Static mixer and syringe plunger combination for viscous biomaterial solutions and uses thereof

Abstract

A system for mixing first and second solutions is provided. The system comprises: i) a syringe proportioner comprising a base adapted to receive first and second input syringes, and a gear assembly to drive a plunger of each of the input syringes to empty solution from each of the input syringes at a predetermined flow rate; ii) a channel comprising first and second inlets and a single outlet, the first and second inlets being adapted to fluidly couple respectively with first and second fluid expelling ends of each of the first and second input syringes, to receive solution from the first and second syringes; and iii) a static mixer comprising a fluid receiving end and a fluid discharging end, wherein the fluid receiving end is adapted to fluidly couple with the outlet of the channel and receive solution therefrom and the fluid discharging end is adapted to discharge fluid, said mixer comprising an internal geometry which provides mixing of a first and second solutions. The system is particularly useful for mixing solutions of different viscosities, for example, a cell suspension with a biomaterial solution to achieve effective mixing while minimizing sheer stress to retain cell viability.

Description

STATIC MIXER AND SYRINGE PLUNGER COMBINATION FOR VISCOUS BIOMATERIAL SOLUTIONS AND USES THEREOF FIELD

[0001] The present disclosure relates to a system for mixing first and second solutions and in particular, to a system comprising a syringe proportioner, a channel and a static mixer for mixing viscous biomaterial solutions and uses thereof.BACKGROUND

[0002] The goal of 3D bioprinting is to create cell-laden synthetic tissues that can perform the functions of native tissues. There are a variety of different 3D bioprinting methods, all of which rely on the additive manufacturing of biocompatible materials known as biomaterials. The viscosity of biomaterials is an important consideration depending on the 3D bioprinting method. In extrusion-based bioprinting, low viscosity is appropriate when the biomaterial is immediately crosslinked or supported in a sacrificial bath, whereas higher viscosity is required when real-time crosslinking or support baths are not desired.

[0003] The incorporation of cells into low viscosity biomaterials has been optimized with commercially available products (CELLINK CELLMIXER, VitroINK Mixing Kit) using static mixers which provide uniform and reproducible mixing of the cell suspension (with water-like rheology) and the low viscosity bioink. The internal geometry of the static mixers in these systems introduces turbulent flow, providing efficient mixing. However, high viscosity biomaterials require much higher flow rates to achieve turbulent flow, which likely introduces cell-damaging shear stress. As such, there is a trade-off between mixing efficiency and cell viability when mixing high viscosity biomaterials using these systems designed for low viscosity. Typical workarounds include manual mixing in open containers using a spatula or a pipette, or repeated plunging of two coupled syringes, both methods of which have poor reproducibility and are not suitable for good manufacturing practice (GMP) workflows.

[0004] The background herein is included solely to explain the context of the disclosure. This is not to be taken as an admission that any of the material referred to was published, known, or part of the common general knowledge as of the priority date.SUMMARY

[0005] Disclosed herein is a system for mixing two solutions, for example, solutions having different viscosities such as mixing an aqueous solution with a viscous solution comprising a biomaterial.

[0006] Thus, in accordance with an aspect, there is provided a validated system for mixing cells, microparticles and other additives in an aqueous solution into viscous biomaterial solutions in defined ratios.

[0007] In another aspect, there is disclosed a system for mixing first and second solutions. The system comprises a syringe proportioner comprising a base adapted to receive first and second input syringes, and a gear assembly to drive a plunger of each of the input syringes to empty solution from each of the input syringes at a predetermined flow rate. The system further comprises a channel comprising first and second inlets and a single outlet, the first and second inlets being adapted to fluidly couple respectively with first and second fluid expelling ends of each of the first and second input syringes, to receive solution from the first and second syringes. The system further comprises a static mixer comprising a fluid receiving end and a fluid discharging end, wherein the fluid receiving end is adapted to fluidly couple with the outlet of the channel and receive solution therefrom and the fluid discharging end is adapted to fluidly couple with an output syringe. The mixer comprises an internal geometry which provides mixing of a first solution from the first input syringe and a second solution from the second input syringe.

