Investigation of Cell Aggregation on the Printing Performance in Inkjet-Based Bioprinting of Cell-Laden Bioink.

During 3D bioprinting, when the gravitational force exceeds the buoyant force, cell sedimentation will be induced, resulting in local cell concentration change and cell aggregation which affect the printing performance. This paper aims at studying and quantifying cell aggregation and its effects on the droplet formation process during inkjet-based bioprinting and cell distribution after inkjet-based bioprinting. The major conclusions of this study are as follows: (1) Cell aggregation is a significant challenge during inkjet-based bioprinting by observing the percentage of individual cells after different printing times. In addition, as polymer concentration increases, the cell aggregation is suppressed. (2) As printing time and cell aggregation increase, the ligament length and droplet velocity generally decrease first and then increase due to the initial increase and subsequent decrease of the viscous effect. (3) As the printing time increases, both the maximum number of cells within one microsphere and the mean cell number have a significant increase, especially for low polymer concentrations such as 0.5% (w/v). In addition, the increased rate is the highest using the lowest polymer concentration of 0.5% (w/v) because of its highest cell sedimentation velocity.

A review on cell damage, viability, and functionality during 3D bioprinting.

Three-dimensional (3D) bioprinting fabricates 3D functional tissues/organs by accurately depositing the bioink composed of the biological materials and living cells. Even though 3D bioprinting techniques have experienced significant advancement over the past decades, it remains challenging for 3D bioprinting to artificially fabricate functional tissues/organs with high post-printing cell viability and functionality since cells endure various types of stress during the bioprinting process. Generally, cell viability which is affected by several factors including the stress and the environmental factors, such as pH and temperature, is mainly determined by the magnitude and duration of the stress imposed on the cells with poorer cell viability under a higher stress and a longer duration condition. The maintenance of high cell viability especially for those vulnerable cells, such as stem cells which are more sensitive to multiple stresses, is a key initial step to ensure the functionality of the artificial tissues/organs. In addition, maintaining the pluripotency of the cells such as proliferation and differentiation abilities is also essential for the 3D-bioprinted tissues/organs to be similar to native tissues/organs. This review discusses various pathways triggering cell damage and the major factors affecting cell viability during different bioprinting processes, summarizes the studies on cell viabilities and functionalities in different bioprinting processes, and presents several potential approaches to protect cells from injuries to ensure high cell viability and functionality.

Cell-laden bioink circulation-assisted inkjet-based bioprinting to mitigate cell sedimentation and aggregation.

Three-dimensional (3D) bioprinting precisely deposits picolitre bioink to fabricate functional tissues and organs in a layer-by-layer manner. The bioink used for 3D bioprinting incorporates living cells. During printing, cells suspended in the bioink sediment to form cell aggregates through cell-cell interaction. The formation of cell aggregates due to cell sedimentation have been widely recognized as a significant challenge to affect the printing reliability and quality. This study has incorporated the active circulation into the bioink reservoir to mitigate cell sedimentation and aggregation. Force and velocity analysis were performed, and a circulation model has been proposed based on iteration algorithm with the time step for each divided region. It has been found that (a) the comparison of the cell sedimentation and aggregation with and without the active bioink circulation has demonstrated high effectiveness of active circulation to mitigate cell sedimentation and aggregation for the bioink with both a low cell concentration of 1 × 106cells ml-1and a high cell concentration of 5 × 106cells ml-1; and (b) the effect of circulation flow rate on cell sedimentation and aggregation has been investigated, showing that large flow rate results in slow increments in effectiveness. Besides, the predicted mitigation effectiveness percentages on cell sedimentation by the circulation model generally agrees well with the experimental results. In addition, the cell viability assessment at the recommended maximum flow rate of 0.5 ml min-1has demonstrated negligible cell damage due to the circulation. The proposed active circulation approach is an effective and efficient approach with superior performance in mitigating cell sedimentation and aggregation, and the resulting knowledge is easily applicable to other 3D bioprinting techniques significantly improving printing reliability and quality in 3D bioprinting.

Effect of topography parameters on cellular morphology during guided cell migration on a graded micropillar surface.

PURPOSE: Guided cell migration refers to the engineering of local cell environment to specifically direct cell migration and has important applications such as utilization in cell sorting and wound healing assays. Graded micropillar surfaces have been utilized for achieving guided cell migration. Topographic parameters such as micropillar diameter and spacing gradient may have effects on the morphology of attached cells. It is critical to understand this interaction between the cells and the underlying microscale structures. METHODS: In this study, a graded micropillar substrate has been fabricated to investigate the effects of the microtopography on the cell morphology in terms of the cell aspect ratio and cell circularity. RESULTS: It is found that 1) with the increase of the micropillar diameter, the cell aspect ratio has no significance change. At the small spacing gradients, the aspect ratio is smaller than that at the large spacing gradients; 2) statistical analysis shows both the micropillar diameter and spacing gradient have no significant effect on the cell aspect ratio compared to the flat surface; 3) the cell circularity at the small micropillar diameters is higher than that at the large micropillar diameters. The cell circularity at the micropillar gradient of 0.1 µm is higher than those at the other micropillar gradients; 4) three microtopographic conditions are considered to have statistically significant effect on the cell circularity compared to the flat surface, including the micropillar diameters of 5 µm and 10 µm and the spacing gradient of 0.1 µm.

Effects of Irgacure 2959 and lithium phenyl-2,4,6-trimethylbenzoylphosphinate on cell viability, physical properties, and microstructure in 3D bioprinting of vascular-like constructs.

Photocrosslinkable polymers such as gelatin methacrylate (GelMA) have various 3D bioprinting applications. These polymers crosslink upon exposure to UV irradiation with the existence of an appropriate photoinitiator. Two photoinitiators, Irgacure 2959 and lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) are commonly used. This study systematically investigates the effects of photoinitiator types on the cell viability, physical properties, and microstructure in 3D bioprinting of GelMA-based cellular constructs. The main conclusions are: (1) during the 3D bioprinting, the cell viability generally decreases as the photoinitiator concentration and printing time increase using both Irgacure 2959 and LAP. At the low photoinitiator concentrations (such as 0.3% and 0.5% (w/v)), the overall cell viability is good within the printing time of 60 min using both Irgacure 2959 and LAP. However, at the high photoinitiator concentrations (such as 0.7% and 0.9% (w/v)), the overall cell viability using LAP is much higher than that using Irgacure 2959 within the printing time of 60 min; (2) after the 3D bioprinting, the photoinitiator types, either Irgacure 2959 or LAP, have negligible effects on the post-printing cell viability after crosslinking; (3) after the 3D bioprinting, GelMA samples cured with Irgacure 2959 have slightly larger pore size, faster degradation rate, and greater swelling ratio compared to those cured with LAP; (4) 3D GelMA-based vascular-like constructs have been fabricated using dynamic optical projection stereolithography, and the measured dimensions have been compared with the designed dimensions showing good shape fidelity.