Name: a multi-cue bioreactor to evaluate the inflammatory and regenerative capacity of biomaterials under flow and stretch
Uploaded: 2020-12-10T13:00:00
Description: Watch this Scientific Journal Video about A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch at JoVE.com

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).

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...

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...

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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >advanced Dulbecco&#8217;s modified EagleMedium (aDMEM)</td>
			<td >Gibco</td>
			<td >12491-015</td>
			<td >cell culture medium for fibroblasts</td>
		</tr>
		<tr >
			<td >Aqua Stabil</td>
			<td >Julabo</td>
			<td >8940012</td>
			<td >prevent microorganism growth in bioreactor-hydraulic reservoir</td>
		</tr>
		<tr >
			<td >Bovine fibrinogen</td>
			<td >Sigma</td>
			<td >F8630</td>
			<td >to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td>
		</tr>
		<tr >
			<td >Bovine thrombin</td>
			<td >Sigma</td>
			<td >T4648</td>
			<td >to prepare fibrinogen gel to seed the cells on the electrospun scaffold</td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Eppendorf</td>
			<td >5804</td>
			<td >to spin down cells and conditioned medium</td>
		</tr>
		<tr >
			<td >Clamp scissor - &#34;kelly forceps&#34;</td>
			<td >Almedic</td>
			<td >P-422</td>
			<td >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)</td>
		</tr>
		<tr >
			<td >CO2 cell culture incubators</td>
			<td >Sanyo</td>
			<td >MCO-170AIC-PE</td>
			<td >for cell culturing</td>
		</tr>
		<tr >
			<td >Compressed air reservoir</td>
			<td >Festo</td>
			<td >CRVZS-5</td>
			<td >smoothing air pressure fluctuations and create time delays for pressure build-up</td>
		</tr>
		<tr >
			<td >Custom Matlab script to calculate the maximum stretches</td>
			<td >Matlab</td>
			<td >R2017. The Mathworks, Natick, MA</td>
			<td >calculate the minimum and maximum outer diameter of the electrospun scaffold</td>
		</tr>
		<tr >
			<td >Data acquisition board</td>
			<td >National Instruments</td>
			<td >BNC-2090</td>
			<td >data processing in between amplifier system and computer</td>
		</tr>
		<tr >
			<td >Ethanol</td>
			<td >VWR</td>
			<td >VWRK4096-9005</td>
			<td >to keep sterile working conditions</td>
		</tr>
		<tr >
			<td >Fetal bovine calf serum (FBS)</td>
			<td >Greiner</td>
			<td >758087</td>
			<td >cell culture medium supplement; serum-supplement</td>
		</tr>
		<tr >
			<td >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</td>
			<td >Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >flow culture chamber compartments (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Glass Pasteur pipet</td>
			<td >Assistant</td>
			<td >HE40567002</td>
			<td >apply vacuum on electrospun scaffold (autoclave at step 1)</td>
		</tr>
		<tr >
			<td >Glass tubes of the flow culture chamber</td>
			<td >Custon made, Equipment &#38; Prototype Center, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >part of the flow culture chamber (clean and store in 70% ethanol, at step 1 and 7)</td>
		</tr>
		<tr >
			<td >GlutaMax</td>
			<td >Gibco</td>
			<td >35050061</td>
			<td >cell culture medium amino acid supplement, minimizes ammonia build-up</td>
		</tr>
		<tr >
			<td >High speed camera</td>
			<td >MotionScope</td>
			<td >M-5</td>
			<td >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)</td>
		</tr>
		<tr >
			<td >High speed camera lens - Micro-NIKKOR 55mm f/2.8 - lens</td>
			<td >Nikon</td>
			<td >JAA616AB</td>
			<td >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)</td>
		</tr>
		<tr >
			<td >Hose clip</td>
			<td >ibidi GmbH</td>
			<td >10821</td>
			<td >block medium flow (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Hydraulic reservoir with 8 screw threads for 8 flow culture chambers</td>
