Restricted accessResearch articleFirst published online 2014-12
A Multicompartment Holder for Spinner Flasks Improves Expansion and Osteogenic Differentiation of Mesenchymal Stem Cells in Three-Dimensional Scaffolds
In the tissue engineering field dynamic culture systems, such as spinner flasks, are widely used due to their ability to improve mass transfer in suspension cell cultures. However, this culture system is often unsuitable to culture cells in three-dimensional (3D) scaffolds. To address this drawback, we designed a multicompartment holder for 3D cell culture, easily adaptable to spinner flasks. Here, the device was tested with human mesenchymal stem cells (MSCs) seeded in 3D porous chitosan scaffolds that were maintained in spinner flasks under dynamic conditions (50 rpm). Standard static culture conditions were used as control. The dynamic conditions were shown to significantly increase MSCs proliferation over 1 week (approximately 6-fold) and to improve cell distribution within the scaffold. Moreover, they also promoted osteogenic differentiation of MSCs, inducing an earlier peak in alkaline phosphatase (ALP) activity, and a more homogenous ALP staining and matrix mineralization in the whole scaffolds, but particularly in the center. Overall, this study shows a new multicompartment holder to culture 3D scaffolds that can broaden the application of spinner flasks.
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References
1.
RungarunlertS., TechakumphuM., PirityM.K., and DinnyesA.Embryoid body formation from embryonic and induced pluripotent stem cells: Benefits of bioreactors. World J Stem Cells, 1, 11, 2009.
2.
YeattsA.B., ChoquetteD.T., and FisherJ.P.Bioreactors to influence stem cell fate: augmentation of mesenchymal stem cell signaling pathways via dynamic culture systems. Biochim Biophys Acta, 1830, 2470, 2013.
3.
dos SantosF., AndradeP.Z., AbecasisM.M., GimbleJ.M., ChaseL.G., CampbellA.M., BoucherS., VemuriM.C., SilvaC.L., and CabralJ.M.Toward a clinical-grade expansion of mesenchymal stem cells from human sources: a microcarrier-based culture system under xeno-free conditions. Tissue Eng Part C Methods, 17, 1201, 2011.
4.
GigoutA., BuschmannM.D., and JolicoeurM.Chondrocytes cultured in stirred suspension with serum-free medium containing pluronic-68 aggregate and proliferate while maintaining their differentiated phenotype. Tissue Eng Part A, 15, 2237, 2009.
5.
LeeT.J., BhangS.H., LaW.G., YangH.S., SeongJ.Y., LeeH., ImG.I., LeeS.H., and KimB.S.Spinner-flask culture induces redifferentiation of de-differentiated chondrocytes. Biotechnol Lett, 33, 829, 2011.
6.
ChenM., WangX., YeZ., ZhangY., ZhouY., and TanW.A modular approach to the engineering of a centimeter-sized bone tissue construct with human amniotic mesenchymal stem cells-laden microcarriers. Biomaterials, 32, 7532, 2011.
7.
GohT.K., ZhangZ.Y., ChenA.K., ReuvenyS., ChoolaniM., ChanJ.K., and OhS.K.Microcarrier culture for efficient expansion and osteogenic differentiation of human fetal mesenchymal stem cells. Biores Open Access, 2, 84, 2013.
8.
PörtnerR., Nagel-HeyerS., GoepfertC., AdamietzP., and MeenenN.M.Bioreactor design for tissue engineering. J Biosci Bioeng, 100, 235, 2005.
9.
TurnerA.E., and FlynnL.E.Design and characterization of tissue-specific extracellular matrix-derived microcarriers. Tissue Eng Part C Methods, 18, 186, 2012.
10.
ChenK.Y., ChungC.M., ChenY.S., BauD.T., and YaoC.H.Rat bone marrow stromal cells-seeded porous gelatin/tricalcium phosphate/oligomeric proanthocyanidins composite scaffold for bone repair. J Tissue Eng Regen Med, 7, 708, 2013.
11.
MukherjeeD.P., SmithD.F., RogersS.H., EmmanualJ.E., JadinK.D., and HayesB.K.Effect of 3D-microstructure of bioabsorbable PGA:TMC scaffolds on the growth of chondrogenic cells. J Biomed Mater Res B Appl Biomater, 88, 92, 2009.
12.
