Name: Forfatter Spotlight: Utvikling av et unikt modulært mikrofysiologisk system for å etterligne menneskelig barrierevev
Uploaded: 2024-02-16T14:10:00
Description: Watch this Scientific Journal Video about Author Spotlight: Developing a Unique Modular Microphysiological System to Mimic Human Barrier Tissue at JoVE.com

Denne forskningen ble delvis finansiert av National Institute of Health under tildelingsnummer R43GM137651, R61HL154249, R16GM146687 og NSF-tilskudd CBET 2150798. Forfatterne takker RIT Machine Shop for aluminiumsformfabrikasjon. Innholdet er utelukkende forfatternes ansvar og representerer ikke nødvendigvis de offisielle synspunktene til National Institutes of Health.

Denne designen kan brukes i forskjellige moduser basert på eksperimentelle krav og sluttbrukerens preferanser. Før hvert eksperiment, konsulter beslutningsflytdiagrammet presentert i figur 2 for å bestemme de nødvendige trinnene og modulene for protokollen. Hvis brukeren for eksempel har til hensikt å opprettholde open well-formatet gjennom et eksperiment for å sammenligne det direkte med Transwell-typesystemet, er mønstersjablongen ikke nødvendig for cellesåing. Kjernemodulen er ko...

En rekonfigurerbar barrierevevsplattform med åpen brønn og mikrofluidisk modus

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Målet med denne protokollen er å utvikle en praktisk metode for å inkorporere strømningsegenskaper i en åpen brønnplattform med en ultratynn nanomembran. I dette designet benyttes en magnetisk låsetilnærming, noe som gjør det mulig å bytte mellom åpen brønn og fluidisk modus under eksperimenter og kombinere fordelene ved begge tilnærmingene. I motsetning til konvensjonelle permanent bundne plattformer, gjør magnetisk låsing at plattformen kan demonteres på praktiske punkter under den eksperimentelle arbei...

