Name: construction and characterization of a novel vocal fold bioreactor
Uploaded: 2014-08-01T13:00:00
Description: Watch this Scientific Journal Video about Construction and Characterization of a Novel Vocal Fold Bioreactor at JoVE.com

Vi takker Dr. Jeffrey Caplan for hans uddannelse og rådgivning om konfokal billedbehandling. Vi takker også Keck Electron Microscopy Lab og Dr. Chaoying Ni for SEM assistance. Dette arbejde er finansieret af National Institutes of Health (NIDCD, R01DC008965 og R01DC011377). ABZ anerkender NSF Integrativ Graduate Uddannelse &amp; Forskning praktik (IGERT) program for finansiering.

1.. Bioreaktor Assembly (Video 1) <ol><li> Lav en aluminiumsform (cirkulær matrice + spacer pin) med forudbestemte indre og ydre dimensioner (fig. 1). </li><li> Brug af mug fra trin 1.1, fabrikere en silikone membran (diameter: 42 mm, tykkelse: 1,5 mm, figur 1) med en indgroet muffe (diameter: 12 mm, tykkelse ~ 0.25 mm, formet af spacer pin i figur 1) midten ved hjælp af en kommercielt tilgængelig siliconeelastomer kit. </li><li> Lav et par af akryl blokke ...

<ol>
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Vellykket engineering af funktionelle vokal fold væv in vitro kræver genskabelse af en vocal fold-lignende mikromiljø at mægle adfærd af multipotente celler. Det er generelt accepteret, at væv eller organ strukturer afspejler de funktioner, de skal udføre. 22. For vokal fold væv er de højfrekvente vibrationer, der opstår under phonation foreslås at være vigtigt for væv modning. I vores undersøgelse, er PCL stilladser bruges til at give et ledbånd-lignende strukturel støtte, mens den v...

