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

Summary

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.

Abstract

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.

Introduction

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 implantation^1,2,3. In regenerative medicine, they can be used to engineer organized tissues in vitro⁴, including vascular tissue^5,6,7, 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 source^10,11, while others emphasize the capacity of mesenchymal stem cells (MSC) to differentiate into chondrocytes^12,13. 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 cells^16,17. 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.

Protocol

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 2.5×10⁵/aggregate, so the magnetic tips must be very thin (750 µm 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.

1. Construction of a device with micro-magnets for aggregate formation (Figure 1A)
   1. Make micro-holes with a 0.8-mm drill through aluminum plates (3 cm in diameter and 6 mm thick).
   2. Insert a magnetic tip (750 µm in diameter) into each hole of the plate.
   3. Place this disk over a permanent neodymium magnet, which ensures magnetization to saturation.
2. Construction of a device for scaffold seeding (Figure 1B)
   1. Cut hard polystyrene into 2.4 cm² squares.
   2. Insert 9 small magnets (3 mm in diameter, 6 mm long) at an equal distance over a surface area of 1.6 cm²_.
   3. Place this device over a permanent neodymium magnet.

2. Stem Cell Labeling

NOTE: Stem cells were labeled with 0.1 mM magnetic nanoparticles for 30 min (2.6 ± 0.2 pg iron/cell) to form aggregates, while they were labeled with 0.2 mM magnetic nanoparticles for 30 min (5 ± 0.4 pg iron/cell) to seed scaffolds. These nanoparticle concentrations and incubation times have been used previously and published for MSC and other cells^18,19, 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 magnetophoresis^19,20.

1. Culture human mesenchymal stem cells (MSC) in complete mesenchymal stem cell growth medium (MSCGM) at 37 °C and 5% CO₂ until near to confluence (~90%).
2. Prepare the magnetic labeling solution by mixing 0.1 or 0.2 mM maghemite citrate-coated iron oxide (γFe₂O₃; core: 8 nm diameter) in serum-free McCoy's 5A medium modified at Roswell Park Memorial Institute (RMPI) without glutamine and containing 5 mM sodium citrate.
3. 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.
4. Incubate for 30 min at 37 °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.
5. Discard the RPMI medium and add 25 mL of complete MSCGM medium per flask. Incubate overnight at 37 °C and 5% CO₂.

3. Magnetic Cell Seeding

1. Freshly prepare the chondrogenic medium using Dulbecco's Modified Eagle Medium (DMEM) high glucose with L-glutamine by adding 50 µM L-ascorbic acid 2-phosphate, 0.1 µM 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-β3).
2. Detach the magnetic cells using 8 mL of 0.05% trypsin-EDTA per 150-cm² culture flask and centrifuge the dissociated cells at 260 × g for 5 min. Aspirate the medium and count the re-suspended cells.
3. Place a glass-bottomed cell culture Petri dish (35 mm) on top of both magnetic devices.
4. 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 µL) containing 2.5×10⁵ 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 °C and 5% CO₂.
5. Form control aggregates following the same protocol and replace the complete medium with chondrogenic medium without TGF-β3.
   1. 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.
   2. At the same time, form aggregates by centrifuging 2.5×10⁵ labeled stem cells at 260 × g for 5 min in 15-mL tubes with 1.5 mL of chondrogenic medium with or without TGF-β3 (for sample and control, respectively).
6. To magnetically seed scaffolds, place each dried scaffold into a Petri dish. Use polysaccharide porous scaffolds made of pullulan/dextran²¹. For each scaffold, dilute 2×10⁶ labeled stem cells in 350 µL of chondrogenic medium without TGF-β3 and carefully pipette the cells onto the scaffold.
   1. Incubate for 5 min at 37 °C to allow for full cell penetration within the scaffold and then gently add 3 mL of chondrogenic medium with or without TGF-β3 (for sample or control, respectively) to the Petri dish.
   2. Incubate the cellularized scaffold on its magnetic device at 37 °C and 5% CO₂ for 4 days to allow for cell migration through the scaffold pores and confinement.
7. At the same time, seed scaffolds with 2×10⁶ labeled stem cells following the same method and incubate without the magnet to obtain uniformly seeded scaffolds as positive controls.

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-β3.

1. In static conditions, keep the cellularized scaffolds or aggregates in the same Petri dish. Change the chondrogenic medium twice a week for 21 days.
2. In dynamic conditions, prepare the bioreactor.
   1. Cut silicone tubing at the appropriate length according to the manufacturer's protocol.
   2. Autoclave all materials: 500-mL culture chamber, tubing, 2-way rotators, and cages.
   3. 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's instructions.
   4. 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.
3. Turn on the peristaltic pump to fill the tubing with chondrogenic medium and to eliminate air bubbles.
4. 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.
5. 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.

5. RNA Extraction and Gene Expression Analysis

NOTE: Prior to RNA extraction, digest the scaffolds with an enzymatic solution.