[0008] Accordingly, provided is a validated system comprising a static mixer for combining and mixing low and high viscosity fluid input streams (e.g., for mixing cells, microparticles and other additives in an aqueous solution into viscous biomaterial solutions) in respective ratios of, for example, 1:3, 1:4, and 1:10, i.e. aqueous solutiombiomaterial solution.

[0009] In some embodiments, the gear assembly of the syringe proportioner comprises a gear train and first and second gear racks respectively comprising first and second input syringe drivers, wherein rotation of the gear train in an actuation direction is configured to engage the first and second gear racks and actuate the first and second input syringe drivers to drive fluid from the first and second input syringes, respectively.

[0010] In some embodiments, the gear train comprises primary, secondary and tertiary gears, wherein actuation of the tertiary gear engages with the primary and secondary gears to engage the first and second gear racks, respectively, actuating the first and second input syringe drivers, wherein the primary gear has a first diameter and the secondary gear has a second diameter.

[0011] In some embodiments, the ratio of the first and second diameters of the primary and secondary gears corresponds to the relative volumes of the first and second solutions, to be dispensed by the syringe proportioner. In some embodiments, the first diameter is greater than the second diameter such that the ratio of the first diameter to the second diameter is selected from, for example, 3:1, 4:1 and 10:1, or other ratios. In other embodiments, the second diameter is greater than the first diameter such that the ratio may be selected from, for example, 1:3, 1:4 and 1:10, or other ratios. In some embodiments, the gears of the gear train are removable.

[0012] In some embodiments, the internal geometry of the mixer comprises one or more obstacles. In some embodiments, the obstacle(s) of the internal geometry of the mixer are in the form of: a chain-like structure; a helical or ribbon structure; a spiral structure; a grid-like structure; a lattice-like structure; or a fin structure. In preferred embodiments, the internal geometry comprises a lattice structure(s).

[0013] In some embodiments, the base of the syringe proportioner is adapted to removably retain the first and second input syringes. In some embodiments, the system comprises two input syringes, a first input syringe retained in the first input syringe retainer for containing a low viscosity fluid (e.g., cell suspension) and a second input syringe retained in the second input syringe retainer for containing a higher viscosity (e.g., biomaterial ink) solution.

[0014] In some embodiments, the system accommodates 1, 3, and 5 mL input syringes.

[0015] In some embodiments, the design is scaled up for compatibility with larger syringes.

[0016] In some embodiments, the channel comprises a Y-shape or T-shape.

[0017] In another aspect of the invention, a method of mixing first and second solutions having different viscosities is provided. The method comprises: i) actuating the gear assembly of a system as described herein to drive solution contained within the first and second input syringes into the static mixer for mixing via the channel, and ii) collecting the mixed solution as it is discharged from fluid discharging end of the static mixer.

[0018] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the disclosure, are given by way of illustration only and the scope of the claims should not be limited by these embodiments, but should be given the broadest interpretation consistent with the description as a whole.DRAWINGS

[0019] Certain embodiments of the disclosure will now be described in greater detail with reference to the attached drawings in which:

[0020] Figure 1A illustrates a perspective view of a first embodiment of a system according to the first aspect of the invention;

[0021] Figure IB illustrates the static mixer of the system of Figure 1A;

[0022] Figure 1C illustrates a portion of the syringe proportioner of the system of Figure 1A;

[0023] Figure ID illustrates a gear assembly for use in the system of Figure 1 A, the gear train having a gear ratio of 10: 1 ;

[0024] Figure IE illustrates a second embodiment of a gear assembly for use in the system of Figure 1 A, the gear train having a gear ratio of 3 : 1 ;

