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

Het onderzoek leidt tot deze resultaten is financiering van de Europese Unie zevende kader programma KP7/2007-2013 onder grant overeenkomst n ° 607051 (BIO-INSPIRE) ontvangen. Wij danken Carolien van Spreuwel-Goossens van Fujifilm Manufacturing Europe B.V., voor de technische bijstand tijdens RCP productie en Werner Stracke van Fraunhofer Instituut voor silicaat onderzoek ISC, voor hulp bij de SEM-analyse.

hBMSCs werden geïsoleerd van het dijbeen hoofd van artrose patiënten een dijbeen hoofd vervanging operatie ondergaan. De procedure werd uitgevoerd onder de goedkeuring van de lokale ethische commissie van de Universiteit van Würzburg en geïnformeerde toestemming van de patiënt. Primaire microvasculaire endotheliale cellen werden geïsoleerd van de voorhuid Biopten van jonge donoren. Hun wettelijke vertegenwoordiger (s) verstrekte volledig geïnformeerde toestemming schriftelijk. De studie werd goedgekeurd door de lo...

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H., Porter, B. D., Garc&#237;a, A. J., Guldberg, R. 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Vis. Exp. (78), e50460 (2013).</li><li>Groeber, F., Kahlig, A., Loff, S., Walles, H., Hansmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bioreactor+system+for+interfacial+culture+and+physiological+perfusion+of+vascularized+tissue+equivalents.">A bioreactor system for interfacial culture and physiological perfusion of vascularized tissue equivalents.</a> Biotechnology Journal. 8, 308-316 (2013).</li><li>Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Walter, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Blood+vessels+and+endothelial+cells.">Blood vessels and endothelial cells.</a> Molecular Biology of the Cells. , (2002).</li><li>Logsdon, E. A., Finley, S. D., Popel, A. S., Gabhann, F. 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Een hoofddoel van microcarrier is de uitbreiding van cellen met behoud van hun differentiatie zodat u cellen naar de plaats van noodzaak. De vertegenwoordigde methode introduceren RCP microcarriers waar cellen konden toevoegen, vermenigvuldigen en koloniseren de microcarriers met hoge celdichtheid. Dit werd waargenomen door live/dead kleuring, waarin meer dan 90% van levensvatbare cellen werden ontdekt, terwijl slechts een paar dode cellen werden verkregen na 7 dagen van dynamische culturen. Ook bevestigd de beelden van ...

