Comparative evaluation of macroscopic carriers for Vero cell expansion: Influence of surface properties, incubation parameters, and nutrient media: Macroscopic carriers for Vero cell expansion

Tolganai Imanbekova; Zhanat Batanova; Zhanat Abdraimova; Karlygash Zikibayeva; Diana Amankeldi

doi:10.62310/liab.v6i2.337

Authors

Tolganai Imanbekova Laboratory of Cellular Biotechnology Research and Production Enterprise “Antigen” Karasai, Almaty, 040905, Republic of Kazakhstan https://orcid.org/0009-0003-0379-6235
Zhanat Batanova Laboratory of Virology Research and Production Enterprise “Antigen” Karasai, Almaty, 040905, Republic of Kazakhstan https://orcid.org/0009-0000-6307-0581
Zhanat Abdraimova Department of Biological Control Research and Production Enterprise “Antigen” Karasai, Almaty, 040905, Republic of Kazakhstan https://orcid.org/0009-0005-9619-6750
Karlygash Zikibayeva Laboratory of Molecular Biology Research and Production Enterprise “Antigen” Karasai, Almaty, 040905, Republic of Kazakhstan https://orcid.org/0009-0001-7737-4217
Diana Amankeldi Laboratory of Cellular Biotechnology Research and Production Enterprise “Antigen” Karasai, Almaty, 040905, Republic of Kazakhstan https://orcid.org/0009-0009-9873-3096

DOI:

https://doi.org/10.62310/liab.v6i2.337

Keywords:

Vero cell culture, Macroscopic carriers, Adhesion, Incubation conditions, Viability

Abstract

The aim of this study was to identify optimal conditions for maximising biomass yield and preserving the viability of Vero cells cultured on different macroscopic carriers. The macroscopic carriers, including cellulose, composite, polystyrene (standard and modified), and polyethylene, were analysed in terms of adhesion, cell mass accumulation, doubling time, and morphological stability as well as the effects of incubation parameters and nutrient medium composition. After 48 h of cultivation, cellulose and composite carriers showed the highest adhesion levels (90-94%), followed by modified and standard polystyrene carriers (76-92%), whereas polyethylene carriers were the least effective (50-55%). Optimal growth was observed at 37°C, 21% O2, and pH 7.2, resulting in a 3.5-fold increase in cell density (4.2 x 105/cm2) and viability of 95-96%. Moderate deviations from these conditions led to reduced cell growth (2.6-3.2-fold; 3.3-4.0 x 105 /cm2) and viability (89-93%). However, extreme deviations (35 °C, 15% O2, pH 6.8-7.0) significantly reduced growth to 2.1-2.4-fold, with cell densities of 2.6-2.8 x 105 /cm2 and viability of 83-85%. The nutrient medium composition significantly influenced culture performance; the addition of L-glutamine (4 mmol/l) resulted in the highest cell biomass (4.6-fold) with final cell density of 9.2×105/cm2 and viability of 95% after 48 h. In contrast, glucose deficiency or serum deprivation reduced cell growth (2.0-2.5-fold) and cell layer uniformity. A comparison of cultivation systems showed that macroscopic carriers provided 20-25% greater adhesion levels and effective surface coverage compared to suspension culture. The study demonstrated that cellulose and composite carriers provided the highest adhesion and cell viability, while optimal growth was achieved at 37 °C, 21% O2, and pH 7.2. Nutrient medium composition, particularly L-glutamine supplementation, further enhanced cell proliferation and final cell density.

