Abstract
Here, we present a novel approach to form hydrogels from yeast whole cell protein. Countless hydrogels are available for sophisticated research, but their fabrication is often difficult to reproduce, with the gels being complicated to handle or simply too expensive. The yeast hydrogels presented here are polymerized using a four-armed, amine reactive crosslinker and show a high chemical and thermal resistance. The free water content was determined by measuring swelling ratios for different protein concentrations, and in a freeze-drying approach, pore sizes of up to 100 μm in the gel could be created without destabilizing the 3D network. Elasticity was proofed to be adjustable with the help of atomic force microscopy by merely changing the amount of used protein. Furthermore, the material was tested for possible cell culture applications; diffusion rates in the network are high enough for sufficient supply of human breast cancer cells and adenocarcinomic human alveolar basal epithelial cells with nutrition, and cells showed high viabilities when tested for compatibility with the material. Furthermore, hydrogels could be functionalized with RGD peptide and the optimal concentration for sufficient cell adhesion was determined to be 150 μM. Given that yeast protein is one of the cheapest and easiest available protein sources and that hydrogels are extremely easy to handle, the developed material has highly promising potential for both sophisticated cell culture techniques as well as for larger scale industrial applications.
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References
Annabi N, Nichol JW, Zhong X, Ji C, Koshy S, Khademhosseini A, Dehghani F (2010) Controlling the porosity and microarchitecture of hydrogels for tissue engineering. Tissue Eng Part B Rev 16:371–383. doi:10.1089/ten.teb.2009.0639
Ashley GW, Henise J, Reid R, Santi DV (2013) Hydrogel drug delivery system with predictable and tunable drug release and degradation rates. Proc Natl Acad Sci U S A 110:2318–2323. doi:10.1073/pnas.1215498110
Bellis SL (2012) Advantages of RGD peptides for directing cell association with biomaterials. Biomaterials 32:4205–4210. doi:10.1016/j.2011.02.029
Bhattarai N, Gunn J, Zhang M (2010) Chitosan-based hydrogels for controlled, localized drug delivery. Adv Drug Deliv Rev 62:83–99. doi:10.1016/j.addr.2009.07.019
Bodenberger N, Paul P, Kubiczek D, Walther P, Gottschalk K, Rosenau F (2016b) A novel cheap and easy to handle protein hydrogel for 3D cell culture applications: a high stability matrix with tunable elasticity and cell adhesion properties. Chemisrty Sel 1:1353–1360. doi:10.1002/slct.201600206
Bodenberger N, Kubiczek D, Abrosimova I, Scharm A, Kipper F, Walther P, Rosenau F (2016a) Evaluation of methods for pore generation and their influence on physio-chemical properties of a protein based hydrogel. Biotech Reports 12(2016b):6–12. doi:10.1016/j.btre.2016.09.001
Bonnier F, Keating ME, Wróbel TP, Majzner K, Baranska M, Garcia-Munoz A, Blanco A, Byrne HJ (2015) Cell viability assessment using the Alamar blue assay: a comparison of 2D and 3D cell culture models. Toxicol in Vitro 29:124–131. doi:10.1016/j.tiv.2014.09.014
Bosman FT, Stamenkovic I (2003) Functional structure and composition of the extracellular matrix. J Pathol 200:423–428. doi:10.1002/path.1437
