Dimetil Sülfoksit, Gliserol ve Metanol’ün HeLa Hücrelerinin Çözdürme Sonrası Hücre Canlılığı Üzerindeki Karşılaştırmalı Etkinliği

Yıl 2024, Cilt: 35 Sayı: 1, 32 - 37, 29.03.2024

Zeyno Nuhoğlu Öztürk Orhan Tokur Abdurrahman Aksoy

https://doi.org/10.36483/vanvetj.1322291

Öz

Kriyoprotektanlar, hücrelerin dondurma işlemi sırasında korunması için kullanılır. Bu maddelerin, konsantrasyonu, tipi ve dondurma-çözdürme koşulları kullanılacak hücre tipine göre değişir. Uygun kriyoprezervasyon yönteminin hücreye özel olarak belirlenmesi oldukça önemlidir. Bu çalışma, farklı kriyoprotektanların performansını karşılaştırarak ve bunların çeşitli dondurma ve saklama koşulları altında etkinliğini değerlendirerek, HeLa hücreleri için en uygun kriyoprezervasyon yöntemine ilişkin bilgiler sağlanması amaçlanmaktadır. Hücre süspansiyonları 5:10:85 (v:v:v) oranında kriyoprotektan + fetal sığır serumu + medyumdan oluşan bir dondurucu besiyerinde donduruldu ve 3 ay (-20 °C), 1 ay (-80 °C) ve 6 ay (-80 °C) koşullarında saklandı. Hücre canlılığı ve geri kazanım oranları, çözülmeden hemen sonra ve çözülmeyi takiben 48 saat sonra, tripan mavisi kullanılarak analiz edildi. Canlılık ve geri kazanım oranları 3 ay -20 °C’de, metanol grubunda daha yüksekti. Gliserol grubunda ise canlılık ve geri kazanım oranları 1 ay -80 °C’de daha iyi performans gösterdi. DMSO grubunda ise bu oranlar, 6 ay -80 °C’de en yüksekti. Metanol grubu -80 °C’deki depolama koşullarında başarısız oldu. Bu çalışma, HeLa hücrelerindeki bu kriyoprotektanların, çözdürme işleminden hemen sonra ve 48 saatlik kültivasyondan sonra hücre canlılığı ve hücre geri kazanım oranları üzerindeki etkisini göstermektedir.

Anahtar Kelimeler

Dondurarak saklama, Donma, HeLa hücreleri, Hücre kültürü teknikleri, Hücre yaşamı

Comparative Efficacy of the Dimethyl Sulfoxide, Glycerol and Methanol on the Post-Thaw Cell Viability of HeLa Cells

Öz

Cryoprotectants are used to protect cells during freezing. The concentration, type, and freeze-thaw conditions of these substances vary depending on the type of cell to be used. It is very important to determine the appropriate cryopreservation method for the particular cell. This study aims to provide insights into the optimal cryopreservation method for HeLa cells by comparing the performance of different cryoprotectants and evaluating their effectiveness under various freezing and storage conditions. Cell suspensions were frozen with a freezing media composed of cryoprotectant + fetal bovine serum + medium at a ratio of 5:10:85 (v:v:v) and stored under the following conditions: 3 months (-20 °C), 1 month (-80 °C), and 6 months (-80 °C). Cell viability and recovery rates were analyzed immediately post-thaw and after 48 h using the trypan blue dye exclusion assay. In 3 months (-20 °C), viability and recovery rates were higher in the methanol group. Glycerol showed better performance in 1 month (-80 °C). DMSO was the most efficient in 6 months (-80 °C). Methanol failed at -80 °C storage temperature. This study demonstrates the effect of these cryoprotectants in HeLa cells on cell viability and cell recovery rates immediately after thawing and after 48 hours of cultivation.

