The authors would like to acknowledge QuinXell Technologies and CellD, particularly Lothar Grannemann and Dominique Ghozlan for their help with the bioreactor. We thank Catherine Le Visage, who provided us with the pullulan/dextran polysaccharide scaffolds. This work was supported by the European Union (ERC-2014-CoG project MaTissE 648779) and by the AgenceNationalede la Recherche (ANR), France (MagStem project ANR-11 SVSE5).

1. Construction of the Magnetic Devices
NOTE: The devices used for cell seeding vary depending on the application (Figure 1). To form aggregates, the number of cells is limited to 2.5&#215;105/aggregate, so the magnetic tips must be very thin (750 &#181;m in diameter). To seed the 1.8 cm2/7 mm-thick scaffolds, the magnets must be larger (3 mm in diameter) and will ensure cell migration through the pores of the scaffold.
<ol>
	<li>Construction of a device with micro-magnets for aggregate formation (Figure 1A)
	<ol>
		<li>Make micro-holes with a 0.8-mm drill through aluminum plates (3 cm in diameter and 6 mm thick).</li>
		<li>Insert a magnetic tip (750 &#181;m in diameter) into each hole of the plate.</li>
		<li>Place this disk over a permanent neodymium magnet, which ensures magnetization to saturation.</li>
	</ol>
	</li>
	<li>Construction of a device for scaffold seeding (Figure 1B)
	<ol>
		<li>Cut hard polystyrene into 2.4 cm2 squares.</li>
		<li>Insert 9 small magnets (3 mm in diameter, 6 mm long) at an equal distance over a surface area of 1.6 cm2.</li>
		<li>Place this device over a permanent neodymium magnet.</li>
	</ol>
	</li>
</ol>
2. Stem Cell Labeling
NOTE: Stem cells were labeled with 0.1 mM magnetic nanoparticles for 30 min (2.6 &#177; 0.2 pg iron/cell) to form aggregates, while they were labeled with 0.2 mM magnetic nanoparticles for 30 min (5 &#177; 0.4 pg iron/cell) to seed scaffolds. These nanoparticle concentrations and incubation times have been used previously and published for MSC and other cells18,19, and it has been determined that nanoparticles impacted neither cell viability nor MSC differentiation capacity. The iron mass incorporated by the stem cells was measured via single-cell magnetophoresis19,20.
<ol>
	<li>Culture human mesenchymal stem cells (MSC) in complete mesenchymal stem cell growth medium (MSCGM) at 37 &#176;C and 5% CO2 until near to confluence (~90%).</li>
	<li>Prepare the magnetic labeling solution by mixing 0.1 or 0.2 mM maghemite citrate-coated iron oxide (&#947;Fe2O3; core: 8 nm diameter) in serum-free McCoy&#39;s 5A medium modified at Roswell Park Memorial Institute (RMPI) without glutamine and containing 5 mM sodium citrate.</li>
	<li>Discard the medium, rinse the cells with serum-free RPMI medium without glutamine, and add 10 mL of iron oxide nanoparticle solution per 150-cm2 culture flask, the minimum volume required to cover all the cells.</li>
	<li>Incubate for 30 min at 37 &#176;C and 5% CO2 and then discard the nanoparticle solution. Rinse for 5 min with serum-free RPMI medium without glutamine to internalize the nanoparticles still attached to the plasma membrane.</li>
	<li>Discard the RPMI medium and add 25 mL of complete MSCGM medium per flask. Incubate overnight at 37 &#176;C and 5% CO2.</li>
</ol>
3. Magnetic Cell Seeding
<ol>
	<li>Freshly prepare the chondrogenic medium using Dulbecco&#39;s Modified Eagle Medium (DMEM) high glucose with L-glutamine by adding 50 &#181;M L-ascorbic acid 2-phosphate, 0.1 &#181;M dexamethasone, 1 mM sodium pyruvate, 0.35 mM L-proline, 1% universal culture supplement containing insulin, human transferrin and selenous acid (ITS-Premix), and 10 ng/mL transforming growth factor-beta 3 (TGF-&#946;3).</li>
