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

Forskning fører til disse resultater har modtaget støtte fra den Europæiske Union syvende ramme program FP7/2007-2013 under grant aftale n ° 607051 (BIO-INSPIRERE). Vi takker Carolien van Spreuwel-Goossens fra Fujifilm fremstilling Europe B.V., for den tekniske bistand under RCP produktion og Werner Stracke fra Fraunhofer-instituttet for silikat forskning ISC, for assistance med SEM analyse.

hBMSCs blev isoleret fra lårbenet hovedet af slidgigt patienter i lårbenet hovedet udskiftning kirurgi. Proceduren blev udført under godkendelse af de lokale etiske udvalg af Universitet Wuerzburg og informeret samtykke fra patienterne. Primære mikrovaskulære endothelial celler blev isoleret fra forhuden biopsier af juvenile donorer. Deres juridiske repræsentant(er) leveres fuldt informeret samtykke skriftligt. Undersøgelsen blev godkendt af de lokale etiske nævn Universitet Wuerzburg (afstemning 182/10).
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Én Hovedformålet med microcarrier er en udvidelse af celler samtidig bevare deres differentiering for at levere celler til stedet, hvor behovet. Metoden repræsenterede indføre RCP microcarriers hvor celler kunne vedhæfte, formere og kolonisere microcarriers med høj celle tæthed. Dette blev observeret af live/døde pletter, hvor mere end 90% af levedygtige celler blev opdaget mens kun få døde celler blev opnået efter 7 dage af dynamiske kulturer. SEM billeder bekræftede ligeledes, at cellerne dækket hele overf...

Et generelt problem i væv ingeniørmæssige anvendelser er at give en høj cellemasse med den korrekte differentiering fænotype ved placering har brug for. Anvendelsen af microcarriers at løse problemet startede i 1967 med stigende betydning for dato på områder som ortopædisk vævsmanipulering for storstilet generation af hud, knogle, brusk og sener1. De tillader håndtering af vedhængende kulturer på måder ligner suspension kulturer2 ved at udvide celler på individuel tredimensionale (3D) substrater. Dermed opleve celler en homogen næringsstofforsyning og celle-matrix interaktioner at føre til bedre vedligeholdelse af i vivo3,4 differentiering som er ofte tabt over tid i 2D nærmer sig5. Et højere overflade-til-volumen forhold - i sidste ende fører til højere celle udbytter6,7, højere gas og næringsstof valutakurser sammenligne med statisk systemer8, mulighed for at regulere og kultur på fysisk stimuli9, og potentialet for optrapning af ekspansion proces7 er yderligere fordele. Flere funktioner såsom diameter, tæthed, porøsitet, overflade afgift og vedhæftning egenskaber10,11 skelne de forskellige kommercielt tilgængelige mikro - og makro-luftfartsselskaber. Men en af de største fordel er deres potentiale som microtissues levering til webstedet defekt eller efterspørgsel.
For applikationer af microcarrier teknologien i knoglen vævsmanipulering, vi illustreret i en tidligere betænkning12 produktion af en ny microcarrier type udgjorde af en rekombinant kollagen jeg peptid (RCP, kommercielt tilgængelige som Cellnest). Denne nye microcarrier tillader GMP-kompatibel op skalering af stillads og celle produktion, som er nødvendig for celle levering i et klinisk scenario. I denne forbindelse tillader tuning af stillads stabilitet, nedbrydning sats og overflade egenskaber gennem korrekt valg af et egnet crosslinking strategi hen til tilpasse teknikken til det valgte program, celle type af interesse eller målrette væv13. Navnlig gør den mulige beskæftigelse af denne microcarrier som en injicerbar celle levering system til terapeutisk anvendelse14 dem særligt interessant i en klinisk indstilling.
I dette papir illustrere vi derfor dyrkningsbaserede proceduren for isolation og udvidelse af menneskelige knoglemarv-afledte mesenchymale stromale celler (hBMSCs) og human dermal mikrovaskulære endothelial celler (HDMECs) på kollagen-jeg-baserede rekombinant peptid-baseret microcarriers, og deres forberedelse til levering i kliniske omgivelser. Derudover beskriver vi tillægsprotokoller nyttig til vedligeholdelse af cellernes levedygtighed efter implantation.
Cellernes levedygtighed efter implantation er faktisk stærkt afhængige af vascularization15,16,17, som sikrer udveksling af ilt og næringsstoffer og letter bortskaffelse. Bioreaktorer udgør en strategi for at overvinde vascularization udfordringer i vævsmanipulering og vedligeholde celle levedygtighed, gennem perfusion af næringssubstratet giver dermed ilt og næringsstoffer18. Her viser vi en in vitro- metode til at evaluere migration evne af mikrovaskulære endotelceller fra RCP-microcarriers til en biomatrix og deres evne til at bidrage til de novo vascularization og angiogenese. Denne biomatrix er et decellularized segment af svin jejunum kaldes BioVaSc (biologisk vaskulariserede stillads), rig på kollagen og elastin og med bevaret vaskulære strukturer, som omfatter en fodring arterie og en dræning vene19 , der har været anvendes til implantation spørgsmål20.