Een algemeen probleem in weefsel waterbouwkundige toepassingen is moeten opleveren van de massa van een hoge cel met het fenotype van de juiste differentiatie op de locatie van. De toepassing van microcarriers aan te pakken dit probleem begon in 1967 met toenemende betekenis toe op gebieden zoals orthopedische weefselengineering voor grootschalige generatie van huid, botten, kraakbeen en pezen1. Zij toestaan dat de behandeling van aanhangend culturen op manieren gelijkend op dat van schorsing culturen2 door cellen op microscale driedimensionale (3D) substraten uit te vouwen. Daardoor ervaren cellen een homogene voedingsstoffen levering en cel-matrix interacties dat leiden tot beter onderhoud van in vivo3,4 differentiatie, die vaak verloren na verloop van tijd in 2D5 benaderingen. Een hogere oppervlak-te-volumeverhouding - uiteindelijk zal leiden tot hogere cel opbrengsten6,7, hogere gas en nutriënten wisselkoersen vergelijken met statische systemen8, de mogelijkheid om te reguleren en de cultuur onderworpen aan fysieke prikkels9, en de mogelijkheden voor schaalvergroting van de uitbreiding proces7 zijn verdere voordelen. Verschillende functies zoals diameter, dichtheid, porositeit, oppervlakte lading en hechting eigenschappen10,11 onderscheid maken tussen de verschillende commercieel beschikbare micro - en macro-dragers. Één van het belangrijkste voordeel is echter hun potentieel als microtissues levering aan site defect of vraag.
Voor toepassingen van de technologie van de microcarrier in bot weefselengineering, we geïllustreerd in een vorige verslag12 de productie van een nieuwe microcarrier type bestaat uit een recombinant collageen ik peptide (RCP, commercieel beschikbaar als Cellnest). Deze nieuwe microcarrier kunt de GMP-conforme een schaal voor de productie van de steiger en cel, als nodig is voor de levering van de cel in een klinische scenario. In dit verband kunt afstemming van de stabiliteit van de steiger, aantasting van het tarief en oppervlakte-eigenschappen door middel van de juiste keuze van een geschikt crosslinking strategie passen de techniek om de geselecteerde toepassing, cell type van belang of target weefsel13. In het bijzonder maakt de potentiële tewerkstelling van deze microcarrier als een injecteerbare cel expresbezorgingssysteem voor therapeutische toepassing14 ze bijzonder interessant in een klinische setting.
In deze paper illustreren we daarom de kweken procedure voor de isolatie en de uitbreiding van de menselijke beenmerg-afgeleide mesenchymale stromale cellen (hBMSCs) en menselijke dermale microvasculaire endotheliale cellen (HDMECs) op collageen ik-gebaseerde recombinant Peptide gebaseerde microcarriers, en hun bereiding voor levering in een klinische setting. Daarnaast beschrijven we aanvullende protocollen nuttig voor het onderhoud van de levensvatbaarheid van de cellen op innesteling.
Levensvatbaarheid van de cellen na implantatie is in feite sterk afhankelijk van vascularisatie15,16,17, die zorgt voor een uitwisseling van zuurstof en voedingsstoffen en afvalstoffen verwijdering vergemakkelijkt. Bioreactoren vormen één aanpak vascularisatie uitdagingen in de weefselkweek en handhaven van de levensvatbaarheid van de cellen, via perfusie van kweekmedium verstrekken waardoor zuurstof en voedingsstoffen18. We illustreren hier, een in vitro -methode om te evalueren van het vermogen van de migratie van microvasculaire endotheliale cellen van de RCP microcarriers een biomatrix en hun vermogen om bij te dragen aan de DOVO vascularisatie en angiogenese. Deze biomatrix is een decellularized segment van varkens jejunum genoemd BioVaSc (biologische gevacuoliseerd steiger), rijk aan collageen en elastine en bewaard met vasculaire structuren, waaronder een voeding slagader en een zuig ader19 dat is geweest implantatie kwesties20aangevraagd.

<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

Weefseltechnologie is een veelbelovend gebied, gericht op het ontwikkelen van oplossingen voor de toenemende vraag op de weefsels en organen voor transplantatiedoeleinden. Het proces voor het genereren van dergelijke weefsels is complex en omvat een passende combinatie van specifieke celtypes, steigers en fysieke of biochemische prikkels om te leiden van de celgroei en differentiatie. Microcarriers vormen een aantrekkelijk instrument uit te breiden van cellen in een driedimensionale (3D) communicatie, omdat ze hogere oppervlak-volume ratio's geven en de in vivo situatie vergeleken met traditionele tweedimensionale methoden nauwer wordt nagebootst. Het vaatstelsel, de levering van zuurstof en voedingsstoffen naar de cellen en het waarborgen van de verwijdering van het afval, vormt een belangrijke bouwsteen wanneer weefsels genereren ontworpen. In feite, mislukken de meeste constructies na wordt geïmplanteerd te wijten aan het ontbreken van vasculaire ondersteuning. In deze studie, presenteren we een protocol voor endothelial cel expansie op recombinante collageen gebaseerde microcarriers onder dynamische omstandigheden in spinner kolf en bioreactoren en leggen we uit hoe u kunt bepalen in dit de levensvatbaarheid van de cellen van de instelling en functionaliteit. Daarnaast stellen wij een methode voor de levering van de cel voor vascularisatie doeleinden zonder extra detachement stappen die nodig zijn. Bovendien bieden wij een strategie om te evalueren van de cel vascularisatie potentiële in een bioreactor perfusie op een decellularized biologische matrix. Wij zijn van mening dat het gebruik van de gepresenteerde methoden kan leiden tot de ontwikkeling van nieuwe cel-gebaseerde therapieën voor een groot scala aan weefsel technische toepassingen in de klinische praktijk.