Den menneskelige vocal fold, sammensat af et epithellag, lamina propria (LP) og vocalis muskel, er en specialiseret blødt væv, der konverterer luftstrømmen fra lungerne i akustiske bølger af lyd produktion. En Vocal folder svinge regelmæssigt under normal phonation, udstiller stammer på op til 30% ved grundlæggende frekvenser, der spænder fra 100 til 300 Hz. 2 Voksen vocal fold LP er en gradient struktur består af en overfladisk (SLP), et mellemprodukt (ILP), og en dyb (DLP) lag. Yderligere klassificering grupper epitel og SLP som slimhinden lag, og kombinerer ILP og DLP i den vokale ledbånd. 3 SLP lag indeholder primært en amorf matrix med tyndt spredte kollagene fibre, mens ledbånd er beriget med moden kollagen og elastin fibre at give tilstrækkelig styrke. 4. Strukturen og mekanik nyfødte vokal folder varierer betydeligt fra deres modne kolleger. Selvom mekanismens regulerer vokal fold udvikling og modning er endnu ikke fuldt forstået, eksperimentelle bevismateriale har peget på de afgørende roller vocalization-afledt mekanisk stress. Adskillige medicinske tilstande, herunder tale misbrug, infektioner, kemiske irritanter og kirurgiske procedurer, kan beskadige vocal fold. Vocal fold lidelser påvirker en anslået 3-9% af den amerikanske befolkning. Nuværende metoder til behandling af vokal fold lidelser er begrænset 5 og en stamcelle-baserede tissue engineering tilgang har vist sig som en lovende strategi for at genoprette vocal fold funktion. Mesenkymale stamceller (MSC) er et velegnet alternativ til de primære vocal fold fibroblaster til vocal fold tissue engineering. 6-9 Stamceller skæbne specifikation og efterfølgende udvikling væv medieret af specifikke niche de bor i, hvoraf den mekaniske tilstand er en afgørende faktor. 10 Mekaniske kræfter er vigtige regulatorer af vævsmorfogenese ennd homeostase, især for væv, der rutinemæssigt udsættes for belastning. 11 Fra en tissue engineering perspektiv, er det blevet påvist, at eksponering for fysiologisk relevante mekaniske stimuleringer fremmer stamceller differentiering og vævsspecifik matrix remodellering. 12-15 Vævskultur bioreaktorer er beregnet til at simulere den ønskede fysiologiske miljø for celle-eller vævsvækst in vitro. For vocal fold tissue engineering, er det især vigtigt at genskabe den mekaniske miljø i phonating vokal folder. En ideel vocal fold bioreaktor skal effektivt levere vibrerende stikord til kultur celler, hvilket giver facile kontrol over frekvens, amplitude og varighed af vibrationer. Titze og kolleger udtænkt en vokal fold bioreaktor (T1 bioreaktor) 16, der kombinerer statisk strækning med høj frekvens (20-200 Hz) svingninger at stimulere cellulær produktion af matrix proteiner. Using denne bioreaktor, Webb og kolleger 17 undersøgte effekten af 10-dages, 100 Hz vibrationer på dermalfibroblaster dyrket i en hyaluronsyre (HA)-baserede hydrogel. Konstruktioner, der udsættes for vibrationer udviste en forhøjet udtryk for HA synthase-2 (HAS2) decorin, fibromodulin og matrixmetalloproteinase-1 (MMP1) i forhold til de statiske kontrol. De stimulerende effekter blev fundet at være tidsafhængig. For nylig, vores gruppe 18 samlet en vocal fold bioreaktor (J1 bioreaktor) ved hjælp af en effektforstærker, en funktion generator, en lukket højttaler og et periferisk forankret silikone membran, der overfører den oscillerende luft til de vedhæftede celler. Neonatale forhudsfibroblaster dyrket i J1 bioreaktoren blev udsat for 1 time af vibration ved 60, 110 eller 300 Hz, med en in-plane stamme af op til 0,05%. QPCR Resultaterne antydede, at ekspression af nogle ECM gener moderat blev ændret som reaktion på de forskellige vibrations frekvenserog amplituder. Disse bioreaktor motiver mens spændende, har flere begrænsninger. For eksempel T1-systemet kræver et stort antal stik og barer til mekanisk kobling, begrænse den maksimale frekvenser opnåelige. Desuden kan celler udsættes for uønsket mekanisk omrøring og væske perturbation komplicerer fortolkning af dataene. Den J1 bioreaktor, på den anden side udviser relativt lav energi konvertering effektivitet og er ikke brugervenligt. Desuden vibrationer ofte løsnes de celle-holdige konstruktioner fra den underliggende siliconemembran. J2 vocal fold bioreaktor rapporteret her, designet baseret på samme princip som J1 version er optimeret til konsistens og reproducerbarhed. De phonation efterligner vibrationer genereres aerodynamisk i individuelt monteret vibrationer kamre hvor MSC-befolkede fiberholdigt poly (ε-caprolacton) (PCL) stilladser er virkningsfuldely sikret. Laser Doppler Vibrometry (LDV) giver brugeren mulighed for at kontrollere den vibrerende profil membranen / stillads forsamling. I vores demonstration MSC&#39;er udsat for 200 Hz sinusformede svingninger med en 1-timers-on-1-h-off (OF) mønstret for i alt 12 timer dagligt i 7 dage. Cellulære reaktioner på de pålagte vibrerende tidskoder undersøges systematisk. Samlet set J2 vocal fold giver den mest brugervenlige funktioner, der giver dynamisk cellekultur undersøgelser, der gennemføres i et high throughput og reproducerbar måde.