[0025] Figure IF illustrates a third embodiment of a gear assembly for use in the system of Figure 1A, the geartrain having a gear ratio of 4:1;

[0026] Figure 2A illustrates a second embodiment of a static mixer for use in the system of Figure 1A, the internal geometry having a ‘cellmixer’ arrangement, comprising an alternating helical geometry;

[0027] Figure 2B illustrates a further view of the static mixer of the system of Figure 1A, the internal geometry having a ‘lattice’ arrangement;

[0028] Figure 2C illustrates a third embodiment of a static mixer for use in the system of Figure 1A, the internal geometry having an ‘interfacial surface generator’ (ISG) arrangement;

[0029] Figure 2D illustrates a fourth embodiment of a static mixer for use in the system of Figure 1A, the internal geometry having a ‘serpentine’ arrangement;

[0030] Figure 3 illustrates experimental rheology and simulated shear stress achieved using mixing systems in accordance with embodiments. Logarithmic plots of measured viscosity (r|) as a function of shear rate (y) for A) PEGDA ink and C) TissuGel biomaterials prepared for three different ink: suspension ratios of 3: 1, 4: 1, and 10:1. Error bars represent standard deviation across independent measurements (n=3). Maximum shear stress in each mixer geometry by computational fluid dynamics simulations using the measured rheological properties of B) PEGDA ink and D) TissuGel for 4: 1 ink: suspension ratio.

[0031] Figure 4 illustrates mixing performance of each mixer geometry. A) Representative images and B) quantified coefficients of variation (CoV) of fluorescence intensity in transverse cross-sections of the mixed outputs using each static mixer geometry at a 4:1 ink: suspension ratio. C) Representative images and D) coefficients of variation (CoV) of fluorescence intensity for 3:1, 4:1, and 10:1 ink: suspension ratios using the lattice mixer. Plotted points represent independent experiments (n=3) with mean and standard deviation. * P<0.05, **** P<0.0001

[0032] Figure 5 illustrates that various static mixers maintain cell viability. A) Representative confocal microscope images of live (green) and dead (red) A549, 3T3, and primary human lung fibroblast (HLF) cells and B) quantified percentage of live cells post-mixing with PEGDA ink using the CELLMIXER and lattice mixer. Plotted points represent independent experiments (n=3) with mean and standard deviation. *** P0.001 viat-test.

[0033] Figure 6 illustrates primary human lung fibroblast distribution using different mixers. A) Representative confocal microscope images of primary humanlung fibroblasts mixed in PEGDA ink at a 4:1 ink: suspension ratio using different mixers. B) Quantified variance of nearest neighbor distances (o2) normalized to the theoretical minimum nearest neighbor distance (oo2). Plotted points represent independent experiments (n=3) with mean and standard deviation. ** P<0.01 viat-test.

[0034] Figure 7 illustrates printing a TissueGel-A549 bioink mixed with a system in accordance with an embodiment. Representative confocal microscope images of A) the centre of printed constructs on days 1, 3, and 7 post-printing and C) an entire printed construct with live (green) and dead (purple) cells. B) Quantified percentage of live cells on days 1, 3, and 7 post-printing. Plotted points represent individually printed constructs with mean and standard deviation. D) 3 -dimensional confocal microscope z-stack image of the centre of a printed construct with colours indicating the depth of spheroids.

[0035] Figure 8A illustrates a perspective view of a second embodiment of a system according to the first aspect of the invention;

[0036] Figure 8B illustrates a top view of the system of Figure 8A;

[0037] Figure 8C illustrates a side view of the system of Figure 8A;

[0038] Figure 9A illustrates a bottom view of the system of Figure 8 A, wherein the back panel is removed to show the gear train, in situ; and

[0039] Figure 9B illustrates a second bottom view of the system of Figure 8A, wherein the gears of the gear train are removed from the body of the syringe proportioner.DETAILED DESCRIPTION

[0040] There is provided herein a system for mixing first and second solutions according to a first aspect of the invention. A first embodiment 100 is illustrated in Figure 1 and 2 and a second embodiment 200 is illustrated in Figures 8 and 9.

