Thermal inkjet bioprinting drastically alters cell phenotype

Abstract

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.

Keywords: differentiation; pluripotency; thermal inkjet bioprinting.

Figure 1.

Confocal microscopy images visualizing staining for OCT-4, Sox-2 and Nanog proteins, as well as DAPI (A). Non-printed control samples (B). Cell aggregates after 50 h stained for OCT-4 (green), Sox-2 (red), and DAPI (blue) (C). Cell aggregates after 50 h stained for OCT-4 (green), Nanog (red) and DAPI (blue) (D). four-split panel of TIB cells plated after 24 h post-printing stained for OCT-4 (green), Sox-2 (red), and DAPI (blue) showing individual colors and all three color channels overlaid. E. four-split panel of TIB cells plated after 24 h post-printing stained for OCT-4 (green), Nanog (red), and DAPI (blue) showing individual colors and all three color channels overlaid. Scale bars are 100 µm except for D.

Figure 2.

TIB fibroblasts were differentiated using a cardiomyocyte differentiation protocol. Cells were stained with a primary troponin I type 3 (cardiac) antibody labeled with Alexa Fluor 647. (A) and (B) a combined channel with cells that spread out from aggregates, as shown in figure 1 (B) and (C) generated by the hanging drop method. (C) Combined blue/red channel showing control samples showing non-printed cells that underwent the differentiation protocol. (D) Combined blue/red channel showing cells that were differentiated immediately after plating into a well without hanging drops.

Figure 3.

Shows TIB fibroblasts expressing YAP (red) and Alpha tubulin (green) after 3 h (A) and 24 h (B) postprinting.

Figure 4.

Differential expression of genes associated with pluripotency-relevant pathways. Genes that should be downregulated and upregulated are in red and green.

Figure 5.

Significantly upregulated gene network diagram at 12 h, depicting the pivotal role of the pluripotency regulating genes FOS, FOXO1, and PIK3.

Chart 1.

Fold-change in the expression of other pluripotency-related genes in TIB vs manually seeded fibroblasts.