	<li>Detach the magnetic cells using 8 mL of 0.05% trypsin-EDTA per 150-cm2 culture flask and centrifuge the dissociated cells at 260 &#215; g for 5 min. Aspirate the medium and count the re-suspended cells.</li>
	<li>Place a glass-bottomed cell culture Petri dish (35 mm) on top of both magnetic devices.</li>
	<li>To magnetically form aggregates, add 3 mL of chondrogenic medium to the Petri dish and gently deposit the smallest volume possible (no more than 8 &#181;L) containing 2.5&#215;105 labeled cells per aggregate (up to 16 aggregates can be deposited). Leave the Petri dish for 20-30 min without moving it, allowing it to form spheroids, and then place the complete device, including the Petri dish containing the 16 aggregates, into the incubator at 37 &#176;C and 5% CO2.</li>
	<li>Form control aggregates following the same protocol and replace the complete medium with chondrogenic medium without TGF-&#946;3.
	<ol>
		<li>To generate the 3D aggregate construct, place 2 aggregates in contact on day 8 to form 8 doublets and initiate the fusion. On day 11, merge 2 doublets to form 4 quadruplets. Finally, fuse the 4 quadruplets on day 15 to obtain the final structure.</li>
		<li>At the same time, form aggregates by centrifuging 2.5&#215;105 labeled stem cells at 260 &#215; g for 5 min in 15-mL tubes with 1.5 mL of chondrogenic medium with or without TGF-&#946;3 (for sample and control, respectively).</li>
	</ol>
	</li>
	<li>To magnetically seed scaffolds, place each dried scaffold into a Petri dish. Use polysaccharide porous scaffolds made of pullulan/dextran21. For each scaffold, dilute 2&#215;106 labeled stem cells in 350 &#181;L of chondrogenic medium without TGF-&#946;3 and carefully pipette the cells onto the scaffold.
	<ol>
		<li>Incubate for 5 min at 37 &#176;C to allow for full cell penetration within the scaffold and then gently add 3 mL of chondrogenic medium with or without TGF-&#946;3 (for sample or control, respectively) to the Petri dish.</li>
		<li>Incubate the cellularized scaffold on its magnetic device at 37 &#176;C and 5% CO2 for 4 days to allow for cell migration through the scaffold pores and confinement.</li>
	</ol>
	</li>
	<li>At the same time, seed scaffolds with 2&#215;106 labeled stem cells following the same method and incubate without the magnet to obtain uniformly seeded scaffolds as positive controls.</li>
</ol>
4. Differentiation into Chondrocytes
NOTE: After 4 days of incubation, remove the magnets and continue the chondrogenic maturation either in a Petri dish (static conditions) or in a bioreactor (dynamic conditions). Negative control samples are matured in static conditions with chondrogenic medium without TGF-&#946;3.
<ol>
	<li>In static conditions, keep the cellularized scaffolds or aggregates in the same Petri dish. Change the chondrogenic medium twice a week for 21 days.</li>
	<li>In dynamic conditions, prepare the bioreactor.
	<ol>
		<li>Cut silicone tubing at the appropriate length according to the manufacturer&#39;s protocol.</li>
		<li>Autoclave all materials: 500-mL culture chamber, tubing, 2-way rotators, and cages.</li>
		<li>Place the bioreactor parts into a sterile microbiological safety station. Connect the tubing to the 2-way rotators and to the culture chamber following the manufacturer&#39;s instructions.</li>
		<li>Carefully transfer the cellularized scaffolds into sterilized cages using a sterile spatula. Place 2 scaffolds per cage. When the cages are ready, insert them into the needles of the lid to keep them from moving during further rotation. Fill the culture chamber with chondrogenic medium and close it with the lid containing the cages.</li>
	</ol>
	</li>
	<li>Turn on the peristaltic pump to fill the tubing with chondrogenic medium and to eliminate air bubbles.</li>
