세포 배양 배지 및 PBS의 구성과 삼투압

목차

1. IMDM의 구성 성분의 역할
2. 일반적인 culture media와 Chemically defined media의 차이점
3. 세포 배양액을 멸균을 위해 autoclave가 아닌 필터링을 하는 이유
4. Filtering의 원리 및 방법
5. 세포 배양시 PBS 역할
6. 삼투압 계산
7. 적용(PBS 삼투압 구하기)
8. Reference

본문내용

개요
이하의 내용은 세포 배양 실험에 있어 필수적으로 사용되는 배양 배지 및 PBS(Phosphate Buffered Saline)의 구성 성분과 그 역할에 대해 서술하고 있다. 특히 IMDM(Iscove’s Modified Dulbecco’s Medium) 배지에 포함된 대표적인 성분의 역할과 세포 성장에 미치는 영향을 알아보았고, 일반적인 배지(culture medium)와 화학적으로 정의된 배지(Chemically defined medium)의 차이점을 비교, 서술하고 있다. 또한, 배지를 멸균하는 데 있어 Autoclave 대신 여과(Filtering)를 사용하는 이유와 그 원리 및 방법을 설명하였다. 마지막으로 PBS의 삼투압 계산을 통해 생리적 조건과의 유사성을 화학적, 수학적으로 알아내었으며, 이를 통해 PBS의 실험적 가치를 논의하였다.

1. IMDM의 구성 성분의 역할
Culture media에는 세포를 구성하는 성분들의 조합과 세포성장과 단백질 생산을 위한 에너지 공급이 필수적이다. 따라서 기본적으로 들어가는 성분은 포도당, 아미노산, 지방, 비타민, 무기염류, pH buffer 가 있고, 추가로 혈청(serum), 펩톤, 성장인자 등을 media에 공급해준다.
Glucose는 대표적인 cell media의 영양분으로 세포 구성 성분을 만들어 주는 에너지원이다. Glucose는 가장 흔하게 사용되는 에너지원이지만 다른 6탄당 물질인 Fructose, galactose, mannose 등이 사용되기도 한다(Jongkwang Hong, Cell Culture Media, lecture slide 19). Glucose는 배지에서 가장 중요한 탄소원이지만, 포도당의 해당과정(glycolysis)에 의해 pyruvate가 형성되고, 이는 TCA회로를 통해 젖산(lactate)로 전환된다. 그럼으로, 세포 배지에는 젖산의 축적이 발생

