PERTANIKA JOURNAL OF TROPICAL AGRICULTURAL SCIENCE

Microcarrier-based cell culture systems have gained significant attention and popularity in tissue engineering and regenerative medicine. In this culture system, tissue cells are grown as a monolayer on the surface of small solid particles called microcarriers (100 to 300 μm), kept suspended in the culture medium by stirring. This technology has paved the way for creating engineered tissues, one of the cutting-edge topics in tissue engineering and regenerative medicine. Microcarrier-based approaches have been proposed for three-dimensional (3D) cell culture in which cellular morphology and functions are maintained in vivo. This paper provides an overview of the optimal characteristics such as microcarriers’ size, shape, density and porosity. Various methods of preparation of microcarriers and surface modification techniques have been elaborated. Recent advances and applications of microcarriers in biotechnology fields, like the production of viral vaccines and recombinant proteins, culture and expansion of stem cells (SC), are described.

• Arifin, M. A., Mel, M., Swan, S. Y., Samsudin, N., Hashim, Y. Z., & Salleh, H. M. (2022). Optimization of ultraviolet/ozone (UVO3) process conditions for the preparation of gelatin coated polystyrene (PS) microcarriers. Preparative Biochemistry & Biotechnology, 52(2), 181–196. https://doi.org/10.1080/10826068.2021.1923031

• Azahar, N. I., Mokhtar, N. M., Mahmood, S., Aziz, M. A. A., & Arifin, M. A. (2023). Evaluation of Piper betle L. extracts and its antivirulence activity towards P. aeruginosa. Jurnal Teknologi, 85(1), 133-140. https://doi.org/10.11113/jurnalteknologi.v85.18892

• Badenes, S. M., Fernandes-Platzgummer, A., Rodrigues, C. A. V., Diogo, M. M., da Silva, C. L., & Cabral, J. M. S. (2016). Microcarrier culture systems for stem cell manufacturing. In J. M. S. Cabral, C. L. de Silva, L. G. Chase & M. M. Diogo (Eds.), Stem Cell Manufacturing (pp. 77–104). Elsevier. https://doi.org/10.1016/b978-0-444-63265-4.00004-2

• Burnett, M. J., & Burnett, A. C. (2020). Therapeutic recombinant protein production in plants: Challenges and opportunities. Plants, People, Planet, 2(2), 121–132. https://doi.org/10.1002/ppp3.10073

• Campos, E., Branquinho, J., Carreira, A. S., Carvalho, A., Coimbra, P., Ferreira, P., & Gil, M. H. (2013). Designing polymeric microparticles for biomedical and industrial applications. European Polymer Journal, 49(8), 2005–2021. https://doi.org/10.1016/j.eurpolymj.2013.04.033

• Cer, E., Gürpınar, Ö. A., Onur, M. A., & Tuncel, A. (2007). Polyethylene glycol-based cationically charged hydrogel beads as a new microcarrier for cell culture. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 80(2), 406–414. https://doi.org/10.1002/jbm.b.30611

• Chen, A. K. L., Reuveny, S., & Oh, S. K. W. (2013). Application of human mesenchymal and pluripotent stem cell microcarrier cultures in cellular therapy: Achievements and future direction. Biotechnology Advances, 31(7), 1032-1046. https://doi.org/10.1016/j.biotechadv.2013.03.006

• Chen, X. Y., Chen, J. Y., Tong, X. M., Mei, J. G., Chen, Y. F., & Mou, X. Z. (2020). Recent advances in the use of microcarriers for cell cultures and their ex vivo and in vivo applications. Biotechnology Letters, 42(1), 1-10. https://doi.org/10.1007/s10529-019-02738-7

• Chevalot, I., Visvikis, A., Nabet, P., Engasser, J. M., & Marc, A. (1994). Production of a membrane-bound proteins, the human gamma-glutamyl transferase, by CHO cells cultivated on microcarriers, in aggregates and in suspension. Cytotechnology, 16(2), 121-129. https://doi.org/10.1007/BF00754614