			<td >Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >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)</td>
		</tr>
		<tr >
			<td >Ibidi pump system (8x) including ibidi pump, PumpControl software, fluidic unit, perfusion set (medium tubing), air pressure tubing, drying bottles with orange silica beads</td>
			<td >ibidi GmbH</td>
			<td >10902</td>
			<td >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.</td>
		</tr>
		<tr >
			<td >Knives (no.10 sterile blades, individual foil pack) and scalpel handle (stainless steel, individually wrapped)</td>
			<td >Swann Morton</td>
			<td >0301; 0933</td>
			<td >to cut the silicone tubing in the correct size for the scaffold and to cut the suture material</td>
		</tr>
		<tr >
			<td >LabVIEW Software</td>
			<td >National Instruments</td>
			<td >version 2018</td>
			<td >to control the stretch applied to the scaffolds</td>
		</tr>
		<tr >
			<td >Laminar flow biosafety cabinet with UV light</td>
			<td >Labconco</td>
			<td >302310001</td>
			<td >to ensure sterile working conditions. The UV is used to decontaminate everything that cannot be autoclaved, or touched after autoclaving</td>
		</tr>
		<tr >
			<td >Large and small petri dishes</td>
			<td >Greiner</td>
			<td >664-160</td>
			<td >for sterile working conditions</td>
		</tr>
		<tr >
			<td >L-ascorbic acid 2-phosphate (vitamin C)</td>
			<td >Sigma</td>
			<td >A8960</td>
			<td >cell culture medium supplement, important for collagen production</td>
		</tr>
		<tr >
			<td >LED light cold source KL2500</td>
			<td >Zeiss</td>
			<td >Schott AG</td>
			<td >to aid in visualization for the time lapse of the scaffolds during monitoring of the stretch</td>
		</tr>
		<tr >
			<td >Luer (female and male) locks and connectors, white luer caps</td>
			<td >ibidi GmbH</td>
			<td >various, see (https://ibidi.com/26-flow-accessories)</td>
			<td >to close or connect parts of the bioreactor and the ibidi pump (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Measuring amplifier (PICAS)</td>
			<td >PEEKEL instruments B.V.</td>
			<td >n.a.</td>
			<td >to amplify the signal from the pressure sensor and feedback to LabView</td>
		</tr>
		<tr >
			<td >Medium reservoir (large syringes 60 mL) and reservoir holders</td>
			<td >ibidi GmbH</td>
			<td >10974</td>
			<td >medium reservoir (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Medium tubing with 4.25 mm outer diameter and 1 mm inner diameter</td>
			<td >Rubber BV</td>
			<td >1805</td>
			<td >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</td>
		</tr>
		<tr >
			<td >Motion Studio Software</td>
			<td >Idtvision</td>
			<td >2.15.00</td>
			<td >to make the high speed time lapse images for stretch monitoring</td>
		</tr>
		<tr >
			<td >Needle (19G)</td>
			<td >BD Microlance</td>
			<td >301700</td>
			<td >together with thin flexible tubing used to fill the hydraulic reservoir with ultrapure water without adding air bubbles</td>
		</tr>
		<tr >
			<td >Needle driver</td>
			<td >Adson</td>
			<td >2429218</td>
			<td >to handle the needle of the nylon suture through the silicone tube (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Paper tissues</td>
			<td >Kleenex</td>
			<td >38044001</td>
			<td >for cleaning of the equipment with 70% ethanol</td>
		</tr>
		<tr >
			<td >Parafilm</td>
			<td >Sigma</td>
			<td >P7793-1EA</td>
			<td >quick fix if leakage occurs</td>
		</tr>
		<tr >
			<td >Penicillin/streptomycin (P/S)</td>
			<td >Lonza</td>
			<td >DE17-602E</td>
			<td >cell culture medium supplement; prevent bacterial contamination</td>
		</tr>
		<tr >
			<td >Phosphate Buffered Saline (PBS)</td>
			<td >Sigma</td>
			<td >P4417-100TAB</td>
			<td >for storage and washing steps (autoclave at step 1)</td>
		</tr>
		<tr >
			<td >Plastic containers (60 mL) with red screw caps</td>
			<td >Greiner</td>
			<td >206202</td>
			<td >to prepare the fibrinogen solution</td>
		</tr>
		<tr >
			<td >Pneumatic cylinder</td>
			<td >Festo</td>
			<td >AEVC-20-10-I-P</td>
