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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</ol>

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><tbody><tr><td>Iron oxide (maghemite) nanoparticules (&amp;gamma;-Fe&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;3&lt;/sub&gt;)</td><td>PHENIX - University Paris 6</td><td>Made and given by C. M&amp;eacute;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 (10&amp;nbsp;M NaOH). Porosity: 185-205&amp;nbsp;&amp;micro;m; Thickness: 7&amp;nbsp;mm; Surface area: 1.8&amp;nbsp;cm&lt;sup&gt;2&lt;/sup&gt;.</td></tr><tr><td>TisXell Regeneration System</td><td>QuinXell Technologies</td><td>QX900-002</td><td>Biaxial bioreactor with 500 mL culture chamber&amp;nbsp;</td></tr><tr><td>Cage for scaffolds: Histosette II M492&amp;nbsp;</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 CaCl&lt;sub&gt;2&lt;/sub&gt; w/o MgCl&lt;sub&gt;2&lt;/sub&gt;</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 (10,000&amp;nbsp;U/mL) / Streptomycin (10,000&amp;nbsp;&amp;micro;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 100 mM</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&amp;nbsp;&amp;deg;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&amp;nbsp;&amp;deg;C</td></tr><tr><td>TGF-beta 3 protein 10&amp;nbsp;&amp;micro;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 1&amp;nbsp;M stock solution diluted in distilled water and store at 4&amp;nbsp;&amp;deg;C</td></tr><tr><td>Pullulanase from &lt;em&gt;Bacillus acidopullulyticus&lt;/em&gt;</td><td>Sigma</td><td>P2986</td><td></td></tr><tr><td>Dextranase from &lt;em&gt;Chaetomium erraticum&lt;/em&gt;</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&amp;nbsp;</td><td>Promega</td><td>C1181</td><td>500 &amp;micro;g/mL: Use diluted 1/2 and put 1 &amp;micro;L per sample</td></tr><tr><td>Recombinant RNAs in ribonuclease inhibitor</td><td>Promega</td><td>N2511</td><td>40 U/&amp;micro;L: put 1 &amp;micro;L per sample</td></tr><tr><td>PCR nucleotide dNTP mix (10&amp;nbsp;mM 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&#039;-TGCATCAGTAC&lt;br/&gt; CCCATTCTATCAT-3&#039;;&lt;br/&gt; 5&#039;-AAGGTGTAATC&lt;br/&gt; CGTCTCCACAGA-3&#039;</td></tr><tr><td>Aggrecan Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-TCTACCGCTGCGAGGTGAT-3&#039;; 3&#039;-TGTAATGGAACACGATGCCTTT-5&#039;</td></tr><tr><td>Collagen II Primer for qPCR</td><td>Eurogentec</td><td></td><td>5&#039;-ACTGGATTGACCCCAACCAA-3&#039;; 3&#039;-TCCATGTTGCAGAAAACCTTCA-5&#039;</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...

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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

9.8K Views.  UMR 7057 CNRS and University Paris Diderot.  Chondrogenese uit stamcellen vereist fine tuning van de kweekomstandigheden. Hier presenteren we een magnetische aanpak voor het condenseren van cellen, een essentiële stap om chondrogenese te initiëren. Bovendien tonen wij aan dat dynamische rijping in een bioreactor zijn mechanische stimulatie van de cellulaire constructen en verbetert kraakbeenachtige extracellulaire matrix productie. 

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

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Chondrogenic Pellet Formation from Cord Blood-derived Induced Pluripotent Stem Cells

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. Currently, efforts are being made to regenerate damaged cartilage with ex vivo expanded chondrocytes or bone marrow-derived mesenchymal stem cells (BMSCs). However, the restricted viability and instability of these cells limit their application in cartilage reconstruction. Human induced pluripotent stem cells (hiPSCs) have received scientific attention as a new alternative for regenerative applications. With unlimited self-renewal ability and multipotency, hiPSCs have been highlighted as a new replacement cell source for cartilage repair. However, obtaining a high quantity of high-quality chondrogenic pellets is a major challenge to their clinical application. In this study, we used embryoid body (EB)-derived outgrowth cells for chondrogenic differentiation. Successful chondrogenesis was confirmed by PCR and staining with alcian blue, toluidine blue, and antibodies against collagen types I and II (COL1A1 and COL2A1, respectively). We provide a detailed method for the differentiation of cord blood mononuclear cell-derived iPSCs (CBMC-hiPSCs) into chondrogenic pellets.