Zoals blijkt uit figuur 1A, verkregen we hoge aantal levensvatbare cellen op de RCP microcarriers na 7 dagen van cultuur, bepaald door het leven/dood kleuring. Deze resultaten werden bevestigd door SEM analyse, waarbij volledig gekoloniseerde microcarriers werden waargenomen rond de poriën, deels overgrowing hen (figuur 1B). Aan de andere kant, resulteerde experimenten waarin cellen waren niet gelijkmatig ontpit in verschillende...

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

Recombinante collageen ik Peptide Microcarriers voor cel expansie en hun mogelijke toepassing als cel levering systeem in een Bioreactor Model

Wij stellen voor een cel expansie protocol betreffende macroporeuze microcarriers en hun gebruik als levering systeem in een bioreactor perfusie om het zonebeheer van een matrix decellularized weefsel. Wij omvatten ook verschillende technieken om te bepalen van celproliferatie en levensvatbaarheid van cellen gekweekt op microcarriers. Bovendien tonen we functionaliteit van cellen na bioreactor culturen.

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

The overall goal of this procedure is to expand the cells on a high available surface area and to further deliver these cells to sites of defect or demand for clinical applications. This method can help answer key questions in the tissue engineering field, such as cell expansion, cell delivery, and vascularization. The main advantage of this technique is that we can expand the cells and deliver them, without any harvesting step, into defect sites, in a biocompatible way.Visual demonstration of this method is critical, as working microcarriers and bioreactors can be complicated, at first, due to the many steps involved. Helping with the procedure will be Jihyoung Choi, a PhD student from our laboratory. One day before the experiments, set up the spinner flasks and prepare them for sterilization by autoclave.Wrap the spinner flasks with aluminum foil around the side caps separately to avoid contamination during unwrapping. Wrap the flasks with one to two layers of aluminum foil for autoclaving to minimize contamination risks after sterilization. Leave the caps of the spinner flasks loose and when possible, sterilize them in an upright position.Next, weigh the required amount of RCP macroporous microcarriers in 20 milliliters of Dulbecco's phosphate buffered saline. Let them hydrate for at least one hour before heat sterilization by autoclave. Use the bioreactor system referenced in text protocol to produce vascularized tissue equivalents.This bioreactor consists of a vessel in which the biomatrix is placed, a medium reservoir, and a pressure bottle. Connect the vessel with the medium reservoir and the pressure bottle through silicone tubes. Leave the caps loose and place the system in an autoclave plastic bag.Close the bag well and place this in a second autoclave plastic bag before sterilizing by autoclave. Let the sterilized RCP macroporous microcarriers settle to the bottom and wash them with 10 milliliters of culture medium. Repeat this process three times.Also wash the spinner flasks with 10 milliliters of culture medium. Next, add 30 milligrams of RCP macroporous microcarriers per spinner flask in eight milliliters of culture medium. Equilibrate the spinner flasks RCP macroporous microcarriers at 37 degrees Celsius and 5%carbon dioxide for 30 minutes before seeding the cells.Then, seed half a million human bone marrow derived mesenchymal stromal cells, or human dermal microvascular endothelial cells per spinner flask in two milliliters of culture medium. Place the spinner flasks on a stirrer plate at 37 degrees Celsius and 5%carbon dioxide and start stirring at 90rpm for five minutes. Then, rest for 55 minutes for a total of one hour static dynamic incubation.Repeat this incubation cycle four times. Now, add 10 milliliters of cell culture medium per spinner flask. Then start continuous stirring at 95rpm while incubating at 37 degrees Celsius and 5%carbon dioxide.One day before starting the bioreactor cultures, cut open the biomatrix longitudinally on the antimesenteric side and fix it in a previously disinfected polycarbonate frame. After detaching and counting the cells, use a two milliliter syringe to inject 10 million HDMECs in one thousand microliters of culture medium through the preserved vasculature of the biomatrix. Inject approximately 700 microliters through the arterial inlet, and approximately 300 microliters through the vein outlet.Incubate the biomatrix with injected cells at 37 degrees Celsius and 5%carbon dioxide for three hours to allow cell attachment. Following incubation, the connection of the biomatrix to the bioreactor has to be done by two individuals, taking care that the arterial and the venous pedicles are connected to the medium flow correctly. Fill the bioreactor system with 350 milliliters of the vascular