<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

Vævsmanipulering er et lovende felt, fokuseret på at udvikle løsninger til den stigende efterspørgsel på væv og organer til transplantation formål. Processen med at skabe sådanne væv er kompleks, og indeholder en passende kombination af specifikke celletyper, stilladser og fysisk eller biokemiske stimuli guide cellevækst og differentiering. Microcarriers repræsenterer en tiltalende redskab til at udvide celler i en tre-dimensionelle (3D) mikromiljø, da de giver større overflade-til volumen nøgletal og efterligne tættere i vivo situation i forhold til traditionelle to-dimensionelle metoder. Det vaskulære system, levering af ilt og næringsstoffer til cellerne og at sikre bortskaffelse, udgør en vigtig byggesten når generering fremstillet ud fra manipuleret væv. I virkeligheden, mislykkes de fleste konstruktioner efter at blive implanteret på grund af mangler vaskulære støtte. I denne undersøgelse, præsenterer vi en protokol i endothelial celle ekspansion på rekombinant kollagen-baseret microcarriers under dynamiske forhold i spinner kolbe og bioreaktorer og vi forklare, hvordan at bestemme i denne indstilling cellernes levedygtighed og funktionalitet. Derudover foreslår vi en metode til celle levering til vascularization formål uden yderligere detachement skridt nødvendigt. Derudover giver vi en strategi for at evaluere celle vascularization potentielle i en perfusion bioreaktor på en decellularized biologiske matrix. Vi mener, at brugen af de præsenterede metoder kan føre til udvikling af nye celle-baserede terapier for en lang række tissue engineering applikationer i den kliniske praksis.

Som vist i figur 1A, opnåede vi høje antallet af levedygtige celler på RCP microcarriers efter 7 dage af kultur, bestemmes af live/døde farvning. Disse resultater er blevet bekræftet af SEM analyse, hvor helt koloniseret microcarriers blev observeret i nærheden af porer, dels tilgroning dem (figur 1B). På den anden side resulterede eksperimenter hvor celler ikke var jævnt seedede i flere tomme microcarriers. Mislykkede fo...

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 celle ekspansion og deres potentielle anvendelse som celle levering System i en bioreaktor Model

Vi foreslår en celle ekspansion protokol om kombineret makroporøs microcarriers og deres anvendelse som levering system i en perfusion bioreaktor til frø et decellularized væv matrix. Vi også omfatter forskellige teknikker til at bestemme celleproliferation og levedygtighed af celler dyrkes på microcarriers. Derudover viser vi funktionalitet af celler efter bioreaktor 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 Culture Systems for Human Cell Culture: Human trofoblasten celler som en model

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

Konstruktion af et Collagen hydrogel til levering af stamcelle-loaded chitosanmikrosfærer

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

Hud tatovering som en ny tilgang til DNA Vaccine Delivery

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

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, Mikrofluid Intracellulær Delivery 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 metode til forberedelse af 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

Dyrkning af pattedvrceller hjælp af en enkelt brug Pneumatic bioreaktorsystem

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 Væv i Komprimeret Collagen: A Cell-holdige Biotransplant for Single-iscenesat rekonstruktionskirurgi 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

Fabrikation af ekstracellulære matrix-afledte Skum og Mikrobærere som Tissue-specifikke cellekultur og Delivery Platforms

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