Abstract
Scaffold cell seeding is a crucial step for the standardization and homogeneous maturation of tissue engineered constructs. This is particularly critical in the context of additively manufactured scaffolds whereby large pore size and high porosity usually impedes the retention of the seeding solution resulting in poor seeding efficacy and heterogeneous cell distribution. To circumvent this limitation, a simple yet efficient cell seeding technique is described in this chapter consisting of preincubating the scaffold in 100% serum for 1 h leading to reproducible seeding. A proof of concept is demonstrated using highly porous melt electrowritten polycaprolactone scaffolds as the cell carrier. As cell density, cell distribution, and differentiation within the scaffold are important parameters, various assays are proposed to validate the seeding and perform quality control of the cellularized construct using techniques such as alizarin red, Sirius red, and immunostaining.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
O’Brien FJ (2011) Biomaterials & scaffolds for tissue engineering. Mater Today 14:88–95. https://doi.org/10.1016/S1369-7021(11)70058-X
Tan L, Ren Y, Kuijer R (2012) A 1-min method for homogenous cell seeding in porous scaffolds. J Biomater Appl 26:877–889. https://doi.org/10.1177/0885328210389504
Bueno EM, Laevsky G, Barabino GA (2007) Enhancing cell seeding of scaffolds in tissue engineering through manipulation of hydrodynamic parameters. J Biotechnol 129:516–531. https://doi.org/10.1016/j.jbiotec.2007.01.005
Thevenot P, Nair A, Dey J et al (2008) Method to analyze three-dimensional cell distribution and infiltration in degradable scaffolds. Tissue Eng Part C Methods 14:319–331. https://doi.org/10.1089/ten.tec.2008.0221
Sikavitsas VI, van den Dolder J, Bancroft GN et al (2003) Influence of the in vitro culture period on the in vivo performance of cell/titanium bone tissue-engineered constructs using a rat cranial critical size defect model. J Biomed Mater Res A 67:944–951. https://doi.org/10.1002/jbm.a.10126
Van den Dolder J, Spauwen PHM, Jansen JA (2003) Evaluation of various seeding techniques for culturing osteogenic cells on titanium fiber mesh. Tissue Eng 9:315–325. https://doi.org/10.1089/107632703764664783
Saini S, Wick TM (2003) Concentric cylinder bioreactor for production of tissue engineered cartilage: effect of seeding density and hydrodynamic loading on construct development. Biotechnol Prog 19:510–521. https://doi.org/10.1021/bp0256519
Li Y, Ma T, Kniss DA et al (2001) Effects of filtration seeding on cell density, spatial distribution, and proliferation in nonwoven fibrous matrices. Biotechnol Prog 17:935–944. https://doi.org/10.1021/bp0100878
Soletti L, Nieponice A, Guan J et al (2006) A seeding device for tissue engineered tubular structures. Biomaterials 27:4863–4870. https://doi.org/10.1016/J.BIOMATERIALS.2006.04.042
Godbey WT, Stacey Hindy BS, Sherman ME, Atala A (2004) A novel use of centrifugal force for cell seeding into porous scaffolds. Biomaterials 25:2799–2805. https://doi.org/10.1016/J.BIOMATERIALS.2003.09.056
van Wachem PB, Stronck JWS, Koers-Zuideveld R et al (1990) Vacuum cell seeding: a new method for the fast application of an evenly distributed cell layer on porous vascular grafts. Biomaterials 11:602–606. https://doi.org/10.1016/0142-9612(90)90086-6
Nasrollahzadeh N, Applegate LA, Pioletti DP (2017) Development of an effective cell seeding technique: simulation, implementation, and analysis of contributing factors. Tissue Eng Part C Methods 23:485–496. https://doi.org/10.1089/ten.tec.2017.0108
