Thermal inkjet bioprinting drastically alters cell phenotype.

Since the first description of inkjet bioprinting of cells in 2003, quantifying the input and measuring the output of the printers has been the hallmark of the field of bioprinting, as it is virtually impossible to characterize cells that are inside the printing orifices or extrusion needles. We will describe here some recent discoveries of cell behavior due to inkjet bioprinting. Primary and immortalized adult dermal fibroblasts were expanded for 2-3 passages upon receiving. The cells were harvested, resuspended in PBS, and bioprinted into a 96-well plate with pluriSTEM media. Cells were then transferred either into precoated 96-well plates or 20µl drops were pipetted for hanging drop culture. IPC differentiation protocols were applied and the induction was begun approximately 45 min after printing. When differentiating aggregates, the initiation happened 45 min after the aggregates were transferred into the 96 wells. Standard immunostaining and RNA sequencing (RNA-Seq) were used to analyze the cell phenotypes. Preliminary results indicate that all cells expressed the three pluripotency markers oct-4, nanog, and sox-2. After applying a cardiomyocyte differentiation protocol, the cells stained positively for troponin-3. The cells also elongated and became more cardiomyocyte-like in their morphology. We analyzed bulk RNA seq data and our preliminary results show 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 shows some pathways are off like the MAPK/p38, MAPK/JNK1-3 which is expected for a pluripotent state. We also have data supporting the activation of the hippo pathway with transcriptional co-activator with PDZ binding motif (TAZ) highly upregulated and yes-associated protein staining the cell body. In addition, GSK3B is off and TGFB1, LIF/PIK3, and AKT1 are on as expected for pluripotency. Examining the gene network of upregulated genes, one can clearly distinguish the pivotal role of FOS, FOXO1, and PIK3 all related to pluripotency. 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.

Assessment of Angiogenesis and Cell Survivability of an Inkjet Bioprinted Biological Implant in an Animal Model.

The rapidly growing field of tissue engineering hopes to soon address the shortage of transplantable tissues, allowing for precise control and fabrication that could be made for each specific patient. The protocols currently in place to print large-scale tissues have yet to address the main challenge of nutritional deficiencies in the central areas of the engineered tissue, causing necrosis deep within and rendering it ineffective. Bioprinted microvasculature has been proposed to encourage angiogenesis and facilitate the mobility of oxygen and nutrients throughout the engineered tissue. An implant made via an inkjet printing process containing human microvascular endothelial cells was placed in both B17-SCID and NSG-SGM3 animal models to determine the rate of angiogenesis and degree of cell survival. The implantable tissues were made using a combination of alginate and gelatin type B; all implants were printed via previously published procedures using a modified HP inkjet printer. Histopathological results show a dramatic increase in the average microvasculature formation for mice that received the printed constructs within the implant area when compared to the manual and control implants, indicating inkjet bioprinting technology can be effectively used for vascularization of engineered tissues.

Bioprinting of Decellularized Porcine Cardiac Tissue for Large-Scale Aortic Models.

Bioprinting is an emerging technique used to layer extrudable materials and cells into simple constructs to engineer tissue or arrive at in vitro organ models. Although many examples of bioprinted tissues exist, many lack the biochemical complexity found in the native extracellular matrix. Therefore, the resulting tissues may be less competent than native tissues-this can be especially problematic for tissues that need strong mechanical properties, such as cardiac or those found in the great vessels. Decellularization of native tissues combined with processing for bioprinting may improve the cellular environment for proliferation, biochemical signaling, and improved mechanical characteristics for better outcomes. Whole porcine hearts were decellularized using a series of detergents, followed by lyophilization and mechanical grinding in order to produce a fine powder. Temperature-controlled enzymatic digestion was done to allow for the resuspension of the decellularized extracellular matrix into a pre-gel solution. Using a commercial extrusion bioprinter with a temperature-controlled printhead, a 1:1 scale model of a human ascending aorta and dog bone shaped structures were printed into a reservoir of alginate and xanthium gum then allowed to crosslink at 37C. The bioengineered aortic construct was monitored for cell adhesion, survival, and proliferation through fluorescent microscopy. The dog bone structure was subjected to tensile mechanical testing in order to determine structural and mechanical patterns for comparison to native tissue structures. The stability of the engineered structure was maintained throughout the printing process, allowing for a final structure that upheld the dimensions of the original Computer-Aided Design model. The decellularized ECM (E = 920 kPa) exhibited almost three times greater elasticity than the porcine cardiac tissue (E = 330 kPa). Similarly, the porcine cardiac tissue displayed two times the deformation than that of the printed decellularized ECM. Cell proliferation and attachment were observed during the in vitro cell survivability assessment of human aortic smooth muscle cells within the extracellular matrix, along with no morphological abnormalities to the cell structure. These observations allow us to report the ability to bioprint mechanically stable, cell-laden structures that serve as a bridge in the current knowledge gap, which could lead to future work involving complex, large-scale tissue models.