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

The research leading to these results has received funding from the European Union Seventh Framework Programme FP7/2007-2013 under grant agreement n&#176; 607051 (BIO-INSPIRE). We thank Carolien van Spreuwel-Goossens from Fujifilm Manufacturing Europe B.V., for the technical assistance during RCP manufacturing, and Werner Stracke from Fraunhofer Institute for Silicate Research ISC, for assistance with the SEM analysis.

hBMSCs were isolated from the femur head of osteoarthritis patients undergoing femur head replacement surgery. The procedure was performed under the approval of the Local Ethics Committee of the University of Wuerzburg and informed consent of the patients. Primary microvascular endothelial cells were isolated from foreskin biopsies of juvenile donors. Their legal representative(s) provided full informed consent in writing. The study was approved by the local ethical board of the University of Wuerzburg (vote 182/10)....

<ol>
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One main goal of microcarrier is the expansion of cells while maintaining their differentiation in order to deliver cells to the place of need. The represented method introduce RCP microcarriers where cells were able to attach, proliferate, and colonize the microcarriers with high cell density. This was observed by live/dead staining, in which more than 90% of viable cells were detected while only few dead cells were obtained after 7 days of dynamic cultures. Likewise, the SEM images confirmed that the cells covered the ...

One general problem in tissue engineering applications is to yield a high cell mass with the correct differentiation phenotype at the location of need. The application of microcarriers to address this issue started in 1967 with increasing significance to date in fields such as orthopaedic tissue engineering for large-scale generation of skin, bone, cartilage, and tendons1. They allow the handling of adherent cultures in ways similar to that of suspension cultures2 by expanding cells on microscale three-dimensional (3D) substrates. Thereby cells experience a homogeneous nutrient supply and cell-matrix interactions that lead to better maintenance of in vivo3,4 differentiation which is often lost over time in 2D approaches5. A higher surface-to-volume ratio - eventually leading to higher cell yields6,7, higher gas and nutrient exchange rates comparing to static systems8, the possibility to regulate and subject the culture to physical stimuli9, and the potential for scaling up of the expansion process7 are further advantages. Several features such as diameter, density, porosity, surface charge, and adhesion properties10,11 distinguish the different commercially available micro- and macro-carriers. However, one of the main advantage is their delivery potential as microtissues to site defect or demand.
For applications of the microcarrier technology in bone tissue engineering, we illustrated in a previous report12 the production of a new microcarrier type constituted of a recombinant collagen I peptide (RCP, commercially available as Cellnest). This new microcarrier allows the GMP-compliant up scaling of scaffold and cell production, as needed for cell delivery in a clinical scenario. In this context, tuning of scaffold stability, degradation rate, and surface properties through proper choice of a suited crosslinking strategy allows to adapt the technique to the selected application, cell type of interest or target tissue13. In particular, the potential employment of this microcarrier as an injectable cell delivery system for therapeutic application14 makes them particularly interesting in a clinical setting.
In this paper, we therefore illustrate the culturing procedure for the isolation and expansion of human bone marrow-derived mesenchymal stromal cells (hBMSCs) and human dermal microvascular endothelial cells (HDMECs) on collagen-I-based recombinant peptide-based microcarriers, and their preparation for delivery in a clinical setting. Furthermore, we describe additional protocols useful for the maintenance of cell viability upon implantation.
Cell viability after implantation is in fact strongly dependent on vascularization15,16,17, which ensures exchange of oxygen and nutrients and facilitates waste removal. Bioreactors constitute one approach to overcome vascularization challenges in tissue engineering and maintain cell viability, through perfusion of culture medium providing thereby oxygen and nutrients18. Here, we illustrate an in vitro method to evaluate the migration capability of microvascular endothelial cells from the RCP microcarriers to a biomatrix and their ability to contribute to the de novo vascularization and angiogenesis. This biomatrix is a decellularized segment of porcine jejunum termed BioVaSc (Biological Vascularized Scaffold), rich in collagen and elastin and with preserved vascular structures, which includes a feeding artery and a draining vein19 that has been applied for implantation issues20.

<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 is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

As shown in Figure 1A, we obtained high number of viable cells on the RCP microcarriers after 7 days of culture, determined by live/dead staining. Those results were confirmed by SEM analysis, in which completely colonized microcarriers were observed around the pores, partly overgrowing them (Figure 1B). On the other hand, experiments in which cells were not evenly seeded resulted in several empty microcarriers. Failed experiment...

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

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

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

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