Vaskulære barrierer tjener som et kritisk grensesnitt som skiller blodrommet fra det omkringliggende vevet. De spiller en kritisk rolle i å bevare homeostase ved å tiltrekke immunceller, kontrollere molekylær permeabilitet og skjerming mot inntrenging av patogener i vevet 1,2. In vitro dyrkningsmodeller er utviklet for å etterligne in vivo mikromiljøet, noe som muliggjør systematiske undersøkelser av faktorer og forhold som påvirker barriereegenskaper i både friske og syke tilstander 3,4.
Den mest brukte tilnærmingen for slike kulturmodeller er den Transwell-lignende "open-well" -konfigurasjonen5, hvor en porøs, sporetset kulturmembran skiller mediefylte rom (figur 1A). I dette formatet kan celler sås på hver side av membranen, og et bredt spekter av eksperimentelle protokoller er utviklet. Imidlertid er disse systemene begrenset i deres evne til å gi væskestrømmene som er avgjørende for å støtte barrieremodning og etterligne immuncellesirkulasjon sett in vivo 5,6. Følgelig kan de ikke brukes til studier som krever dynamiske strømninger som introduserer legemiddeldoser, mekanisk stimulering eller væskeinduserte skjærspenninger 6,7,8.
For å overvinne begrensningene i åpne brønnsystemer, har mikrofluidiske plattformer som kombinerer porøse kulturmembraner med individuelt adresserbare fluidiske kanaler blitt utviklet9. Disse plattformene gir presis kontroll over væskeruting, perfusjon og innføring av kjemiske forbindelser, kontrollert skjærstimulering og dynamisk celleaddisjonskapasitet 7,10,11,12,13. Til tross for de avanserte mulighetene som tilbys av mikrofluidiske plattformer, har de ikke sett utbredt adopsjon i biovitenskapslaboratorier på grunn av komplekse mikrofluidiske protokoller og deres inkompatibilitet med etablerte eksperimentelle arbeidsflyter 4,10,14.
For å bygge bro over gapet mellom disse teknologiene, presenterer vi en protokoll som bruker et magnetisk rekonfigurerbart, modulbasert system. Dette systemet kan enkelt byttes mellom åpen brønn og mikrofluidisk modus basert på eksperimentets spesifikke behov. Plattformen har en åpen brønn-enhet, kjent som m-μSiM (modulært mikrofysiologisk system aktivert av en silisiummembran), med en 100 nm tykk kulturmembran (nanomembran). Denne nanomembranen har høy porøsitet (15%) og glasslignende gjennomsiktighet, som illustrert i figur 1B. Det skiller fysisk det øverste rommet fra en bunnkanal, noe som muliggjør molekylær transport over fysiologiske lengdeskalaer15. I motsetning til konvensjonelle sporetsede membraner, som har kjente utfordringer i avbildning av levende celler med lysfeltavbildning, muliggjør nanomembranens gunstige optiske og fysiske egenskaper klar visualisering av celler på hver side av membranoverflaten 15,16,17.
Den nåværende protokollen skisserer fabrikasjon av spesialiserte så- og strømningsmoduler og forklarer den magnetiske rekonfigureringen av plattformen. Den demonstrerer hvordan plattformen kan brukes til å etablere endotelbarrierer under både statiske og dynamiske forhold. Denne demonstrasjonen avslører at endotelceller justerer seg langs strømningsretningen, med en oppregulering av skjærfølsomme genmål under skjærstimulering.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 x 0.86 Micro Flow tubes</td>
			<td>Langer Instruments</td>
			<td>WX10-14 &#38; DG Series</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mm Disposable Biopsy Punches, Integra Miltex</td>
			<td>VWR</td>
			<td>95039-090</td>
			<td></td>
		</tr>
		<tr>
			<td>1x PBS 7.4 pH</td>
			<td>ThermoFisher Scientific</td>
			<td>10010023</td>
			<td></td>
		</tr>
		<tr>
			<td>20 GAUGE IT SERIES DISPENSING TIP</td>
			<td>Jensen Global</td>
			<td>JG20-1.5X</td>
			<td></td>
		</tr>
		<tr>
			<td>21 GAUGE NT PREMIUM SERIES ANGLED DISPENSING TIP</td>
			<td>Jensen Global</td>
			<td>JG21-1.0HPX-90</td>
			<td></td>
		</tr>
		<tr>
			<td>3M 467 MP Pressure senstitive adhesive (PSA)</td>
			<td>DigiKey</td>
			<td>3M9726-ND</td>
			<td></td>
		</tr>
		<tr>
			<td>3M 468 MP Pressure senstitive adhesive (PSA)</td>
			<td>DigiKey</td>
			<td>3M9720-ND</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 488 conjugated phalloidin</td>
			<td>ThermoFisher Scientific</td>