	<li>Place and secure the filled chamber into the motor of the bioreactor and turn on the computer, which controls the rotations both of the arm and of the chamber.</li>
	<li>Apply a rotation speed of 5 rotations per min (rpm) on both the arm and the chamber. Adjust the peristaltic pump at a flow rate of 10 rpm for continuous feeding of the cellularized scaffolds.</li>
</ol>
5. RNA Extraction and Gene Expression Analysis
NOTE: Prior to RNA extraction, digest the scaffolds with an enzymatic solution.
<ol>
	<li>Prepare 1 mL of enzymatic solution by adding 100 &#181;L of pullulanase (40 U/mL) and 50 &#181;L of dextranase (60 mg/mL) to 850 mL of serum-free DMEM medium.</li>
	<li>Rinse the scaffolds twice with serum-free DMEM medium, discard the medium, and add 800 &#181;L of the enzymatic solution per scaffold. Incubate for 15-30 min at 37 &#176;C under gentle agitation.</li>
	<li>When the scaffold is completely dissolved, transfer the solution containing the cells to a 1.5-mL tube, centrifuge at 300 &#215; g for 10 min, carefully aspirate the medium, rinse twice with sterile 1&#215; phosphate-buffered saline (PBS), centrifuge at 300 &#215; g for 10 min, and re-suspend the cells in the RNA isolation solution.</li>
	<li>To extract the RNA from the aggregates, place the spheroids in the RNA isolation solution and completely crush them using a homogenizer before performing RNA extraction.</li>
	<li>Isolate the RNA using a kit for total RNA extraction according to the manufacturer&#39;s instructions.</li>
	<li>Synthesize complementary DNA from 400 ng of total RNA using reverse transcriptase according to the manufacturer&#39;s instructions, using 250-ng random primers, 1 &#181;L of dNTP mix (10 mM each), and 40 U/mL RNase inhibitor; the final volume of reaction is 20 &#181;L. At the end of the reaction, add 80 &#181;L of distilled water to obtain a final volume of 100 &#181;L.</li>
	<li>For quantitative polymerase chain reaction (PCR), use a PCR mix containing a fluorescent reagent to quantify the relative expression of the genes of interest, such as aggrecan (AGC) and collagen II (Col II), with 10&#215; diluted cDNA. Normalize the levels of gene expression with the reference gene Ribosomal Protein, Large, P0 (RPLP0). Perform the calculations with the 2-&#916;&#916;CT formula, where &#916;&#916;CT = &#916;CT of differentiated condition - mean &#916;CT of control condition, and each &#916;CT represents the CT of the gene of interest - the CT of the reference gene (RPLP0).</li>
	<li>Determine statistical measurements as the mean values &#177; the standard error of the mean (SEM). Perform the analysis with n &#8805; 2 independent experiments. Use Student&#39;s t-test to analyze the statistical differences between centrifuged pellets and magnetic fusions (* p &#60; 0.05). Determine the significance with a Kruskal-Wallis test (one-way ANOVA nonparametric) to analyze statistical differences between differentiated scaffolds and with the control scaffold (* p &#60; 0.05).</li>
</ol>
6. Histological Analysis
<ol>
	<li>Rinse the cellularized scaffolds or aggregates with sterile 1&#215; PBS, fix them in 10% formalin solution for 1 h at room temperature, and rinse with 1&#215; PBS.</li>
	<li>Remove the PBS, embed the samples in optimal cutting temperature compound (OCT), and freeze them in an isopentane bath immersed in liquid nitrogen. Store the sample at -20 &#176;C. Cut the samples with a cryostat to obtain 8-&#181;m cryosections for the aggregates or 12-&#181;m for the cellularized scaffolds.</li>
	<li>Stain the cryosections with 0.5% toluidine blue solution for 2 min, rinse in tap water, dehydrate with 100% ethanol, clarify using toluene, and mount the slides with a mounting medium for light microscopy.</li>
</ol>