• Chia, M. Y., Chung, W. Y., Wang, C. H., Chang, W. H., & Lee, M. S. (2018). Development of a high-growth enterovirus 71 vaccine candidate inducing cross-reactive neutralizing antibody responses. Vaccine, 36(9), 1167-1173. https://doi.org/10.1016/j.vaccine.2018.01.041

• Clainche, T. L., Moisan, A., Coll, J. L., & Martel-Frachet, V. (2021). The disc-shaped microcarriers: A new tool for increasing harvesting of adipose-derived mesenchymal stromal cells. Biochemical Engineering Journal, 174, Article 108082. https://doi.org/10.1016/j.bej.2021.108082

• Clapp, K. P., Castan, A., & Lindskog, E. K. (2018). Upstream processing equipment. In G. Jagschies, E. Lindskog, K. Łącki & P. Galliher (Eds.), Biopharmaceutical Processing: Development, Design, and Implementation of Manufacturing Processes (pp. 457-476). Elsevier. https://doi.org/10.1016/B978-0-08-100623-8.00024-4

• Clara-Trujillo, S., Marín-Payá, J. C., Cordón, L., Sempere, A., Ferrer, G. G., & Ribelles, J. L. G. (2019). Biomimetic microspheres for 3D mesenchymal stem cell culture and characterization. Colloids and Surfaces B: Biointerfaces, 177, 68-76. https://doi.org/10.1016/j.colsurfb.2019.01.050

• Croughan, M. S., Hamel, J. F. P., & Wang, D. I. (1988). Effects of microcarrier concentration in animal cell culture. Biotechnology and Bioengineering, 32(8), 975-982. https://doi.org/10.1002/bit.260320805

• Dashtimoghadam, E., Fahimipour, F., Tongas, N., & Tayebi, L. (2020). Microfluidic fabrication of microcarriers with sequential delivery of VEGF and BMP-2 for bone regeneration. Scientific Reports, 10(1), Article 11764. https://doi.org/10.1038/s41598-020-68221-w

• Ding, S. L., Liu, X., Zhao, X. Y., Wang, K. T., Xiong, W., Gao, Z. L., Sun, C. Y., Jia, M. X., Li, C., Gu, Q., & Zhang, M. Z. (2022). Microcarriers in application for cartilage tissue engineering: Recent progress and challenges. Bioactive Materials, 17, 81-108. https://doi.org/10.1016/j.bioactmat.2022.01.033

• Eisenkraetzer, D. (2014). 6.1 Bioreactors for animal cell culture. In H. Hauser & R. Wagner (Eds.), Animal Cell Biotechnology (pp. 389–426). De Gruyter. https://doi.org/10.1515/9783110278965.389

• Fliedl, L., & Kaisermayer, C. (2014). Scalable transient gene expression in adherent mammalian cells using polyethylenimine. In R. Pörtner (Ed.), Animal Cell Biotechnology: Methods and Protocols (pp. 29–34). Springer https://doi.org/10.1007/978-1-62703-733-4_3

• Frey, S. J., Hoffman, A. S., Hubbell, J. A., & Kane, R. S. (2020). Surface-immobilized biomolecules. In Biomaterials Science (pp. 539-551). Academic Press. https://doi.org/10.1016/b978-0-12-816137-1.00036-2

• Goodwin, T. J., McCarthy, M., Cohrs, R. J., & Kaufer, B. B. (2015). 3D tissue-like assemblies: A novel approach to investigate virus–cell interactions. Methods, 90, 76–84. https://doi.org/10.1016/j.ymeth.2015.05.010

• Govindarajan, T., & Shandas, R. (2014). A survey of surface modification techniques for next-generation shape memory polymer stent devices. Polymers, 6(9), 2309–2331. https://doi.org/10.3390/polym6092309