			<td>A12379&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Applied Biosystems&#160;TaqMan Fast Advanced Master Mix</td>
			<td>Thermo Fisher Scientific</td>
			<td>4444556</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA), Fraction V, 98%, Reagent grade, Alfa Aesar, Size = 10 g</td>
			<td>VWR</td>
			<td>AAJ64100-09</td>
			<td></td>
		</tr>
		<tr>
			<td>Clear Scratch- and UV-Resistant Cast Acrylic Sheet</td>
			<td>McMaster-Carr</td>
			<td>8560K171</td>
			<td>12&#34; x 12&#34; x 1/16&#34;</td>
		</tr>
		<tr>
			<td>Clear Scratch- and UV-Resistant Cast Acrylic Sheet</td>
			<td>McMaster-Carr</td>
			<td>8589K31</td>
			<td>12&#34; x 12&#34; x 3/32&#34;</td>
		</tr>
		<tr>
			<td>Clear Scratch- and UV-Resistant Cast Acrylic Sheet</td>
			<td>McMaster-Carr</td>
			<td>8560K191</td>
			<td>12&#34; x 12&#34; x 7.64&#34;</td>
		</tr>
		<tr>
			<td>Corning Fibronectin, Human, 1 mg</td>
			<td>Corning</td>
			<td>47743-728</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover Glasses, Globe Scientific, L x W = 24 x 60 mm</td>
			<td>VWR</td>
			<td>10118-677</td>
			<td></td>
		</tr>
		<tr>
			<td>DOW SYLGARD 184 SILICONE ENCAPSULANT CLEAR 0.5 KG KIT</td>
			<td>Ellsworth Adhesives</td>
			<td>4019862</td>
			<td></td>
		</tr>
		<tr>
			<td>EGM-2 Endothelial Cell Growth Medium-2 BulletKit</td>
			<td>Lonza</td>
			<td>CC-3162</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixture A1&#38;A2</td>
			<td>SiMPore Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixture B1&#38;B2</td>
			<td>SiMPore Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>High Capacity cDNA Reverse Transcription Kit with RNase Inhibitor</td>
			<td>Thermo Fisher Scientific</td>
			<td>4374966</td>
			<td></td>
		</tr>
		<tr>
			<td>Human umbilical vein endothelial cells (HUVEC)</td>
			<td>ThermoFisher Scientific</td>
			<td>C0035C</td>
			<td></td>
		</tr>
		<tr>
			<td>LIVE/DEAD Cell Imaging Kit (488/570)</td>
			<td>Thermo Fisher Scientific</td>
			<td>R37601</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular Probes Hoechst 33342, Trihydrochloride, Trihydrate</td>
			<td>Thermo Fisher Scientific</td>
			<td>H3570</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel-plated magnets (4.75 mm diameter, 0.34 kg pull force)</td>
			<td>K&#38;J Magnetics</td>
			<td>D31</td>
			<td>3/16&#34; dia.&#160;x 1/16&#34; thick</td>
		</tr>
		<tr>
			<td>Paraformaldehyde, 4% w/v aq. soln., methanol free, Alfa Aesar</td>
			<td>Fisher Scientific</td>
			<td>aa47392-9M</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic Pump</td>
			<td>Langer Instruments</td>
			<td>BQ50-1J-A</td>
			<td></td>
		</tr>
		<tr>
			<td>Photoresist SU-8 developer solution</td>
			<td>Fisher Scientific</td>
			<td>NC9901158</td>
			<td></td>
		</tr>
		<tr>
			<td>PVDF syringe filters</td>
			<td>PerkinElmer</td>
			<td>2542913</td>
			<td></td>
		</tr>
		<tr>
			<td>Silicon wafer</td>
			<td>University wafer,USA</td>
			<td>1196</td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 3050</td>
			<td>Fisher Scientific</td>
			<td>NC0702369</td>
			<td></td>
		</tr>
		<tr>
			<td>Target gene: eNOS (Hs01574659_m1)</td>
			<td>ThermoFisher Scientific</td>
			<td>4331182</td>
			<td></td>
		</tr>
		<tr>
			<td>Target gene: GAPDH (Hs02786624_g1)</td>
			<td>ThermoFisher Scientific</td>
			<td>4331182</td>
			<td></td>
		</tr>
		<tr>
			<td>Target gene: KLF2 (Hs00360439_g1)</td>
			<td>ThermoFisher Scientific</td>
			<td>4331182</td>
			<td></td>
		</tr>
		<tr>
			<td>Thermo Scientific&#160;Pierce 20x PBS Tween 20</td>
			<td>Thermo Fisher Scientific</td>
			<td>28352</td>
			<td></td>
		</tr>
		<tr>
			<td>Transport Tube Sample White caps, 5 mL, Sterile</td>
			<td>VWR</td>
			<td>100500-422</td>
			<td></td>
		</tr>
		<tr>
			<td>TRI-reagent</td>
			<td>ThermoFisher Scientific</td>
			<td>AM9738</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrathin Nanoporous Membrane Chip</td>
			<td>SiMPore Inc.</td>
			<td>NPSN100-1L</td>
			<td>The design is&#160; compatible with all of SiMPore membranes</td>
		</tr>
		<tr>
			<td>uSiM component 1</td>
			<td>SiMPore Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>uSiM component 2</td>
			<td>SiMPore Inc.</td>
			<td>NA</td>
			<td></td>
		</tr>
	</tbody>
</table>

