Our results demonstrate the protective effects of alginate shear-thinning hydrogels on cells experiencing clinically relevant syringe needle flow. During syringe needle flow, cells experience three types of mechanical forces that can lead to cell disruption: (i) a pressure drop across the cell, (ii) shearing forces due to linear shear flow, and (iii) stretching forces due to extensional flow. In our experimental design, we specifically assay for physical disruption of the cell membrane using ethidium homodimer-1, which is a high-affinity nucleic acid stain that cannot diffuse across an intact lipid bilayer, thereby allowing direct quantification of cells that experience cell membrane damage in response to these mechanical forces.
Extensional flow during ejection is likely the leading cause of acute cell death during syringe needle flow. Pressure drop is not the main cause because we observed that pressure drop did not correlate with cell death in our studies (, ). In our flow experiments, peak pressure drop for the most protective alginate gel formulation (G
Pa) was 292.69
kPa (2.9269 bars, ), while a more compliant alginate gel formulation (G'
Pa) resulted in a lower peak pressure drop of 215.40
kPa (2.1540 bars, ) but significantly fewer viable cells (). The pressures measured in our most protective alginate hydrogel (~3 bars) are similar in scale to those previously reported to be well tolerated in studies of hydrocyclone cell separation protocols.36
We next considered the role of shear forces in inducing mechanical disruption of the cell membrane. The maximum shear rate during syringe needle flow occurs at the wall of the needle and was calculated to be 26,800
. A linear shear rate of similar magnitude (17,240
, the maximum rate experimentally possible) was directly applied to cells within a PBS cell carrier using a rheometer. Acute viabilities for both linearly sheared and not sheared cells were greater than 90% and were statistically similar (); therefore, shearing alone does not significantly disrupt the cell membrane. Taken together, we conclude that the pressure drop and linear shear flow are not the main causes of acute cell death during syringe needle flow.
Extensional flow occurs when there is an abrupt change in the flow geometry causing a dramatic increase in linear velocity. The change in cross-sectional diameter from 3.170 to 0.185
mm as the fluid transitions from the syringe to the needle results in a 294-fold increase in linear velocity (). Previous experiments have shown that cells undergo significant stretching and deformation in extensional flows, leading to cell death.37,38
Further, extensional flow is commonly used to stretch and fragment DNA for high throughput sequencing.39,40
HUVEC in a saline carrier subjected to syringe needle flow (which includes extensional and linear shear flows) resulted in 58.7% viability () while cells subjected to linear shear flow alone resulted in 89.1% viability (). This suggests the leading contributor to acute cell death during syringe needle flow is mechanical disruption caused by extensional flow.
We hypothesized that cell encapsulation within hydrogel cell carriers may provide mechanical protection that prevents the damage caused by extensional flow during injection procedures. To systematically test this hypothesis, we designed a range of alginate hydrogels with varying viscoelastic properties and assessed their protective capabilities. We identified a specific alginate hydrogel formulation (G'
Pa) that significantly improved the acute cell viability of four different cell types (, , and ). In comparison, noncrosslinked alginate solutions were not cell protective and resulted in low cell viability comparable to PBS-only cell carriers (). Therefore, cell protection is a result of the mechanical properties of the alginate hydrogel and not the biochemical properties of the alginate biopolymers.
Upon ejection, our alginate hydrogels may be experiencing “shear banding” along the inner wall of the needle. During shear banding, a layer of hydrogel near the walls undergoes shear thinning to form a fluid while the rest of the hydrogel remains intact. This layer of shear-thinned fluid acts as a lubricant, allowing the rest of the intact hydrogel to slip through the needle in a process known as “plug flow”. Many noncovalently crosslinked hydrogels have been reported to undergo plug flow.41,42
One requirement for plug flow is the rapid shear thinning of the hydrogel into a viscous fluid as demonstrated by our alginate hydrogels (). We hypothesize that this plug flow behavior may be the mechanism by which cells are rescued from the damaging effects of extensional flow by alginate hydrogels. During plug flow, a portion of the hydrogel may retain its structural integrity and not become shear-thinned. Cells encapsulated within these hydrogel plugs may be shielded from deformation by extensional flow and shear by linear flow. Changing the alginate hydrogel formulation by altering the degree of crosslinking or the polymer molecular weight may impact the ability to undergo plug flow. This mechanical protection strategy relies only on the mechanical flow properties and is independent of cell properties. Therefore, this cell protection strategy should be broadly applicable to multiple cell types. Consistent with this idea, the alginate formulation that produced a hydrogel with G'
Pa provided the most cell protection for all four cell types tested ().
In conclusion, stem cell transplantations are notoriously inefficient due to the low viability of transplanted cells. Currently, to overcome this low transplantation efficiency, a large quantity of cells must be transplanted to increase the likelihood of a successful procedure.1,16
Our studies demonstrate a novel strategy to protect transplanted cells from the mechanical forces experienced during syringe needle flow that may reduce the stem cell concentration required for successful transplantation. Using fewer cells to achieve a similar number of transplanted, viable cells would greatly reduce the cost, time, and effort required for transplantation protocols.