Byrne B (2015) Pichia pastoris as an expression host for membrane protein structural biology. Curr Opin Struct Biol 32C:9–17. doi:10.1016/j.sbi.2015.01.005
Chung C, Lampe KJ, SCH (2008) Tetrakis (hydroxylmethyl) phosphonium chloride as a covalent crosslinking agent for cell encapsulation within protein-based hydrogels. Biomacromolecules 13(12):3912–3916
Ciofalo V, Barton N, Kreps J, Coats I, Shanahan D (2006) Safety evaluation of a lipase enzyme preparation, expressed in Pichia pastoris, intended for use in the degumming of edible vegetable oil. Regul Toxicol Pharmacol 45:1–8. doi:10.1016/j.yrtph.2006.02.001
De Jong SJ, De Smedt SC, Demeester J, Van Nostrum CF, Kettenes-van den Bosch JJ, Hennink WE (2001) Biodegradable hydrogels based on stereocomplex formation between lactic acid oligomers grafted to dextran. J Control Release 72:47–56. doi:10.1016/S0168-3659(01)00261-9
Duan H, Umar S, Xiong R, Chen J (2011) New strategy for expression of recombinant hydroxylated human-derived gelatin in Pichia pastoris KM71. J Agric Food Chem 59:7127–7134. doi:10.1021/jf200778r
Fang J, Mehlich A, Koga N, Huang J, Koga R, Gao X, Hu C, Jin C, Rief M, Kast J, Baker D, Li H (2013) Forced protein unfolding leads to highly elastic and tough protein hydrogels. Nat Commun 4:2974. doi:10.1038/ncomms3974
Fechine GJM, Barros JAG, Catalani LH (2004) Poly(N-vinyl-2-pyrrolidone) hydrogel production by ultraviolet radiation: new methodologies to accelerate crosslinking. Polymer (Guildf) 45:4705–4709. doi:10.1016/j.polymer.2004.05.006
Gonçalves MAD, Pinto VD, Costa RAS, Dias RCS, Hernándes-Ortiz JC, Costa MRPFN (2013) Stimuli-responsive hydrogels synthesis using free radical and RAFT polymerization. Macromol Symp 333:41–54. doi:10.1002/masy.201300045
Geckil H, Xu F, Zhang X, Moon SJ, Demirci U (2011) Engineering hydrogels as extracellular matrix mimics. Nanomedicine 5:469–484. doi:10.2217/nnm.10.12.Engineering
Hennink WE, van Nostrum CF (2012) Novel crosslinking methods to design hydrogels. Adv Drug Deliv Rev 64:223–236. doi:10.1016/j.addr.2012.09.009
Hoare TR, Kohane DS (2008) Hydrogels in drug delivery: progress and challenges. Polymer (Guildf) 49:1993–2007. doi:10.1016/j.polymer.2008.01.027
Ito F, Usui K, Kawahara D, Suenaga A, Maki T, Kidoaki S, Suzuki H, Taiji M, Itoh M, Hayashizaki Y, Matsuda T (2010) Reversible hydrogel formation driven by protein-peptide-specific interaction and chondrocyte entrapment. Biomaterials 31:58–66. doi:10.1016/j.biomaterials.2009.09.026
Jeong JH, Schmidt JJ, Cha C, Kong H (2010) Tuning responsiveness and structural integrity of a pH responsive hydrogel using a poly(ethylene glycol) cross-linker. Soft Matter 6:3930. doi:10.1039/c0sm00094a
Jonker AM, Lo DWPM, Hest JCM Van (2012) Peptide- and protein-based hydrogels. doi: 10.1021/cm202640w
Li J, Suo Z, Vlassak JJ (2014) Stiff, strong, and tough hydrogels with good chemical stability. J Mater Chem B 2:6708–6713. doi:10.1039/C4TB01194E
Li R-K, Chen P, Ng TB, Yang J, Lin J, Yan F, Ye X-Y (2015) Highly efficient expression and characterization of a β-mannanase from Bacillus subtilis in Pichia pastoris. Biotechnol Appl Biochem 62:64–70. doi:10.1002/bab.1250
Li XJ, Valadez AV, Zuo P, Nie Z (2012) Microfluidic 3D cell culture: potential application for tissue-based bioassays. Bioanalysis 4:1509–1525. doi:10.4155/bio.12.133
Mason BN, Califano JP, Reinhart-king CA (2012) Engineering biomaterials for regenerative medicine. 19–38. doi: 10.1007/978–1–4614-1080-5