transforming static barrier tissue models into dynamic microphysiological systems

Mikrofysiologiske systemer er miniatyriserte cellekulturplattformer som brukes til å etterligne strukturen og funksjonen til humant vev i en laboratorieinnstilling. Imidlertid har disse plattformene ikke fått utbredt adopsjon i biovitenskapslaboratorier der åpne brønner, membranbaserte tilnærminger tjener som gullstandarden for etterligning av vevsbarrierer, til tross for manglende væskestrømningsfunksjoner. Dette problemet kan primært tilskrives inkompatibiliteten til eksisterende mikrofysiologiske systemer med standardprotokoller og verktøy utviklet for åpne brønnsystemer.
Her presenterer vi en protokoll for å skape en rekonfigurerbar membranbasert plattform med en åpen brønnstruktur, strømningsforbedringsevne og kompatibilitet med konvensjonelle protokoller. Dette systemet benytter en magnetisk monteringstilnærming som muliggjør reversibel veksling mellom åpen brønn og mikrofluidisk modus. Med denne fremgangsmåten har brukerne fleksibilitet til å starte et eksperiment i åpent brønnformat ved hjelp av standardprotokoller og legge til eller fjerne flytfunksjoner etter behov. For å demonstrere den praktiske bruken av dette systemet og dets kompatibilitet med standardteknikker, ble et endotelcellemonolag etablert i et åpent brønnformat. Systemet ble rekonfigurert for å introdusere væskestrøm og deretter byttet til åpent brønnformat for å utføre immunfarging og RNA-ekstraksjon. På grunn av sin kompatibilitet med konvensjonelle åpne brønnprotokoller og strømningsforbedringsevne, forventes denne rekonfigurerbare designen å bli tatt i bruk av både ingeniør- og biovitenskapslaboratorier.

Vascular barriers serve as a critical interface that separates the blood compartment from the surrounding tissue. They play a critical role in preserving homeostasis by attracting immune cells, controlling molecular permeability, and shielding against the intrusion of pathogens into the tissue1,2. In vitro culture models have been developed to mimic the in vivo microenvironment, enabling systematic investigations into the factors and conditions that impact barrier properties in both healthy and diseased states3,4.
The most widely used approach for such culture models is the Transwell-like &#34;open-well&#34; configuration5, where a porous, track-etched culture membrane separates media-filled compartments (Figure 1A). In this format, cells can be seeded on either side of the membrane, and a wide range of experimental protocols has been developed. However, these systems are limited in their ability to provide the fluid flows essential for supporting barrier maturation and mimicking immune cell circulation seen in vivo5,6. Consequently, they cannot be used for studies requiring dynamic flows that introduce drug doses, mechanical stimulation, or fluid-induced shear stresses6,7,8.
To overcome the limitations of open-well systems, microfluidic platforms that combine porous culture membranes with individually addressable fluidic channels have been developed9. These platforms offer precise control over fluid routing, perfusion, and the introduction of chemical compounds, controlled shear stimulation, and dynamic cell addition capabilities7,10,11,12,13. Despite the advanced capabilities provided by microfluidic platforms, they have not seen widespread adoption in bioscience laboratories due to complex microfluidic protocols and their incompatibility with established experimental workflows4,10,14.
To bridge the gap between these technologies, we present a protocol that employs a magnetically reconfigurable, module-based system. This system can be easily switched between open-well and microfluidic modes based on the specific needs of the experiment. The platform features an open-well device, known as m-&#181;SiM (modular microphysiological system enabled by a silicon membrane), with a 100 nm thick culture membrane (nanomembrane). This nanomembrane possesses high porosity (15%) and glass-like transparency, as illustrated in Figure 1B. It physically separates the top compartment from a bottom channel, allowing for molecular transport across physiological length scales15. Unlike conventional track-etched membranes, which have known challenges in imaging live cells with bright-field imaging, the nanomembrane&#39;s favorable optical and physical properties enable clear visualization of cells on either side of the membrane surface15,16,17.
The present protocol outlines the fabrication of specialized seeding and flow modules and explains the magnetic reconfiguration of the platform. It demonstrates how the platform can be employed to establish endothelial barriers under both static and dynamic conditions. This demonstration reveals that endothelial cells align along the flow direction, with an upregulation of shear-sensitive gene targets under shear stimulation.

