Abstract
There are many techniques that allow in vitro interactions among cells and their environment to be monitored, including molecular, biochemical and immunochemical techniques. Traditional techniques for the analysis of cells often require fixation or lysis from substrates; however, use of such destructive methods is not feasible where the expanded cell cultures are required to be used for clinical implantation. Several studies have previously highlighted the potential of Raman spectroscopy to provide useful information on key biochemical markers within cells. As such, we highlight the capability of Raman spectroscopy with different laser spot sizes for use as a non-invasive, rapid, and specific method to perform in situ analysis of primary bovine aortic endothelial cells (BAECs). Raman spectra were collected from both individual live cells cultured on fused silica substrates and on clusters of live cells placed on fused silica substrates, measured at 532 and 785 nm. The results obtained show notable spectral differences in DNA/RNA region indicative of the relative cytoplasm and nucleus contributions. Raman spectra of cell clusters show slight variations in the intensity of the phenylalanine peak (1004 cm−1) indicating variations in protein contribution. These spectra also highlight contributions from other cellular components such as, proteins, lipids, nucleic acids and carbohydrates, respectively.
Similar content being viewed by others
References
Langer R, Vacanti JP. Tissue engineering. Science. 1993;260(5110):920–6.
Ikada Y. Tissue engineering: fundamentals and applications. Amsterdam: Elsevier Science Ltd.; 2006.
Polak JM, Bishop AE. Stem cells and tissue engineering: past, present, and future. In: Annals of the New York Academy of Sciences, Skeletal Development and Remodeling in Health, Disease, and Aging, vol. 1068; 2006. p. 352–66.
Notingher I, et al. In situ non-invasive spectral discrimination between bone cell phenotypes used in tissue engineering. J Cell Biochem. 2004;92(6):1180–92.
Mather ML, Morgan SP, Crow J. Meeting the needs of monitoring in tissue engineering. Regen Med. 2007;2:145–60.
Notingher I. Raman spectroscopy cell-based biosensors. Optic Biosen. 2007;7(8):1343–58.
Notingher I, Hench LL. Raman microspectroscopy: a noninvasive tool for studies of individual living cells in vitro. Expert Rev Med Devices. 2006;3:215–34.
Notingher I, Verrier S, Haque S, Polak JM, Hench LL. Spectroscopic study of human lung epithelial cells (A549) in culture: living cells versus dead cells. Biopolymers. 2003;72(4):230–40.
Swain RJ, Stevens MM. Raman microspectroscopy for non-invasive biochemical analysis of single cells. Biochem Soc Trans. 2007;035(3):544–9.
Yang Y, Dubois A, Qin X, Li J, Haj A. Investigation of optical coherence tomography as an imaging modality in tissue engineering. Phys, Med Bio. 2006;51:1649–59.
Krafft C. Bioanalytical applications of Raman spectroscopy. Anal Bioanal Chem. 2004;378(1):60–2.
Puppels GJ, Garritsen HS, Segers-Nolten GM, de Mul FF, Greve J. Raman microspectroscopic approach to the study of human granulocytes. Biophys J. 1991;60(5):1046–56.
Notingher I, Hench LL. In situ characterisation of living cells by Raman spectroscopy. Spectroscopy. 2002;16(2):43–51.
Short KW, Carpenter S, Freyer JP, Mourant JR. Raman spectroscopy detects biochemical changes due to proliferation in Mammalian Cell Cultures. Biophys J. 2005;88(6):4274–88.
Swain RJ, Jell G, Stevens MM. Non-invasive analysis of cell cycle dynamics in single living cells with Raman micro-spectroscopy. J Cell Biochem. 2008;104(4):1427–38.
Uzunbajakava N, Manen HWJV, Otto C. Raman microscopy on single cells: imaging of apoptosis and phagocytosis at high resolution. GIT Lab J Eur. 2004;8(3):22–5.
Verrier S, Notingher I, Polak JM, Hench LL. In situ monitoring of cell death using Raman microspectroscopy. Biopolymers. 2004;74(1–2):157–62.
Jell G, Notingher I, Tsigkou O, Notingher P, Polak JM, Hench LL, Stevens MM. Bioactive glass-induced osteoblast differentiation: a noninvasive spectroscopic study. J Biomed Mater Res Part A. 2008;86A(1):31–40.
Notingher I, Bisson I, Polak JM, Hench LL. In situ spectroscopic study of nucleic acids in differentiating embryonic stem cells. Vib Spectrosc. 2004;35(1–2):199–203.
Notingher I, Jell G, Notingher P, Bisson I, Tsigkou O, Polak JM, Stevens MM, Hench LL. Multivariate analysis of Raman spectra for in vitro non-invasive studies of living cells. J Mol Struct. 2005;744–747:179–85.
Gentleman E, Swain RJ, Evans ND, Boonrungsiman S, Jell G, Ball MD, Shean TAV, Oyen ML, Porter A, Stevens MM. Comparative materials differences revealed in engineered bone as a function of cell-specific differentiation. Nat Mater. 2009;8(9):763–70.