• Gümüşderelioğlu, M., Çakmak, S., Timuçin, H. Ö., & Çakmak, A. S. (2013). Thermosensitive Phema Microcarriers: ATRP synthesis, characterization, and usabilities in cell cultures. Journal of Biomaterials Science, Polymer Edition, 24(18), 2110–2125. https://doi.org/10.1080/09205063.2013.827104

• Guo, J., Li, K., Ning, C., & Liu, X. (2020). Improved cellular bioactivity by heparin immobilization on polycarbonate film via an aminolysis modification for potential tendon repair. International Journal of Biological Macromolecules, 142, 835–845. https://doi.org/10.1016/j.ijbiomac.2019.09.136

• Heathman, T. R. J., Nienow, A. W., Rafiq, Q. A., Coopman, K., Kara, B., & Hewitt, C. J. (2018). Agitation and aeration of stirred-bioreactors for the microcarrier culture of human mesenchymal stem cells and potential implications for large-scale bioprocess development. Biochemical Engineering Journal, 136, 9–17. https://doi.org/10.1016/j.bej.2018.04.011

• Holmes, C., & Tabrizian, M. (2015). Surface functionalization of Biomaterials. In A. Vishwakarma, P. Sharpe, S. Shi & M. Ramlingam (Eds.), Stem Cell Biology and Tissue Engineering in Dental Sciences (pp. 187–206). Academic Press. https://doi.org/10.1016/b9780-12-397157-9.00016-3

• Hossain, K. M. Z., Patel, U., & Ahmed, I. (2015). Development of microspheres for biomedical applications: A Review. Progress in Biomaterials, 4(1), 1–19. https://doi.org/10.1007/s40204-014-0033-8

• Huang, L., Abdalla, A. M. E., Xiao, L., & Yang, G. (2020). Biopolymer-based microcarriers for three-dimensional cell culture and engineered tissue formation. International Journal of Molecular Sciences, 21(5), Article 1895. https://doi.org/10.3390/ijms21051895

• Huang, L., Xiao, L., Jung Poudel, A., Li, J., Zhou, P., Gauthier, M., Liu, H., Wu, Z., & Yang, G. (2018). Porous chitosan microspheres as microcarriers for 3D cell culture. Carbohydrate Polymers, 202, 611–620. https://doi.org/10.1016/j.carbpol.2018.09.021

• Ismail, N. A., Abd Aziz, M. A., Hisyam, A., & Abidin, M. A. (2021). Separation of samarium from medium rare earth mixture using multi-stage counter-current extraction. Chemical Engineering Communications, 208(5), 764–774. https://doi.org/10.1080/00986445.2020.1746654

• Kankala, R. K., Zhao, J., Liu, C., Song, X., Yang, D., Zhu, K., Wang, S., Zhang, Y. S., & Chen, A. (2019). Highly porous microcarriers for minimally invasive in situ skeletal muscle cell delivery. Small, 15(25), Article 1901397. https://doi.org/10.1002/smll.201901397

• Kiesslich, S., Losa, J. P. V. C., Gélinas, J. F., & Kamen, A. A. (2020). Serum-free production of rVSV-Zebov in Vero Cells: Microcarrier Bioreactor versus scale-XTM hydro fixed-bed. Journal of Biotechnology, 310, 32–39. https://doi.org/10.1016/j.jbiotec.2020.01.015

• Kuang, P., & Constant, K. (2015). Increased wettability and surface free energy of polyurethane by ultraviolet ozone treatment. In M. Aliofkhazraei (Ed.), Wetting and Wettability (pp. 85-102). InTech. https://doi.org/10.5772/60798

• Kumar, A., & Starly, B. (2015). Large scale industrialized cell expansion: Producing the critical raw material for Biofabrication processes. Biofabrication, 7(4), Article 044103. https://doi.org/10.1088/1758-5090/7/4/044103