1. Prepare 1 mL of enzymatic solution by adding 100 µL of pullulanase (40 U/mL) and 50 µL of dextranase (60 mg/mL) to 850 mL of serum-free DMEM medium.
2. Rinse the scaffolds twice with serum-free DMEM medium, discard the medium, and add 800 µL of the enzymatic solution per scaffold. Incubate for 15-30 min at 37 °C under gentle agitation.
3. When the scaffold is completely dissolved, transfer the solution containing the cells to a 1.5-mL tube, centrifuge at 300 × g for 10 min, carefully aspirate the medium, rinse twice with sterile 1× phosphate-buffered saline (PBS), centrifuge at 300 × g for 10 min, and re-suspend the cells in the RNA isolation solution.
4. 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.
5. Isolate the RNA using a kit for total RNA extraction according to the manufacturer's instructions.
6. Synthesize complementary DNA from 400 ng of total RNA using reverse transcriptase according to the manufacturer's instructions, using 250-ng random primers, 1 µL of dNTP mix (10 mM each), and 40 U/mL RNase inhibitor; the final volume of reaction is 20 µL. At the end of the reaction, add 80 µL of distilled water to obtain a final volume of 100 µL.
7. 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× diluted cDNA. Normalize the levels of gene expression with the reference gene Ribosomal Protein, Large, P0 (RPLP0). Perform the calculations with the 2^-ΔΔCT formula, where ΔΔCT = ΔCT of differentiated condition - mean ΔCT of control condition, and each ΔCT represents the CT of the gene of interest - the CT of the reference gene (RPLP0).
8. Determine statistical measurements as the mean values ± the standard error of the mean (SEM). Perform the analysis with n ≥ 2 independent experiments. Use Student's t-test to analyze the statistical differences between centrifuged pellets and magnetic fusions (* p < 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 < 0.05).

6. Histological Analysis

1. Rinse the cellularized scaffolds or aggregates with sterile 1× PBS, fix them in 10% formalin solution for 1 h at room temperature, and rinse with 1× PBS.
2. 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 °C. Cut the samples with a cryostat to obtain 8-µm cryosections for the aggregates or 12-µm for the cellularized scaffolds.
3. 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.

Results

First, aggregates can be individually formed using micro-magnets by depositing 2.5×10⁵ 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 quadruplet...

Discussion

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 exposure^19,22. However, several studies have shown no impact on cellular physiology when encapsulated iron nanoparticles were...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Iron oxide (maghemite) nanoparticules (γ-Fe2O3)	PHENIX - University Paris 6	Made and given by C. Ménager	Mean diameter of 8.1 nm and negative surface charge
Polysaccharide Pullulan/Dextran scaffolds	LIOAD - University Nantes	Made and given by C. Le Visage	Prepared from a 75:25 mixture of pullulan/dextran in alkaline conditions (10 M NaOH). Porosity: 185-205 µm; Thickness: 7 mm; Surface area: 1.8 cm2.
TisXell Regeneration System	QuinXell Technologies	QX900-002	Biaxial bioreactor with 500 mL culture chamber
Cage for scaffolds: Histosette II M492	VWR	720-0909
Mesenchymal Stem Cell (MSC)	Lonza	PT-2501	Three independant batches have been used
MSCGM BulletKit medium	Lonza	PT-3001	For the complete medium, add the provided BulletKit (containing serum, glutamine and antibiotics) to the MSCGM medium
DMEM with Glutamax I	Life Technologies	31966-021	No sodium pyruvate, no HEPES
RPMI medium 1640, no Glutamine	Life Technologies	31870-025	No sodium pyruvate, no HEPES
PBS w/o CaCl2 w/o MgCl2	Life Technologies	14190-094
0.05% Trypsin-EDTA (1x)	Life Technologies	25300-054
Penicillin (10,000 U/mL) / Streptomycin (10,000 µg/mL)	Life Technologies	15140-122
ITS Premix Universal Culture Supplement (20x)	Corning	354352
Sodium pyruvate solution 100 mM	Sigma	S8636
L-Ascorbic Acid 2-phosphate	Sigma	A8960	Prepare the concentrated solution (25 mM) in distilled water extemporaneously
L-Proline	Sigma	P5607	Prepare the 175 mM stock solution diluted in distilled water and store at 4 °C
Dexamethasone	Sigma	D4902	Prepare the 1 mM stock solution diluted in Ethanol 100% and store at -20 °C
TGF-beta 3 protein 10 µg	Interchim	30R-AT028
Tri-sodium citrate	VWR	33615.268	Prepare the 1 M stock solution diluted in distilled water and store at 4 °C
Pullulanase from Bacillus acidopullulyticus	Sigma	P2986
Dextranase from Chaetomium erraticum	Sigma	D0443
NucleoSpin RNA Extraction Kit	Macherey-Nagel	740955.5
SuperScript II Reverse Transcriptase	Life Technologies	18064-014
Random Primer - Hexamer	Promega	C1181	500 µg/mL: Use diluted 1/2 and put 1 µL per sample
Recombinant RNAs in ribonuclease inhibitor	Promega	N2511	40 U/µL: put 1 µL per sample
PCR nucleotide dNTP mix (10 mM each)	Roche	10842321
SyBr Green PCR Master Mix	Life Technologies	4368708
Step One Plus Real-Time PCR System	Life Technologies	4381792
Formalin solution 10% neutral buffered	Sigma	HT5012
OCT solution	VWR	361603E
Isopentane	Sigma	M32631
Toluidine blue O	VWR	1.15930.0025
Ethanol absolute	VWR	20821.310
Toluene	VWR	1.08323.1000
Mounting medium Pertex	Histolab	840
RPLP0 Primer for qPCR	Eurogentec		5'-TGCATCAGTAC CCCATTCTATCAT-3'; 5'-AAGGTGTAATC CGTCTCCACAGA-3'
Aggrecan Primer for qPCR	Eurogentec		5'-TCTACCGCTGCGAGGTGAT-3'; 3'-TGTAATGGAACACGATGCCTTT-5'
Collagen II Primer for qPCR	Eurogentec		5'-ACTGGATTGACCCCAACCAA-3'; 3'-TCCATGTTGCAGAAAACCTTCA-5'