Name: a microphysiologic platform for human fat: sandwiched white adipose tissue
Uploaded: 2018-08-15T14:00:00
Description: Watch this Scientific Journal Video about A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue at JoVE.com

Forfatterne vil gerne anerkende den institutionelle støtte fra LSU Health Sciences Center, som finansieres projektet.

Alle opgaver blev udført i tilslutning til protokollerne #8759 og #9189, som er godkendt af den interne Ratingmetode Office LSUHSC-NO. Alle dyr arbejde blev udført i tilslutning til protokollen #3285 godkendt af Kontoret for IACUC på LSUHSC-nr.
1. såning af Sandwiching celle ark
Bemærk: Se figur 1.
<ol><li>Seed ADSCs på ca 80% confluency i vævskultur plader (6 cm eller 6-godt plader). For hver brønd af SWAT ønskede frø 1 kon...

<ol>
	<li>Cawley, J., Meyerhoefer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+medical+costs+of+obesity:+An+instrumental+variables+approach.">The medical costs of obesity: An instrumental variables approach.</a> Journal of Health Economics. 31 (1), 219-230 (2012).</li><li>Pi-Sunyer, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Medical+Risks+of+Obesity.">The Medical Risks of Obesity.</a> Postgraduate Medicine. 121 (6), 21-33 (2009).</li><li>Smith, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+studies+of+human+subcutaneous+adipose+tissue+in+vitro.">Morphologic studies of human subcutaneous adipose tissue in vitro.</a> Anatomical Record. 169 (1), 97-104 (1971).</li><li>Sugihara, H., Yonemitsu, N., Miyabara, S., Yun, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cultures+of+unilocular+fat+cells:+characteristics+of+growth+in+vitro+and+changes+in+differentiation+properties.">Primary cultures of unilocular fat cells: characteristics of growth in vitro and changes in differentiation properties.</a> Differentiation. 31 (1), 42-49 (1986).</li><li>Sugihara, H., Yonemitsu, N., Toda, S., Miyabara, S., Funatsumaru, S., Matsumoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unilocular+fat+cells+in+three-dimensional+collagen+gel+matrix+culture.">Unilocular fat cells in three-dimensional collagen gel matrix culture.</a> The Journal of Lipid Research. 29, 691-697 (1988).</li><li>Hazen, S. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+National+Institutes+of+Health.+Microphysiological+Systems+Program+focuses+on+a+critical+challenge+in+the+drug+discovery+pipeline.">The National Institutes of Health. Microphysiological Systems Program focuses on a critical challenge in the drug discovery pipeline.</a> Stem Cell Research and Therapy. 4, (2013).</li><li>Wikswo, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+relevance+and+potential+roles+of+microphysiological+systems+in+biology+and+medicine.">The relevance and potential roles of microphysiological systems in biology and medicine.</a> Experimental Biology and Medicine. 239 (9), 1061-1072 (2014).</li><li>. NIH-CASIS Coordinated Microphysiological Systems Program for Translational Research in Space (#RFA-TR-18-001) Available from: <a target="_blank" href="https://grants.nih.gov/grants/guide/rfa-files/RFA-TR-18-001.html">https://grants.nih.gov/grants/guide/rfa-files/RFA-TR-18-001.html</a> (2017)</li><li>Lau, F. H., Vogel, K., Luckett, J. P., Hunt, M., Meyer, A., Rogers, C. L., Tessler, O., Dupin, C. L., St Hilaire, H., Islam, K. N., Frazier, T., Gimble, J. M., Scahill, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sandwiched+White+Adipose+Tissue:+A+Microphysiological+System+of+Primary+Human+Adipose+Tissue.">Sandwiched White Adipose Tissue: A Microphysiological System of Primary Human Adipose Tissue.</a> Tissue Engineering Part C: Methods. 24 (3), (2018).</li><li>Ebara, M., Hoffman, J. M., Stayton, P. S., Hoffman, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+modification+of+microfluidic+channels+by+UV-mediated+graft+polymerization+of+non-fouling+and+'smart'+polymers.">Surface modification of microfluidic channels by UV-mediated graft polymerization of non-fouling and 'smart' polymers.</a> Radiation Physics and Chemistry. 76, 1409-1413 (2007).</li><li>Lin, J. B., Isenberg, B. C., Shen, Y., Schorsch, K., Sazonova, O. V., Wong, J. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermo-responsive+poly(N-isopropylacrylamide)+grafted+onto+microtextured+poly(dimethylsiloxane)+for+aligned+cell+sheet+engineering.">Thermo-responsive poly(N-isopropylacrylamide) grafted onto microtextured poly(dimethylsiloxane) for aligned cell sheet engineering.</a> Colloids and Surfaces B: Biointerfaces. 99, 108-115 (2012).</li><li>Turner, W. S., Sandhu, N., McCloskey, K. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Engineering:+Construction+of+a+Multicellular+3D+Scaffold+for+the+Delivery+of+Layered+Cell+Sheets.">Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets.</a> Journal of Visualized Experiments. (92), e51044 (2014).</li><li>Bunnell, B., Flaat, M., Gagliardi, C., Patel, B., Ripoll, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adipose-derived+stem+cells:+Isolation,+expansion+and+differentiation.">Adipose-derived stem cells: Isolation, expansion and differentiation.</a> Methods. 45 (2), 115-120 (2008).</li><li>Akiyama, Y., Kikuchi, A., Yamato, M., Okano, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrathin+poly(N-isopropylacrylamide)+grafted+layer+on+polystyrene+surfaces+for+cell+adhesion/detachment+control.">Ultrathin poly(N-isopropylacrylamide) grafted layer on polystyrene surfaces for cell adhesion/detachment control.</a> Langmuir. 20 (13), 5506-5511 (2004).</li><li>Fried, S. K., Russell, C. D., Grauso, N. L., Brolin, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipoprotein+lipase+regulation+by+insulin+and+glucocorticoid+in+subcutaneous+and+omental+adipose+tissues+of+obese+women+and+men.">Lipoprotein lipase regulation by insulin and glucocorticoid in subcutaneous and omental adipose tissues of obese women and men.</a> Journal of Clinical Investigation. 92 (5), 2191-2198 (1993).</li><li>Keipert, S., Jastroch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brite/beige+fat+and+UCP1+-+is+it+thermogenesis?.">Brite/beige fat and UCP1 - is it thermogenesis?.</a> Biochimica et Biophysica Acta. 1837 (7), 1075-1082 (2014).</li></ol>

