Cell viability is an important factor to consider in cell transplantation. Cells lose their function during isolation, preservation, and infusion. The infusion pro-cess is one of the causes of viability loss in hepatocyte transplantation. Viability loss depends on cell quality, catheter size, and applied flow rate [69]. The last two factors are represented by wall shear stress, which will increase proportionally with decreased diameter and increased velocity. The cross-sectional area of the channel is influenced by the diameter of the catheter and the fluid velocity influenced by flow rate of the infusion. Meyburg et al. [69] showed that catheter size and flow rate of the infusion are important for hepatocyte viability because of shear stress. How-ever, the relationship between viability loss and shear stress remains unexplained.
Sedimentation during infusion process is also important for considering the effect
Figure 2.3: Cell viability loss. White box and whisker graph describe vertical syringe orientation viability loss. Gray box and whisker graph describe that for horizontal syringe orientation. p Values for vertical and horizontal comparison for 1.3, 2.7, 4.6, and 5.4 Pa are 0.004, 0.04, 0.08, and 0.14, respectively. Bar and square inside box denote mean and median value.
of shear stress because flow trajectories of the cells are influenced by sedimentation and the strength of the shear stress is defined by the location of the trajectory. In this study, we investigated the relationship between shear stress and sedimentation effect during infusion with different syringe orientations. Viability loss showed no apparent correlation with shear stress. However, difference in viability losses be-tween syringe orientations showed statistical differences bebe-tween the horizontal and vertical syringe orientations under low-shear stress conditions. These differences in viability loss must be derived from sedimentation occurring during cell infusion because the density of hepatocytes is higher than that of the medium and the ve-locity of the cells is low under these low-shear stress conditions. We will consider sedimentation effects in detail in the following discussion.
The differences of the viability loss between the horizontal and the vertical ori-entation can be estimated from sedimori-entation phenomena. The higher density of cells, at 1.1 g/cm3, than that of the medium, at 1.007 g/cm3, causes cells to settle on walls of the delivery channel (syringe, tube, and microchannel). From calculated estimates of the sedimentation effect, the sedimentation velocity was 0.015 mm/min for this combination of isolated hepatocytes and medium under the experimental temperature conditions. This velocity depends on the diameter of the cell, and the velocity of clustered cells will be greater than that of isolated cells. In this condition, a biased cell distribution occurs in the microchannel. A detailed distribution of the cells settling inside the delivery channel was estimated by sedimentation calculation.
Figure 2.4 shows the histograms of the cell distribution calculated using sedimenta-tion velocity during the elapsed time of the infusion process without any cell volume consideration under each shear stress condition. An ideal Poiseuille velocity profile and an ideal shear stress profile of the two dimensional channel are illustrated in Figure 4. Maximum shear stress occurs at the wall of the channel and minimum shear stress at its center. The percentages of cells settling on the wall are 60%, 30%, 20%, and 16% for 1.3, 2.7, 4.6, and 5.4 Pa, respectively, in the horizontal orienta-tion. These estimates predict that >60% of cells will experience high shear stress
near the bottom wall at 1.3 Pa. Non-uniform, biased distributions of cells under every shear stress condition are present, and the non-uniform distribution decreases in proportion to increasing velocity inside the delivery channel.
Figure 2.4: Correlation of velocity and shear stress inside infusion line, followed by cell distribution for particular shear stress condition inside microchannel. (A), (B), (C), and (D), respectively, denote 1.3, 2.7, 4.6, and 5.4 Pa cell distribution condition for horizontal orientation.
In the horizontal orientation, cells settling inside the delivery channel form a biased cell distribution pattern in supplying cells to the microchannel. Some cells are distributed near the wall in the higher shear stress region and a few cells near the centerline in the lower shear stress region, with cell distributions for each shear stress condition as mentioned above. This cell distribution pattern produces a higher viability loss compared with that in the vertical orientation, which is uniform.
The cell distribution pattern changes from biased to uniform as an effect of changing the supply flow from the horizontal to the vertical direction. Sedimenta-tion is made parallel to the syringe centerline and cells become distributed evenly along the fluid flow. This pattern continues inside the tube and a uniform cell dis-tribution is created in supplying the cells to the microchannel. On entering the microchannel, cells are exposed to shear uniformly, resulting in lower viability loss compared with the horizontal orientation. Our results suggest that removing the sedimentation effect is important not only to prevent aggregation but also to
im-prove the maintenance of cell viability by preventing cells from experiencing high shear stress.
The advantage of the vertical syringe orientation, besides safely delivering a uniform cell distribution into the microchannel or catheter, is its elimination of the necessity of gently shaking or rotating the syringe during the infusion process to prevent sedimentation at low cell concentrations. At high cell concentrations, we must consider a partial concentration difference effect on viability during infusion, and interaction between cells is also not negligible. We will in future investigate the concentration effect on cell viability during infusion. This study did not investigate the effect of shear stress on cell clusters or effect of cell interaction during flow on viability loss. These effects as well as sedimentation, flow pattern, and shear-induced particle migration, await further detailed investigation on the microscopic scale.