참고자료

· Jongkwang Hong, Cell Culture Media, lecture slide 5, “Biological Engineering” (lecture, Yonsei University Mirae Campus, Wonju, March 20, 2025).
· Y. S. Tsao et al., "Monitoring Chinese Hamster Ovary Cell Culture by the Analysis of Glucose and Lactate Metabolism," Journal of Biotechnology 118, no. 3 (August 22, 2005): 316–27, https://doi.org/10.1016/j.jbiotec.2005.05.016
· HiMedia Laboratories. TCL013: 7.5% Sodium Bicarbonate Solution – Technical Data Sheet. Revision 03/2022. Accessed March 20, 2025. https://www.himedialabs.com/media/TD/TCL013.pdf
· Hakhyun Ka, Cell Media Preparation, lecture slide 8, “Cell Culture Laboratory” (lecture, Yonsei University Mirae Campus, Wonju, March 20, 2025)
· SAFC Biosciences. L-Glutamine Solution 200 mM: Technical Data Sheet. Issued December 2006. Accessed March 20, 2025. https://www.safcbiosciences.com
· Thermo Fisher Scientific. "Gibco™ L-Glutamine (200 mM) Supplement." Accessed March 20, 2025. https://www.thermofisher.com/order/catalog/product/kr/ko/A2916801.
· Merck. "Base Ingredients of Microbiology Media." Accessed March 20, 2025. https://www.sigmaaldrich.com/US/en/technical-documents/technical-article/microbiological-testing/microbial-culture-media-preparation/base-ingredients-of-microbiology-media.
· Bonar, Pamela T., and Joseph R. Casey. "Plasma Membrane Cl⁻/HCO₃⁻ Exchangers: Structure, Mechanism and Physiology." Channels (Austin) 2, no. 5 (September-October 2008): 337–45. https://doi.org/10.4161/chan.2.5.6899.
· National Center for Biotechnology Information. "PubChem Compound Summary for CID 4766, Phenolsulfonphthalein." PubChem. Accessed March 20, 2025. https://pubchem.ncbi.nlm.nih.gov/compound/Phenolsulfonphthalein.
· Sigma-Aldrich. Phenol Red Sodium Salt – Product Information Sheet. Accessed March 20, 2025. https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/product/documents/367/102/p5530pis.pdf.
· SAFC Biosciences. Iscove’s Modified Dulbecco’s Medium – Product Information Sheet. Issued December 2006. Accessed March 20, 2025. https://www.safcbiosciences.com.
· Whitford, William G., Mats Lundgren, and Alain Fairbank. “Cell Culture Media in Bioprocessing.” In Biopharmaceutical Processing, edited by Günter Jagschies, Eva Lindskog, Karol Łącki, and Parrish Galliher, 147–62. Amsterdam: Elsevier, 2018. https://doi.org/10.1016/B978-0-08-100623-8.00008-6.
· Arora, Meenakshi. “Cell Culture Media: A Review.” MATER METHODS 3 (2013): 175. University of Pittsburgh Medical Center, United States. Last modified July 8, 2023. https://dx.doi.org/10.13070/mm.en.3.175.
· Sigma-Aldrich. “Types of Media in Microbiology.” Sigma-Aldrich. Accessed March 20, 2025. https://www.sigmaaldrich.com/KR/ko/technical-documents/technical-article/microbiological-testing/microbial-culture-media-preparation/types-of-media-in-microbiology.
· Wang, H., J. Guo, X. Chen, and H. He. “The Metabolomics Changes in Luria-Bertani Broth Medium under Different Sterilization Methods and Their Effects on Bacillus Growth.” Metabolites 13, no. 8 (August 18, 2023): 958. https://doi.org/10.3390/metabo13080958.
· Pioli, Peter D. “LB Media Recipe.” Western Michigan University Homer Stryker M.D. School of Medicine. Last edited September 13, 2019. Accessed March 20, 2025. https://med.wmich.edu/sites/default/files/Pioli_Lab_LB_Media_Recipe.pdf.
· Michaels, I. 1948. “The Sterilisation of Sodium Bicarbonate Solutions.” Quarterly Journal of Pharmacy and Pharmacology 21 (3): 231–39.
· Leitzen, Sarah, Matthias Vogel, Michael Steffens, Thomas Zapf, Christa Elisabeth Müller, and Martin Brandl. 2021. "Quantification of Degradation Products Formed during Heat Sterilization of Glucose Solutions by LC-MS/MS: Impact of Autoclaving Temperature and Duration on Degradation" Pharmaceuticals 14, no. 11: 1121. https://doi.org/10.3390/ph14111121
· Wang, Xiao-Jia, and Ke-Cheng Hsiao. 1995. “Sugar Degradation during Autoclaving: Effects of Duration and Solution Volume on Breakdown of Glucose.” Physiologia Plantarum 94 (1): 46–49. https://doi.org/10.1111/j.1399-3054.1995.tb00947.x.
· Sandal, I., A. Bhattacharya, U. Saini, D. Kaur, S. Sharma, A. Gulati, J. K. Kumar, N. Kumar, J. Dayma, P. Das, B. Singh, and P. S. Ahuja. “Chemical Modification of L-Glutamine to Alpha-Amino Glutarimide on Autoclaving Facilitates Agrobacterium Infection of Host and Non-Host Plants: A New Use of a Known Compound.” BMC Chemical Biology 11 (May 31, 2011): 1. https://doi.org/10.1186/1472-6769-11-1.
· Yenny Mayerly Herrera-Ardila et al., “Effect of Heat Treatment on Vitamin Content during the Manufacture of Food Products at Industrial Scale,” DYNA 89, no. 223 (2022): 130, https://doi.org/10.15446/dyna.v89n223.99775.
· Robert John Evans and Helen A. Butts, "Inactivation of Amino Acids by Autoclaving," Science 109, no. 2840 (June 3, 1949): 569, https://doi.org/10.1126/science.109.2840.569.
· "Separation and Purification," Encyclopedia Britannica, accessed March 20, 2025, https://www.britannica.com/science/separation-and-purification.
· "Syringe Filters," Sigma-Aldrich, accessed March 20, 2025, https://www.sigmaaldrich.com/KR/ko/technical-documents/technical-article/analytical-chemistry/filtration/syringe-filters."
· Sigma-Aldrich. "Phosphate Buffered Saline, Tablets, High-Purity, P4417."
· https://www.sigmaaldrich.com/KR/ko/product/sigma/p4417
· Cold Spring Harbor Laboratory Press. Phosphate-Buffered Saline (PBS). 2006.
· Brown, Theodore L., H. Eugene LeMay Jr., and Bruce E. Bursten. Chemistry: The Central Science. 10th ed. Upper Saddle River, NJ: Pearson Education, 2006.
· Raymond Chang, General Chemistry: The Essential Concepts, 5th ed. (New York: McGraw-Hill, 2008), 445–446.
· Rasouli, Mehdi. "Basic Concepts and Practical Equations on Osmolality: Biochemical Approach." Clinical Biochemistry 49, no. 12 (2016): 936–941. https://doi.org/10.1016/j.clinbiochem.2016.06.001.
· HiMedia Laboratories Pvt. Ltd., Iscove's Modified Dulbecco's Medium (IMDM): With L-Glutamine and 25mM HEPES Buffer, Without Sodium Bicarbonate, Product Code: AT070 (Mumbai: HiMedia Laboratories Pvt. Ltd., 2011).