Abstract
Objectives
To develop a rapid and simple method to fabricate intact, robust cell sheets from common cell culture dishes by combination of a macromolecular crowding (MMC) reagent and vitamin C.
Results
It was found that 3T3 fibroblasts or human bone marrow mesenchymal stem cells (hBMSCs) and their secreted cell derived extracellular matrices could be easily detached as intact cell sheets under gently pipetting after treated by MMC and vitamin C for 4 days. This method also allowed fabrication of functional multi-layered hepatic cell sheets by culturing 10 × 104 cells/cm2 HepG2 cells on top of confluent 3T3 fibroblast layers. What’s more, MMC induced hBMSC cell sheets demonstrated 1.9 times larger area and 1.6 times greater cell number than that of cell sheets harvested from temperature-responsive cell culture dishes.
Conclusion
MMC based method make it possible to fabricate various types of cell sheets more conveniently, economically, and thus may facilitate wide application of cell sheet technology.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Data availability
The datasets generated during the current study are available from the corresponding author on reasonable request.
References
Chen C, Loe F, Blocki A, Peng Y, Raghunath M (2011) Applying macromolecular crowding to enhance extracellular matrix deposition and its remodeling in vitro for tissue engineering and cell-based therapies. Adv Drug Deliv Rev 63:277–290. https://doi.org/10.1016/j.addr.2011.03.003
Enomoto J, Mochizuki N, Ebisawa K, Osaki T, Kageyama T, Myasnikova D, Nittami T, Fukuda J (2016) Engineering thick cell sheets by electrochemical desorption of oligopeptides on membrane substrates. Regen Ther 3:24–31. https://doi.org/10.1016/j.reth.2015.12.003
Gao B, Sakaguchi K, Matsuura K, Ogawa T, Kagawa Y, Kubo H, Shimizu T (2020) In vitro production of human ballooned hepatocytes in a cell sheet-based three-dimensional model. Tissue Eng Part A 26:93–101. https://doi.org/10.1089/ten.tea.2019.0101
Gao B, Sakaguchi K, Ogawa T, Kagawa Y, Kubo H, Shimizu T (2021) Functional analysis of induced human ballooned hepatocytes in a cell sheet-based three dimensional model. Tissue Eng Regen Med 18:217–224. https://doi.org/10.1007/s13770-020-00297-x
Gonçalves AI, Rodrigues MT, Gomes ME (2017) Tissue-engineered magnetic cell sheet patches for advanced strategies in tendon regeneration. Acta Biomater 63:110–122. https://doi.org/10.1016/j.actbio.2017.09.014
Imashiro C, Hirano M, Morikura T, Fukuma Y, Ohnuma K, Kurashina Y, Miyata S, Takemura K (2020) Detachment of cell sheets from clinically ubiquitous cell culture vessels by ultrasonic vibration. Sci Rep 10:9468. https://doi.org/10.1038/s41598-020-66375-1
Kim K, Ohashi K, Utoh R, Kano K, Okano T (2012) Preserved liver-specific functions of hepatocytes in 3D co-culture with endothelial cell sheets. Biomaterials 33:1406–1413. https://doi.org/10.1016/j.biomaterials.2011.10.084
Kim K, Utoh R, Ohashi K, Kikuchi T, Okano T (2017) Fabrication of functional 3D hepatic tissues with polarized hepatocytes by stacking endothelial cell sheets in vitro. J Tissue Eng Regen Med 11:2071–2080. https://doi.org/10.1002/term.2102
Kumar P, Satyam A, Fan X, Collin E, Rochev Y, Rodriguez BJ, Gorelov A, Dillon S, Joshi L, Raghunath M, Pandit A, Zeugolis DI (2015) Macromolecularly crowded in vitro microenvironments accelerate the production of extracellular matrix-rich supramolecular assemblies. Sci Rep 5:8729. https://doi.org/10.1038/srep08729
Marinkovic M, Sridharan R, Santarella F, Smith A, Garlick JA, Kearney CJ (2021) Optimization of extracellular matrix production from human induced pluripotent stem cell-derived fibroblasts for scaffold fabrication for application in wound healing. J Biomed Mater Res A 109:1803–1811. https://doi.org/10.1002/jbm.a.37173
Matsuda N, Shimizu T, Yamato M, Okano T (2007) Tissue engineering based on cell sheet technology. Adv Mater 19:3089–3099. https://doi.org/10.1002/adma.200701978
Miyamoto D, Sakai Y, Huang Y, Yamasaki C, Tateno C, Hasegawa H, Murai T, Hara T, Adachi T, Soyama A, Hidaka M, Ito S, Kanetaka K, Eguchi S (2021) Functional changes of co-cultured hepatocyte sheets subjected to continuous liver regeneration stimulation in cDNA-uPA/SCID mouse: differences in transplantation sites. Regen Ther 18:7–11. https://doi.org/10.1016/j.reth.2021.02.004