<table border="0" cellpadding="0" cellspacing="0" width="853">
	<tbody>
		<tr height="16">
			<td height="16" style="height:17px;width:248px;">silicone elastomer kit</td>
			<td style="width:188px;">Dow Corning</td>
			<td style="width:124px;">Sylgard 184</td>
			<td style="width:293px;">cure the membrane at 100 C for 2 hr</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PCL</td>
			<td>Sigma Aldrich</td>
			<td>440744-500G</td>
			<td>Mn ~ 80 kDa, dissolve overnight</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">chloroform</td>
			<td>Sigma Aldrich</td>
			<td>C7559-5VL</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">human bone marrow-derived MSCs</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td>received with passage 2</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">MSC maintenance media</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td style="width:293px;">10% FBS in basal media supplemented 
			with L-glutamine, gentamicin and amphotericin</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Accutase cell dissociation reagent</td>
			<td>Life Technologies</td>
			<td>A11105-01</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">ethanol</td>
			<td>Sigma Aldrich</td>
			<td>E7023-500ML</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">fibronectin</td>
			<td>Sigma Aldrich</td>
			<td>F2006-1MG</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">MMP1 DuoSet ELISA kit</td>
			<td>R&#38;D systems</td>
			<td>DY901</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HA ELISA kit</td>
			<td>Echelon Biosciences</td>
			<td>&#160;K-1200&#160;&#160;</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PBS</td>
			<td>Life Technologies</td>
			<td>14190-136</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">propidium iodide&#160;</td>
			<td>Life Technologies</td>
			<td>P1304MP</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Syto-13&#160;</td>
			<td>Life Technologies</td>
			<td>S7575</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">QuantiTect reverse transcription kit&#160;</td>
			<td>Qiagen</td>
			<td>205311</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">SYBR Green PCR master mix</td>
			<td>Life Technologies</td>
			<td>4309155</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">replacement speaker</td>
			<td style="width:188px;">DAYTON audio 
			(via Parts Express)</td>
			<td>DS90-8</td>
			<td>paper cone, full range (80-13000 Hz), 85dB</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Ergo Micro torque screwdriver</td>
			<td>Mountz</td>
			<td># 020377</td>
			<td>torque range: 20-120 cN.m</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">stereo speaker selector</td>
			<td>RadioShack</td>
			<td>40-244</td>
			<td>maximum power handling 50 W</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">function generator&#160;</td>
			<td>Agilent&#160;</td>
			<td>33220A</td>
			<td>frequency range 1 &#181;Hz- 20 MHz</td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">power amplifier&#160;</td>
			<td>PYLE audio</td>
			<td>PylePro PT2400</td>
			<td style="width:293px;">frequency response: 10 Hz-50 kHz, two speaker 
			channels</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">cell culture incubator&#160;</td>
			<td>Thermo Fisher&#160;</td>
			<td>Steri-Cult 3307</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">syringe pump&#160;</td>
			<td>New Era Pump Systems</td>
			<td>NE-300</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">High voltage power supply</td>
			<td>Spellman</td>
			<td>CZE 1000R</td>
			<td>output voltage: 0-30 kV</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">scanning electron microscope&#160;</td>
			<td>JEOL-USA</td>
			<td>JSM-7400F</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">desk gold sputter coater</td>
			<td>Denton Vacuum</td>
			<td>DSK00V-0013</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Doppler laser vibrometer&#160;</td>
			<td>Polytec</td>
			<td>PDV-100</td>
			<td>non-contact velocity measurement (0-22 kHz)</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">PCR sequence detection system&#160;</td>
			<td>Applied Biosystems</td>
			<td>ABI7300</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">multiphoton confocal microscope</td>
			<td>Zeiss</td>
			<td>Zeiss 510Meta NLO</td>
			<td></td>
		</tr>
		<tr height="33">
			<td height="33" style="height:33px;">UV-VIS Spectrophotometer&#160;</td>
			<td style="width:188px;">NanoDrop Products 
			via Thermo Scientific</td>
			<td>ND-2000</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">VibSoft Data Acquisition Software</td>
			<td>Polytec</td>
			<td></td>
			<td>acquisition bandwidth up to 40 MHz</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Origin 8.5 data analysis software&#160;</td>
			<td>OriginLab</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">qbasePlus qPCR data analysis software&#160;</td>
			<td>Biogazelle</td>
			<td>V2.3</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">aluminium alloy&#160;</td>
			<td>McMaster-Carr</td>
			<td>Alloy 6061</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">acrylic blocks</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="27">
			<td height="27" style="height:27px;">polycarbonate anti-humidity chamber</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td style="width:293px;">Impact-Resistant&#160;Polycarbonate</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">screws&#160;</td>
			<td>McMaster-Carr</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">electronic cable/wire</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">medical grade PVC tubing</td>
			<td>US Plastic Corp.</td>
			<td>Tygon S-50-HL</td>
			<td>clear, biocompatible</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">10 mL syringe&#160;</td>
			<td>Becton Dickinson</td>
			<td>309604</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">21 G blunt ended needle</td>
			<td>Small Parts</td>
			<td>NE-213PL-25</td>
			<td>1-1/2&#34; length</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">Alligator clip adapters&#160;</td>
			<td>RadioShack</td>
			<td>270-354</td>
			<td>fully insulated</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">8 mm biopsy punch</td>
			<td><a href="http://www.sklarcorp.com/ProductListUS.aspx?DepartmentID=10&amp;CategoryID=96">Sklar&#160;Surgical Instruments</a></td>
			<td>96-1152</td>
			<td>sterile, disposable</td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">12 mm biopsy punch</td>
			<td>Acuderm (via Fisher Scientific)</td>
			<td style="width:124px;">NC9998681</td>
			<td></td>
		</tr>
		<tr height="16">
			<td height="16" style="height:17px;">tissue culture flasks</td>
			<td>Corning</td>
			<td></td>
			<td>cell culture treated</td>
		</tr>
	</tbody>
</table>