endothelial growth factor, 1%penicillin streptomycin, and incubate at 37 degrees Celsius and 5%carbon dioxide.Place the frame inside the vessel of the bioreactor. Then, carefully connect the arterial and venous pedicles to the vessel. Start perfusion by connecting the bioreactor system to a peristaltic pump.Set the pressure at 10 millimeters of mercury amplitude plus minus one. Increase the pressure stepwise every hour until reaching a pressure of 100 millimeters of mercury amplitude plus minus 20. Mix 330 microliters of 0.4%Collagen R solution, 170 microliters of 0.1%acetic acid, and 50 microliters of medium M199 in a 15 milliliter Falcon tube.Keep the mixture on ice. Then, take the RCP microcarriers from the spinner flasks. Remove the culture medium completely.Next, add the volume of sodium hydroxide required to neutralize the collagen mixture. Once neutralized, immediately mix the mixture with the RCP microcarriers. It is important to allow gelation of the collagen gel before starting medium flow.Otherwise, the gel will detach and the microcarriers will be washed away from the lumen of the biomatrix. Add the collagen gel RCP microcarrier mixture on the lumen of the biomatrix. Allow gelation for 30 minutes.Then, connect the bioreactor to the peristaltic pump and set up the pressure at 100 millimeters of mercury amplitude plus minus 20. Finally, perform analysis and read outs of bioreactor cultures as described in the text protocol. Representative results of cell proliferation on microcarriers is shown by live dead staining in which many living cells can be observed on the microcarriers after seven days of dynamic cultures.These results were confirmed by scanning electron microscopy analysis showing completely colonized microcarriers after seven days of dynamic cultures. HDMECs kept their functionality after seven days of dynamic cultures on RCP microcarriers. Here, bright field microscopy reveals sprouts growing from the colonized microcarriers when cultured on a collagen gel.RFP labeled HDMECs were observed in the vascular structures of the biomatrix after 21 days of bioreactor cultures, indicating their migration from the RCP microcarriers to the biomatrix. Furthermore, after 21 days of bioreactor cultures, the scanning electron microscopy image shows that the microcarriers were in close proximity to the biomatrix. Interestingly, they are not colonized by cells anymore.After watching this video, you should have a good understanding on how to expand the cells on microcarriers to prepare them in an injectable cell delivery system for therapeutic application. Once mastered, this technique can be done in four hours. This procedure represents a promising strategy in regenerative medicine to obtain and maximize cell expansion and to deliver cells to implantation sites where they could improve vascularization for tissue engineered grafts.The implications of this technique extend toward vascularization because it can be used on any cell type for a tissue specific application in a clinical scenario. This method can also provide understanding into the potential employment of microcarriers as an injectible cell delivery system for therapeutic purposes. Don't forget that working with primary cells can be extremely hazardous, and precautions such as wearing gloves and lab coat, and working in a biosafety cabinet should always be taken when performing this procedure.After its development, this technique paved the way for researchers in the field of tissue engineering to explore cell migration in vitro which could help to understand or predict in vivo situations.

Research

JoVE Journal

Bioengineering

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

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

Roterende celkweek systemen for Human Cultuur van de Cel: Human trofoblast cellen als een 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

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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

Huid tatoeëren als een nieuwe benadering voor DNA-vaccin Levering

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

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Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

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Analysis of Cell Migration within a Three-dimensional Collagen Matrix

Analyse van cel-migratie binnen Driedimensionale Collagen Matrix

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

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

Gehakt Tissue in Compressed Collageen: A-cel met Biotransplant voor Single-geënsceneerde Reconstructieve Repair

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

Fabricage van extracellulaire matrix-afgeleide schuimen en microdragers Weefsel-specifieke Cell Cultuur en Delivery Platforms

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