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The authors have nothing to disclose.

First, because the techniques presented here rely on the internalization of magnetic nanoparticles, one important issue is the outcome of the nanoparticles once they localize within the cells. It is true that iron nanoparticles may trigger potential toxicity or impaired differentiation capacity depending on their size, coating, and time of exposure19,22. However, several studies have shown no impact on cellular physiology when encapsulated iron nanoparticles were...

Magnetic nanoparticles are already used in the clinic as contrast agents for magnetic resonance imaging (MRI), and their therapeutic applications keep expanding. For example, it has recently been shown that labeled cells can be manipulated in vivo using an external magnetic field and can be directed and/or maintained at a defined site of implantation1,2,3. In regenerative medicine, they can be used to engineer organized tissues in vitro4, including vascular tissue5,6,7, bone8, and cartilage9.
Articular cartilage is immersed in an avascular environment, making repairs of the extracellular matrix components very limited when damages occur. For this reason, research is currently focused on the engineering of hyaline cartilage replacements that can be implanted at the defect site. In order to produce an autologous replacement, some research groups are exploring the use of autologous chondrocytes as a cell source10,11, while others emphasize the capacity of mesenchymal stem cells (MSC) to differentiate into chondrocytes12,13. In previous studies recapitulated here, we selected MSC, as their bone marrow sampling is fairly simple and does not require the sacrifice of healthy chondrocytes, which risk losing their phenotype14.
An early step essential to initiating the chondrogenic differentiation of stem cells is their condensation. Cell aggregates are commonly formed using either centrifugation or micromass culture15; however, these condensation methods neither present the potential to create cell clusters within thick scaffolds nor the potential to control the fusion of aggregates. In this paper, we describe an innovative approach to condensing stem cells using MSC magnetic labeling and magnetic attraction. This technique has been proven to form scaffold-free 3D constructs via the fusion of aggregates with one another to obtain a millimeter-scale cartilaginous tissue9. Magnetic seeding of thick and large scaffolds has also allowed the possibility of increasing the size of the engineered tissue, designing a shape more readily useful for implantation, and diversifying the potential for clinical applications in cartilage repair. Here, we detail the protocol for the magnetic seeding of MSC into porous scaffolds composed of natural polysaccharides, pullulan, and dextran, scaffolds previously used to confine stem cells16,17. Chondrogenic differentiation was finally performed in a bioreactor to ensure continuous nutrient and gas diffusion into the matrix core of the scaffolds seeded with a high density of cells. Besides providing nutrients, chondrogenic growth factors, and gas to the cells, the bioreactor offered mechanical stimulation. Overall, the magnetic technology used to confine stem cells, combined with dynamic maturation in a bioreactor, can markedly improve chondrogenic differentiation.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Iron&#160; oxide (maghemite) nanoparticules ( &#947;-Fe2O3)</td>
			<td>PHENIX - University Paris 6</td>
			<td>Made and given by C. M&#233;nager</td>
			<td>Mean diameter of 8.1 nm and negative surface charge</td>
		</tr>
		<tr>
			<td>Polysaccharide Pullulan/Dextran scaffolds</td>
			<td>LIOAD - University Nantes</td>
			<td>Made and given by C. Le Visage</td>
			<td>Prepared from a 75:25 mixture of pullulan/dextran in alkaline conditions (10M NaOH). Porosity: 185-205&#181;m; Thickness: 7mm; Surface area: 1.8cm2.</td>
		</tr>
		<tr>
			<td>TisXell Regeneration System</td>
			<td>QuinXell Technologies</td>
			<td>QX900-002</td>
			<td>Biaxial bioreactor with 500 mL culture chamber&#160;</td>
		</tr>
		<tr>
			<td>Cage for scaffolds: Histosette II M492&#160;</td>
			<td>VWR</td>
			<td>720-0909</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesenchymal Stem Cell (MSC)</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td>Three independant batches have been used</td>