Alvarez-Barreto JF, Linehan SM, Shambaugh RL, Sikavitsas VI (2007) Flow perfusion improves seeding of tissue engineering scaffolds with different architectures. Ann Biomed Eng 35:429–442. https://doi.org/10.1007/s10439-006-9244-z
Beşkardeş IG, Aydın G, Bektaş Ş et al (2018) A systematic study for optimal cell seeding and culture conditions in a perfusion mode bone-tissue bioreactor. Biochem Eng J 132:100–111. https://doi.org/10.1016/J.BEJ.2018.01.006
Campos Marín A, Brunelli M, Lacroix D (2018) Flow perfusion rate modulates cell deposition onto scaffold substrate during cell seeding. Biomech Model Mechanobiol 17:675–687. https://doi.org/10.1007/s10237-017-0985-4
Cao T, Ho K-H, Teoh S-H (2003) Scaffold design and in vitro study of osteochondral coculture in a three-dimensional porous polycaprolactone scaffold fabricated by fused deposition modeling. Tissue Eng 9:103. https://doi.org/10.1089/10763270360697012
Jukes JM, Moroni L, Van Blitterswijk CA, De Boer J (2008) Critical steps toward a tissue-engineered cartilage implant using embryonic stem cells. Tissue Eng Part A 14:135–147. https://doi.org/10.1089/ten.a.2006.0397
Woodfield TBF, Malda J, de Wijn J et al (2004) Design of porous scaffolds for cartilage tissue engineering using a three-dimensional fiber-deposition technique. Biomaterials 25:4149–4161. https://doi.org/10.1016/J.BIOMATERIALS.2003.10.056
Vaquette C, Ivanovski S, Hamlet SM, Hutmacher DW (2013) Effect of culture conditions and calcium phosphate coating on ectopic bone formation. Biomaterials 34:5538–5551. https://doi.org/10.1016/j.biomaterials.2013.03.088
Jinyoon J, Song SH, Lee DS, Park TG (2004) Immobilization of cell adhesive RGD peptide onto the surface of highly porous biodegradable polymer scaffolds fabricated by a gas foaming/salt leaching method. Biomaterials 25:5613–5620. https://doi.org/10.1016/j.biomaterials.2004.01.014
Wang W, Caetano G, Ambler WS et al (2016) Enhancing the hydrophilicity and cell attachment of 3D printed PCL/graphene scaffolds for bone tissue engineering. Materials (Basel) 9:992. https://doi.org/10.3390/ma9120992
Truong YB, Glattauer V, Briggs KL et al (2012) Collagen-based layer-by-layer coating on electrospun polymer scaffolds. Biomaterials 33:9198–9204. https://doi.org/10.1016/j.biomaterials.2012.09.012
Jordan AM, Viswanath V, Kim SE, Pokorski JK, Korley LTJ (2016) Processing and surface modification of polymer nanofibers for biological scaffolds: a review. J Mater Chem B 4:5958–5974. https://doi.org/10.1039/c6tb01303a
Blaudez F, Ivanovski S, Ipe D, Vaquette C (2020) A comprehensive comparison of cell seeding methods using highly porous melt electrowriting scaffolds. Mater Sci Eng C 117:111282. https://doi.org/10.1016/j.msec.2020.111282
Brown TD, Dalton PD, Hutmacher DW (2011) Direct writing by way of melt electrospinning. Adv Mater 23:5651–5657. https://doi.org/10.1002/adma.201103482
Acknowledgments
This study has been financially supported by the Australian National Health Medical Research Council (grant number: APP1086181).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Blaudez, F., Vaquette, C., Ivanovski, S. (2023). Cell Seeding on 3D Scaffolds for Tissue Engineering and Disease Modeling Applications. In: Seymour, G.J., Cullinan, M.P., Heng, N.C., Cooper, P.R. (eds) Oral Biology. Methods in Molecular Biology, vol 2588. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-2780-8_28
Download citation
DOI: https://doi.org/10.1007/978-1-0716-2780-8_28
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-2779-2
Online ISBN: 978-1-0716-2780-8
eBook Packages: Springer Protocols