDTA (Teknisciences) for 10 minutes to separate the cells followed by centrifugation at 300 g for 10 minutes. Cells recovered from supernatants and scaffolds were incubated with Calcein AM 2.5 nM (Thermo-Fisher) for 15 minutes to label live cells and 4 μM Ethidium homodimer-1 (Thermo-Fisher) to label dead cells. Cells were immediately analyzed using a BD FACSMelody™ flow cytometer (BD Biosciences) and data was processed using FlowJo™ v9 software (Ashland, OR, USA). For mature tissues, they are first removed from the scaffold and placed in digested accutase (Sigma-Aldrich) containing 5.7 U/mL collagenase H (Sigma-Aldrich) with agitation at 300 rpm for 30 min at 37 °C Cells were filtered through a 40 μm cell filter (Fisher Scientific) and centrifuged at 300×g for 10 minutes. Finally, cells were analyzed as described below.
Statistical analysis was performed using GraphPad Prism 9.0 software (GraphPad, San Diego, CA, USA). The data, presented as box and whisker plots with maximum and minimum values, were analyzed using two-way ANOVA and Tukey’s multiple comparisons test or Kruskal-Wallis’s test and Dunn’s multiple comparisons test. Data presented as mean and standard deviation (SD) scatterplots were analyzed using the Kruskal-Wallis test and Dunn’s multiple comparison test or Welch’s t-test. SD stacked bar data were analyzed using the Kruskal-Wallis test and Dunn’s multiple comparison test. A P value <0.05 was considered statistically significant.
The study was approved by our institutional ethics committee (CHU de Québec Ethical Research Committee; protocol numbers 1115 C and 1115 D). For more information, please contact (gurecherche@chuq.qc.ca). All experiments were carried out in accordance with the “Three Councils National Policy Guidelines: Ethical Conduct in Human Research” and were approved by the ethics committee of the CHU de Quebec-Université Laval.
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