		</tr>
		<tr>
			<td>MSCGM BulletKit medium</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td>For the complete medium, add the provided BulletKit (containing serum, glutamine and antibiotics) to the MSCGM medium</td>
		</tr>
		<tr>
			<td>DMEM with Glutamax I</td>
			<td>Life Technologies</td>
			<td>31966-021</td>
			<td>No sodium pyruvate, no HEPES</td>
		</tr>
		<tr>
			<td>RPMI medium 1640, no Glutamine</td>
			<td>Life Technologies</td>
			<td>31870-025</td>
			<td>No sodium pyruvate, no HEPES</td>
		</tr>
		<tr>
			<td>PBS w/o CaCl2 w/o MgCl2</td>
			<td>Life Technologies</td>
			<td>14190-094</td>
			<td></td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA (1x)</td>
			<td>Life Technologies</td>
			<td>25300-054</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin (10.000U/mL)/Streptomicin (10.000&#181;g/mL)</td>
			<td>Life Technologies</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>ITS Premix Universal Culture Supplement (20x)</td>
			<td>Corning</td>
			<td>354352</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium pyruvate solution 100mM</td>
			<td>Sigma</td>
			<td>S8636</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid 2-phosphate</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td>Prepare the concentrated solution (25 mM) in distilled water extemporaneously</td>
		</tr>
		<tr>
			<td>L-Proline</td>
			<td>Sigma</td>
			<td>P5607</td>
			<td>Prepare the 175 mM stock solution diluted in distilled water and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Dexamethasone</td>
			<td>Sigma</td>
			<td>D4902</td>
			<td>Prepare the 1 mM stock solution diluted in Ethanol 100% and store at -20&#176;C</td>
		</tr>
		<tr>
			<td>TGF-beta 3 protein 10&#181;g</td>
			<td>Interchim</td>
			<td>30R-AT028</td>
			<td></td>
		</tr>
		<tr>
			<td>Tri-sodium citrate</td>
			<td>VWR</td>
			<td>33615.268</td>
			<td>Prepare the 1M stock solution diluted in distilled water and store at 4&#176;C</td>
		</tr>
		<tr>
			<td>Pullulanase from Bacillus acidopullulyticus</td>
			<td>Sigma</td>
			<td>P2986</td>
			<td></td>
		</tr>
		<tr>
			<td>Dextranase from Chaetomium erraticum</td>
			<td>Sigma</td>
			<td>D0443</td>
			<td></td>
		</tr>
		<tr>
			<td>NucleoSpin RNA Extraction Kit</td>
			<td>Macherey-Nagel</td>
			<td>740955.5</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase</td>
			<td>Life Technologies</td>
			<td>18064-014</td>
			<td></td>
		</tr>
		<tr>
			<td>Random Primer - Hexamer&#160;</td>
			<td>Promega</td>
			<td>C1181</td>
			<td>500 &#181;g/mL: Use diluted 1/2 and put 1 &#181;L per sample</td>
		</tr>
		<tr>
			<td>Recombinant RNAsin ribonuclease inhibitor</td>
			<td>Promega</td>
			<td>N2511</td>
			<td>40 U/&#181;L: put 1 &#181;L per sample</td>
		</tr>
		<tr>
			<td>PCR nucleotide dNTP mix (10mM each)</td>
			<td>Roche</td>
			<td>10842321</td>
			<td></td>
		</tr>
		<tr>
			<td>SyBr Green PCR Master Mix</td>
			<td>Life Technologies</td>
			<td>4368708</td>
			<td></td>
		</tr>
		<tr>
			<td>Step One Plus Real-Time PCR System</td>
			<td>Life Technologies</td>
			<td>4381792</td>
			<td></td>
		</tr>
		<tr>
			<td>Formalin solution 10% neutral buffered</td>
			<td>Sigma</td>
			<td>HT5012</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT solution</td>
			<td>VWR</td>
			<td>361603E</td>
			<td></td>
		</tr>
		<tr>
			<td>Isopentane</td>
			<td>Sigma</td>
			<td>M32631</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine blue O</td>
			<td>VWR</td>
			<td>1.15930.0025</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol absolute</td>
			<td>VWR</td>
			<td>20821.310</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluene</td>
			<td>VWR</td>
			<td>1.08323.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting medium Pertex</td>
			<td>Histolab</td>
			<td>840</td>
			<td></td>
		</tr>
		<tr>
			<td>RPLP0 Primer for qPCR</td>
			<td>Eurogentec</td>
			<td></td>
			<td>5&#39;-TGCATCAGTAC 
			CCCATTCTATCAT-3&#39; ; 
			5&#39;-AAGGTGTAATC 
			CGTCTCCACAGA-3&#39;</td>
		</tr>
		<tr>
			<td>Aggrecan Primer for qPCR</td>
			<td>Eurogentec</td>
			<td></td>
			<td>5&#39;-TCTACCGCTGCGAGGTGAT-3&#39; ; 3&#39;-TGTAATGGAACACGATGCCTTT-5&#39;</td>
		</tr>
		<tr>
			<td>Collagen II Primer for qPCR</td>
			<td>Eurogentec</td>
			<td></td>
			<td>5&#39;-ACTGGATTGACCCCAACCAA-3&#39; ; 3&#39;-TCCATGTTGCAGAAAACCTTCA-5&#39;</td>
		</tr>
	</tbody>
</table>