ÇetinD., KahramanA.S., and GümüşderelioğluM.Novel pHEMA-gelatin SPHs as bone scaffolds in dynamic cultures. J Mater Sci Mater Med, 23, 2803, 2012.
13.
KimH.J., KimU.J., LeiskG.G., BayanC., GeorgakoudiI., and KaplanD.L.Bone regeneration on macroporous aqueous-derived silk 3-D scaffolds. Macromol Biosci, 7, 643, 2007.
14.
MeinelL., KarageorgiouV., FajardoR., SnyderB., Shinde-PatilV., ZichnerL., KaplanD., LangerR., and Vunjak-NovakovicG.Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow. Ann Biomed Eng, 32, 112, 2004.
15.
MygindT., StiehlerM., BaatrupA., LiH., ZouX., FlyvbjergA., KassemM., and BüngerC.Mesenchymal stem cell ingrowth and differentiation on coralline hydroxyapatite scaffolds. Biomaterials, 28, 1036, 2007.
16.
SongK., LiuT., CuiZ., LiX., and MaX.Three-dimensional fabrication of engineered bone with human bio-derived bone scaffolds in a rotating wall vessel bioreactor. J Biomed Mater Res A, 86, 323, 2008.
17.
JanjaninS., LiW.J., MorganM.T., ShantiR.M., and TuanR.S.Mold-shaped, nanofiber scaffold-based cartilage engineering using human mesenchymal stem cells and bioreactor. J Surg Res, 149, 47, 2008.
18.
BarriasC., and GoncalvesR.M.Container, useful in spinner flask assembly and kit for culturing cell samples in three-dimensional matrix, comprises chambers for containing cell samples, where one of the walls of chambers are perforated to allow culture media and a lid; Patent Number(s): W02013043072-A1 (2011).
19.
AntunesJ.C., PereiraC.L., MolinosM., Ferreira-da-SilvaF., DessìM., GloriaA., AmbrosioL., GonçalvesR.M., and BarbosaM.A.Layer-by-layer self-assembly of chitosan and poly(γ-glutamic acid) into polyelectrolyte complexes. Biomacromolecules, 12, 4183, 2011.
20.
AmaralI.F., SampaioP., and BarbosaM.A.Three-dimensional culture of human osteoblastic cells in chitosan sponges: the effect of the degree of acetylation. J Biomed Mater Res A, 76, 335, 2006.
21.
BidarraS.J., BarriasC.C., BarbosaM.A., SoaresR., AmédéeJ., and GranjaP.L.Phenotypic and proliferative modulation of human mesenchymal stem cells via crosstalk with endothelial cells. Stem Cell Res, 7, 186, 2011.
22.
FolkmanJ., and MosconaA.Role of cell shape in growth control. Nature, 273, 345, 1978.
23.
BarriasC.C., and GonçalvesR.M.Multi-compartment holder for 3D cell culture under dynamic conditions. Provisional patent application UK no.11163979, 2011.
24.
BidarraS.J., BarriasC.C., BarbosaM.A., SoaresR., and GranjaP.L.Immobilization of human mesenchymal stem cells within RGD-grafted alginate microspheres and assessment of their angiogenic potential. Biomacromolecules, 11, 1956, 2010.
25.
PatelS.D., PapoutsakisE.T., WinterJ.N., and MillerW.M.The lactate issue revisited: novel feeding protocols to examine inhibition of cell proliferation and glucose metabolism in hematopoietic cell cultures. Biotechnol Prog, 16, 885, 2000.
26.
ZhuB., BaileyS.R., and Mauli AgrawalC.Calcification of primary human osteoblast cultures under flow conditions using polycaprolactone scaffolds for intravascular applications. J Tissue Eng Regen Med, 6, 687, 2012.
27.
AmaralI.F., UngerR.E., FuchsS., MendonçaA.M., SousaS.R., BarbosaM.A., PêgoA.P., and KirkpatrickC.J.Fibronectin-mediated endothelialisation of chitosan porous matrices. Biomaterials, 30, 5465, 2009.
28.
BarbosaJ.N., AmaralI.F., AguasA.P., and BarbosaM.A.Evaluation of the effect of the degree of acetylation on the inflammatory response to 3D porous chitosan scaffolds. J Biomed Mater Res A, 93, 20, 2010.
29.