Denne protokol beskriver brugen af ADSCs til sandwich menneskelige hvidt fedtvæv; menneskelige ADSC cellelinjer kan isoleres via veletablerede protokoller15. Dog kan systemet tilpasses til individualiseret forskning krav (f.eks. ved hjælp af 3T3L-1 celler til sandwich mus WAT). Denne proces indebærer håndtering primære humant væv. Standard sikkerhedsforanstaltninger bør være ansat; håndtere humane væv som BSL-2 patogener (fx HIV, HepC). Kun håndtere væv direkte under en BSC. B...

Fedtvæv er den primære organ af fedme, som varetager direkte årlige medicinske omkostninger mellem 147 milliarder dollars og 210 milliarder dollar i den amerikanske1. Ophobningen af fedtvæv bidrager også til andre førende dødsårsager som hjertesygdom, type II sukkersyge og visse former for kræft2. In vitro kultur modeller er afgørende for metaboliske undersøgelser og udvikling af lægemidler, men aktuelle forskning modeller af fedtvæv har store mangler. Adipocytter er skrøbelige, livlig, og uhelbredeligt differentierede celler, der ikke vil overholde celle kultur plast, og derfor kan ikke være kulturperler konventionel celle kultur metoder. Siden 1970 ' erne, er flere metoder blevet brugt i forsøg på at overvinde disse hindringer, herunder brugen af glas coverslips loft, suspension kultur, og kultur ekstracellulære matricer3,4,5, 6 , 7. men disse metoder har været præget af celledød og dedifferentiation, og de bruges typisk til ikke mere end en to-ugers undersøgelse periode. Desuden, disse modeller forsøg ikke sammenfatte de indfødte adipøst mikromiljø som de ikke bevare intakt ECM, interaktioner mellem adipocytter og stromale støtte celler, og heller ikke de kontraktile styrker celler øve på hinanden i in vivo WAT.
I mangel af en guld-standard primære adipøst kultur metode, har fedt forskning påberåbt sig primært differentieret pre adipocytter (diffAds). DiffAds er multilocular, vedhængende og metabolisk aktive. Derimod primære hvid adipocytter er ensfarvet, nonadherent, og viser relativt lavt stofskifte. Den manglende nuværende adipøst kultur modeller til at sammenfatte fysiologi af sunde modne fedtvæv er sandsynligvis en vigtig faktor i mangel af FDA-godkendt medicin, der er direkte målrettet adipocytter. I virkeligheden, er manglen på fysiologisk in vitro- orgel modeller et stort problem på tværs af de fleste organer og sygdom.
I sit oplæg, annoncere oprettelsen af sin Microphysiological Systems (MPS) program, rapporterede National Institutes of Health (NIH), at 2013 succesrate på tværs af alle menneskelige farmaceutiske kliniske forsøg var kun 18% for fase II og 50% for fase III kliniske forsøg8. MPS-programmet er designet til at tage direkte fat i vitro monokultur manglende evne til at model menneskelige fysiologi. NIH definerer MPSs som kultur systemer består af menneskelig primær eller stamceller i flercellede 3D konstruktioner, at sammenfatte orgel funktion. I modsætning til reduktionistiske modeller af homogen, udødeliggjort cellekulturer, bør MPSs præcist model celle-celle, narkotika-celle, stof-stof og orgel-drug interaktioner9. I modsætning til kortsigtede primære kultur metoder dikterer NIH standarder MPS bæredygtighed over 4 uger i kulturen8. Yderligere oplysninger om MPS-programmet kan findes på NIH'S RFAs (#RFA-TR-18-001)10.
