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Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Engineering

En Multi-Cue Bioreactor til at evaluere inflammatoriske og regenerative kapacitet Biomaterialer under Flow og Stretch

Published: December 10, 2020
doi:
10.3791/61824
, , , , , , ,
1Department of Biomedical Engineering,Eindhoven University of Technology, 2Institute for Complex Molecular Systems (ICMS),Eindhoven University of Technology

Summary

Målet med denne protokol er at udføre en dynamisk co-kultur af menneskelige makrofager og myofibroblaster i rørformede elektropun stilladser til at undersøge materiale-drevet væv regenerering, ved hjælp af en bioreaktor, som gør det muligt afkobling af shear stress og cyklisk stretch.

Abstract

Brugen af resorbable biomaterialer til at fremkalde regenerering direkte i kroppen er en attraktiv strategi fra et translationelt perspektiv. Sådanne materialer fremkalder en inflammatorisk reaktion ved implantation, som er drivkraften bag efterfølgende resorption af materialet og regenerering af nyt væv. Denne strategi, også kendt som in situ vævsteknik, forfølges for at opnå hjerte-kar-udskiftninger såsom væv-manipuleret vaskulære grafts. Både de inflammatoriske og regenerative processer bestemmes af de lokale biomekaniske signaler på stilladset (dvs. stræk og forskydningsstress). Her beskriver vi i detaljer brugen af en specialudviklet bioreaktor, der unikt muliggør afkobling af stræk- og forskydningsstress på et rørformet stillads. Dette giver mulighed for systematisk og standardiseret evaluering af den inflammatoriske og regenerative kapacitet af rørformede stilladser under påvirkning af velkontrollerede mekaniske belastninger, som vi demonstrerer på grundlag af et dynamisk co-kultur eksperiment ved hjælp af menneskelige makrofager og myofibroblasts. De vigtigste praktiske skridt i denne fremgangsmåde — konstruktion og opsætning af bioreaktoren, forberedelse af stilladser og cellesåning, anvendelse og vedligeholdelse af stræk- og forskydningsflow og prøvehøst til analyse — gennemgås i detaljer.

Introduction

Kardiovaskulær vævsteknik (TE) forfølges som en alternativ behandlingsmulighed til de aktuelt anvendte permanente hjerte-kar-proteser (f.eks. vaskulære grafts, hjerteklapproteser), som er suboptimale for store kohorter af patienter1,2,3,4. Meget eftertragtede applikationer omfatter vævsfremstillede vaskulære grafts (TEVGs)5,6 og hjerteklapper (TEHVs)7,8. Oftest gør kardiovaskulære TE-metoder brug af resorbable biomaterialer (enten naturlige eller syntetiske), der tjener som et lærerigt stillads til det nye væv, der skal dannes. Dannelsen af nyt væv kan enten konstrueres fuldstændigt in vitro ved at så stilladset med celler og dyrkning i en bioreaktor inden implantation (in vitro TE)9,10,11eller direkte in situ, hvor det syntetiske stillads implanteres uden forudgående dyrkning for at fremkalde dannelsen af nyt væv direkte i kroppen (in situ TE)12,13,14. For både in vitro- og in situ-kar-te-tilgange er vellykket funktionel regenerering overvejende afhængig af både værtens immunrespons på den implanterede konstruktion og passende biomekanisk belastning.

Betydningen af biomekanisk belastning for hjerte-kar-TE er velkendt15. I tilfælde af hjerte-kar-implantater udsættes de celler, der befolker stilladset, for cyklisk stræk og forskydningsspændinger, der opstår som følge af det hæmodynamiske miljø. Talrige undersøgelser har rapporteret den stimulerende virkning af (cyklisk) strækning på dannelsen af matrixkomponenter, såsom kollagen16,17,18,19, glycosaminoglycans (GAGs)20, og elastin21,22, af forskellige celletyper. For eksempel viste Huang et al. at toårig strækning forhøjede depositionen og organiseringen af kollagen og elastin i in vitro TEVGs ved hjælp af en vaskulær bioreaktor23. Mens vægten typisk ligger på stræk som den dominerende belastning, gør disse undersøgelser ofte brug af flowdrevne bioreaktorer, hvor prøven også udsættes for forskydningsflow. Selv om relativt lidt er kendt om den isolerede indflydelse af forskydning understreger på væv dannelse og betændelse i 3D, nogle data er tilgængelige. For eksempel viste Hinderer et al. og Eoh et al., at forskydningsflow ud over en 3D-stilladsmikrostruktur var vigtig for dannelsen af moden elastin af menneskelige vaskulære glatte muskelceller i et in vitro-modelsystem24,25. Alt i alt illustrerer disse resultater relevansen af både cyklisk stræk og forskydningsstress for kardiovaskulær TE.