3d magnetic stem cell aggregation and bioreactor maturation for cartilage regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

First, aggregates can be individually formed using micro-magnets by depositing 2.5&#215;105 labeled stem cells (Figure 2A). These single aggregates (~0.8 mm in size) can then be fused into larger structures thanks to sequential, magnetically induced fusion. For instance, on day 8 of chondrogenic maturation, aggregates were placed in contact in pairs to form doublets; quadruplets were assembled on day 11 by merging 2 doublets; and finally, on day 15, the 4 quadr...

Watch this Scientific Journal Video about 3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration at JoVE.com

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

3d-magnetic-stem-cell-aggregation-bioreactor-maturation-for-cartilage

Research

JoVE Journal

Bioengineering

9.7K Views.  UMR 7057 CNRS and University Paris Diderot. Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Video: 3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells (hESC) to Different Lineages

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture indefinitely and differentiated to any cell types in the body. Various types of biomaterials have also been used in stem cell cultures to provide a microenvironment mimicking the stem cell niche1-3. The latter is important for promoting cell-to-cell interaction, cell proliferation, and differentiation into specific lineages as well as tissue organization by providing a three-dimensional (3D) environment4 such as encapsulation. The principle of cell encapsulation involves entrapment of living cells within the confines of semi-permeable membranes in 3D cultures2. These membranes allow for the exchange of nutrients, oxygen and stimuli across the membranes, whereas antibodies and immune cells from the host that are larger than the capsule pore size are excluded5. Here, we present an approach to culture and differentiate hESC DA neurons in a 3D microenvironment using alginate microcapsules. We have modified the culture conditions2 to enhance the viability of encapsulated hESC. We have previously shown that the addition of p160-Rho-associated coiled-coil kinase (ROCK) inhibitor, Y-27632 and human fetal fibroblast-conditioned serum replacement medium (hFF-CM) to the 3D platform significantly enhanced the viability of encapsulated hESC in which the cells expressed definitive endoderm marker genes1. We have now used this 3D platform for the propagation of hESC and efficient differentiation to DA neurons. Protein and gene expression analyses after the final stage of DA neuronal differentiation showed an increased expression of tyrosine hydroxylase (TH), a marker for DA neurons, &gt;100 folds after 2 weeks. We hypothesized that our 3D platform using alginate microcapsules may be useful to study the proliferation and directed differentiation of hESC to various lineages. This 3D system also allows the separation of feeder cells from hESC during the process of differentiation and also has potential for immune-isolation during transplantation in the future.