Vi har udviklet en simpel, roman, fleksibel, og billig adipøst MPS betegnes "klemt inde hvidt fedtvæv" (SWAT)11. Vi overvinde den naturlige opdrift af adipocytter af "sandwiching" hakket primære fedtvæv mellem ark af fedt-afledt stromale celler (ADSCs) (figur 1). Den resulterende 3D konstruktion sammenfatter celle-celle-kontakt og de indfødte adipøst mikromiljø af omkring modne adipocytter med en naturlig adipocyt støtte celle population. SWAT er blevet valideret ved at demonstrere 8-ugers levedygtighed, reaktion på eksogene signalering, adipokine sekretion og engraftment i en dyremodel.

<table border="0" cellpadding="0" cellspacing="0" width="817">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:219px;">10x HBSS</td>
			<td style="width:92px;">Thermofisher</td>
			<td style="width:115px;">14185052</td>
			<td style="width:393px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Gelatin</td>
			<td>Sigma-aldrich</td>
			<td>G9391</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Collagenase</td>
			<td>Sigma-aldrich</td>
			<td>C5138</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Adenosine</td>
			<td>Sigma-aldrich</td>
			<td>A9251</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">DMEM</td>
			<td>Thermofisher</td>
			<td>11995065</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">M199 Media</td>
			<td>Thermofisher</td>
			<td>11043023</td>
			<td>Phenol red-free&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">250&#181;m Mesh Filter</td>
			<td>Pierce</td>
			<td>87791</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">0.2&#181;m Syringe Filter</td>
			<td>Celltreat</td>
			<td>229747</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">5mL Luer-Lok syringes&#160;</td>
			<td>BD</td>
			<td>309646</td>
			<td></td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">Metal Washers</td>
			<td></td>
			<td></td>
			<td style="width:393px;">These are simple metal washers and can be bought at any hardware store. They simply add leight weight to the backs of the plugers to ensure even contact between cells and gelatin, while being easy to stock and sterilize. Approximate mass: 6.3g&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Heated Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Incubated Orbital Shaker</td>
			<td>VWR</td>
			<td>10020-988</td>
			<td style="width:393px;">Samples should be pitched at 45&#176; angle to facilitate collagenase digestion</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Heat Block</td>
			<td></td>
			<td></td>
			<td style="width:393px;">Set to 37-40&#176;C and placed under Biosafety Cabinet</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Water Bath</td>
			<td></td>
			<td></td>
			<td style="width:393px;">Set to ~75&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Specialized Plastics</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="180">
			<td height="180" style="height:180px;">Upcell Dishes 6cm of 6-multiwell</td>
			<td>Nunc</td>
			<td>174902&#160; or 174901</td>
			<td style="width:393px;">These are commerically available pNIPAAm-coated dishes which can be used to grow the upper sheet of ADSCs. Alternatively, pNIPAAm-coated plates can be produced in-lab. diameter: &#60;6cm for 6cm dish, &#60;3.5cm for 6-well plate; approximate mass: 6.7g for 6cm dish, 5.1g for 6-well plate</td>
		</tr>
		<tr height="160">
			<td height="160" style="height:160px;">Plastic Plunger Apparatus</td>
			<td></td>
			<td></td>
			<td style="width:393px;">These can be fashioned to fit within desired pNIPAAm-coated plastics (multiwell plates, petri dishes). They are comprised of a simple stem attached to a circular disk. They can be produced in-lab or by any facility that can fashion acrylic plastics</td>
		</tr>
	</tbody>
</table>