			<td >to actuate the Teflon bellow (clean with a paper tissue with 70% ethanol at step 1 and 7)</td>
		</tr>
		<tr >
			<td >Polycaprolactone bisurea (PCL-BU) tubular scaffolds (3 mm inner diameter, 200 &#181;m wall thickness, 20 mm length)</td>
			<td >SyMO-Chem, Eindhoven, The Netherlands</td>
			<td >n.a.</td>
			<td >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</td>
		</tr>
		<tr >
			<td >Pressure conduit without holes (for static control)</td>
			<td >Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >to mount electrospun tubes on silicon tubing (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Pressure sensor and transducer</td>
			<td >BD</td>
			<td >TC-XX and P 10 EZ</td>
			<td >the air pressure going to the pneumatic actuated pump is raised until it reaches the set pressure</td>
		</tr>
		<tr >
			<td >Proportional air pressure control valve and pressure sensor</td>
			<td >Festo</td>
			<td >MPPES-3-1/8-2-010, 159596</td>
			<td >provides compressed air to the pneumatic actuated pump</td>
		</tr>
		<tr >
			<td >Roswell Park Memorial Institute 1640 (RPMI-1640)</td>
			<td >Gibco</td>
			<td >A1049101</td>
			<td >cell culture medium for monocyte/macrophage</td>
		</tr>
		<tr >
			<td >Safe lock Eppendorf tubes (1.5 mL)</td>
			<td >Eppendorf</td>
			<td >30120086</td>
			<td >multiple applications (autoclave at step 1)</td>
		</tr>
		<tr >
			<td >Sodium dodecyl sulfate solution 20%</td>
			<td >Sigma</td>
			<td >5030</td>
			<td >Used to clean materials, at a concentration of 0.1%.&#160;&#160;</td>
		</tr>
		<tr >
			<td >Silicone O-rings</td>
			<td >Technirub</td>
			<td >1250S</td>
			<td >to prevent leakage (autoclave at step 1, step 7)</td>
		</tr>
		<tr >
			<td >Silicone tubing (2.8 mm outer diameter, 400 um wall thickness)</td>
			<td >Rubber BV</td>
			<td >1805</td>
			<td >to mount the electrospun tubes on the pressure conduits (autoclave at step 1)</td>
		</tr>
		<tr >
			<td >Sterile tube (15 mL)</td>
			<td >Falcon</td>
			<td >352095</td>
			<td >multiple applications</td>
		</tr>
		<tr >
			<td >Suture, 5-0 prolene with pre-attached taper point needle</td>
			<td >Ethicon, Johnson&#38;Johnson</td>
			<td >EH7404H</td>
			<td >Prolene suture wire 5-0 (75cm length, TF taper point needle, 1/2 circle, 13 mm needle length)</td>
		</tr>
		<tr >
			<td >Syringe (24 mL)</td>
			<td >B. Braun Melsungen AG</td>
			<td >2057932</td>
			<td >to add the ultrapure water or medium to the hydraulic reservoir or flow culture chamber</td>
		</tr>
		<tr >
			<td >Syringe filter (0.2 &#181;m)</td>
			<td >Satorius</td>
			<td >17597-K</td>
			<td >to filter the fibrinogen solution</td>
		</tr>
		<tr >
			<td >T150 cell culture flask with filter cap</td>
			<td >Nunc</td>
			<td >178983</td>
			<td >to degas culture medium</td>
		</tr>
		<tr >
			<td >T75 Cell culture flask with filter cap</td>
			<td >Nunc</td>
			<td >156499</td>
			<td >to culture static control samples</td>
		</tr>
		<tr >
			<td >Teflon bellow</td>
			<td >Custom made, Department of Biomedical Engineering, Eindhoven University of Technology</td>
			<td >n.a.</td>
			<td >to load the hydraulic reservoir (clean outside with a paper tissue with 70% ethanol at step 1 and 7)</td>
		</tr>
		<tr >
			<td >Tray (stainless steel)</td>
			<td >PolarWare</td>
			<td >15-248</td>
			<td >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)</td>
		</tr>
		<tr >
			<td >Tweezers</td>
			<td >Wironit</td>
			<td >4910</td>
			<td >sterile handling of individual parts (autoclave at step 1 and 7)</td>
		</tr>
		<tr >
			<td >Ultrapure water</td>
			<td >Stakpure</td>
			<td >Omniapure UV 18200002</td>
			<td >to correct for medium evaporation, mixed with aqua stabil mixed and used as hydraulic fluid. (autoclave ultrapure water at step 1)</td>
		</tr>
		<tr >
			<td >UV light</td>
			<td >Philips</td>
			<td >TUV 30W/G30 T8</td>
			<td >for decontamination of grafts and bioreactor parts before seeding</td>
		</tr>
	</tbody>
</table>