Forfatter Spotlight: Utvikling av et unikt modulært mikrofysiologisk system for å etterligne menneskelig barrierevev

Transformere statiske barrierevevsmodeller til dynamiske mikrofysiologiske systemer

We're developing modular microphysiological systems that mimic human barrier tissues. Our system is unique because it can be reconfigured from a static open well culture to a flow-enhanced system. This flexibility allows the platform to be used in both bioscience and engineering-focused laboratories.We present a protocol for creating a reconfigurable open well platform with flow-enhanced culture and lifestyle imaging. It is compatible with conventional bioscience protocols. Our reconfigurable design allows users to switch between the open well and microfluidic modes during an experiment, and allows the user to conduct each step using a format they are comfortable with.

Den åpne brønnkjernemodulen er opprinnelig plassert i et spesifikt hulrom skapt av et nedre hus og en deksel, som illustrert i figur 6A. Deretter settes strømningsmodulen, som inkluderer en mikrokanal og tilgangsporter, inn i brønnen til kjernemodulen. Strømningsmodulen er forsvarlig forseglet mot silisiumstøttelaget til membranen på grunn av den magnetiske tiltrekningskraften mellom magneter innebygd i nedre og øvre hus, som vist i figur 6B. For å eval...

Watch this Scientific Journal Video about Author Spotlight: Developing a Unique Modular Microphysiological System to Mimic Human Barrier Tissue at JoVE.com

Denne protokollen beskriver en rekonfigurerbar membranbasert cellekulturplattform som integrerer det åpne brønnformatet med væskestrømningsfunksjoner. Denne plattformen er kompatibel med standardprotokoller og muliggjør reversible overganger mellom åpen brønn og mikrofluidisk kulturmodus, imøtekomme behovene til både ingeniør- og biovitenskapslaboratorier.

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. ...

Vi utvikler modulære mikrofysiologiske systemer som etterligner menneskelige barrierevev. Systemet vårt er unikt fordi det kan rekonfigureres fra en statisk åpen brønnkultur til et strømningsforsterket system. Denne fleksibiliteten gjør at plattformen kan brukes i både biovitenskap og ingeniørfokuserte laboratorier.Vi presenterer en protokoll for å skape en rekonfigurerbar åpen brønnplattform med strømningsforbedret kultur og livsstilsavbildning. Den er kompatibel med konvensjonelle biovitenskapsprotokoller. Vår rekonfigurerbare design lar brukerne bytte mellom åpen brønn og mikrofluidisk modus under et eksperiment, og lar brukeren utføre hvert trinn ved hjelp av et format de er komfortable med.

transforming-static-barrier-tissue-models-into-dynamic

Research

JoVE Journal

Bioengineering

Transforming Static Barrier Tissue Models into Dynamic Microphysiological Systems

Microphysiological systems are miniaturized cell culture platforms used to mimic the structure and function of human tissues in a laboratory setting. However, these platforms have not gained widespread adoption in bioscience laboratories where open-well, membrane-based approaches serve as the gold standard for mimicking tissue barriers, despite lacking fluid flow capabilities. This issue can be primarily attributed to the incompatibility of existing microphysiological systems with standard protocols and tools developed for open-well systems.
Here, we present a protocol for creating a reconfigurable membrane-based platform with an open-well structure, flow enhancement capability, and compatibility with conventional protocols. This system utilizes a magnetic assembly approach that enables reversible switching between open-well and microfluidic modes. With this approach, users have the flexibility to begin an experiment in the open-well format using standard protocols and add or remove flow capabilities as needed. To demonstrate the practical usage of this system and its compatibility with standard techniques, an endothelial cell monolayer was established in an open-well format. The system was reconfigured to introduce fluid flow and then switched to the open-well format to conduct immunostaining and RNA extraction. Due to its compatibility with conventional open-well protocols and flow enhancement capability, this reconfigurable design is expected to be adopted by both engineering and bioscience laboratories.

This protocol describes a reconfigurable membrane-based cell culture platform that integrates the open-well format with fluid flow capabilities. This platform is compatible with standard protocols and allows for reversible transitions between open-well and microfluidic culture modes, accommodating the needs of both engineering and bioscience laboratories.