• Lagreca, E., Onesto, V., Di Natale, C., La Manna, S., Netti, P. A., & Vecchione, R. (2020). Recent advances in the formulation of PLGA microparticles for controlled drug delivery. Progress in Biomaterials, 9(4), 153–174. https://doi.org/10.1007/s40204-020-00139-y

• Lai, J. Y., & Ma, D. H. K. (2017). Ocular biocompatibility of gelatin microcarriers functionalized with oxidized hyaluronic acid. Materials Science and Engineering: C, 72, 150–159. https://doi.org/10.1016/j.msec.2016.11.067

• Laput, O. A., Vasenina, I. V., Shapovalova, Y. G., Ochered’ko, A. N., Chernyavskii, A. V., Kudryashov, S. V., & Kurzina, I. A. (2022). Low-temperature barrier discharge plasma modification of scaffolds based on polylactic acid. ACS Applied Materials & Interfaces, 14(37), 41742–41750. https://doi.org/10.1021/acsami.2c11027

• Levato, R., Planell, J. A., Mateos-Timoneda, M. A., & Engel, E. (2015). Role of ECM/peptide coatings on SDF-1α triggered mesenchymal stromal cell migration from microcarriers for cell therapy. Acta Biomaterialia, 18, 59–67. https://doi.org/10.1016/j.actbio.2015.02.008

• Li, J., Lam, A. T. L., Toh, J. P., Reuveny, S., Oh, S. K. W., & Birch, W. R. (2017). Tunable volumetric density and porous structure of spherical poly-ε-caprolactone microcarriers, as applied in human mesenchymal stem cell expansion. Langmuir, 33(12), 3068–3079. https://doi.org/10.1021/acs.langmuir.7b00125

• Luo, X., Niu, Y., Fu, X., Lin, Q., Liang, H., Liu, L., & Li, N. (2021). Large-scale microcarrier culture of Chinese perch brain cell for viral vaccine production in a stirred bioreactor. Vaccines, 9(9), Article 1003. https://doi.org/10.3390/vaccines9091003

• Ma, Z., Gao, C., Ji, J., & Shen, J. (2002). Protein immobilization on the surface of poly-Llactic acid films for improvement of cellular interactions. European Polymer Journal, 38(11), 2279–2284. https://doi.org/10.1016/s0014-3057(02)00119-2

• Maillot, C., Isla, N. D., Loubiere, C., Toye, D., & Olmos, E. (2022). Impact of microcarrier concentration on mesenchymal stem cell growth and death: Experiments and modeling. Biotechnology and Bioengineering, 119(12), 3537–3548. https://doi.org/10.1002/bit.28228

• Mattiasson, B. (2018). Immobilized cells and organelles: Volume I. CRC Press. https://doi.org/10.1201/9781351073394

• Mattos, D. A., Silva, M. V., Gaspar, L. P., & Castilho, L. R. (2015). Increasing vero viable cell densities for yellow fever virus production in stirred-tank bioreactors using serum-free medium. Vaccine, 33(35), 4288–4291. https://doi.org/10.1016/j.vaccine.2015.04.050

• May, C. P. (2016). The study and fabrication of a novel thermally responsive microcarrier for cell culture application [Unpublish doctoral thesis]. University of Nottingham, England.

• Meiser, I., Majer, J., Katsen-Globa, A., Schulz, A., Schmidt, K., Stracke, F., Koutsouraki, E., Witt, G., Keminer, O., Pless, O., Gardner, J., Claussen, C., Gribbon, P., Neubauer, J. C., & Zimmermann, H. (2021). Droplet-based vitrification of adherent human induced pluripotent stem cells on alginate microcarrier influenced by adhesion time and matrix elasticity. Cryobiology, 103, 57-69. https://doi.org/10.1016/j.cryobiol.2021.09.010

• Merten, O. W. (2015). Advances in cell culture: Anchorage dependence. Philosophical Transactions of the Royal Society B: Biological Sciences, 370(1661), Article 20140040. https://doi.org/10.1098/rstb.2014.0040

