Saturday September 25, 2021 - 15:50 to 17:05
Giuseppe Pettinato, United States has been granted the CTRMS Congress Scientific Awards
Noninvasive Monitoring of Liver Organoid Differentiation with Raman Spectroscopy
Giuseppe Pettinato1, Mark F. Coughlan1, Xuejun Zhang1, Liming Chen1, Maria Glyavina1, Yuri N. Zakharov1, Lei Zhang1, Le Qiu1, Robert A. Fisher1,2, Lev T. Perelman1.
1Center for Advanced Biomedical Imaging and Photonics, Div. of Gastroenterology, Dept. of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, United States; 2SP Global, Chantilly, VA, United States
Introduction: Engineered tissue represents a great promise for treating liver diseases1-3. Differentiated liver organoids have shown to be an interesting alternative to the native liver-derived hepatocytes4-7. Controlling the progression of differentiation in live cells could have important implications for obtaining fully functional terminally differentiated liver organoids. In this study, in order to monitor the progression of differentiating organoids consisted of HLA-Class II knockout human induced pluripotent stem cells (hiPSCs) interlaced with endothelial cells toward functional liver organoids, we have complemented the conventional real-time PCR measurements of hepatic markers and coagulation factors with the noninvasive Raman spectroscopy.
Methods: Generation of human embryoid bodies (hEBs) using HLA-Class II knockout hiPSCs interlaced with endothelial cells was achieved using ROCKi/Spin-free technology we developed earlier8. Raman spectra were collected in live organoids using a Raman spectroscopy system consisted of a 785 nm excitation diode laser and a portable Raman spectrometer. We also performed clinically relevant assays for measuring hepatic function ex vivo.
Results: Changes in the Raman spectra of organoids over the course of 16 days, spanning four consecutive stages of differentiation, exhibit peaks that can be associated with CER1, AFP, and creatinine (Fig. 1a-c). In addition, gene expression of several hepatic markers, as well as coagulation factors, was detected by real-time PCR, showing a progression in the maturation of the liver organoids (Fig. 1d).
Discussion/Conclusions: Real-time monitoring of differentiating hiPSC organoids represents an excellent tool to assess the differentiation progresses in live cells. Our results demonstrated the potential of Raman spectroscopy to complement the conventional techniques and provide information on the hepatic differentiation noninvasively. This type of monitoring could be important for successful clinical translation.