En anden vigtig determinant for succes eller svigt af TE implantater er værtens immunrespons på den implanterede graft26. Dette er især vigtigt for materialedrevne in situ TE-strategier, som faktisk er afhængige af den akutte inflammatoriske reaktion på stilladset for at kickstarte de efterfølgende processer med cellulær tilstrømning og endogen vævsdannelse og remodeling27. Makrofaget er en kritisk initiator for funktionel vævsgendannelse, som er blevet vist ved flere undersøgelser28,29,30. Svarende til sårheling, regenerering af væv er omfattet af parakrine signalering mellem makrofager og vævsproducerende celler såsom fibroblaster og myofibroblasts31,32,33. Ud over at koordinere nye væv deposition, makrofager er involveret i aktiv resorption af udenlandske stillads materiale34,35. Som sådan er in vitro-makrofagets reaktion på et biomateriale blevet identificeret som en prædiktiv parameter for implantaternes in vivo-succes36,37,38.

Makrofagets respons på et implanteret stillads afhænger af stilladsdesignfunktioner som materialesammensætning og mikrostruktur35,39,40. Ud over stilladsegenskaber påvirkes makrofagets reaktion på et stillads og deres krydstale med myofibroblaster også af hæmodynamiske belastninger. For eksempel viste cyklisk strækning sig at være en vigtig modulator af makrofagfostype41,42,43,44 og sekretionen af cytokiner43,44,45,46 i 3D elektrospun stilladser. Ved hjælp af et co-kultur system af makrofager og vaskulære glatte muskelceller, Battiston et al. vist, at tilstedeværelsen af makrofager førte til øgede niveauer af elastin og GAGs, og at moderate niveauer af cyklisk stretch (1,07-1,10) stimuleret deposition af kollagen I og elastin47. I tidligere værker har vi vist, at forskydningsstress er en vigtig determinant for monocytrekruttering i 3D elektrospun stilladser48,49, og at både forskydningsstress og cyklisk stræk påvirker parakrinesignalering mellem menneskelige monocytter og mesenchymale stromale celler50. Fahy et al. påviste, at forskydningsstrømmen øgede udskillelsen af proinflammatoriske cytokiner ved menneskelige monocytter51.

Samlet set viser ovenstående beviser, at en passende forståelse af og kontrol over hæmodynamiske belastninger er afgørende for kardiovaskulær TE, og at det er vigtigt at overveje den inflammatoriske reaktion for at opnå dette. Talrige bioreaktorer er tidligere blevet beskrevet for in vitro52,53,54,55,56,57,58 eller ex vivo59,60,61 kultur af hjerte-kar-væv. Men alle disse systemer er designet til at efterligne de fysiologiske hæmodynamiske belastningsforhold så meget som muligt. Selv om dette er meget værdifuldt med henblik på at skabe hjerte-kar-væv in vitro eller opretholde ex vivo kulturer, sådanne systemer ikke giver mulighed for systematiske undersøgelser af de enkelte virkninger af de enkelte signaler. Dette skyldes, at anvendelsen af både cyklisk stræk og forskydningsbelastning i disse bioreaktorer er drevet af det samme trykstrøm, som i sig selv forbinder dem. Mens mikrosystemer, der giver mulighed for nøjagtig multi-cue mekanisk manipulation er blevet beskrevet for 2D substrater62 eller 3D hydrogel opsætninger63,64, sådanne opsætninger ikke giver mulighed for inkorporering af elastomeriske 3D biomateriale stilladser.