• Minati, L., Migliaresi, C., Lunelli, L., Viero, G., Dalla Serra, M., & Speranza, G. (2017). Plasma assisted surface treatments of biomaterials. Biophysical Chemistry, 229, 151–164. https://doi.org/10.1016/j.bpc.2017.07.003

• Mohamad, N. R., Marzuki, N. H., Buang, N. A., Huyop, F., & Wahab, R. A. (2015). An overview of technologies for immobilization of enzymes and surface analysis techniques for immobilized enzymes. Biotechnology & Biotechnological Equipment, 29(2), 205–220. https://doi.org/10.1080/13102818.2015.1008192

• Mozaffari, A., Gashti, M. P., Mirjalili, M., & Parsania, M. (2021). Argon and argon–oxygen plasma surface modification of gelatin nanofibers for tissue engineering applications. Membranes, 11(1), Article 31. https://doi.org/10.3390/membranes11010031

• Nikolova, M. P., & Chavali, M. S. (2019). Recent advances in biomaterials for 3D scaffolds: A review. Bioactive Materials, 4, 271–292. https://doi.org/10.1016/j.bioactmat.2019.10.005

• Omrani, M. M., Kumar, H., Mohamed, M. G., Golovin, K., S. Milani, A., Hadjizadeh, A., & Kim, K. (2020). Polyether ether ketone surface modification with plasma and gelatin for enhancing cell attachment. Journal of Biomedical Materials Research Part B: Applied Biomaterials, 109(5), 622–629. https://doi.org/10.1002/jbm.b.34726

• Ornelas-González, A., González-González, M., & Rito-Palomares, M. (2021). Microcarrier-based stem cell bioprocessing: GMP-grade culture challenges and future trends for regenerative medicine. Critical Reviews in Biotechnology, 41(7), 1081–1095. https://doi.org/10.1080/07388551.2021.1898328

• Özçam, A. E., Efimenko, K., & Genzer, J. (2014). Effect of ultraviolet/ozone treatment on the surface and bulk properties of poly (dimethyl siloxane) and poly (vinylmethyl siloxane) networks. Polymer, 55(14), 3107–3119. https://doi.org/10.1016/j.polymer.2014.05.027

• Park, Y., Chen, Y., Ordovas, L., & Verfaillie, C. M. (2014). Hepatic differentiation of human embryonic stem cells on microcarriers. Journal of Biotechnology, 174, 39–48. https://doi.org/10.1016/j.jbiotec.2014.01.025

• Pörtner, R. (2015). Bioreactors for mammalian cells. In M. Al-Rubeai (Ed.), Animal Cell Culture (pp. 89–135). Springer. https://doi.org/10.1007/978-3-319-10320-4_4

• Rafiq, Q. A., Ruck, S., Hanga, M. P., Heathman, T. R. J., Coopman, K., Nienow, A. W., Williams, D. J., & Hewitt, C. J. (2018). Qualitative and quantitative demonstration of beadto-bead transfer with bone marrow-derived human mesenchymal stem cells on microcarriers: Utilising the phenomenon to improve culture performance. Biochemical Engineering Journal, 135, 11–21. https://doi.org/10.1016/j.bej.2017.11.005

• Ravikumar, M. N. V. (2016). Handbook of polyester drug delivery systems. CRC Press.

