Inkjet Cell Printing Research Articles

Development of Cyroprotectant Agent-free Cell Cryopreservation by Superflash Freezing of Micro-droplets

Cell cryopreservation is the only way to stop biological activity and is widely used to maintain backups of cells. Conventionally, at least a cryoprotectant agent (CPA) must be added to avoid forming damaging ice crystals and to vitrify water on the inside and outside of the cell. We developed a CPA-free cryopreservation method by superflash freezing based on inkjet cell printing, in that cells were ejected as micro-droplets and deposited on the liquid nitrogen-cooled substrate. The method cryopreserved mouse fibroblast cells at a viability almost as high as the conventional method using dimethyl sulfoxide. To improve the reproducibility and viability further, the thawing process is important as well as the cooling process. Next, we also developed an automatic thawing apparatus that transports the vitrified cells rapidly into a prewarmed medium using a spring hinge. The experimental results indicate that the combination of superflash freezing with the rapid thawing process using the apparatus would become a possible approach to circumvent the problems associated with the addition of CPAs.

Cryoprotectant-free cryopreservation of mammalian cells by superflash freezing

Cryopreservation is widely used to maintain backups of cells as it enables the semipermanent storage of cells. During the freezing process, ice crystals that are generated inside and outside the cells can lethally damage the cells. All conventional cryopreservation methods use at least one cryoprotective agent (CPA) to render water inside and outside the cells vitreous or nanocrystallized (near-vitrification) without forming damaging ice crystals. However, CPAs should ideally be avoided due to their cytotoxicity and potential side effects on the cellular state. Herein, we demonstrate the CPA-free cryopreservation of mammalian cells by ultrarapid cooling using inkjet cell printing, which we named superflash freezing (SFF). The SFF cooling rate, which was estimated by a heat-transfer stimulation, is sufficient to nearly vitrify the cells. The experimental results of Raman spectroscopy measurements, and observations with an ultrahigh-speed video camera support the near-vitrification of the droplets under these conditions. Initially, the practical utility of SFF was demonstrated on mouse fibroblast 3T3 cells, and the results were comparable to conventional CPA-assisted methods. Then, the general viability of this method was confirmed on mouse myoblast C2C12 cells and rat primary mesenchymal stem cells. In their entirety, the thus-obtained results unequivocally demonstrate that CPA-free cell cryopreservation is possible by SFF. Such a CPA-free cryopreservation method should be ideally suited for most cells and circumvent the problems typically associated with the addition of CPAs.

Single-Cell Analysis Using Drop-on-Demand Inkjet Printing and Probe Electrospray Ionization Mass Spectrometry.

This study describes a novel method for single-cell analysis and lipid profiling by combining drop-on-demand inkjet cell printing and probe electrospray ionization mass spectrometry (PESI-MS). Through inkjet sampling of a cell suspension, droplets with single cells were generated, precisely dripped onto a tungsten-made electrospray ionization needle, and immediately sprayed under a high-voltage electric field. Lipid fingerprints of single cells were obtained by a mass spectrometry (MS) detector. A homemade magnetic stirring device was applied to the cell suspension reservoir, which controlled the homogeneous distribution of cells in liquid and improved the single-cell-droplet percentage by 43.8%. Eight types of single cells were screened in our platform and further differentiated by principal component analysis based on cellular surface phospholipids. Thus, this study successfully provides a facile method for the direct MS profiling of single-cell lipids by PESI-MS.

A novel approach for precisely controlled multiple cell patterning in microfluidic chips by inkjet printing and the detection of drug metabolism and diffusion.

In this work we report the use of inkjet printing as a precise and convenient means for microscale cell patterning in microfluidic chips followed by cell co-culture, stimulation and analysis. A self-made inkjet printing device was manufactured with adjustable parameters, which was capable of multiple cell printing within biocompatible materials. Sodium alginate was used as a printing matrix for cell encapsulation, and precisely distributed cell arrays on glass slides were obtained by accurate software controlled printing. By covering a PDMS layer with the corresponding microchannels onto the cell array substrate and subsequently injecting an ion cross-linking reagent, the cells containing alginate arrays gelated immediately and were immobilized on the bottom of the microchip, which could be utilized for cell culture and analysis. HepG2 cells and U251 cells were successfully co-patterned in the microchip and used for drug metabolism and diffusion experiment to imitate the in vivo situation, as a means to ascertain the capability of the system for precise microscale cell patterning in a microchip. The prodrug tegafur was metabolized by HepG2 cells into the active anticancer compound 5-fluorouracil and this produced an adverse gradient effect on U251 cells according to the distance from the HepG2 cells. The developed approach presented a feasible way to integrate inkjet cell printing and microfluidic chips for the first time, which is proved to be capable of spatially controlled printing of multiple kinds of cells into a microchip for cell culture, stimulation and analysis, which could be applied to tissue engineering, drug testing and related areas. We envision that the approach will help significantly increase the cell patterning efficacy in microfluidic chips as well as reduce the extent of laborious experimental work.

Tissue Engineering: Three‐Dimensional Human Tissue Chips Fabricated by Rapid and Automatic Inkjet Cell Printing (Adv. Healthcare Mater. 4/2013)

Rapid and automatic tissue engineering technology for 3D-human tissue chips consisting of over 400 simplified microtissues is described by Mitsuru Akashi and co-workers on page 534. The 3D-liver micro-arrays consisting of hepatocytes and endothelial cells are prepared by combination of hierarchical cell manipulation and inkjet cell printing technologies. The 3D-human tissue chips will be useful as an innovative technology for pharmaceutical assays.

Three‐Dimensional Human Tissue Chips Fabricated by Rapid and Automatic Inkjet Cell Printing

Three‐Dimensional Human Tissue Chips Fabricated by Rapid and Automatic Inkjet Cell Printing

3D-Human Tissue Chips Fabricated by Inkjet Cell Printing

3D-Human Tissue Chips Fabricated by Inkjet Cell Printing

Supporting Biomaterials for Articular Cartilage Repair.

Orthopedic surgeons and researchers worldwide are continuously faced with the challenge of regenerating articular cartilage defects. However, until now, it has not been possible to completely mimic the biological and biochemical properties of articular cartilage using current research and development approaches. In this review, biomaterials previously used for articular cartilage repair research are addressed. Furthermore, a brief discussion of the state of the art of current cell printing procedures mimicking native cartilage is offered in light of their use as future alternatives for cartilage tissue engineering. Inkjet cell printing, controlled deposition cell printing tools, and laser cell printing are cutting-edge techniques in this context. The development of mimetic hydrogels with specific biological properties relevant to articular cartilage native tissue will support the development of improved, functional, and novel engineered tissue for clinical application.