Presenting on “Cells in Machines and Things we Discovered Along the Way”

Thomas Boland, PhD, professor of metallurgical, materials and biomedical engineering and director of biomedical engineering program at The University of Texas at El Paso, will speak on Thursday, March 2, 2023 at 10:00 am CST in Whitaker 218.

Abstract: When we first described inkjet bioprinting (TIB) of cells in 2003, the devices were treated like a black-box.  Quantifying the input and measuring the output has become the hallmark of not only our group but, indeed, the entire field of bioprinting. Most of this is understandable, as it is virtually impossible to characterize cells that are inside the printing orifices, extrusion needles, or drops ejected from ribbons. Also understandable is then that we may be missing out on important information about cell biology, for example, what may be happening inside the printer or shortly after the bioprinting event. We will describe here some recent discoveries of cell behavior due to inkjet bioprinting.

For these experiments, we use primary adult dermal fibroblasts which were expanded for 2-3 passages upon receiving. The cells were harvested and resuspended in PBS and bioprinted into a 96-well plate with pluriSTEM media. Cells were then transferred either into precoated 96-well plates or 20 µL drops were pipetted for hanging drop culture using a multichannel pipettor. IPC to differentiation protocols was applied and the induction was begun approximately 45 mins after TIB for the cells that were transferred to the plate immediately after printing. When differentiating aggregates, the initiation happened 45 mins after the aggregates were transferred into the 96 wells. Preliminary results indicate that all cells expressed the 3 pluripotency markers oct-4, nanog, and sox-2. After differentiation protocols,  the cells stained positively for tropinin-3 for the cardiomyocyte differentiation protocol.  The cells also elongated and became more cardiomyocyte-like in their morphology.  We analyzed bulk RNA seq data and our preliminary results showed upregulation of some genes that have been implicated as stem cell markers: EPCAM, LEFTY1, ZFP42, and TEX19. In addition, differential expression of genes associated with pluripotency-relevant pathways show some pathways are off like the MAPK/p38, MAPK/JNK1-3 which would be expected for a pluripotent state, but at this time the activation of the hippo pathway is inconclusive with TAZ highly upregulated but YAP slightly downregulated. Similarly, GSK3B is off and TGFB1, LIF/PIK3, and AKT1 are on as expected for pluripotency, but SMAD2/3 and CTNNB1. Examining the gene network of upregulated genes, one can clearly distinguish the pivotal role of FOS, FOXO1, and PIK3 all related to pluripotency. In conclusion, bioprinted fibroblasts will at least temporarily adopt a more primitive or dedifferentiated state, reminiscent of pluripotency. While immunochemistry shows the classic transcription factors required for pluripotency, gene expression shows a more nuanced picture of the transformations that occur upon printing. Understanding these transformations, even if temporary will be crucial when trying to build tissues using bioprinting technologies.

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