[0041] Turning to Figure 1A, the system 100 comprises a syringe proportioner 102. With reference to the embodiment of Figures 1 and 2, the syringe proportioner 102 comprises a base 106. In this embodiment, the base 106 comprises a substantiallyrectangular prism, however, as would be appreciated, the base 106 may assume any other suitable configurations.

[0042] In this non-limiting embodiment, the base 106 is adapted to receive first and second input syringes 108, 110 in a first input syringe retainer 112 and a second input syringe retainer 114, respectively. The first input syringe retainer 112 is configured to retain the first input syringe 108 in position within the base 106, illustrated in situ in Figure 1A, and the second input syringe retainer 110 is configured to retain the second input syringe in position within the base 106, also shown in situ in Figure 1A.

[0043] In this embodiment, the first input syringe retainer 112 comprises four posts 116 configured to be upstanding from the base 106. Similarly, the second input syringe retainer 114 comprises four posts 116 configured to be upstanding from the base 106. The configuration of the posts 116 is illustrated in Figures 1A and 1C. In limited embodiments, each post may be removably retained on the base. Alternatively, each post may be fixed to the base, such as through heat treatment or other means of fixing, e.g. via an adhesive, or may be integrally formed with the base. In the embodiment of Figures 1 and 2, each post 116 comprises a stainless-steel dowel pin with a 4 mm diameter and a 30 mm length, although other materials and dimensions are envisaged in other embodiments. In other embodiments, each input syringe retainer may comprise any suitable number of upstanding posts, such as a pair of upstanding posts provided that their function to retain the syringe in position within the base is preserved. The first and second input syringe retainers 112, 114 are configured to contact the flange 118 of the first and second input syringes 108, 110, respectively.

[0044] In other embodiments, the first and second input syringe retainers may each comprise any suitable fixing mechanism or retaining means for retaining a syringe on the base, such as for retaining the flange of the syringe. A non-exhaustive list of suitable mechanisms includes: an interference fit (also known as a press fit or friction fit) within a slot or groove, a clip, a strap, one or more screws, hook and loop fastener or an adhesive or tape. In some embodiments, each input syringe may be retained atop the base, such as in the embodiments of Figures 1, 2, 8 and 9. In other embodiments,at least a portion of each input syringe may be retained within the base, such as within a housing or casing of the base or within a groove formed within the surface of the base.

[0045] It is envisaged that the system loo may be compatible with any suitable syringe size and shape. In particular, it is envisaged that the first and second input syringes 108, no may have a size of 1 mL, 3 mL or 5 mL, although embodiments of the device may be compatible with other syringe sizes. The input syringe retainers are designed accordingly to retain the particular sized syringes selected for use with the system.

[0046] In the non-limiting embodiment of Figures 1 and 2, the first input syringe retainer 112 is parallel to and spaced apart on the base 106 from the second input syringe retainer 114. In this way, the first input syringe 108 is retained on the first input syringe retainer 112 parallel to, and facing the same direction as, the second input syringe 110 retained on the second input syringe retainer 114. In this embodiment, a first input syringe 108 is removably retained within the first input syringe retainer 112. Similarly, a second input syringe 110 is removably retained within the second input syringe retainer 114. In this way, spent syringes may be refilled or otherwise replaced. In other embodiments, the first and second input syringe retainers may be configured differently, e.g. arranged in a non-parallel manner provided that the use of the input syringes, including their insertion into and removal, within the retainer is not adversely impacted.

