Magnetic Stirring Device for Limiting the Sedimentation of Cells inside Microfluidic Devices.

In experiments considering cell handling in microchannels, cell sedimentation in the storage container is a key problem because it affects the reproducibility of the experiments. Here, a simple and low-cost cell mixing device (CMD) is presented; the device is designed to prevent the sedimentation of cells in a syringe during their injection into a microfluidic channel. The CMD is based on a slider crank device made of 3D-printed parts that, combined with a permanent magnet, actuate a stir bar placed into the syringe containing the cells. By using A549 cell lines, the device is characterized in terms of cell viability (higher than 95%) in different mixing conditions, by varying the oscillation frequency and the overall mixing time. Then, a dedicated microfluidic experiment is designed to evaluate the injection frequency of the cells within a microfluidic chip. In the presence of the CMD, a higher number of cells are injected into the microfluidic chip with respect to the static conditions (2.5 times), proving that it contrasts cell sedimentation and allows accurate cell handling. For these reasons, the CMD can be useful in microfluidic experiments involving single-cell analysis.

Microcapillary cell extrusion deposition with picolitre dispensing resolution.

Extrusion-based cell deposition has become a prominent technique for expanding bioprinting applications. However, the associated print resolution in the order of nanolitre or above has been a limiting factor. The demand for improving print resolution towards the scale of a single cell has driven the development of precision nozzle extrusion, although the benefits gained remain ambiguous. Here, aided by in situ imaging, we investigated the dynamics of cell organisation through an extrusion-based microcapillary tip with picolitre precision through in-air or immersion deposition. The microcapillary extrusion setup, termed 'Picodis', was demonstrated by generating droplets of colouring inks immersed in an immiscible medium. Next, using 3T3 fibroblast cells as an experimental model, we demonstrated the deposition of cell suspension, and pre-aggregated cell pellets. Then, the dynamic organisation of cells within the microcapillary tip was described, along with cell ejection and deposition upon exiting the tip opening. The vision-assisted approach revealed that when dispersed in a culture medium, the movements of cells were distinctive based on the flow profiles and were purely driven by laminar fluid flow within a narrow tip. The primary process limitations were cell sedimentation, aggregation and compaction, along with trapped air bubbles. The use of picolitre-level resolution microcapillary extrusion, although it provides some level of control for a small number of cells, does not necessarily offer a reliable method when a specified number of cells are required. Our study provides insights into the process limitations of high-resolution cell ink extrusion, which may be useful for optimising biofabrication processes of cell-laden constructs for biomedical research. Supplementary information: The online version contains supplementary material available at 10.1007/s42242-022-00205-3.

Numerical assessment of recellularization conditions to vessel occlusion.

To ensure the functional properties of an organ generated by the process of decellularization and recellularization, the initial density and distribution of seeding cells in the parenchymal space should be maximized. However, achieving a uniform distribution of cells across the entire organ is not straightforward because of vessel occlusion. This study assessed vessel occlusion during recellularization under different conditions. A combination of the electrical analog permeability (EPA) model, computational fluid dynamics (CFD), and discrete element method (DEM) was employed to describe the vessel occlusion phenomenon. In particular, realistic flow distributions in vascular trees of the decellularized organ were indicated by the EPA model. The cell suspension flow was modeled by a coupled CFD-DEM model, whereby living cells were presented as a discrete phase (solved by the DEM solver), and the culture medium was modeled as the fluid phase (solved by CFD solver). The cell suspension velocity was reduced up to 47% after decellularization, which directly affected cell movement. Simulation results also indicate that the occurrence of vessel occlusion was promoted by gravity direction in the asymmetric bifurcation and increased as the cell concentration increased. The assessment of vessel occlusion under different conditions was quantitatively investigated. The model provides insights into the dynamics of cells in the vessel compartment, allowing for the selection of optimum seeding parameters for the recellularization process.

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.

Contributions of Red Blood Cell Sedimentation in a Driving Syringe to Blood Flow in Capillary Channels.

The erythrocyte sedimentation rate (ESR), which has been commonly used to detect physiological and pathological diseases in clinical settings, has been quantified using an interface in a vertical tube. However, previous methods do not provide biophysical information on blood during the ESR test. Therefore, it is necessary to quantify the individual contributions in terms of viscosity and pressure. In this study, to quantify RBC sedimentation, the image intensity (Ib) and interface (ß) were obtained by analyzing the blood flow in the microfluidic channels. Based on threshold image intensity, the corresponding interfaces of RBCs (Ib > 0.15) and diluent (Ib < 0.15) were employed to obtain the viscosities (µb, µ0) and junction pressures (Pb, P0). Two coefficients (CH1, CH2) obtained from the empirical formulas (µb = µ0 [1 + CH1], Pb = P0 [1 + CH2]) were calculated to quantify RBC sedimentation. The present method was then adopted to detect differences in RBC sedimentation for various suspended blood samples (healthy RBCs suspended in dextran solutions or plasma). Based on the experimental results, four parameters (µ0, P0, CH1, and CH2) are considered to be effective for quantifying the contributions of the hematocrit and diluent. Two coefficients exhibited more consistent trends than the conventional ESR method. In conclusion, the proposed method can effectively detect RBC sedimentation.