Anahtar Kelimeler

Cell culture techniques, Cell survival, Cryopreservation, Freezing, HeLa cells

Kaynakça

• ATCC (2022). American Type Culture Collection Animal Cell Culture Guide. Date of access: 9 April 2023. Access address: https://www.atcc.org/resources/culture-guides/animal-cell-culture-guide.
• Baust JG, Corwin WL, Baust JM (2011). Cell Preservation Technology. Moo-Young M (Ed). Comprehensive Biotechnology (pp. 179-190). Elsevier BV.
• Baust JM, Campbell LH, Harbell JW (2017). Best practices for cryopreserving, thawing, recovering, and assessing cells. In Vitro Cell. Dev- An, 53 (10), 855-871.
• Baust JM, Van B, Baust JG (2000). Cell viability improves following inhibition of cryopreservation-induced apoptosis. In Vitro Cell. Dev- An, 36 (4), 262-270.
• Bryant SJ, Awad MN, Elbourne A et al. (2022). Deep eutectic solvents as cryoprotective agents for mammalian cells. J Mater Chem B, 10 (24), 4546-4560.
• Bumbat M, Wang M, Liang W et al. (2020). Effects of Me(2)SO and Trehalose on the Cell Viability, Proliferation, and Bcl-2 Family Gene (BCL-2, BAX, and BAD) Expression in Cryopreserved Human Breast Cancer Cells. Biopreserv Biobank, 18 (1), 33-40.
• Chow-Shi-Yee M, Grondin M, Ouellet F, Averill-Bates DA (2020). Control of stress-induced apoptosis by freezing tolerance-associated wheat proteins during cryopreservation of rat hepatocytes. Cell Stress Chaperones, 25 (6), 869-886.
• Elliott GD, Wang SP, Fuller BJ (2017). Cryoprotectants: A review of the actions and applications of cryoprotective solutes that modulate cell recovery from ultra-low temperatures. Cryobiology, 76, 74-91.
• Freshney RI. (2015). Cryopreservation. Freshney RI (Ed). Culture of animal cells: a manual of basic technique and specialized applications (pp. 317-334). John Wiley & Sons, Inc.
• Fujisawa R, Mizuno M, Katano H et al. (2019). Cryopreservation in 95% serum with 5% DMSO maintains colony formation and chondrogenic abilities in human synovial mesenchymal stem cells. BMC Musculoskelet Disord, 20 (1), 316.
• Gao S, Ogawa M, Takami A et al. (2020). Practical and Safe Method of Long-term Cryopreservation for Clinical Application of Human Adipose-derived Mesenchymal Stem Cells Without a Programmable Freezer or Serum. Cryoletters, 41 (6), 337-343.
• Gomez-Lechon MJ, Lahoz A, Jimenez N, Castell JV, Donato MT (2006). Cryopreservation of rat, dog and human hepatocytes: influence of preculture and cryoprotectants on recovery, cytochrome P450 activities and induction upon thawing. Xenobiotica, 36 (6), 457-472.
• Gonzalez Porto SA, Domenech N, Gonzalez Rodriguez A et al. (2018). The addition of albumin improves Schwann cells viability in nerve cryopreservation. Cell Tissue Bank, 19 (4), 507-517.
• Gupta V, Sengupta M, Prakash J, Tripathy BC (2017). Animal cell culture and cryopreservation. Gupta V (Ed). Basic and Applied Aspects of Biotechnology. (pp. 59-75) Singapore, Springer.
• Moss AC, Higgins AZ (2016). Investigating the potential for cryopreservation of human granulocytes with concentrated glycerol. Cryobiology, 72 (3), 290-293.
• Murray KA, Gibson MI (2020). Post-Thaw Culture and Measurement of Total Cell Recovery Is Crucial in the Evaluation of New Macromolecular Cryoprotectants. Biomacromolecules, 21 (7), 2864-2873.
• Murray KA, Gibson MI (2022). Chemical approaches to cryopreservation. Nat Rev Chem, 6 (8), 579-593.
• Myagmarjav B, Liu B (2022). Cryopreservation of HEP-G2 cells attached to substrates: the benefit of sucrose and trehalose in combination with dimethyl sulfoxide. Cryoletters, 43 (3), 175-182.
• Pereira J, Ferraretto X, Patrat C, Meddahi-Pelle A (2019). Dextran-Based Hydrogel as a New Tool for BALB/c 3T3 Cell Cryopreservation Without Dimethyl Sulfoxide. Biopreserv Biobank, 17 (1), 2-10.
• Poisson JS, Acker JP, Briard JG, Meyer JE, Ben RN (2019). Modulating Intracellular Ice Growth with Cell-Permeating Small-Molecule Ice Recrystallization Inhibitors. Langmuir, 35 (23), 7452-7458.
• Reuther T, Kettmann C, Scheer M et al. (2006). Cryopreservation of osteoblast-like cells: viability and differentiation with replacement of fetal bovine serum in vitro. Cells Tissues Organs, 183 (1), 32-40.
• Sevim ET, Arat S (2021). Combining dimethyl sulphoxide (DMSO) with different cryoprotectants ensures better cartilage cell cryopreservation. Cryoletters, 42 (4), 220-226.
• Shinde P, Khan N, Melinkeri S, Kale V, Limaye L. (2019). Freezing of dendritic cells with trehalose as an additive in the conventional freezing medium results in improved recovery after cryopreservation. Transfusion, 59 (2), 686-696.
• Stevenson DJ, Morgan C, Goldie E, Connel G (2004). Cryopreservation of viable hepatocyte monolayers in cryoprotectant media with high serum content: metabolism of testosterone and kaempherol post-cryopreservation. Cryobiology, 49 (2), 97-113.
• Tamagawa S, Sakai D, Schol J et al. (2022). N-acetylcysteine attenuates oxidative stress-mediated cell viability loss induced by dimethyl sulfoxide in cryopreservation of human nucleus pulposus cells: A potential solution for mass production. JOR spine, 5 (4), e1223.
• Thirumala S, Gimble JM, Devireddy RV (2010). Evaluation of Methylcellulose and Dimethyl Sulfoxide as the Cryoprotectants in a Serum-Free Freezing Media for Cryopreservation of Adipose-Derived Adult Stem Cells. Stem Cells Dev, 19 (4), 513-522.
• Vian AM, Higgins AZ (2014). Membrane permeability of the human granulocyte to water, dimethyl sulfoxide, glycerol, propylene glycol and ethylene glycol. Cryobiology, 68 (1), 35-42.
• Weinberg A, Song LY, Wilkening C et al. (2009). Optimization and limitations of use of cryopreserved peripheral blood mononuclear cells for functional and phenotypic T-cell characterization. Clin Vaccine Immunol, 16 (8), 1176-1186.
• Liu X, Pan Y, Liu F et al. (2021). A review of the material characteristics, antifreeze mechanisms, and applications of cryoprotectants (CPAs). J Nanomater, 1-14.
• Yamatoya K, Nagai Y, Teramoto N et al. (2022). Cryopreservation of undifferentiated and differentiated human neuronal cells. Regen Ther, 19, 58-68.