• Recek, N., Resnik, M., Motaln, H., Lah-Turnšek, T., Augustine, R., Kalarikkal, N., Thomas, S., & Mozetič, M. (2016). Cell adhesion on polycaprolactone modified by plasma treatment. International Journal of Polymer Science, 2016, Article 7354396. https://doi.org/10.1155/2016/7354396

• Reddy, M. S., Ponnamma, D., Choudhary, R., & Sadasivuni, K. K. (2021). A comparative review of natural and synthetic biopolymer composite scaffolds. Polymers, 13(7), Article 1105. https://doi.org/10.3390/polym13071105

• Samsudin, N., Hashim, Y. Z., Arifin, M. A., & Salleh, H. M. (2018). Surface modification of microcporous of polycaprolactone (PCL) microcarrier to improve Microcarrier biocompatibility. International Journal on Advanced Science, Engineering and Information Technology, 8(4–2), Article 1642. https://doi.org/10.18517/ijaseit.8.4-2.7060

• Saralidze, K., Koole, L. H., & Knetsch, M. L. W. (2010). Polymeric microspheres for medical applications. Materials, 3(6), 3537–3564. https://doi.org/10.3390/ma3063537

• Sengupta, P., & Prasad, B. L. V. (2018). Surface modification of polymers for tissue engineering applications: Arginine acts as a sticky protein equivalent for viable cell accommodation. ACS Omega, 3(4), 4242-4251. https://doi.org/10.1021/acsomega.8b00215

• Shahrifi, B. H., Mohammadi, M., Manoochehri, M., & Atashi, A. (2020). Mechanical and biological properties of polycaprolactone/fibrin nanocomposite adhesive produced by electrospinning method. Bulletin of Materials Science, 43(1), Article 135. https://doi.org/10.1007/s12034-020-02111-9

• Shi, X., Cui, L., Sun, H., Jiang, N., Heng, L., Zhuang, X., Gan, Z., & Chen, X. (2019). Promoting cell growth on porous PLA microspheres through simple degradation methods. Polymer Degradation and Stability, 161, 319–325. https://doi.org/10.1016/j.polymdegradstab.2019.01.003

• Shirokaze, J., Yanagida, K., Shudo, K., Konomoto, K., Kamiya, K., & Sagara, K. (1995). IL-4 production using macroporous microcarrier. In E. C. Beuvery, J. B. Griffiths & W. P. Zeijlemaker (Eds.), Animal Cell Technology: Developments Towards the 21st Century (pp. 877–881). Springer. https://doi.org/10.1007/978-94-011-0437-1_141

• Sia, Y. S., Azahar, N. I., Aziz, M. A. A., & Arifin, M. A. (2023). Sequential adaptation to Serum-free medium for Vero cells cultivation on ultraviolet/ozone (UVO) treated microcarrier. Materials Today: Proceedings. https://doi.org/10.1016/j.matpr.2023.08.031

• Silva, A. C., Roldão, A., Teixeira, A., Fernandes, P., Sousa, M. F., & Alves, P. M. (2015). Cell immobilization for the production of viral vaccines. In M. Al-Rubeai (Ed.), Animal Cell Engineering (pp. 541–563). Springer. https://doi.org/10.1007/978-3-319-10320-4_17

• Silva, C. L. D., Carmelo, J. G., Fernandes-Platzgummer, A., Weber, J. L., Bear, M., Hervy, M., Diogo, M. M., & Cabral, J. S. (2014). Scalable production of human mesenchymal stem/stromal cells in microcarrier-based culture systems. Cytotherapy, 16(4), S101-S102. https://doi.org/10.1016/j.jcyt.2014.01.377

• Suzuki, H., Kasai, K., Kimura, Y., & Miyata, S. (2021). UV/ozone surface modification combined with atmospheric pressure plasma irradiation for cell culture plastics to improve pluripotent stem cell culture. Materials Science and Engineering: C, 123, Article 112012. https://doi.org/10.1016/j.msec.2021.112012

• Syromotina, D. S., Surmenev, R. A., Surmeneva, M. A., Boyandin, A. N., Epple, M., Ulbricht, M., Oehr, C., & Volova, T. G. (2016). Oxygen and ammonia plasma treatment of poly(3-hydroxybutyrate) films for controlled surface zeta potential and improved cell compatibility. Materials Letters, 163, 277–280. https://doi.org/10.1016/j.matlet.2015.10.080

