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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

Abstract

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach—the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis—are discussed in detail.

Introduction

Cardiovascular tissue engineering (TE) is being pursued as an alternative treatment option to the currently used permanent cardiovascular prostheses (e.g., vascular grafts, heart valve replacements), which are suboptimal for large cohorts of patients1,2,3,4. Much sought-after applications include tissue-engineered vascular grafts (TEVGs)5,6 and heart valves (TEHVs)7,8. Most often, cardiovascular TE methodologies make use of r....

Protocol

In the studies described in this protocol, primary human macrophages isolated from peripheral blood buffy coats and human myofibroblasts isolated from the saphenous vein after coronary by-pass surgery have been used44. The buffy coats were obtained from healthy, anonymized volunteers who provided written informed consent, which was approved by the Sanquin Research Institutional Medical Ethical Committee. The use of human vena saphena cells (HVSCs) was in accordance to the “Code Proper S.......

Representative Results

This bioreactor was developed to study the individual and combined effects of shear stress and cyclic stretch on vascular tissue growth and remodeling in 3D biomaterial scaffolds. The design of the bioreactor allows for culturing up to eight vascular constructs under various loading conditions (Figure 1A). The vascular constructs are positioned in a flow culture chamber (Figure 1B) in which both the circumferential stretch and WSS can be independently controlled.......

Discussion

The bioreactor described herein allows for the systematic evaluation of the contributions of the individual and combined effects of shear stress and cyclic stretch on inflammation and tissue regeneration in tubular resorbable scaffolds. This approach also enables a large variety of analyses to be performed on vascular constructs, as exemplified in the representative results section. These results show the distinctive impact of the different hemodynamic loading regimes (i.e., different combinations of shear and stretch) o.......

Acknowledgements

This study is financially supported by ZonMw as part of the LSH 2Treat program (436001003) and the Dutch Kidney Foundation (14a2d507). N.A.K. acknowledges support from the European Research Council (851960). We gratefully acknowledge the Gravitation Program “Materials Driven Regeneration”, funded by the Netherlands Organization for Scientific Research (024.003.013).

....