Missirlis D, Spatz JP (2014) Combined effects of PEG hydrogel elasticity and cell-adhesive coating on fibroblast adhesion and persistent migration. Biomacromolecules 15:195–205. doi:10.1021/bm4014827
Moreira Teixeira LS, Feijen J, van Blitterswijk CA, Dijkstra PJ, Karperien M (2012) Enzyme-catalyzed crosslinkable hydrogels: emerging strategies for tissue engineering. Biomaterials 33:1281–1290. doi:10.1016/j.biomaterials.2011.10.067
Okay O (2009) General properties of hydrogels. Springer Ser Chem Sensors Biosens 6:1–15. doi:10.1007/b100321
Park H, Guo X, Temenoff JS, Tabata Y, Caplan AI, Kasper FK, Mikos AG, Box PO (2010) Effect of swelling ratio of injectable hydrogel composites on chondrogenic differentiation of encapsulated rabbit marrow mesenchymal stem cells in vitro. Biomacromolecules 10:541–546. doi:10.1021/bm801197m.Effect
Pfister PM, Wendlandt M, Neuenschwander P, Suter UW (2007) Surface-textured PEG-based hydrogels with adjustable elasticity: synthesis and characterization. Biomaterials 28:567–575. doi:10.1016/j.biomaterials.2006.09.016
Raic A, Rödling L, Kalbacher H, Lee-Thedieck C (2014) Biomimetic macroporous PEG hydrogels as 3D scaffolds for the multiplication of human hematopoietic stem and progenitor cells. Biomaterials 35:929–940. doi:10.1016/j.biomaterials.2013.10.038
Raja STK, Thiruselvi T, Mandal AB, Gnanamani A (2015) pH and redox sensitive albumin hydrogel: a self-derived biomaterial. Sci Rep 5:15977. doi:10.1038/srep15977
Ruoslahti E, Pierschbacher MD (1987) New perspectives in cell adhesion: RGD and integrins. Science 238:491–497. doi:10.1126/science.2821619
Rutz AL, Hyland KE, Jakus AE, Burghardt WR, Shah RN (2015) A multimaterial bioink method for 3D printing tunable, cell-compatible hydrogels. Adv Mater 27:1607–1614. doi:10.1002/adma.201405076
Spohner SC, Müller H, Quitmann H, Czermak P (2015) Expression of enzymes for the usage in food and feed industry with Pichia pastoris. J Biotechnol 202:118–134. doi:10.1016/j.jbiotec.2015.01.027
Van Vlierberghe S, Dubruel P, Schacht E (2011) Biopolymer-based hydrogels as scaffolds for tissue engineering applications: a review. Biomacromolecules 12:1387–1408. doi:10.1021/bm200083n
Wang Y, Halls C, Zhang J, Matsuno M, Zhang Y, Yu O (2011) Stepwise increase of resveratrol biosynthesis in yeast Saccharomyces cerevisiae by metabolic engineering. Metab Eng 13:455–463. doi:10.1016/j.ymben.2011.04.005
Weinacker D, Rabert C, Zepeda AB, Figueroa CA, Pessoa A, Faras JG (2013) Applications of recombinant Pichia pastoris in the healthcare industry. Brazilian J Microbiol 44:1043–1048. doi:10.1590/S1517-83822013000400004
Acknowledgements
N.B. and F.R. thank the “Baden-Württemberg Stiftung” and the “Bundesministerium für Bildung und Forschung” (BMBF) for their financial support in the framework “Bioinspired Materials Synthesis.”
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This study was funded by the Baden-Württemberg Stiftung in the program “Bioinspirierte Materialsynthese” (BioMatS-14).
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Bodenberger, N., Kubiczek, D., Paul, P. et al. Beyond bread and beer: whole cell protein extracts from baker’s yeast as a bulk source for 3D cell culture matrices. Appl Microbiol Biotechnol 101, 1907–1917 (2017). https://doi.org/10.1007/s00253-016-7982-x
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DOI: https://doi.org/10.1007/s00253-016-7982-x