construction and characterization of a novel vocal fold bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To emulate the dynamic environment of vocal folds, a novel vocal fold bioreactor capable of producing vibratory stimulations at fundamental phonation frequencies is constructed and characterized. The device is composed of a function generator, a power amplifier, a speaker selector and parallel vibration chambers. Individual vibration chambers are created by sandwiching a custom-made silicone membrane between a pair of acrylic blocks. The silicone membrane not only serves as the bottom of the chamber but also provides a mechanism for securing the cell-laden scaffold. Vibration signals, generated by a speaker mounted underneath the bottom acrylic block, are transmitted to the membrane aerodynamically by the oscillating air. Eight identical vibration modules, fixed on two stationary metal bars, are housed in an anti-humidity chamber for long-term operation in a cell culture incubator. The vibration characteristics of the vocal fold bioreactor are analyzed non-destructively using a Laser Doppler Vibrometer (LDV). The utility of the dynamic culture device is demonstrated by culturing cellular constructs in the presence of 200-Hz sinusoidal vibrations with a mid-membrane displacement of 40 &#181;m. Mesenchymal stem cells cultured in the bioreactor respond to the vibratory signals by altering the synthesis and degradation of vocal fold-relevant, extracellular matrix components. The novel bioreactor system presented herein offers an excellent in vitro platform for studying vibration-induced mechanotransduction and for the engineering of functional vocal fold tissues.

PCL stilladser fremstillet ved electrospinning indeholder mikronstørrelse interstitielle porer og tilfældigt sammenfiltrede fibre med en gennemsnitlig diameter på 4,7 um (figur 4A). Ved en højere forstørrelse, nanoskala riller og porerne er synlige på de individuelle fibre (figur 4B). Coating af stilladser med fibronektin forbedrer hydrofilitet og letter den indledende celleadhæsion / spredning på PCL stillads (upubliceret observation). Sinusformede ...

Watch this Scientific Journal Video about Construction and Characterization of a Novel Vocal Fold Bioreactor at JoVE.com

Construction and Characterization of a Novel Vocal Fold Bioreactor

A novel vocal fold bioreactor capable of delivering physiologically relevant, vibratory stimulation to cultured cells is constructed and characterized. This dynamic culture device, when combined with a fibrous poly(&#949;-caprolactone) scaffold, creates a vocal fold-mimetic environment that modulates the behaviors of mesenchymal stem cells.

Konstruktion og karakterisering af en roman Vocal Fold bioreaktor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

Research

JoVE Journal

Bioengineering

Engineering Biological-Based Vascular Grafts Using a Pulsatile Bioreactor

Much effort has been devoted to develop and advance the methodology to regenerate functional small-diameter arterial bypasses. In the physiological ...

Synthetic, Multi-Layer, Self-Oscillating Vocal Fold Model Fabrication

Syntetisk, Multi-Layer, Self-Oscillerende Vocal Fold Model Fabrication

Sound for the human voice is produced via flow-induced vocal fold vibration. The vocal folds consist of several layers of tissue, each with differing ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

Design af en Biaksial Mekanisk Loading bioreaktor for Tissue Engineering

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Operation of a Benchtop Bioreactor

Drift af Benchtops bioreaktor

Fermentation systems are used to provide an optimal growth environment for many different types of cell cultures. The ability afforded by fermentors to ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

Human primat Lung Decellularization og Recellularization bruge en specialiseret Large-orgel bioreaktor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

Undersøgelse af tre-dimensionelle Flow Separation induceret af en model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Karakterisering af leukocyt-blodpladerigt fibrin, A Novel biomateriale

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

A Novel Bioreactor for High Density Cultivation of Diverse Microbial Communities

A Novel bioreaktor for High Density Dyrkning af Diverse mikrobielle samfund

A novel reactor design, coined a high density bioreactor (HDBR), is presented for the cultivation and study of high density microbial communities. Past ...

Construction and Setup of a Bench-scale Algal Photosynthetic Bioreactor with Temperature, Light, and pH Monitoring for Kinetic Growth Tests

Konstruktion og opsætning af en Bench-skala Algal Photosynthetic Bioreactor med temperatur, lys og pH-overvågning for kinetiske væksttest

The optimal design and operation of photosynthetic bioreactors (PBRs) for microalgal cultivation is essential for improving the environmental and economic ...

Preparation and Characterization of Novel HDL-mimicking Nanoparticles for Nerve Growth Factor Encapsulation

Fremstilling og karakterisering af hidtil ukendte HDL-imiterende nanopartikler til nervevækstfaktorindkapsling

The objective of this article is to introduce preparation and characterization methods for nerve growth factor (NGF)-loaded, high-density, lipoprotein ...