SantosS.G., LamghariM., AlmeidaC.R., OliveiraM.I., NevesN., RibeiroA.C., BarbosaJ.N., BarrosR., MacielJ., MartinsM.C., GonçalvesR.M., and BarbosaM.A.Adsorbed fibrinogen leads to improved bone regeneration and correlates with differences in the systemic immune response. Acta Biomater, 9, 7209, 2013.
30.
Di MartinoA., SittingerM., and RisbudM.V.Chitosan: a versatile biopolymer for orthopaedic tissue-engineering. Biomaterials, 26, 5983, 2005.
31.
EibesG., dos SantosF., AndradeP.Z., BouraJ.S., AbecasisM.M., da SilvaC.L., and CabralJ.M.Maximizing the ex vivo expansion of human mesenchymal stem cells using a microcarrier-based stirred culture system. J Biotechnol, 146, 194, 2010.
32.
FerrariC., BalandrasF., GuedonE., OlmosE., ChevalotI., and MarcA.Limiting cell aggregation during mesenchymal stem cell expansion on microcarriers. Biotechnol Prog, 28, 780, 2012.
33.
RafiqQ.A., BrosnanK.M., CoopmanK., NienowA.W., and HewittC.J.Culture of human mesenchymal stem cells on microcarriers in a 5 l stirred-tank bioreactor. Biotechnol Lett, 35, 1233, 2013.
34.
WeberC., PohlS., PörtnerR., WallrappC., KassemM., GeigleP., and CzermakP.Expansion and harvesting of hMSC-TERT. Open Biomed Eng J, 1, 38, 2007.
35.
StiehlerM., BüngerC., BaatrupA., LindM., KassemM., and MygindT.Effect of dynamic 3-D culture on proliferation, distribution, and osteogenic differentiation of human mesenchymal stem cells. J Biomed Mater Res A, 89, 96, 2009.
36.
HewittC.J., LeeK., NienowA.W., ThomasR.J., SmithM., and ThomasC.R.Expansion of human mesenchymal stem cells on microcarriers. Biotechnol Lett, 33, 2325, 2011.
37.
SchopD., JanssenF.W., BorgartE., de BruijnJ.D., and van Dijkhuizen-RadersmaR.Expansion of mesenchymal stem cells using a microcarrier-based cultivation system: growth and metabolism. J Tissue Eng Regen Med, 2, 126, 2008.
38.
LamghariM., AmaralI.F., SousaS.R., SampaioP., and BarbosaM.A.Rat bone marrow stromal cell osteogenic differentiation and fibronectin adsorption on chitosan membranes: the effect of the degree of acetylation. J Biomed Mater Res A, 75, 387, 2005.
39.
HoltorfH.L., SheffieldT.L., AmbroseC.G., JansenJ.A., and MikosA.G.Flow perfusion culture of marrow stromal cells seeded on porous biphasic calcium phosphate ceramics. Ann Biomed Eng, 33, 1238, 2005.
40.
ZhangZ.Y., TeohS.H., TeoE.Y., Khoon ChongM.S., ShinC.W., TienF.T., ChoolaniM.A., and ChanJ.K.A comparison of bioreactors for culture of fetal mesenchymal stem cells for bone tissue engineering. Biomaterials, 31, 8684, 2010.
41.
FeketeN., RojewskiM.T., LotfiR., and SchrezenmeierH.Essential components for ex vivo proliferation of mesenchymal stromal cells. Tissue Eng Part C Methods, 20, 129, 2013.
42.
YourekG., McCormickS.M., MaoJ.J., and ReillyG.C.Shear stress induces osteogenic differentiation of human mesenchymal stem cells. Regen Med, 5, 713, 2010.
43.
PattappaG., HeywoodH.K., de BruijnJ.D., and LeeD.A.The metabolism of human mesenchymal stem cells during proliferation and differentiation. J Cell Physiol, 226, 2562, 2011.
44.
BilgenB., and BarabinoG.A.Location of scaffolds in bioreactors modulates the hydrodynamic environment experienced by engineered tissues. Biotechnol Bioeng, 98, 282, 2007.
45.
BilgenB., Chang-MateuI.M., and BarabinoG.A.Characterization of mixing in a novel wavy-walled bioreactor for tissue engineering. Biotechnol Bioeng, 92, 907, 2005.
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