a multi-cue bioreactor to evaluate the inflammatory and regenerative capacity of biomaterials under flow and stretch

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.

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...

Watch this Scientific Journal Video about A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch at JoVE.com

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

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.

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

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

So far, it has been really difficult to identify the cause and effect relationships between hemodynamics and vascular tissue regeneration. And this has because it's challenging to control the individual mechanical loads. This bioreactor enables us to mechanistically investigate the individual and combined effects of sheer stress and cyclic stretch on the regenerative potential of a broad variety of tissue engineered vascular grafts.Demonstrating this procedure will be Suzanne Koch, a PhD candidate in our group. And Dr.Tamar Wissing who is a postdoctoral researcher from our laboratory. To mount the electro spun scaffold onto the silicone tubing, thread a 4-0 prolene suture into one end of a piece of silicone tubing and out of the other.Make a small knot on both sides of the tube leaving approximately 10 centimeters of wire on both knots and make a third knot on top of the two. After removing a suture needle, trim the edges of the silicone tubing into a triangular shape and make a final knot at the end of the two 10 centimeter wires. Dip the electro spun scaffold in 30%ethanol and place the scaffold over one end of free suture wire.Next, gently stretch both sides of the silicone tubing and the 10 centimeter suture knot while second researcher uses tweezers with a smooth inner tip to gently slide the electro spun scaffold over the tubing. Slowly release the stretch on the silicone tubing while simultaneously smoothing the electro spun scaffold with the tweezers. And dip the scaffold and silicone tubing in ultra pure water two times.Construct the bottom compartment and make sure that the O-ring is properly placed. Place an adaptor bushing in the upper part of the bottom compartment and place the pressure conduit with holes through the bottom compartment. Lace the silicone O-ring around the lower end of the pressure conduit to prevent leakage.And screw the lower part of the bottom compartment to the upper part of the bottom compartment to secure the pressure conduit. Make sure that the lower engraved groove of the pressure conduit is approximately three to five millimeters above the edge of the adapter bushing of the bottom compartment. Pull the silicone tube with the electro spun scaffold over the pressure conduit and make a nod and the suture wire at the lower end of the electro spun scaffold, at the location of the engraved groove on the pressure conduit.Make a second nod at the opposite side to tightly secure the silicone tubing with the electro spun graft. And placing a scissor clamp equipped with a rule at the upper end of the silicone tubing, pull the scissor clamp upwards in a consistent manner and gently pull on the electric spawn scaffold to remove any wrinkles. Use a suture wire to make two knots at both ends of the scaffold at the upper engraved groove of the pressure conduit.When both knots have been made, release the scissor clamp and use a knife to remove any excess silicone tubing. Screw the nose cones on the screw thread of the pressure conduits for the samples that will be dynamically loaded. Dip the pressure conduit, tubing, and scaffold one time in 30%ethanol and two times in ultrapure water to pre-wet the setup.Place the glass tube over the pressure conduit and push gently on the bottom compartment to secure the glass tube in place. Then place the flow straightener, a silicone O-ring, and adapter bushing in the top compartment. And secure the compartment over the open end of the glass tube.Remove the male lure plug from the flow outlet, work in a sterile laminar flow cabinet. Open the white lure cap and place an ethanol soak tissue in front of the flow outlet. Deconstruct the flow culture chamber by taking off the glass tube with the top compartment.Place a vacuum pasture pipe add onto the electro spun scaffold to remove as much medium as possible. And add fibrinogen solution at a one-to-one ratio with the thrombin supplemented cell suspension. Pipette the solution up and down one time and immediately drip the solution over the full length of the scaffold.When all of the cells have been delivered, slowly move the scaffold from left to right and up and down to achieve an even distribution of the cells. When both sides of the scaffold have been seeded, carefully reconstruct the flow culture chamber by putting the glass tube and top compartment back. And place the flow culture chamber into the incubator to let the fiber solidify.To couple the bioreactor to the pump system, place the flow culture chamber on one of the eight screw threads on the bioreactor base. And place a host clip on the medium tubing. Remove the white lure cap, covering the flow inlet of the top compartment of the flow culture chamber and remove the female lure coupler of the medium tubing.Connect the medium tubing with one side of the flow inlet on the top compartment. And the other side with the flow outlet at the bottom compartment. Transfer the complete setup from the laminar flow cabinet to the incubator.And connect a fluidic units and the bioreactor base to the air pressure tubing and electric cable. Start the software and initialize the pumps. Start the medium flow for the samples one by one.Then change the strain pump parameters to the desired settings and start the strain. Monitor the stretch applied to the scaffold every other day. The monitoring of stretch and wall shear stress over longterm culture periods shows that these values can be maintained at relatively constant levels over a period of up to 20 days.After three days of hemodynamic loading, immunofluorescent staining reveals a homogenous distribution of monocyte derived macrophages and myofibroblasts throughout the scaffold. After 20 days of co-culture, cyclic stretch results in the deposition of more numerous and thicker collagen type 1 fibers. While in the combined hemodynamic load group, the cyclic stretch effect is overruled by sheer stress resulting in a less pronounced collagen type 1 deposition.After eight days of macrophage monoculture, fiber erosion and fiber cleavage are observed in all of the hemodynamic loading regimes with the most pronounced resorption observed in the static group, and the least pronounced resorption observed in the sheer stress group. Both cyclic stretch and a sheer stress impact the cytokine secretion profile of the co-culture setup. Interestingly, the combined effects of both loads show either dominance of one of the two loads or synergistic effects of both loads.Co-culture experiments also show that the mechanical environment and resulting loading dependent inflammatory environments modulate the phenotype of the myofibroblasts. Furthermore, the gene expression patterns of contractile marker alpha smooth muscle actin correlate with protein synthesis. The most important thing when performing this protocol is that's what the application of stretch, it's essential that your setup is leak-free.

