Noninvasive measurement of viable cell number in tissue-engineered constructs in vitro, using 1H nuclear magnetic resonance spectroscopy.

Noninvasive monitoring of tissue-engineered constructs is of critical importance for accurate characterization of constructs and their remodeling in vitro and in vivo. This study investigated the utility of (1)H NMR spectroscopy to noninvasively quantify viable cell number in tissue-engineered substitutes in vitro. Agarose disk-shaped constructs containing betaTC3 cells were employed as the model tissue-engineered system. Two construct prototypes containing different initial cell numbers were monitored by localized, water-suppressed 1H NMR spectroscopy over the course of 13 days. (1)H NMR measurements of the total choline resonance at 3.2 ppm were compared with results from the traditional cell viability assay MTT and with insulin secretion rates. Results show a strong linear correlation between total choline and MTT (R (2) = 0.86), and between total choline and insulin secretion rate (R (2) = 0.90). Overall, this study found noninvasive measurement of total choline to be an accurate and nondestructive assay for monitoring viable betaTC3 cell numbers in tissue-engineered constructs. The applicability of this method to in vivo monitoring is also discussed.

[1]  A. Sambanis,et al.  Towards the development of artificial endocrine tissues: 31P NMR spectroscopic studies of immunoisolated, insulin‐secreting AtT‐20 cells , 1995, Biotechnology and bioengineering.

[2]  S. J. Holt,et al.  Microculture tetrazolium assays: a comparison between two new tetrazolium salts, XTT and MTS. , 1995, Journal of immunological methods.

[3]  H. Degani,et al.  Variations in energy and phospholipid metabolism in normal and cancer human mammary epithelial cells. , 1996, Anticancer research.

[4]  A. Sambanis,et al.  In vitro monitoring of total choline levels in a bioartificial pancreas: (1)H NMR spectroscopic studies of the effects of oxygen level. , 2000, Journal of magnetic resonance.

[5]  F. Podo.  Tumour phospholipid metabolism , 1999, NMR in biomedicine.

[6]  T. Ternynck,et al.  Quantitation of 5-bromo-2-deoxyuridine incorporation into DNA: an enzyme immunoassay for the assessment of the lymphoid cell proliferative response. , 1985, Journal of immunological methods.

[7]  J. Acker,et al.  In situ assessment of cell viability. , 1998, Cell transplantation.

[8]  R. Schreiber,et al.  Identification of an Interferon- Receptor Chain Sequence Required for JAK-1 Binding (*) , 1996, The Journal of Biological Chemistry.

[9]  J P Freyer,et al.  Cellular energetics measured by phosphorous nuclear magnetic resonance spectroscopy are not correlated with chronic nutrient deficiency in multicellular tumor spheroids. , 1991, Cancer research.

[10]  U A Stock,et al.  Tissue engineering: current state and prospects. , 2001, Annual review of medicine.

[11]  S. Williams,et al.  Immortalization and transformation are associated with specific alterations in choline metabolism. , 1996, Cancer research.

[12]  R. Kozłowski,et al.  The use of ATP bioluminescence as a measure of cell proliferation and cytotoxicity. , 1993, Journal of immunological methods.

[13]  T. Mosmann.  Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. , 1983, Journal of immunological methods.