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References

Bastin G, Chotteau V, Wouwer AV. (2021). Metabolic flux analysis of Vero cells under various culture conditions. Processes 9(12): 2097. https://doi.org/10.3390/pr9122097

Chen Y, Shen Y, Fang C, Chan T, Wu S, Wang J, Wu S, Liu C. (2024). Enhanced production of recombinant coxsackievirus A16 using a serum-free HEK293A suspension culture system for bivalent enterovirus vaccine development. Vaccine X 20: 100559. https://doi.org/10.1016/j.jvacx.2024.100559

Eckhardt D, Mueller J, Friedrich J, Klee J, Sardlishvili I, Walter LE, Fey S, Czermak P, Salzig D. (2024). Production of oncolytic measles virus in Vero cells: Impact of culture medium and multiplicity of infection. Viruses 16(11): 1740. https://doi.org/10.3390/v16111740

Fang Z, Lyu J, Li J, Li C, Zhang Y, Guo Y, Wang Y, Zhang Y, Chen K. (2022). Application of bioreactor technology for cell culture-based viral vaccine production: Present status and future prospects. Frontiers in Bioengineering and Biotechnology 10: 921755. https://doi.org/10.3389/fbioe.2022.921755

Ge C, Selvaganapathy PR, Geng F. (2023). Advancing our understanding of bioreactors for industrial-sized cell culture: Health care and cellular agriculture implications. American Journal of Physiology – Cell Physiology 325(3): C580-C591. https://doi.org/10.1152/ajpcell.00408.2022

Gobel S, Pelz L, Reichl U, Genzel Y. (2022). Upstream processing for viral vaccines – Process intensification. In: Kamen A, Cervera L, editors, Bioprocessing of viral vaccines. CRC Press, Boca Raton. Pp. 137-173. https://doi.org/10.1201/9781003229797-6

Guo H, Ding X, Hua D, Liu M, Yang M, Gong Y, Ye N, Chen X, He J, Zhang Y, Xu X, Li J. (2024). Enhancing dengue virus production and immunogenicity with CelcradleTM bioreactor: A comparative study with traditional cell culture methods. Vaccines 12(6): 563. https://doi.org/10.3390/vaccines12060563

Hesley DC, Spatafore D, Shingler J, McNeely JP, Thompson R, Troutman MC, Baron EKB, Sabia M, Lee CH, Ploeger K, Wagner JM. (2025). Rapid bioreactor process optimization and scale-up for production of a measles vector COVID-19 vaccine candidate. Biotechnology Progress 41(3): e70004. https://doi.org/10.1002/btpr.70004

Huang R, Wang K, Flamm MH, Vazquez J, Gercke C, Ton C, Whitmer T, Mathis PK, Ploeger KJM, Rameez S. (2024). Development and qualification of 3 L scale-down model for large scale vaccine process on Vero cell culture using microcarriers. Biotechnology and Bioengineering 121(11): 3402-3414. https://doi.org/10.1002/bit.28785

Khaidarov S, Burashev Y, Kozhabergenov N, Usserbayev B, Melisbek A, Shirinbekov M, Moldakaryzova A, Beisenova A, Mustafaeva A, Kydyrbaeva A. (2024). Targeted in vitro confirmation of the antiviral activity of Tenvir (Tenofovir) drug against the SARS-CoV-2 virus variant B in Kazakhstan and identifying NSP12 in the viral genome. Eurasian Journal of Applied Biotechnology 2: 49-60. https://doi.org/10.11134/btp.2.2024.6

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): 1003. https://doi.org/10.3390/vaccines9091003

Mohammed J, Mengesha A, Hurisa B, Mulugeta D, Waldetensai A, Tesera Y, Gemechu W, Bizuneh A, Getachaw D, Desalegn A. (2021). Sequential adaptation of Vero cell lines in serum free medium for fixed rabies virus propagation. International Journal of Applied Science – Research and Review 8(6): 20. https://www.primescholars.com/articles/sequential-adaptation-of-vero-cell-lines-in-serum-free-medium-for-fixed-rabies-virus-propagation-95451.html

Nakhanov A, Zhapparova G, Bulatov Y, Terebay A, Seidakhmetova B, Sametova Z, Turyskeldi S. (2024). Culture of sheep poxvirus in a 3D Vero cells culture model. Fundamental and Experimental Biology 11429(2): 49-56. https://doi.org/10.31489/2024bmg2/49-56

