Recently, 3D bioprinting techniques have been broadly recognized as a promising tool to fabricate functional tissues and organs. The bioink used for 3D bioprinting consists of biological materials and cells. Because of the dominant gravitational force, the suspended cells in the bioink sediment resulting in the accumulation and aggregation of cells. This study primarily focuses on the quantification of cell sedimentation-induced cell aggregation during and after inkjet-based bioprinting. The major conclusions are summarized as follows: (1) as the printing time increases from 0 min to 60 min, the percentage of the cells forming cell aggregates at the bottom of the bioink reservoir increases significantly from 3.6% to 54.5%, indicating a severe cell aggregation challenge in 3D bioprinting, (2) during inkjet-based bioprinting, at the printing time of only 15 min, more than 80% of the cells within the nozzle have formed cell aggregates. Both the individual cells and cell aggregates tend to migrate to the vicinity of the nozzle centerline mainly due to the weak shear-thinning properties of the bioink, and (3) after the bioprinting process, the mean cell number per microsphere increases significantly from 0.38 to 1.05 as printing time increases from 0 min to 15 min. The maximum number of cells encapsulated within one microsphere is ten, and 29.8% of the microspheres with cells encapsulated have contained small or large cell aggregates at the printing time of 15 min.
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 (1) 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 × 106 cells/ml and a high cell concentration of 5 × 106 cells/ml; and (2) 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 has 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.
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.
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