Name: recombinant collagen i peptide microcarriers for cell expansion and their potential use as cell delivery system in a bioreactor model
Uploaded: 2018-02-07T15:00:00
Description: Watch this Scientific Journal Video about Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model at JoVE.com

Forskningen førte til disse resultatene har mottatt finansiering fra den europeiske Union syvende Framework program FP7/2007-2013 under grant avtalen n ° 607051 (BIO-INSPIRERE). Vi takker Carolien van Spreuwel-Goossens fra Fujifilm produksjon Europa B.V. for teknisk assistanse under RCP produksjon og Werner Stracke fra Fraunhofer Institute for Silicate forskning ISC, hjelp med SEM analyse.

hBMSCs ble isolert fra femur hodet av slitasjegikt pasienter som gjennomgår femur hodet erstatning kirurgi. Prosedyren ble utført under godkjenning av den lokale etikk av universitetet i Würzburg og samtykke av pasientene. Primære mikrovaskulær endotelceller ble isolert fra foreskin biopsies av juvenile givere. Deres forvalter(e) gitt full samtykke skriftlig. Studien ble godkjent av lokale etiske styret ved Universitetet i Würzburg (stemme 182/10).
1. isolering av hBMSCs og HDMECs
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<ol>
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En Hovedmålet med microcarrier er utvidelsen av celler mens deres differensiering for å levere celler til stedet trenger. Metoden representert introdusere RCP microcarriers der cellene kunne knytte sprer og kolonisere microcarriers med høyt tetthet. Dette ble observert av live/døde flekker, der mer enn 90% av levedyktige cellers ble oppdaget mens bare noen døde celler ble innhentet etter 7 dager med dynamisk kulturer. Likeledes, SEM bildene bekreftet at cellene dekket hele overflaten av microcarriers etter 7 dager a...

Et generelt problem i vev engineering programmer er å gi en høy cellemasse med riktig differensiering fenotypen der trenger. Bruk av microcarriers for å løse dette problemet startet i 1967 med økende betydning langt i felt som ortopediske tissue engineering for store generasjon av hud, bein, brusk og sener1. De tillater håndtering av tilhenger kulturer på måter ligner på suspensjon kulturer2 utvide celler i Mikroskala tredimensjonale (3D) underlag. Dermed oppleve celler en homogen nærings levering og celle-matrix interaksjoner som fører til bedre vedlikehold av i vivo3,4 differensiering er ofte tapt over tid i 2D tilnærminger5. Høyere overflate-til-volum forholdet - til slutt fører til høyere celle gir6,7, høyere gass og næringsstoffer valutakurser sammenligne statisk systemer8, muligheten til å regulere og underlagt kulturen fysisk stimuli9og potensialet for å skalere opp av ekspansjon prosessen7 er ytterligere fordeler. Flere funksjoner som diameter, tetthet, porøsitet, overflate kostnad og vedheft egenskaper10,11 skille ulike kommersielt tilgjengelig mikro - og makro-bærere. Men er en av de viktigste fordelen deres leveringsmidler potensial som microtissues nettstedet defekt eller etterspørsel.
For programmer av microcarrier teknologi i bein vev engineering, vi illustrert i en tidligere rapport12 produksjon av en ny microcarrier type utgjorde av en rekombinant kollagen jeg peptid (RCP, kommersielt tilgjengelig som Cellnest). Denne nye microcarrier kan GMP-kompatibel opp skalering av stillaset og celle produksjon, behov for cellen levering i klinisk scenario. I denne sammenheng kan tuning av stillaset stabilitet, degradering rate og overflateegenskaper gjennom riktig valg av en egnet crosslinking strategi tilpasse teknikken til det valgte programmet, celle type interesse eller målrette vev13. Spesielt gjør potensielle ansettelse av dette microcarrier som en injeksjon celle levering system for terapeutisk program14 dem spesielt interessant i en klinisk setting.
I dette papiret illustrere vi derfor culturing prosedyren for isolasjon og utvidelse av menneskelig bein margtransplantasjon-avledet mesenchymal stromal celler (hBMSCs) og menneskelige dermal mikrovaskulær endotelceller (HDMECs) på kollagen-jeg-baserte rekombinant peptid-basert microcarriers og sine forberedelser for levering i en klinisk setting. I tillegg beskriver vi ytterligere protokoller nyttig for vedlikehold av cellen levedyktighet ved implantasjon.
Cellen levedyktighet etter implantasjon er sterkt avhengig av endometrial blodkar15,16,17, som sikrer utveksling av oksygen og næringsstoffer og forenkler fjerning. Bioreaktorer utgjør en tilnærming for å overvinne endometrial blodkar utfordringer i tissue engineering og vedlikeholde celle levedyktighet, gjennom perfusjon av kultur medium gir dermed oksygen og næringsstoffer18. Her viser vi en i vitro metode for å vurdere migrasjon muligheten av mikrovaskulær endotelceller fra RCP-microcarriers en biomatrix og deres evne til å bidra til de novo endometrial blodkar og angiogenese. Denne biomatrix er en decellularized del av svin jejunum kalt BioVaSc (biologiske Vascularized stillas), rik på kollagen og elastin og med bevart vaskulære strukturer, som inkluderer en fôring arterie og en drenering blodåre19 som er brukes for implantasjon problemer20.