Here, we propose a protocol for chondrogenic differentiation from cord blood mononuclear cell-derived human induced pluripotent stem cells.

Human articular cartilage lacks the ability to repair itself. Cartilage degeneration is thus treated not by curative but by conservative treatments. ...

Developmental Biology

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 ...

Implantation of Ferumoxides Labeled Human Mesenchymal Stem Cells in Cartilage Defects

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and functional tissue. Matrix associated stem cell implants (MASI) aim to regenerate cartilage defects due to arthritic or traumatic joint injuries. Adult mesenchymal stem cells (MSCs) have the ability to differentiate into cells of the chondrogenic lineage and have shown promising results for cell-based articular cartilage repair technologies. Autologous MSCs can be isolated from a variety of tissues, can be expanded in cell cultures without losing their differentiation potential, and have demonstrated chondrogenic differentiation in vitro and in vivo1, 2.
In order to provide local retention and viability of transplanted MSCs in cartilage defects, a scaffold is needed, which also supports subsequent differentiation and proliferation. The architecture of the scaffold guides tissue formation and permits the extracellular matrix, produced by the stem cells, to expand. Previous investigations have shown that a 2% agarose scaffold may support the development of stable hyaline cartilage and does not induce immune responses3.
Long term retention of transplanted stem cells in MASI is critical for cartilage regeneration. Labeling of MSCs with iron oxide nanoparticles allows for long-term in vivo tracking with non-invasive MR imaging techniques4.
This presentation will demonstrate techniques for labeling MSCs with iron oxide nanoparticles, the generation of cell-agarose constructs and implantation of these constructs into cartilage defects. The labeled constructs can be tracked non-invasively with MR-Imaging.

Goal of the presentation is to demonstrate a highly reproducible method to generate matrix associated stem cell implants in cartilage defects, which can be visualized with MR imaging. Stem cells are labeled with FDA-approved Ferumoxides, mixed with agarose, implanted into cartilage defects and imaged with a 7T MR scanner.

The field of tissue engineering integrates the principles of engineering, cell biology and medicine towards the regeneration of specific cells and ...

Biology

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 ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Construction, Characterization, and Regenerative Application of Self-Assembled Human Mesenchymal Stem Cell Aggregates

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources and are emerging as promising candidates for regenerative medicine, especially for regeneration of the tooth, bone, cartilage, and skin. The self-assembled approach of MSC aggregation, which notably constructs cell clusters mimicking the developing mesenchymal condensation, allows high-density stem cell delivery along with preserved cell-cell interactions and extracellular matrix (ECM) as the microenvironment niche. This method has been shown to enable efficient cell engraftment and survival, thus promoting the optimized application of exogenous MSCs in tissue engineering and safeguarding clinical organ regeneration. This paper provides a detailed protocol for the construction and characterization of self-assembled aggregates based on umbilical cord mesenchymal stem cells (UCMSCs), as well as an example of the cranial bone regenerative application. The implementation of this procedure will help guide the establishment of an efficient MSC transplantation strategy for tissue engineering and regenerative medicine.

This study describes a method to construct aggregates based on the self-assembly of human mesenchymal stem cells and identifies the morphological and histological characteristics for the regenerative treatment of cranial bone defects.

Mesenchymal stem cells (MSCs), characterized by their self-renewal ability and multilineage differentiation potential, can be derived from various sources ...

Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited self-repairing capacity and, most importantly, no definitive treatments, tendinopathy still influences patients&#8217; life quality negatively. Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both the development of tendinopathy, and functional and structural restoration after tendinopathy. Thus, a method that can in vitro mimic the in vivo differentiation of TDSCs into tendon cells would be useful. Here, the present protocol describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissues. There are seven stages of the present protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The effectiveness was demonstrated by histology. The entire procedure takes less than 3 weeks. To promote extracellular matrix deposition, 4.4 mg/mL ascorbic acid was used in the stimulation culture medium. A separated chamber with a linear motor provides accurate mechanical loading and is portable and easily adjusted, which is applied for the bioreactor. The loading regime in the present protocol was 6% strain, 0.25 Hz, 8 h, followed by 16 h rest for 6 days. This protocol could mimic cell differentiation in the tendon, which is helpful for the investigation of the pathological process of tendinopathy. Moreover, the tendon-like tissue is potentially used to promote tendon healing in tendon injury as an engineered autologous graft. To sum up, the present protocol is simple, economic, reproducible and valid.

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited ...

Medicine

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...

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

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