Nakamura A, Akahane M, Shigematsu H, Tadokoro M, Morita Y, Ohgushi H, Dohi Y, Imamura T, Tanaka Y (2010) Cell sheet transplantation of cultured mesenchymal stem cells enhances bone formation in a rat nonunion model. Bone 46:418–424. https://doi.org/10.1016/j.bone.2009.08.048
Park S, Jung S, Choi M, Lee M, Choi B, Koh W, Lee S, Hong J (2021) Gelatin MAGIC powder as nutrient-delivering 3D spacer for growing cell sheets into cost-effective cultured meat. Biomaterials 278:121155. https://doi.org/10.1016/j.biomaterials.2021.121155
Sakai Y, Koike M, Kawahara D, Hasegawa H, Murai T, Yamanouchi K, Soyama A, Hidaka M, Takatsuki M, Fujita F, Kuroki T, Eguchi S (2018) Controlled cell morphology and liver-specific function of engineered primary hepatocytes by fibroblast layer cell densities. J Biosci Bioeng 126:249–257. https://doi.org/10.1016/j.jbiosc.2018.02.006
Sasaki D, Matsuura K, Seta H, Haraguchi Y, Okano T, Shimizu T (2018) Contractile force measurement of human induced pluripotent stem cell-derived cardiac cell sheet-tissue. PLoS ONE 13:e0198026. https://doi.org/10.1371/journal.pone.0198026
Satyam A, Kumar P, Fan X, Gorelov A, Rochev Y, Joshi L, Peinado H, Lyden D, Thomas B, Rodriguez B, Raghunath M, Pandit A, Zeugolis D (2014) Macromolecular crowding meets tissue engineering by self-assembly: a paradigm shift in regenerative medicine. Adv Mater 26:3024–3034. https://doi.org/10.1002/adma.201304428
Sekine H, Shimizu T, Dobashi I, Matsuura K, Hagiwara N, Takahashi M, Kobayashi E, Yamato M, Okano T (2011a) Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection. Tissue Eng Part A 17:2973–2980. https://doi.org/10.1089/ten.tea.2010.0659
Sekine W, Haraguchi Y, Shimizu T, Umezawa A, Okano T (2011b) Thickness limitation and cell viability of multi-layered cell sheets and overcoming the diffusion limit by a porous-membrane culture insert. J Biochip Issue Chip 1:1–7. https://doi.org/10.4172/2153-0777.S1-007
Takahashi H, Okano T (2019) Thermally-triggered fabrication of cell sheets for tissue engineering and regenerative medicine. Adv Drug Deliver Rev 138:276–292. https://doi.org/10.1016/j.addr.2019.01.004
Tracy LE, Minasian RA, Caterson EJ (2016) Extracellular matrix and dermal fibroblast function in the healing wound. Adv Wound Care 5:119–136. https://doi.org/10.1089/wound.2014.0561
Wei F, Qu C, Song T, Ding G, Fan Z, Liu D, Liu Y, Zhang C, Shi S, Wang S (2012) Vitamin C treatment promotes mesenchymal stem cell sheet formation and tissue regeneration by elevating telomerase activity. J Cell Physiol 227:3216–3224. https://doi.org/10.1002/jcp.24012
Weinstein GD, Boucek RJ (1960) Collagen and elastin of human dermis. J Invest Dermatol 35:227–229. https://doi.org/10.1038/jid.1960.109
Xiao E, He L, Wu Q, Li J, He Y, Zhao L, Chen S, An J, Liu Y, Chen C, Zhang Y (2017) Microbiota regulates bone marrow mesenchymal stem cell lineage differentiation and immunomodulation. Stem Cell Res Ther 8:213. https://doi.org/10.1186/s13287-017-0670-7
Funding
This research was financially supported by Guangdong Academy of Sciences (Grant No. 2020GDASYL-20200102005; Grant No. 2021GDASYL-20210102004; Grant No. 2022GDASZH-2022010110), Science and Technology Planning Project of Guangdong Province (Grant No. 2020B1111560001) and Project funded by China Postdoctoral Science Foundation (Grant No. 2021M690746).
Author information
Authors and Affiliations
Contributions
SG performed experiments and wrote the manuscript. SW and GL prepared materials, performed experiments and data analysis. JX performed experiments. BG conceived, designed the experiments and modify the manuscript. All authors read and approved the final manuscript.
Corresponding author
Ethics declarations
Conflict of interest
The authors have declared that no conflicts of interest.
Consent to participate
Informed consent was obtained from all individual participants included in the study.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Information
Below is the link to the electronic supplementary material.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Guan, S., Wu, S., Li, G. et al. Macromolecular crowding facilitates rapid fabrication of intact, robust cell sheets. Biotechnol Lett 45, 57–67 (2023). https://doi.org/10.1007/s10529-022-03336-w
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10529-022-03336-w