Her præsenterer vi anvendelsen af et rørformet bioreaktorsystem, der unikt muliggør afkobling af forskydningsstress og cyklisk stræk og hjælper med mekanisk at undersøge deres individuelle og kombinerede virkninger. Dette system giver mulighed for testning af en bred vifte af vævsfremstillede vaskulære grafts (f.eks syntetisk eller naturlig oprindelse, forskellige mikroarkitekturer, forskellige porøsiteter). For effektivt at afkoble anvendelsen af forskydningsstress og stræk er de nøglebegreber, som bioreaktoren bruger, (1) adskillelse af styringen af forskydningsstress og stræk ved hjælp af forskellige pumpesystemer og (2) stimulering af stilladserne på en ‘inside-out’ måde med beregningsmæssigt drevne dimensioner. Flow påføres på ydersiden af det rørformede stillads ved hjælp af en strømningspumpe, mens stilladsets omkredssstrækning induceres ved at udvide et silikonerør, hvor stilladset er monteret ved hjælp af en separat stammepumpe. Dimensionerne af silikonerøret og glasrøret, der indeholder konstruktionen, er omhyggeligt udvalgt og valideret ved hjælp af beregningsmæssige væskedynamiksimuleringer for at sikre, at forskydningsspændingen på stilladset (på grund af flow) og omkredsstrækningen (på grund af rørudvidelse) ikke påvirker hinanden væsentligt. Dette inside-out design har flere praktiske rationaler. Hvis strækningen påføres af det luminale væsketryk (svarende til fysiologisk belastning), kræver det i sagens natur, at prøvedesignet er lækagefrit. Desuden vil det tryk, der kræves for at strække prøven, blive fuldstændig bestemt af prøvestivheden, som kan variere mellem prøverne og inden for en prøve over tid, hvilket gør det vanskeligt at kontrollere strækningen. Denne bioreaktor monterer vævsfremstillet graft omkring et silikonerør og giver mulighed for vægforskydningsbelastning (WSS) på graftens ydervæg og presser graften indefra. På denne måde kan der sikres lige belastningsforhold mellem prøver og inden for prøver over tid, og desuden må prøverne være utætte, som det er almindeligt for porøse vaskulære stilladser19. Denne inside-out bioreaktor er specielt beregnet til systematiske undersøgelser af virkningerne af shear og / eller stretch, snarere end konstruktionen af en indfødt-lignende blodkar in vitro, som traditionelle vaskulære bioreaktor opsætninger er mere egnede. Se figur 1A-B for tegningerne af bioreaktordesign og den tilsvarende tabel 1 for en funktionel beskrivelse og begrundelse bag bioreaktorens hovedkomponenter.

Brugen af bioreaktoren demonstreres på grundlag af en række nylige undersøgelser fra vores gruppe, hvor vi undersøgte de individuelle og kombinerede påvirkninger af forskydningsstress og cyklisk strækning på inflammation og vævsdannelse i resorbable elektrospun stilladser til in situ hjerte-kar-væv19,43,44. Til det formål brugte vi menneskelige makrofager og myofibroblaster enten i mono- eller i co-kultur til at simulere de forskellige faser af in situ regenerativ kaskade. Vi har vist, at cytokin sekretion af menneskelige makrofager er tydeligt påvirket af både cyklisk stræk og forskydning stress, påvirker matrix deposition og organisation af menneskelige myofibroblasts i disse stilladser, både via parakrine signalering og direkte kontakt19,43,44. Især viste disse undersøgelser, at i tilfælde af kombineret anvendelse af forskydningsstress og stræk er virkningerne på vævsdannelse og betændelse enten domineret af en af de to belastninger, eller der er synergistiske virkninger af begge belastninger. Disse resultater illustrerer relevansen af afkobling af begge belastninger for at få en bedre forståelse af det mekaniske miljøs bidrag til TE-processer. Denne forståelse kan anvendes til systematisk at optimere stilladsdesignparametre i relevante hæmodynamiske belastningsregimer. Derudover kan de mekanistiske data fra sådanne velkontrollerede miljøer tjene som input til numeriske modeller, der udvikles for at forudsige forløbet af in situ-vævsombygning, som for nylig rapporteret for TEVGs65 eller TEHVs66, for yderligere at forbedre prædiktiv kapacitet.