Huang M, Karashima T, Yamamoto K, Hamaguchi H. Molecular-level investigation of the structure, transformation, and bioactivity of single living fission yeast cells by time- and space-resolved Raman spectroscopy. Biochemistry. 2005;44(30):10009–19.
Huang Y-S, Karashima T, Yamamoto M, Hamaguchi H. Molecular-level pursuit of yeast mitosis by time-and space-resolved Raman spectroscopy. J Raman Spectrosc. 2003;34(1):1–3.
Booyse FM, Sedlak BJ, Rafelson ME. Culture of arterial endothelial cells: characterization and growth of bovine aortic cells. Thromb Diath Haemorrh. 1975;34(3):825–39.
Schwartz SM. Selection and characterization of bovine aortic endothelial cells. In Vitro. 1978;12(12):966–80.
Boyd AR, Burke GA, Meenan BJ. Monitoring cellular behaviour using Raman spectroscopy for tissue engineering and regenerative medicine applications. J Mater Sci: Mater Med. 2010;21(8):2317–24.
Uzunbajakava N, Lenferink ATM, Kraan YM, Willekens B, Vrensen GFJM, Greve J, Otto C. Nonresonant raman imaging of protein distribution in single human cells. Biopolymers. 2003;72(1):1–9.
Chan JW, Taylor DS, Thompson DL. The effect of cell fixation on the discrimination of normal and leukemia cells with laser tweezers Raman spectroscopy. Biopolymers. 2009;91(2):132–9.
Nohe A, Hassel S, Ehrlich M, Neubauer F, Sebald W, Henis YI, Knaus P. The mode of bone morphogenetic protein (BMP) receptor oligomerization determines different BMP-2 signaling pathways. J Biol Chem. 2002;277:5330–8.
Notingher I, Hench LL. A bio-photonics system for rapid in vitro testing of cells and ceramics. Key Eng Mater. 2005;284286:531–6.
Notingher I, Selvakumaran J, Hench LL. New detection system for toxic agents based on continuous spectroscopic monitoring of living cells. Biosens Bioelectron. 2004;20(4):780–9.
Lakshmi RJ, Kartha VB, Murali Krishna C, Solomon JG, Ullas G, Uma Devi P. Tissue Raman spectroscopy for the study of radiation damage: brain irradiation of mice. Radiat Res. 2009;157(2):175–82.
Naumann D. FT-infrared and FT-Raman spectroscopy in biomedical research. In: Gremlich HU, Yan B editors. Infrared and Raman spectroscopy of biological materials, New York: Marcel Dekker; 2001. p. 323–377.
Carter EA, Edwards HGM. Bioloigcal Applications of Raman spectroscopy. In: Gremlich HU, Yan B, editors. Infrared and Raman Spectroscopy of Biological Materials. New York: Marcel Dekker; 2001. p. 421–476.
Omberg KMO, Jill C, Zhang SL, Freyer JP, Mourant JR, Schoonover JR. Raman spectroscopy and factor analysis of tumorigenic and non-tumorigenic cells. Appl Spectros. 2002;56(7):813–9.
Overman SA, Aubrey KL, Reilly KE, Osman O, Hayes SJ, Serwer P, Thomas GJ Jr. Conformation and interactions of the packaged double-stranded DNA genome of bacteriophage T7. Biospectroscopy. 1998;4(S5):S47–56.
Kendall C, Stone N, Shepherd N, Geboes K, Warren B, Bennett R, Barr H. Raman spectroscopy, a potential tool for the objective identification and classification of neoplasia in Barrett’s oesophagus. J Pathol. 2003;200(5):602–9.
Mahadevan-Jensen A, Richards-Kortum R. Raman spectroscopy for the detection of cancers and precancers. J Biomed Opt. 1996;1(1):31–70.
Borchman D, Tang D, Yappert MC. Lipid composition, membrane structure relationships in lens and muscle sarcoplasmic reticulum membranes. Biospectroscopy. 1999;5(3):151–67.
Rajani C, Kincaid JR, Petering DH. Raman spectroscopy of an O(2)-Co(II)bleomycin-calf thymus DNA adduct: alternate polymer conformations. Biophys Chem. 2001;94(3):219–36.
Acknowledgments
The authors would like to express their gratitude to the EPSRC for funding this project (EP/C534247/1 (Remedi) Regenerative Medicine—A New Industry). The authors would also like to thank the Department of Education and Learning Northern Ireland (DEL NI) for additional funding for the project and Horiba Scientific for the use of the Confocal XploRa Raman Microscope.
Author information
Authors and Affiliations
Corresponding author
Additional information
An erratum to this article can be found at http://dx.doi.org/10.1007/s10856-011-4383-7
Rights and permissions
About this article
Cite this article
Boyd, A.R., McManus, L.L., Burke, G.A. et al. Raman spectroscopy of primary bovine aortic endothelial cells: a comparison of single cell and cell cluster analysis. J Mater Sci: Mater Med 22, 1923–1930 (2011). https://doi.org/10.1007/s10856-011-4371-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10856-011-4371-y