• Tavassoli, H., Alhosseini, S. N., Tay, A., Chan, P. P. Y., Weng Oh, S. K., & Warkiani, M. E. (2018). Large-scale production of stem cells utilizing microcarriers: A biomaterials engineering perspective from academic research to commercialized products. Biomaterials, 181, 333–346. https://doi.org/10.1016/j.biomaterials.2018.07.016

• Tham, C. Y., Hamid, Z. A., Ahmad, Z. A., & Ismail, H. (2014). Surface engineered poly (lactic acid) (PLA) microspheres by chemical treatment for drug delivery system. Key Engineering Materials, 594–595, 214–218. https://doi.org/10.4028/www.scientific.net/kem.594-595.214

• Tharmalingam, T., Sunley, K., Spearman, M., & Butler, M. (2011). Enhanced production of human recombinant proteins from CHO cells grown to high densities in macroporous microcarriers. Molecular Biotechnology, 49(3), 263–276. https://doi.org/10.1007/s12033011-9401-y

• Thompson, M., Giuffre, A., McClenny, C., & Dyke, M. V. (2020). A keratin-based microparticle for cell delivery. Journal of Biomaterials Applications, 35(6), 579–591. https://doi.org/10.1177/0885328220951892

• Trabelsi, K., Zakour, M. B., & Kallel, H. (2019). Purification of rabies virus produced in vero cells grown in serum free medium. Vaccine, 37(47), 7052–7060. https://doi.org/10.1016/j.vaccine.2019.06.072

• Tsai, A. C., Jeske, R., Chen, X., Yuan, X., & Li, Y. (2020). Influence of microenvironment on mesenchymal stem cell therapeutic potency: From planar culture to microcarriers. Frontiers in Bioengineering and Biotechnology, 8, Article 640. https://doi.org/10.3389/fbioe.2020.00640

• Verma, A., Verma, M., & Singh, A. (2020). Animal tissue culture principles and applications. In S. Verma & A. Singh (Eds.), Animal Biotechnology (pp. 269–293). Academic Press. https://doi.org/10.1016/b978-0-12-8117101.00012-4

• Wezel, V. A. L. (1967). Growth of cell-strains and primary cells on microcarriers in homogeneous culture. Nature, 216(5110), 64–65. https://doi.org/10.1038/216064a0

• Wieland, F., Bruch, R., Bergmann, M., Partel, S., Urban, G. A., & Dincer, C. (2020). Enhanced protein immobilization on polymers — A plasma surface activation study. Polymers, 12(1), Article 104. https://doi.org/10.3390/polym12010104

• Yang, L., Zhang, J., He, J., Zhang, J., & Gan, Z. (2016). Fabrication, hydrolysis and cell cultivation of microspheres from cellulose-graft-poly(L-lactide) copolymers. RSC Advances, 6(21), 17617–17623. https://doi.org/10.1039/c5ra25993b

• Yusilawati, A. N., Maizirwan, M., Hamzah, M. S., Ng, K. H., & Wong, C. S. (2010). Surface modification of polystyrene beads by ultraviolet/ozone treatment and its effect on gelatin coating. American Journal of Applied Sciences, 7(6), 724–731. https://doi.org/10.3844/ajassp.2010.724.731

• Zheng, P., Yao, Q., Mao, F., Liu, N., Xu, Y., Wei, B., & Wang, L. (2017). Adhesion, proliferation and osteogenic differentiation of mesenchymal stem cells in 3D printed poly-εcaprolactone/hydroxyapatite scaffolds combined with bone marrow clots. Molecular Medicine Reports, 16(4), 5078–5084. https://doi.org/10.3892/mmr.2017.7266

• Zhou, A., Ye, Z., Zhou, Y., & Tan, W. (2019). Bioactive poly(ε-caprolactone) microspheres with tunable open pores as microcarriers for tissue regeneration. Journal of Biomaterials Applications, 33(9), 1242–1251. https://doi.org/10.1177/0885328218825371