Red Blood Cell Sedimentation Index Using Shear Stress of Blood Flow in Microfluidic Channel.

Red blood cell sedimentation has been used as a promising indicator of hematological diseases and disorders. However, to address several issues (i.e., syringe installation direction, blood on-off flow control, image-based quantification, and hemodilution) raised by the previous methods, it is necessary to devise a new method for the effective quantification of red blood cell sedimentation under a constant blood flow. In this study, the shear stress of a blood flow is estimated by analyzing an interface in a co-flowing channel to quantify the red blood cell sedimentation in blood syringes filled with blood (hematocrit = 50%). A red blood cell sedimentation index is newly suggested by analyzing the temporal variations in the shear stress. According to the experimental investigation, the sedimentation index tends to decrease at a higher flow rate. A higher level of hematocrit has a negative influence on the sedimentation index. As a performance demonstration of the present method, the red blood cell sedimentation processes of various test bloods were quantitatively compared in terms of the shear stress, image intensity, and sedimentation velocity. It was found that the proposed index provided a more than 10-fold increase in sensitivity over the previous method (i.e., image intensity). Additionally, it provided more consistent results than another conventional sedimentation method (sedimentation velocity). In conclusion, the present index can be effectively adopted to monitor the red blood cell sedimentation in a 10-min blood delivery.

Comparison of LC-MS/MS and chemiluminescent immunoassays for immunosuppressive drugs reveals organ dependent variation in blood cyclosporine a concentrations.

INTRODUCTION: Life-long monitoring of immunosuppressive drugs (ISDs) in blood is essential after organ transplantation. However, the ISD concentrations vary depending on the assay employed. ISDs are strongly bound to cytoplasmic proteins in erythrocytes in circulation. Therefore, the relatively rapid sedimentation of blood cells in whole blood samples may affect the results when using liquid handling robots. METHODS: We used 1115 blood samples from outpatients and ward patients with kidney (n = 373), liver (n = 101), heart (n = 29) and bone marrow (n = 155) transplant. Whole blood samples were pretreated by protein precipitation. Alternatively, the samples were hemolyzed by freezing prior to precipitation. ISDs were analyzed by a 2-plexing liquid chromatography tandem mass spectrometry (LC-MS/MS) assay and commercial chemiluminescent microparticle immunoassays (CMIA). RESULTS: The difference between the two sample preparation practices was negligible (<2%). Overall, the measured ISD concentrations in patient samples were lower by LC-MS/MS than by CMIA. The difference was the largest (20.2%) and the smallest (9.1%) in samples from liver and from heart transplant patients, respectively. CONCLUSIONS: CMIA overestimates blood ISD concentrations as compared to LC-MS/MS. The extent of the difference was found to be organ transplant dependent. The ISDs can be quantitatedeither from intact or hemolyzed blood samples.

Hydrogel Bioink with Multilayered Interfaces Improves Dispersibility of Encapsulated Cells in Extrusion Bioprinting.

One of the challenges for extrusion bioprinting using low-viscosity bioinks is the fast gravity-driven sedimentation of cells. Cells in a hydrogel bioink that features low viscosity tend to settle to the bottom of the bioink reservoir, and as such, their bioprintability is hindered by association with the inhomogeneous cellularized structures that are deposited. This is particularly true in cases where longer periods are required to print complex or larger tissue constructs. Increasing the bioink's viscosity efficiently retards sedimentation but gives rise to cell membranolysis or functional disruption due to increased shear stress on the cells during the extrusion process. Inspired by the rainbow cocktail, we report the development of a multilayered modification strategy for gelatin methacryloyl (GelMA) bioink to manipulate multiple liquid interfaces, providing interfacial retention to retard cell sedimentation in the bioink reservoir. Indeed, the interfacial tension in our layer-by-layer bioink system, characterized by the pendant drop method, was found to be exponentially higher than the sedimental pull (ΔGravity-Buoyancy = ∼10-9 N) of cells, indicating that the interfacial retention is crucial for preventing cell sedimentation across the adjacent layers. It was demonstrated that the encapsulated cells displayed better dispersibility in constructs bioprinted using the multilayered GelMA bioink system than that of pristine GelMA where the index of homogeneity of the cell distribution in the multilayered bioink was 4 times that of the latter.