[0047] The syringe proportioner 102 further comprises a gear assembly 120 comprising a gear train 122 and first and second gear racks 124, 126. The first gear rack 124 comprises a first input syringe driver 128 configured to drive a plunger 108A of a first syringe 108 retained in the first input syringe retainer 112. Similarly, the second gear rack 126 comprises a second input syringe driver 130 configured to drive a plunger 110A of a second syringe no retained in the second input syringe retainer 114. The first gear rack 124 comprises an elongate member 132 comprising a plurality of teeth 134 along a gear facing surface 136. The elongate member 132 further comprises a plunger abutting head 138 at an end thereof configured to abut the plunger

[0048] Similarly, the second gear rack 126 comprises an elongate member 140 comprising a plurality of teeth 142 along a gear facing surface 144. The elongate member 140 further comprises a plunger abutting head 146 at an end thereof configured to abut a plunger 110A of a second syringe 110 retained in the second input syringe retainer 114.

[0049] The base 106 comprises a first gear rack recess 148 configured to house the first gear rack 124 with a clearance fit and a second gear rack recess 150 configured to house the second gear rack 126 with a clearance fit, as illustrated in Figures 1A and 1C. In this way, the first and second gear racks 124, 126 can slide within their respective gear rack recesses 148, 150. As illustrated in Figures ID to IF, the shape and configuration of the second gear rack 126 may vary, as discussed in more detail herein.

[0050] The gear assembly 120 is configured to drive a plunger 108A, 110A of each of the input syringes 108, 110 to empty solution from each of the input syringes at a predetermined flow rate. In this embodiment, the gear train 122 comprises a primary gear 152, a secondary gear 154 and a tertiary gear 156, in which the secondary gear 154 and tertiary gear 156 form a compound gear. In this embodiment, all three gears are spur gears. The primary gear 152 has a first diameter and is configured to engage with the plurality of teeth 134 of the gear facing surface 136 of the first gear rack 124. Similarly, the secondary gear 154 has a second diameter and is configured to engage with the plurality of teeth 142 of the gear facing surface 144 of the second gear rack 126. The tertiary gear 156 has a third diameter and is not configured to engage directly with the teeth 134, 142 of either the first or second gear racks 124, 126.

[0051] In this embodiment, the primary gear 152 is configured to engage directly with the tertiary gear 156, such that rotation of the tertiary gear 156 causes rotation of the primary gear 152. Embodiments wherein further gears couple but separate the primary gear from the tertiary gear are envisaged. Further, the secondary gear 154 is configured to be coupled concentrically to the tertiary gear 156. In Figures IE and IF, the secondary gear 154 is mounted concentrically to the tertiary gear 156, such that they share the same axis of rotation, thereby forming a compound gear. In Figure ID, the secondary gear 154 is coupled concentrically to the tertiary gear 156 viaa post 158 so as to be spaced from the tertiary gear 156. In other embodiments, the secondary gear and tertiary gear may be in direct contact. As the secondary gear 154 is coupled to the tertiary gear 156, rotation of the tertiary gear 156 causes rotation of the secondary gear 154.

[0052] In this embodiment, the primary gear 152 and secondary gear 154 are located between the first gear rack 124 and second gear rack 126. The first gear rack 124 and second gear rack 126 are arranged on the base 106 such that the respective gear facing surface 136, 144 of each gear rack 124, 126 are facing one another with the primary gear 152 and secondary gear 154 therebetween. An alternative arrangement is illustrated and described herein in relation to the embodiment 200 of Figures 8 and 9.

[0053] In the embodiment of Figure 1A, the primary gear 152 is coupled via gear teeth 152A to the plurality of teeth 134 of the first input syringe driver 128 and the secondary gear 154 is coupled via a plurality of teeth 154A to the plurality of teeth 142 of the second input syringe drive 130. In this embodiment, the primary and secondary gears 152, 154 are configured to directly contact the first and second input syringe drivers 128, 130, respectively. Embodiments wherein further gears couple but separate the primary and secondary gears from the first input syringe driver and the second input syringe driver, respectively, are envisaged.