a microphysiologic platform for human fat: sandwiched white adipose tissue

Hvidt fedtvæv (WAT) spiller en afgørende rolle i reguleringen af vægt og everyday sundhed. Alligevel er der stadig betydelige begrænsninger til rådighed primære kultur modeller, som alle har undladt at trofast sammenfatte den adipøst mikromiljø eller udvide WAT levedygtighed ud over to uger. Manglen på en pålidelig primære kultur model hindrer alvorligt forskning i WAT metabolisme og udvikling af lægemidler. Til dette formål har vi udnyttet NIHS standarder for et microphysiologic system til at udvikle en ny platform for WAT primære kultur kaldet "SWAT" (klemt hvidt fedtvæv). Vi overvinde den naturlige opdrift af adipocytter af sandwiching hakket WAT klynger mellem ark af fedt-afledt stromale celler. I denne konstruktion er WAT prøver levedygtige over otte uger i kultur. SWAT fastholder intakt ECM, celle til celle kontakter og fysiske belastninger i vivo WAT betingelser; Derudover fastholder SWAT en robust transcriptional profil, følsomhed over for eksogene kemiske signaler, og hele væv funktion. SWAT repræsenterer en enkel, reproducerbare og effektiv metode til primær adipøst kultur. Potentielt, er det en generelt gældende platform for forskning i WAT fysiologi, Patofysiologi, stofskifte og farmaceutisk udvikling.

Levedygtighed af SWAT var oprindeligt vurderet af serielle brightfield billeddannelse af enkelte WAT klynger (n = 12) over ca. 7,6 uger. Klynger forblev sikrede på plads på éncellelag i hele denne tid. Morfologiske ændringer blev observeret med individuelle adipocytter vridning lidt eller skiftende positioner. Imidlertid adipocytter heller ikke blive multilocular over tid, med angivelse af en mangel på dedifferentiation, eller gjorde de udviser synlige tegn på celledød som cellulæ...

Watch this Scientific Journal Video about A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue at JoVE.com

A Microphysiologic Platform for Human Fat: Sandwiched White Adipose Tissue

Hvidt fedtvæv (WAT) er kritisk mangler i dens nuværende primære kultur modeller, hindrer farmakologiske udvikling og metaboliske undersøgelser. Vi præsenterer her, en protokol for at producere et fedt microphysiological system af sandwiching WAT mellem ark af stromale celler. Denne konstruktion giver en stabil og fleksibel platform for primære WAT kultur.

En Microphysiologic Platform for menneskelige fedt: klemt inde hvidt fedtvæv

White adipose tissue (WAT) plays a crucial role in regulating weight and everyday health.  Still, there are significant limitations to available primary ...

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