Alginate Microcapsule as a 3D Platform for Propagation and Differentiation of Human Embryonic Stem Cells hESC to Different Lineages

We have optimized a microencapsulation technique as an effective 3D platform for propagation and differentiation of embryonic stem cells to endoderm and dopaminergic (DA) neurons. It also provides an opportunity for immune-isolation of cells from the host during transplantation. This platform can be adapted for other cell types.

Human embryonic stem cells (hESC) are emerging as an attractive alternative source for cell replacement therapy since they can be expanded in culture ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Nonhuman Primate Lung Decellularization and Recellularization Using a Specialized Large-organ Bioreactor

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an attractive alternative to classic organ transplantation. This technology utilizes an organ&#39;s natural biological extracellular matrix (ECM) as a scaffold onto which autologous or stem/progenitor cells may be seeded and cultured in such a way that facilitates regeneration of the original tissue. The natural ECM is isolated by a process called decellularization. Decellularization is accomplished by treating tissues with a series of detergents, salts, and enzymes to achieve effective removal of cellular material while leaving the ECM intact. Studies conducted utilizing decellularization and subsequent recellularization of rodent lungs demonstrated marginal success in generating pulmonary-like tissue which is capable of gas exchange in vivo. While offering essential proof-of-concept, rodent models are not directly translatable to human use. Nonhuman primates (NHP) offer a more suitable model in which to investigate the use of bioartificial organ production for eventual clinical use.
The protocols for achieving complete decellularization of lungs acquired from the NHP rhesus macaque are presented. The resulting acellular lungs can be seeded with a variety of cells including mesenchymal stem cells and endothelial cells. The manuscript also describes the development of a bioreactor system in which cell-seeded macaque lungs can be cultured under conditions of mechanical stretch and strain provided by negative pressure ventilation as well as pulsatile perfusion through the vasculature; these forces are known to direct differentiation along pulmonary and endothelial lineages, respectively. Representative results of decellularization and cell seeding are provided.

Whole-organ decellularization produces natural biological scaffolds that may be used for regenerative medicine. The description of a nonhuman primate model of lung regeneration in which whole lungs are decellularized and then seeded with adult stem cells and endothelial cells in a bioreactor that facilitates vascular circulation and liquid media ventilation is presented.

There are an insufficient number of lungs available to meet current and future organ transplantation needs. Bioartificial tissue regeneration is an ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol adhesive through a metal stencil, the overall amount of adhesive used in assembling paper microfluidic devices can be significantly reduced. We show on a simple 4-layer planar paper microfluidic device that the optimal adhesive application technique and device construction style depends heavily on desired performance characteristics. By moderately increasing the overall area of a device, it is possible to dramatically decrease the wicking time and increase device success rates while also reducing the amount of adhesive required to keep the device together. Such adhesive application also causes the adhesive to form semi-permanent bonds instead of permanent bonds between paper layers, enabling single-use devices to be non-destructively disassembled after use. Nonplanar 3D origami devices also benefit from the semi-permanent bonds during folding, as it reduces the likelihood that unrelated faces may accidently stick together. Like planar devices, nonplanar structures see reduced wicking times with patterned adhesive application vs uniformly applied adhesive.

We demonstrate the use of patterned aerosol adhesives to construct 3D paper microfluidic devices. This method of adhesive application forms semi-permanent bonds between layers, enabling single-use devices to be non-destructively disassembled after use and to ease folding complex nonplanar structures.

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...