Protocol

I de undersøgelser, der er beskrevet i denne protokol, er primære menneskelige makrofager isoleret fra perifere blod buffy frakker og humane myofibroblasts isoleret fra den saphenous vene efter koronar by-pass kirurgi er blevet brugt44. De buffy frakker blev opnået fra sunde, anonymiserede frivillige, der gav skriftligt informeret samtykke, som blev godkendt af Sanquin Research Institutional Medical Ethical Committee. Brugen af humane vena saphena celler (HVSCs) var i overensstemmelse med “Code…

Representative Results

Denne bioreaktor blev udviklet til at studere de individuelle og kombinerede virkninger af forskydningsstress og cyklisk stræk på vaskulær vævsvækst og remodeling i 3D biomateriale stilladser. Bioreaktorens design gør det muligt at dyrke op til otte vaskulære konstruktioner under forskellige belastningsforhold (figur 1A). De vaskulære konstruktioner er placeret i et flowkulturkammer (Figur 1B), hvor både omkredsen og WSS kan styres uafhængigt. Det øve…

Discussion

Den bioreaktor, der er beskrevet heri, giver mulighed for systematisk evaluering af bidragene fra de individuelle og kombinerede virkninger af forskydningsstress og cyklisk strækning på inflammation og vævsgendannelse i rørformede resorbable stilladser. Denne fremgangsmåde gør det også muligt at foretage en lang række analyser af vaskulære konstruktioner, som eksemplificeret i afsnittet om repræsentative resultater. Disse resultater viser den karakteristiske virkning af de forskellige hæmodynamiske belastnings…

Disclosures

The authors have nothing to disclose.

Acknowledgements

Denne undersøgelse er økonomisk støttet af ZonMw som en del af LSH 2Treat program (436001003) og den hollandske Kidney Foundation (14a2d507). N.A.K. anerkender støtte fra Det Europæiske Forskningsråd (851960). Vi anerkender taknemmeligt gravitationsprogrammet “Materials Driven Regeneration”, finansieret af Den Nederlandske Organisation for Videnskabelig Forskning (024.003.013).

