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Tissue Engineering

3D-Bioprinting Coupled with Electroporation for Building Genetically Heterogeneous Heart Tissues

(J-398) 3D-Bioprinting Coupled with Electroporation for Building Genetically Heterogeneous Heart Tissues

Thursday, October 12, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row J - Poster # 398

Presenting Author(s)

MF

Mahya Fazel-Zarandi

Undergraduate Student
Princeton University
Princeton, New Jersey, United States

Co-Author(s)

AL

Aric Lu

Graduate Student (G6)
Harvard University, United States
ML

Margaret Lashutka

Undergraduate Student
The Ohio State University, United States
CM

Carlos Marquez

Graduate Student (G3)
Harvard University, United States

Primary Investigator(s)

JL

Jennifer Lewis

Hansjörg Wyss Professor of Biologically Inspired Engineering
Harvard University and the Wyss Institute for Biologically Inspired Engineering, United States

Introduction:: Ventricular heart muscle is composed of multiple cell types with distinct patterns of gene expression. These primarily include contractile cardiomyocytes but also a minority of conducting cardiomyocytes (e.g., Purkinje fibers) that are vital to the heart’s function. One current challenge in building heart tissues is the difficulty of building genetically heterogeneous tissues that contain the various cell types associated with the heart. In this paper, we have coupled 3D bioprinting with electroporation, which is achieved by loading agarose microparticles as the ink for printing into an electrode-containing core-shell nozzle. The design can be used to force the expression of transcription factors to reprogram contractile cardiomyocytes into conducting cardiomyocytes for making iPSC-derived heart cells, which is a major advance in printing heterogeneous cardiac tissue for organ repair.

Materials and Methods::

The Nozzle

The core-shell nozzle was designed on SOLIDWORKS and 3-D printed with HTM140 on Aureus. It consists of 2 eccentric electrodes: an inner electrode with a length of 1.5inch, an inner diameter of 0.33mm, and an outer diameter of 0.65mm, and an outer electrode with a length of 0.5inch, an inner diameter of 1.54mm, and an outer diameter of 1.83mm. Human embryonic kidney cell lines (HEK 293) along with the gel for printing and green fluorescence protein (GFP) were loaded in a syringe attached to the nozzle, and moved into the space between the 2 electrodes. An electrical connection on the way provided the voltage for electroporation. Live/Dead analysis was performed on the samples.

The Microparticles

We considered 2 types of microparticles as the ink for bioprinting: agarose and alginate. The agarose microparticles were produced using extrusion fragmentation by being passed through a series of nozzles. The alginate microparticles were produced using a Syringe Pump. Once created, the microparticles were crosslinked in 2mM calcium chloride. We tested 4 different concentrations of agarose: 0.5, 1, 1.5, and 2%. For alginate, we only tested a concentration of 2% since this was determined to be an optimal concentration for our purposes based on experiments carried out by others in the lab. The microparticles were imaged under light microscope, and their diameter was determined using ImageJ. DNA binding analysis was performed by running DNA samples on home-made agarose and alginate gels. Conductivity of the microparticles was tested using a Cell Conductivity Probe.

Results, Conclusions, and Discussions::

We successfully 3-D printed the nozzle and tested it by extruding 1% solid agarose, but the agarose was fractured upon exiting. To address this, we explored using microparticles (MPs) as ink for printing. We analyzed different concentrations of agarose MPs and found that the 1% agarose MPs had the optimal diameter for our nozzle (mean diameter of 98.24 ± 21 um), so we used them for subsequent experiments (see the figures section for the sizing data of all concentrations).

Agarose's neutral charge makes it suitable for electroporation, unlike other inks that bind DNA and hinder cell entry. Our data confirmed agarose's lower DNA affinity compared to alginate, as a sample of 20 uL GFP moved for about 3.5cm in the agarose gel while moving for about 1.1cm in the alginate gel. In addition, our results show that the 1% agarose MPs have a conductivity of 1982 uS/cm while the 2% alginate MPs have a conductivity of 2637 uS/cm. While the 2% alginate MPs possess higher electrical conductivity, their large size makes them unsuitable for use with our nozzle design. Considering size and conductivity, we chose 1% agarose MPs as our bioprinting ink.

We combined the nozzle and MPs to introduce a GFP gene into HEK 293 cells. The results demonstrated successful electroporation, with varying efficiencies based on voltage and pulse duration (see the figures section for details). A 4ms pulse at 56V yielded the highest electroporation rate. Although electroporation can harm cells, Live/Dead analysis showed over 80% cell viability after electroporation with 50-70V currents for 4ms.

Our innovation enables bio-printing of heterogeneous organs by combining 3D-printing with electroporation. This system allows TF expression within stem cell-derived tissues, enabling the transformation of one cell type into another, thus creating genetically heterogeneous organs. Future work may involve testing the system on induced pluripotent stem cells (iPSCs) and using specific TFs like ETV1 to reprogram cardiomyocytes. The nozzle design could be further optimized for increased cell viability and counteracting water electrolysis during printing, possibly by coating the electrodes with platinum black.

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