(L-459) 3D Bioprinted Vascularized Tissue Model for Studying Cellular Senescence

Introduction:: Patients diagnosed with cancer are subjected to aggressive treatment, including surgical interventions followed by chemo and/or radiotherapy. Doxorubicin (DOX), a potent chemotherapeutic drug, is often of choice for treatment. Unfortunately, this life-saving pharmaceutical intervention in the fight against cancer induces cardiac toxicity and, subsequently, heart failure, yet the detailed molecular mechanism of cardiac toxicity is not well understood. The toxicity in cardiac tissue can often lead to impairment of quality of life or, in some severe cases, could be lethal. Unfortunately, it is also not established yet why some people have more or less severe responses due to gaps in understanding of the detailed mechanism of DOX-associated cytotoxicity on cardiac tissue and the lack of potential biomarkers that could help with better prediction for severity of side effects for each individual patient. Previous studies have indicated that DOX and many other chemotherapeutics are strongly associated with cellular senescence. Senescent cells are metabolically active and can acquire a destructive, pro-inflammatory senescence-associated secretory phenotype (SASP) contributing to multiple age-related diseases. Importantly, SASP includes proinflammatory cytokines, chemokines, proteases, and a variety of circulating, exosomal microRNAs (miRNAs). The shift of the expression levels of different senescence-specific miRNAs, which take part in turning the fate of the cell from pro-apoptotic into senescent, can be fatal for other cells.


Materials and Methods:: Cellink BioX6 bioprinter was used to print 3D vascularized cardiovascular tissue constructs composed of cardiomyocytes, fibroblasts, and Human Umbilical Vein Endothelial Cells (HUVECs). 3D bioprinting allows the study of the process of senescence and the impact of DOX on the increased accumulation of senescent cells by producing a 3D organoid resembling a young, healthy organ, pre-aging phenotype characterized by the increased number of senescent cells or an old organoid, which could be achieved by controlled input of different percentages of senescent cells during the 3D organoid bioprinting process. RNA was extracted using Trizol administered through a syringe in order to flush out the lysed cells, followed by the addition of chloroform to promote phase separation and ethanol precipitation. cDNA for downstream qRT-PCR was prepared using the TaqMan Advanced microRNA (miRNA) cDNA synthesis kit. Cell viability was assessed using MTS (a calorimetric method for detecting the number of viable cells) by measuring the production of formazan. The formazan product generated by metabolically active cells produces a color that can be detected at an absorbance of 490 nm using a spectrophotometer.


Results, Conclusions, and Discussions:: A microfluidic-based CELLINK Vaskit system has been used to print and maintain 3D bioprinted vascularized tissue constructs, as shown in Figure 1a. First, a thin layer of 10% GelMA as the bed was cast and air-dried for 10 to 15 min. Next, Pluronic F127 was used to print vascular channels having a diameter of ~ 0.8 mm. Finally, the remaining space was filled with 10% GelMA mixed with cardiomyocytes and fibroblasts and crosslinked under UV light for ~ 5 mins. Pluronic F127 was liquefied by placing it at 4ºC and evacuated. Then, the channels were seeded with HUVECs. Cells were flushed in the channels at a range of 3.2 ̶ 8.38 x 10⁶ cells/ml using a syringe. The channels present in the construct allowed for the transport and diffusion of oxygenated perfused media to the cellular environment to maintain the cells. A sample fluorescence image is presented in Fig. 1b, which shows the cellular viability in the channels after 16 days in the Vaskit bioreactor system. Before performing experiments with DOX, preliminary experiments were performed in which co-cultures with young and old HUVECs were achieved by plating HUVECs P5 and HUVECs P11 in a 6-well plate. Once cells adhered to the bottom of each respective well, the constructs were added and co-cultured for seven days prior to lysis. The optical absorbance from the MTS assay measured at the wavelength of 490 nm was greater than 1, confirming the cells were viable after seven days of co-culture. qRT-PCR analysis, as shown in Figure 2 of senescence-associated miRNA, miR-146a-5p demonstrated a relative 2.2 fold-increase in expression and an observed decrease in pro-apoptosis miR-217 in cells co-cultured with old HUVECs when compared with cells co-cultured with young HUVECs.

Cells within the bioprinted constructs displayed the potential to interact with and be influenced by cultured cells in the same well. Co-culturing of bioprinted tissue construct with different age senescent cells can be utilized to study cellular interactions resembling in vivo parabiosis to understand the impact of senescent cells on young, healthy cells.




Acknowledgements (Optional): :

References (Optional): :