<table>
	<tbody>
		<tr>
			<td>3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT)</td>
			<td>Serva Electrophoresis GmbH</td>
			<td>20395.01</td>
			<td></td>
		</tr>
		<tr>
			<td>4&#8217;,6-Diamidino-2-phenylindoldihydrochloride (DAPI)</td>
			<td>Sigma-Aldrich</td>
			<td>D9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid 100%</td>
			<td>Sigma-Aldrich</td>
			<td>533,001</td>
			<td></td>
		</tr>
		<tr>
			<td>Analytical balance Kern EG 2200-2NM</td>
			<td>Kern &#38; Sohn GmbH</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ascorbate-2-phosphate</td>
			<td>Sigma-Aldrich</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioreactor</td>
			<td>Chair of Tissue Engineering and Regenerative Medicine, Wuerzburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bright field microscope Axiovert 40C</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cellnest</td>
			<td>Fujifilm</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes (15 mL, 50 mL)</td>
			<td>Greiner Bio-One</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen R solution 0,4%</td>
			<td>Serva Electrophoresis GmbH</td>
			<td>47254.01</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s Phosphate Buffered Saline</td>
			<td>Sigma-Aldrich</td>
			<td>D8537</td>
			<td>Modified, without calcium chloride and magnesium chloride</td>
		</tr>
		<tr>
			<td>Eosin 1%</td>
			<td>Morphisto</td>
			<td>10177.01000</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%</td>
			<td>Carl Roth GmbH</td>
			<td>T171.4</td>
			<td>Denatured</td>
		</tr>
		<tr>
			<td>Fetal calf serum (FCS)</td>
			<td>Bio&#38;SELL</td>
			<td>FCS.ADD.0500</td>
			<td>not heat-inactivated</td>
		</tr>
		<tr>
			<td>Fluorescence microscope BZ-9000</td>
			<td>Keyence</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Haematoxylin</td>
			<td>Morphisto</td>
			<td>10231.01000</td>
			<td></td>
		</tr>
		<tr>
			<td>Hexamethyldisilazane</td>
			<td>Sigma-Aldrich</td>
			<td>440191</td>
			<td>Reagent grade, &#8805;99%</td>
		</tr>
		<tr>
			<td>Incubator for bioreactor</td>
			<td>Chair of Tissue Engineering and Regenerative Medicine, Wuerzburg, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Live/Dead Cell Double Staining Kit</td>
			<td>Fluka</td>
			<td>04511KT-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic stirrer plate</td>
			<td>2Mag</td>
			<td>80002</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Sigma-Aldrich</td>
			<td>M0650</td>
			<td>10X</td>
		</tr>
		<tr>
			<td>Microplate reader 
			Tecan Infinite M200</td>
			<td>Tecan</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Needle 21G 16mm</td>
			<td>VWR</td>
			<td>613-5389</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain from papaya latex</td>
			<td>Sigma-Aldrich</td>
			<td>P4762</td>
			<td>lyophilized powder, &#8805; 10 units/mg protein</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Carl Roth GmbH</td>
			<td>6642.6</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>Sigma-Aldrich</td>
			<td>P4333</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Ismatec</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Quanti-iT PicoGreen dsDNA assay kit</td>
			<td>Thermo Fischer Scientific</td>
			<td>P7589</td>
			<td></td>
		</tr>
		<tr>
			<td>Histofix 4%</td>
			<td>Carl Roth GmbH</td>
			<td>P087</td>
			<td></td>
		</tr>
		<tr>
			<td>Scanning Electron Microscope Supra 25</td>
			<td>Carl Zeiss AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide solution 1.0 N</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td></td>
		</tr>
		<tr>
			<td>Spinner flasks (25 mL)</td>
			<td>Wheaton</td>
			<td>356879</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe 1 mL</td>
			<td>VWR</td>
			<td>720-2561</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue culture flasks (25 cm2, 75 cm2, 150 cm2)</td>
			<td>TPP Techno Plastik Products AG</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue 0.4%</td>
			<td>Sigma-Aldrich</td>
			<td>T8154</td>
			<td></td>
		</tr>
		<tr>
			<td>VascuLife VEGF-Mv</td>
			<td>Lifeline cell technology</td>
			<td>LL-0005</td>
			<td></td>
		</tr>
	</tbody>
</table>

recombinant collagen i peptide microcarriers for cell expansion and their potential use as cell delivery system in a bioreactor model