Research

JoVE Journal

Engineering

Measuring Material Microstructure Under Flow Using 1-2 Plane Flow-Small Angle Neutron Scattering

Måling Materiale Mikrostruktur Under Flow Brug 1-2 Plane Flow-Small Angle neutronspredning

A new small-angle neutron scattering (SANS) sample environment optimized for studying the microstructure of complex fluids under simple shear flow is ...

Experimental Protocol to Investigate Particle Aerosolization of a Product Under Abrasion and Under Environmental Weathering

Eksperimentel protokol til Undersøge Partikel aerosolisering af et produkt under Slid og Under Environmental Weathering

The present article presents an experimental protocol to investigate particle aerosolization of a product under abrasion and under environmental ...

Design and Use of a Full Flow Sampling System (FFS) for the Quantification of Methane Emissions

Design and Use of a Full Flow Sampling System FFS for the Quantification of Methane Emissions

Design og brug af en Full Flow Sampling System (FFS) til kvantificering af Metan emissioner

The use of natural gas continues to grow with increased discovery and production of unconventional shale resources. At the same time, the natural gas ...

A Robotic Platform to Study the Foreflipper of the California Sea Lion

En robot platform til at studere den Foreflipper af California Sea Lion

The California sea lion (Zalophus californianus), is an agile and powerful swimmer. Unlike many successful swimmers (dolphins, tuna), they generate most ...

Solvent Bonding for Fabrication of PMMA and COP Microfluidic Devices

Opløsningsmiddel Bonding til Fabrikation af PMMA og COP mikrofluidenheder

Thermoplastic microfluidic devices offer many advantages over those made from silicone elastomers, but bonding procedures must be developed for each ...

In Situ Visualization of the Phase Behavior of Oil Samples Under Refinery Process Conditions

In Situ Visualization of the Phase Behavior of Oil Samples Under Refinery Process Conditions

In situ Visualisering af fase Behavior af olieprøver Under raffinaderierne til raffinering Betingelser

To help address production issues in refineries caused by the fouling of process units and lines, we have developed a setup as well as a method to ...

Scanning Light Scattering Profiler (SLPS) Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

Scanning Light Scattering Profiler SLPS Based Methodology to Quantitatively Evaluate Forward and Backward Light Scattering from Intraocular Lenses

Scanning Light Scattering Profiler (SLPS) -baseret metode til kvantitativ evaluering af fremadrettet og bagudlysudspredning fra intraokulære linser

The scanning light scattering profiler (SLSP) methodology has been developed for the full-angle quantitative evaluation of forward and backward light ...

A Novel Biaxial Testing Apparatus for the Determination of Forming Limit under Hot Stamping Conditions

A Novel Biaksial Test Apparat til bestemmelse af Forming Limit under stempling Betingelser

The hot stamping and cold die quenching process is increasingly used to form complex shaped structural components of sheet metals. Conventional ...

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy (ATOM)

Microfluidic Imaging Flow Cytometry by Asymmetric-detection Time-stretch Optical Microscopy ATOM

Mikrofluidisk Imaging Flow Cytometry ved asymmetrisk detektion Time-stretch Optical Microscopy (ATOM)

Scaling the number of measurable parameters, which allows for multidimensional data analysis and thus higher-confidence statistical results, has been the ...

Flow-assisted Dielectrophoresis: A Low Cost Method for the Fabrication of High Performance Solution-processable Nanowire Devices

Flow-assisteret dielektroforesis: En billig metode til fremstilling af højtydende løsning-bearbejdes Nanotråd enheder

Flow-assisted dielectrophoresis (DEP) is an efficient self-assembly method for the controllable and reproducible positioning, alignment, and selection of ...