Petty RD, Sutherland LA, Hunter EM, Cree IA. (1995). Comparison of MTT and ATP-based assays for the measurement of viable cell number. Journal of Bioluminescence and Chemiluminescence 10(1): 29-34. https://doi.org/10.1002/bio.1170100105

Rauer SB, Stüwe L, Steinbeck L, De Toledo MAS, Fischer G, Wennemaring S, Marschick J, Koschmieder S, Wessling M, Linkhorst J. (2024). Cell adhesion and local cytokine control on protein-functionalized PNIPAM-co-AAC hydrogel microcarriers. Small 21(2): 2404183. https://doi.org/10.1002/smll.202404183

Rhazi H, Safini N, Mikou K, Alhyane M, Tadlaoui KO, Lin X, Venkatesan NP, Elharrak M. (2021). Production of small ruminant morbillivirus, rift valley fever virus and lumpy skin disease virus in CelCradleTM-500A bioreactors. BMC Veterinary Research 17: 93. https://doi.org/10.1186/s12917-021-02801-4

Robitaille J, Manceur A, Rodenbrock A, Loignon M. (2024). Bioprocess development and bioreactor scale-up for the production of recombinant lentiviral viral vectors in HEK293 suspension cell culture. In: Basu S, Ranjan A, Sur S, editors, Technologies in Cell Culture-A Journey From Basics to Advanced Applications. IntechOpen, London, UK. https://doi.org/10.5772/intechopen.114000

Saliev T, Tanabayeva S, Ussebayeva N, Izmailova S, Umbayev B, Akhanov G, Akhmad N, Fakhradiyev I. (2024). In vitro cytotoxicity and antiviral activity of aminocaproic acid against SARS-CoV-2. Journal of Advanced Pharmacy Education and Research 14(3): 1-8. https://doi.org/10.51847/uespvwavbt

Sene M, Xia Y, Kamen AA. (2022). Overview of recent advances in Vero cells genomic characterization and engineering for high-throughput vaccine manufacturing. Clinical and Translational Discovery 2(2): e40. https://doi.org/10.1002/ctd2.40

Sene M, Xia Y, Kamen AA. (2023). Comparative transcriptomic analyses of a Vero cell line in suspension versus adherent culture conditions. International Journal of Cell Biology 2023: 364689. https://doi.org/10.1155/2023/9364689

Sha M. (2021). Vero cell-based vaccine production: Cell lines, media and bioreactor options. Eppendorf White Paper No. 23. https://eu-assets.contentstack.com/v3/assets/blt0a48a1f3edca9eb0/bltcdd188ae3268bcc5/658d75a5f15a1204073c6053/Eppendorf_Bioprocess-Cell-based-vaccine-production_Vero-Cell-based-Vacc.pdf

Sharma R. (2021). Development of a novel bioreactor and systems for suspension cell culture in biopharmaceutical production. PhD thesis. University of Cape Town, Cape Town, South Africa. http://hdl.handle.net/11427/36112

Shen CF, Burney E, Gilbert R, Elahi SM, Parato K, Loignon M. (2023). Development, optimization, and scale-up of suspension Vero cell culture process for high titer production of oncolytic herpes simplex virus-1. Biotechnology Journal 19(1): 2300244. https://doi.org/10.1002/biot.202300244

Sia YS, Azahar NI, Aziz MA, Arifin MA. (2023). Sequential adaptation to serum-free medium for Vero cells cultivation on ultraviolet/ozone (UVO) treated microcarrier. Materials Today Proceedings 107: 92-98. https://doi.org/10.1016/j.matpr.2023.08.031

Swan SY, Hairunnaja MA, Samsuddin N, Mahmood S, Aziz MA, Arifin MA. (2024). A review on the development of microcarriers for cell culture applications. Pertanika Journal of Science & Technology 32(5): 1939-1962. https://doi.org/10.47836/pjst.32.5.01

Zinnecker T, Reichl U, Genzel Y. (2024). Innovations in cell culture-based influenza vaccine manufacturing – From static cultures to high cell density cultivations. Human Vaccines & Immunotherapeutics 20(1). https://doi.org/10.1080/21645515.2024.2373521