Tissue engineering er et lovende felt, fokusert på å utvikle løsninger for den økende etterspørselen på vev og organer om transplantasjon formål. Prosessen for å generere slike vev er kompleks, og inkluderer en passende kombinasjon av spesifikke celletyper, stillaser og fysisk eller biokjemiske stimuli å veilede cellevekst og differensiering. Microcarriers representerer et tiltalende for å utvide celler i en tredimensjonal (3D) microenvironment, siden de gir høyere overflate-til volum prosenter og etterligne nærmere i vivo situasjonen sammenlignet med tradisjonelle todimensjonal metoder. Karsystemet, oksygen og næringsstoffer til cellene og sikre fjerning, utgjør en viktig byggesten når genererer konstruert vev. Faktisk mislykkes de fleste konstruksjoner etter blir implantert på grunn av manglende vaskulær støtte. Vi presenterer en protokoll for endothelial celle ekspansjon på rekombinant kollagen-baserte microcarriers under dynamiske forhold i spinner kolbe og bioreaktorer i denne studien, og vi forklare hvordan du finner i denne innstillingen celle levedyktighet og funksjonalitet. Dessuten, foreslår vi en metode for cellen levering for endometrial blodkar formål uten ytterligere avdeling trinnene som er nødvendig. Videre yter vi en strategi for å evaluere den cellen endometrial blodkar potensielle i en perfusjonsmåling bioreactor på et decellularized biologisk matrise. Vi mener at bruken av metodene presentert kan føre til utvikling av nye cellen-basert behandling for et stort utvalg av vev utvikling programmer i klinisk praksis.

Som vist i figur 1A, fikk vi mange levedyktige cellers på RCP microcarriers etter 7 dager av kultur, bestemmes av live/døde flekker. Disse resultatene ble bekreftet av SEM analyse, som helt koloniserte microcarriers ble observert rundt porene, delvis gjengroing dem (figur 1B). På den annen side, resulterte eksperimenter der celler ikke var jevnt frø i flere tomme microcarriers. Mislykkede eksperimentene er preget av et unorma...

Watch this Scientific Journal Video about Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model at JoVE.com

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Rekombinant kollagen I peptid Microcarriers for celleutvidelse og deres mulige bruk som cellen levering System i en Bioreactor modellen

Vi foreslår en celle ekspansjon protokoll på macroporous microcarriers og deres bruk som levering system i en perfusjonsmåling bioreactor å frø en decellularized vev matrise. Vi har også forskjellige teknikker for å finne celle spredning og levedyktigheten til celler kultivert på microcarriers. Videre viser vi funksjonaliteten til cellene etter bioreactor kulturer.

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

Research

JoVE Journal

Bioengineering

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

Roterende Cell Kultur Systems for Human Cell Culture: Human trophoblast Cells som en modell

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Constructing a Collagen Hydrogel for the Delivery of Stem Cell-loaded Chitosan Microspheres

Konstruere en Kollagen Hydrogel for levering av Stem Cell-lastet Chitosan Mikrokuler

Multipotent stem cells have been shown to be extremely useful in the field of regenerative medicine1-3. However, in order to use these cells effectively ...

Skin Tattooing As A Novel Approach For DNA Vaccine Delivery

Skin tatovering som en ny tilnærming for DNA vaksine levering

Nucleic acid-based vaccination is a topic of growing interest, especially plasmid DNA (pDNA) encoding immunologically important antigens. After the ...

Microfluidic Picoliter Bioreactor for Microbial Single-cell Analysis: Fabrication, System Setup, and Operation

Microfluidic pikoliter bioreaktor for Microbial Encellede Analyse: Fabrikasjon, System Setup, og drift

In this protocol the fabrication, experimental setup and basic operation of the recently introduced microfluidic picoliter bioreactor (PLBR) is described ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Cell klemme som en robust, Microfluidic Intracellulær Levering Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

An Improved Method for the Preparation of Type I Collagen From Skin

En forbedret fremgangsmåte for fremstillingen av type I kollagen fra hud

Soluble type 1 collagen (COL1) is used extensively as an adhesive substrate for cell cultures and as a cellular scaffold for regenerative applications. ...

Cultivation of Mammalian Cells Using a Single-use Pneumatic Bioreactor System

Dyrking av pattedyrceller med en enkelt-bruk Pneumatisk Bioreaktor System

Recent advances in mammalian, insect, and stem cell cultivation and scale-up have created tremendous opportunities for new therapeutics and personalized ...

Minced Tissue in Compressed Collagen: A Cell-containing Biotransplant for Single-staged Reconstructive Repair

Hakket Tissue i Compressed Kollagen: A Cell holdig Biotransplant for Single-iscenesatt rekonstruktiv Repair

Conventional techniques for cell expansion and transplantation of autologous cells for tissue engineering purposes can take place in specially equipped ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Fabrikasjon av ekstracellulære matrise-avledet Skum og Mikrocarriers som Tissue-spesifikke cellekultur og Delivery plattformer

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...