Under high-shear conditions, 5.4 Pa in both orientations may result in uniform cell distribution with a viability loss lower than that for the 4.6 Pa condition. At 5.4 Pa, the cells move to a lower shear stress region near the centerline because of a phenomenon called shear-induced particle migration. This mechanism may explain the appropriate conditions of 4.2 F suggested by Meyburg et al [69].
In conclusion, our result show that besides shear stress, sedimentation and cell distribution have important roles in viability loss. Sedimentation occurring in a hor-izontal syringe orientation can be eliminated by changing the syringe orientation to vertical. Vertical orientation markedly reduces viability losses compared with hori-zontal orientation and is proposed for improvement of the transplantation process.
Removing the sedimentation effect is important to improve the maintenance of cell viability by preventing cells from experiencing high shear stress. It can be applied in hepatocyte transplantation and other cell therapy applications that use the same delivery process.
Critical location of cell viability loss during the cell injection
process in hepatocyte transplantation using a
rectangular microchannel
[111]3.1 Introduction
Cell transplantation is a promising alternative therapy to treat end-stage dis-eases of an organ. The shortage of functional donor organs for transplantation has initiated a cell transplantation trend. The fundamental therapeutic concept of cell transplantation is the replacement of dysfunctional cells by functional live cells to treat a disease [73]. Cell transplantations for disease therapy in preclinical and clinical trials include bone marrow [113], neurons [43], islets [94], and hepatocytes [19].
Hepatocyte transplantation can cure inborn error diseases, biliary atresia, cir-rhosis, fulminant hepatic failure, and viral hepatitis (cirrhosis) [32]. The benefits
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of hepatocyte transplantation are that cells can be received by multiple recipients through simple administration using cryopreserved cells, and the cells can be re-peatedly transplanted [25]. Hepatocyte transplantation also reduces costs compared with organ transplantation [25, 107]. Transplantation of hepatocytes is a promis-ing treatment for liver diseases because of various administration choices and cost considerations. However, its translation to clinical practice has some problems.
Some of the problems in hepatocyte transplantation are a limited supply of donor livers, isolation of high-quality cells, viability loss upon cryopreservation, low cell engraftment and proliferation, and allograft rejection [25]. Important considerations for hepatocyte transplantation are its long-term efficacy [18] and therapeutic benefits [25]. As a basic quality indicator of cells, the viability of hepatocytes is measured after cell isolation or before cell injection [98].
Viability measurement post-injection for purposes other than cell transplantation has been reported by some research groups. Studies have reported the effects of cell injection by a needle or cannula on the viabilities of embryonic rat brains cells [114], fibroblasts [5], mesenchymal stem cells [2, 36, 117, 65, 4], neural stem cells [88] and hepatocytes [69]. Improvement of cell injection is desirable to increase the quantities of functional live cells delivered to the target organ, and mechanistic investigation of the phenomena may facilitate development of the cell injection process.
Hepatocyte apoptosis [21], anoikis [96], necrosis [12], and necroptosis might oc-cur in cell transplantation. However, mechanically induced accidental cell death [30], which is considered as the primary cause of hepatocyte viability loss during cell injection for transplantation, is not clearly defined. Knowledge of mechanically induced accidental cell death is necessary for improvement of the cell injection pro-cess because the duration of the event is faster than programmed cell death such as apoptosis, anoikis, necrosis, and necroptosis. Identification of mechanically induced accidental cell death is necessary to achieve safe cell injection conditions.
Safe clinical conditions for hepatocyte transplantation have been determined empirically by cell injection experiments using various catheter sizes and flow rates
[69]. Safe cell injection conditions for clinical application are suggested to be a flow rate of less than 2 mL/minute using a catheter of 4.2 F [69]. However, wall shear stress in the 4.2 F catheter at a flow rate of 2 mL/minute is higher than that in 5 and 6.6 F catheters at the same flow rate with respective values of 1.3, 0.5, and 0.3 Pa [110].
Shear stress-induced hepatocyte viability loss has not yet been evaluated, espe-cially in terms of a mechanical cause during cell injection. The cell injection problem of sedimentation in the syringe is a clinical obstacle for cell delivery in transplan-tation. In practice, gently shaking and rotating the syringe are usually undertaken to prevent sedimentation [70]. Therefore, observations of the effects of sedimen-tation and shear stress on cell viability loss may further our understanding of the phenomena.
The effect of shear stress and sedimentation have been investigated at macro-scale using a rectangular microchannel model [110]. This results were found, the vertical syringe orientation reduced viability loss during hepatocyte delivery and removed the sedimentation effect in the syringe. Avoiding sedimentation can pre-vent a hepatocyte from experiencing high shear stress located near the wall of the delivery line. However, the process of hepatocyte viability loss at the near wall of a microchannel has not been verified experimentally.
This study identified the critical location of hepatocyte viability loss. It was determined whether the hepatocyte viability loss was caused by shear stress on a cell in suspension or near the wall of the delivery line. Therefore, this study determined the critical location of live hepatocyte loss by comparing the live cell distribution at the upstream and downstream in a microchannel. The distribution difference in the microchannel identified the cell loss location, which is preliminary information to understand the processes of viability loss in the delivery line during hepatocyte injection.