Materials

NameCompanyCatalog NumberComments
advanced Dulbecco’s modified EagleMedium (aDMEM)Gibco12491-015cell culture medium for fibroblasts
Aqua StabilJulabo8940012prevent microorganism growth in bioreactor-hydraulic reservoir
Bovine fibrinogenSigmaF8630to prepare fibrinogen gel to seed the cells on the electrospun scaffold
Bovine thrombinSigmaT4648to prepare fibrinogen gel to seed the cells on the electrospun scaffold
CentrifugeEppendorf5804to spin down cells and conditioned medium
Clamp scissor - "kelly forceps"AlmedicP-422clamp the silicone tubing and apply pre-stretch to the scaffold so the scaffold can be sutured into the engraved groove (autoclave at step 1, step 7)
CO2 cell culture incubatorsSanyoMCO-170AIC-PEfor cell culturing
Compressed air reservoirFestoCRVZS-5smoothing air pressure fluctuations and create time delays for pressure build-up
Custom Matlab script to calculate the maximum stretchesMatlabR2017. The Mathworks, Natick, MAcalculate the minimum and maximum outer diameter of the electrospun scaffold
Data acquisition boardNational InstrumentsBNC-2090data processing in between amplifier system and computer
EthanolVWRVWRK4096-9005to keep sterile working conditions
Fetal bovine calf serum (FBS)Greiner758087cell culture medium supplement; serum-supplement
Flow culture chamber compartments, consisting of a pressure conduit with engraved grooves and small holes to apply pressure on silicone tubing, a screw thread, nose cone, top compartment with flow inlet and bottom compartment flow outlet, adapter bushingCustom made, Department of Biomedical Engineering, Eindhoven University of Technologyn.a.flow culture chamber compartments (autoclave at step 1, step 7)
Glass Pasteur pipetAssistantHE40567002apply vacuum on electrospun scaffold (autoclave at step 1)
Glass tubes of the flow culture chamberCuston made, Equipment & Prototype Center, Eindhoven University of Technologyn.a.part of the flow culture chamber (clean and store in 70% ethanol, at step 1 and 7)
GlutaMaxGibco35050061cell culture medium amino acid supplement, minimizes ammonia build-up
High speed cameraMotionScopeM-5to monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)
High speed camera lens - Micro-NIKKOR 55mm f/2.8 - lensNikonJAA616ABto monitor the stretch during culture; time-lapse photographs of the scaffolds are captured at a frequency of 30 Hz for 6 sec (i.e. 3 stretch cycles)
Hose clipibidi GmbH10821block medium flow (autoclave at step 1, step 7)
Hydraulic reservoir with 8 screw threads for 8 flow culture chambersCustom made, Department of Biomedical Engineering, Eindhoven University of Technologyn.a.to apply pressure to the silicone mounted constructs (clean outside with a paper tissue with 70% ethanol, rinse reservoir with 70% ethanol followed by demi water, at step 1 and 7)
Ibidi pump system (8x) including ibidi pump, PumpControl software, fluidic unit, perfusion set (medium tubing), air pressure tubing, drying bottles with orange silica beadsibidi GmbH10902set up used to control the flow in the flow culture chambers. Note 1: the ibidi pumps were modified by the manufacturer to enable 200 mbar capacity. Note 2: can be replaced by pump system of other manufacturer, as long as same flow regimes can be applied.
Knives (no.10 sterile blades, individual foil pack) and scalpel handle (stainless steel, individually wrapped)Swann Morton0301; 0933to cut the silicone tubing in the correct size for the scaffold and to cut the suture material
LabVIEW SoftwareNational Instrumentsversion 2018to control the stretch applied to the scaffolds
Laminar flow biosafety cabinet with UV lightLabconco302310001to ensure sterile working conditions. The UV is used to decontaminate everything that cannot be autoclaved, or touched after autoclaving
Large and small petri dishesGreiner664-160for sterile working conditions
L-ascorbic acid 2-phosphate (vitamin C)SigmaA8960cell culture medium supplement, important for collagen production
LED light cold source KL2500ZeissSchott AGto aid in visualization for the time lapse of the scaffolds during monitoring of the stretch
Luer (female and male) locks and connectors, white luer capsibidi GmbHvarious, see (https://ibidi.com/26-flow-accessories)to close or connect parts of the bioreactor and the ibidi pump (autoclave at step 1, step 7)
Measuring amplifier (PICAS)PEEKEL instruments B.V.n.a.to amplify the signal from the pressure sensor and feedback to LabView
Medium reservoir (large syringes 60 mL) and reservoir holdersibidi GmbH10974medium reservoir (autoclave at step 1, step 7)
Medium tubing with 4.25 mm outer diameter and 1 mm inner diameterRubber BV1805to allow for a larger flow rate, the ibidi medium tubing with larger diameter is used. Note: the part of medium tubing guided through the fluidic unit valves are the same as the default ibidi medium tubing
Motion Studio SoftwareIdtvision2.15.00to make the high speed time lapse images for stretch monitoring
Needle (19G)BD Microlance301700together with thin flexible tubing used to fill the hydraulic reservoir with ultrapure water without adding air bubbles
Needle driverAdson2429218to handle the needle of the nylon suture through the silicone tube (autoclave at step 1, step 7)
Paper tissuesKleenex38044001for cleaning of the equipment with 70% ethanol
ParafilmSigmaP7793-1EAquick fix if leakage occurs
Penicillin/streptomycin (P/S)LonzaDE17-602Ecell culture medium supplement; prevent bacterial contamination
Phosphate Buffered Saline (PBS)SigmaP4417-100TABfor storage and washing steps (autoclave at step 1)
Plastic containers (60 mL) with red screw capsGreiner206202to prepare the fibrinogen solution
Pneumatic cylinderFestoAEVC-20-10-I-Pto actuate the Teflon bellow (clean with a paper tissue with 70% ethanol at step 1 and 7)
Polycaprolactone bisurea (PCL-BU) tubular scaffolds (3 mm inner diameter, 200 µm wall thickness, 20 mm length)SyMO-Chem, Eindhoven, The Netherlandsn.a.produced using electrospinning from 15% (w/w) chloroform (Sigma; 372978) polymer solutions. See Van Haaften et al Tissue Engineering Part C (2018) for more details
Pressure conduit without holes (for static control)Custom made, Department of Biomedical Engineering, Eindhoven University of Technologyn.a.to mount electrospun tubes on silicon tubing (autoclave at step 1, step 7)
Pressure sensor and transducerBDTC-XX and P 10 EZthe air pressure going to the pneumatic actuated pump is raised until it reaches the set pressure
Proportional air pressure control valve and pressure sensorFestoMPPES-3-1/8-2-010, 159596provides compressed air to the pneumatic actuated pump
Roswell Park Memorial Institute 1640 (RPMI-1640)GibcoA1049101cell culture medium for monocyte/macrophage
Safe lock Eppendorf tubes (1.5 mL)Eppendorf30120086multiple applications (autoclave at step 1)
Sodium dodecyl sulfate solution 20%Sigma5030Used to clean materials, at a concentration of 0.1%.  
Silicone O-ringsTechnirub1250Sto prevent leakage (autoclave at step 1, step 7)
Silicone tubing (2.8 mm outer diameter, 400 um wall thickness)Rubber BV1805to mount the electrospun tubes on the pressure conduits (autoclave at step 1)
Sterile tube (15 mL)Falcon352095multiple applications
Suture, 5-0 prolene with pre-attached taper point needleEthicon, Johnson&JohnsonEH7404HProlene suture wire 5-0 (75cm length, TF taper point needle, 1/2 circle, 13 mm needle length)
Syringe (24 mL)B. Braun Melsungen AG2057932to add the ultrapure water or medium to the hydraulic reservoir or flow culture chamber
Syringe filter (0.2 µm)Satorius17597-Kto filter the fibrinogen solution
T150 cell culture flask with filter capNunc178983to degas culture medium
T75 Cell culture flask with filter capNunc156499to culture static control samples
Teflon bellowCustom made, Department of Biomedical Engineering, Eindhoven University of Technologyn.a.to load the hydraulic reservoir (clean outside with a paper tissue with 70% ethanol at step 1 and 7)
Tray (stainless steel)PolarWare15-248for easy transport of the fluidic culture chambers and the bioreactor from incubator to laminar flow cabinet and back (clean with a paper tissue with 70% ethanol before and after use)
TweezersWironit4910sterile handling of individual parts (autoclave at step 1 and 7)
Ultrapure waterStakpureOmniapure UV 18200002to correct for medium evaporation, mixed with aqua stabil mixed and used as hydraulic fluid. (autoclave ultrapure water at step 1)
UV lightPhilipsTUV 30W/G30 T8for decontamination of grafts and bioreactor parts before seeding

References

  1. Chlupác, J., Filová, E., Bacáková, L. Blood vessel replacement: 50 years of development and tissue engineering paradigms in vascular surgery. Physiological Research. 58, 119-139 (2009).
  2. Huygens, S. A., et al.

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