Materials

advanced Dulbecco’s modified EagleMedium (aDMEM) Gibco 12491-015 cell culture medium for fibroblasts
Aqua Stabil Julabo 8940012 prevent microorganism growth in bioreactor-hydraulic reservoir
Bovine fibrinogen Sigma F8630 to prepare fibrinogen gel to seed the cells on the electrospun scaffold
Bovine thrombin Sigma T4648 to prepare fibrinogen gel to seed the cells on the electrospun scaffold
Centrifuge Eppendorf 5804 to spin down cells and conditioned medium
Clamp scissor – "kelly forceps" Almedic P-422 clamp 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 incubators Sanyo MCO-170AIC-PE for cell culturing
Compressed air reservoir Festo CRVZS-5 smoothing air pressure fluctuations and create time delays for pressure build-up
Custom Matlab script to calculate the maximum stretches Matlab R2017. The Mathworks, Natick, MA calculate the minimum and maximum outer diameter of the electrospun scaffold
Data acquisition board National Instruments BNC-2090 data processing in between amplifier system and computer
Ethanol VWR VWRK4096-9005 to keep sterile working conditions
Fetal bovine calf serum (FBS) Greiner 758087 cell 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 bushing Custom made, Department of Biomedical Engineering, Eindhoven University of Technology n.a. flow culture chamber compartments (autoclave at step 1, step 7)
Glass Pasteur pipet Assistant HE40567002 apply vacuum on electrospun scaffold (autoclave at step 1)
Glass tubes of the flow culture chamber Custon made, Equipment & Prototype Center, Eindhoven University of Technology n.a. part of the flow culture chamber (clean and store in 70% ethanol, at step 1 and 7)
GlutaMax Gibco 35050061 cell culture medium amino acid supplement, minimizes ammonia build-up
High speed camera MotionScope M-5 to 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 – lens Nikon JAA616AB to 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 clip ibidi GmbH 10821 block medium flow (autoclave at step 1, step 7)
Hydraulic reservoir with 8 screw threads for 8 flow culture chambers Custom made, Department of Biomedical Engineering, Eindhoven University of Technology n.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 beads ibidi GmbH 10902 set 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 Morton 0301; 0933 to cut the silicone tubing in the correct size for the scaffold and to cut the suture material
LabVIEW Software National Instruments version 2018 to control the stretch applied to the scaffolds
Laminar flow biosafety cabinet with UV light Labconco 302310001 to ensure sterile working conditions. The UV is used to decontaminate everything that cannot be autoclaved, or touched after autoclaving
Large and small petri dishes Greiner 664-160 for sterile working conditions
L-ascorbic acid 2-phosphate (vitamin C) Sigma A8960 cell culture medium supplement, important for collagen production
LED light cold source KL2500 Zeiss Schott AG to aid in visualization for the time lapse of the scaffolds during monitoring of the stretch
Luer (female and male) locks and connectors, white luer caps ibidi GmbH various, 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 holders ibidi GmbH 10974 medium reservoir (autoclave at step 1, step 7)
Medium tubing with 4.25 mm outer diameter and 1 mm inner diameter Rubber BV 1805 to 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 Software Idtvision 2.15.00 to make the high speed time lapse images for stretch monitoring
Needle (19G) BD Microlance 301700 together with thin flexible tubing used to fill the hydraulic reservoir with ultrapure water without adding air bubbles
Needle driver Adson 2429218 to handle the needle of the nylon suture through the silicone tube (autoclave at step 1, step 7)
Paper tissues Kleenex 38044001 for cleaning of the equipment with 70% ethanol
Parafilm Sigma P7793-1EA quick fix if leakage occurs
Penicillin/streptomycin (P/S) Lonza DE17-602E cell culture medium supplement; prevent bacterial contamination
Phosphate Buffered Saline (PBS) Sigma P4417-100TAB for storage and washing steps (autoclave at step 1)
Plastic containers (60 mL) with red screw caps Greiner 206202 to prepare the fibrinogen solution
Pneumatic cylinder Festo AEVC-20-10-I-P to 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 Netherlands n.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 Technology n.a. to mount electrospun tubes on silicon tubing (autoclave at step 1, step 7)
Pressure sensor and transducer BD TC-XX and P 10 EZ the air pressure going to the pneumatic actuated pump is raised until it reaches the set pressure
Proportional air pressure control valve and pressure sensor Festo MPPES-3-1/8-2-010, 159596 provides compressed air to the pneumatic actuated pump
Roswell Park Memorial Institute 1640 (RPMI-1640) Gibco A1049101 cell culture medium for monocyte/macrophage
Safe lock Eppendorf tubes (1.5 mL) Eppendorf 30120086 multiple applications (autoclave at step 1)
Sodium dodecyl sulfate solution 20% Sigma 5030 Used to clean materials, at a concentration of 0.1%.  
Silicone O-rings Technirub 1250S to prevent leakage (autoclave at step 1, step 7)
Silicone tubing (2.8 mm outer diameter, 400 um wall thickness) Rubber BV 1805 to mount the electrospun tubes on the pressure conduits (autoclave at step 1)
Sterile tube (15 mL) Falcon 352095 multiple applications
Suture, 5-0 prolene with pre-attached taper point needle Ethicon, Johnson&Johnson EH7404H Prolene suture wire 5-0 (75cm length, TF taper point needle, 1/2 circle, 13 mm needle length)
Syringe (24 mL) B. Braun Melsungen AG 2057932 to add the ultrapure water or medium to the hydraulic reservoir or flow culture chamber
Syringe filter (0.2 µm) Satorius 17597-K to filter the fibrinogen solution
T150 cell culture flask with filter cap Nunc 178983 to degas culture medium
T75 Cell culture flask with filter cap Nunc 156499 to culture static control samples
Teflon bellow Custom made, Department of Biomedical Engineering, Eindhoven University of Technology n.a. to load the hydraulic reservoir (clean outside with a paper tissue with 70% ethanol at step 1 and 7)
Tray (stainless steel) PolarWare 15-248 for 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)
Tweezers Wironit 4910 sterile handling of individual parts (autoclave at step 1 and 7)
Ultrapure water Stakpure Omniapure UV 18200002 to correct for medium evaporation, mixed with aqua stabil mixed and used as hydraulic fluid. (autoclave ultrapure water at step 1)
UV light Philips TUV 30W/G30 T8 for decontamination of grafts and bioreactor parts before seeding
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BioreactorMulti-cueInflammatoryRegenerativeBiomaterialsFlowStretchHemodynamicsSheer StressCyclic StretchElectrospun ScaffoldSilicone TubingSuturePressure ConduitO-ringAdapter Bushing

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Koch, S. E., van Haaften, E. E., Wissing, T. B., Cuypers, L. A. B., Bulsink, J. A., Bouten, C. V. C., Kurniawan, N. A., Smits, A. I. P. M. A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch. J. Vis. Exp. (166), e61824, doi:10.3791/61824 (2020).
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