[0054] Rotation of the tertiary gear 156 triggers rotation of the primary gear 152. A plurality of teeth 156A of the tertiary gear 156 engage with the teeth 152A of the primary gear 152, causing rotation of the primary gear 152 in the opposite direction to the direction of rotation of the tertiary gear 156. In this way, the rotation motion of the primary gear 152 is translated into linear motion of the first gear rack 124, actuating the first input syringe driver 128. The plunger abutting head 138 of the first gear rack 124 is drawn closer to the first input syringe retainer 112, driving the plunger 108A of a first input syringe 108 retained in the first input syringe retainer 112 and causing fluid to be ejected from the syringe 108. Rotation of the tertiary gear 156 therefore causes fluid to be driven from the first input syringe 108 retained in the first input syringe retainer 112.

[0055] Rotation of the tertiary gear 156 simultaneously triggers rotation of the secondary gear 154 in the same direction, as the gears are coupled to one another. In this way, the rotation motion of the secondary gear 154 is translated into linear motion of the second gear rack 126, actuating the second input syringe driver 130. The plunger abutting head 146 of the second gear rack 126 is drawn closer to the second input syringe retainer 114, driving the plunger 110A of a second input syringe 110 retained in the second input syringe retainer 114 and causing fluid to be ejected from the second input syringe 110. Rotation of the tertiary gear 156 of the compound gear therefore causes fluid to be driven from the second input syringe 110 retained in the second input syringe retainer 114 simultaneously to fluid being driven from the first input syringe 108 retained in the first input syringe retainer 114.

[0056] In this way, the gear train is coupled to the first input syringe driver 128 and the second input syringe driver 130, wherein rotation of the compound gear of the gear train 122 in an actuation direction actuates the first input syringe driver 128 and the second input syringe driver 130 simultaneously, driving fluid from the first and second input syringes 108, 110 at the same time and at a predetermined flow rate. Rotation of the gears 152, 154, 156 in their respective actuation directions is therefore configured to engage the first and second gear racks 124, 126 and actuate the first and second input syringe drivers 128, 130 to drive fluid from the first and second input syringes 108, no, respectively.

[0057] The predetermined flow rate may be determined, in part, by the ratio of the diameters of the primary gear 152, secondary gear 154 and tertiary gear 156. In particular, the relationship between the first diameter of the primary gear 152 and the second diameter of the secondary gear 154 acts to proportion or deposit fluid from each input syringe 108, 110 at the predetermined rate. The ratio may be calculated by second diameter: first diameter (i.e. the ratio of the diameter of the secondary gear 154 to the diameter of the primary gear 152). As will be appreciated in the art, when the first diameter equals the second diameter, the flow rate from the first and second input syringes will be equal. A decrease in the first diameter relative to the second diameter (or an increase in the second diameter relative to the first diameter) will result in an increase in the flow rate of fluid expelled from the first input syringe. The greater thedecrease in the first diameter relative to the second diameter, the greater the flow rate of fluid expelled from the first input syringe.

[0058] In some embodiments, the first diameter and the second diameter are equal, i.e. when the predetermined ratio is 1:1. In other embodiments, the second diameter is greater than the first diameter. In select embodiments, the first diameter is greater than the second diameter. Preferably, the third diameter is greater than both the first diameter and second diameter, although embodiments where this is not the case are envisaged. A non-exhaustive list of ratios (i.e. the ratio of the diameter of the secondary gear to the diameter of the primary gear) includes 3 : 1 , 4 : 1 and 10:1, although it is appreciated that any suitable ratio may be used, for example, 1:3, 1:4 or 1:10.

[0059] The first input syringe 108 may contain a first solution, such as a biomaterial solution or biomaterial ink, and the second input syringe 110 may contain a second solution, such as an aqueous cell suspension. In this way, employing the gear assembly of Figures ID to IF, the first input syringe driver 128 is configured to be driven at a faster rate than the second input syringe driver 130, such that fluid in the first input syringe 108 is ejected at a faster rate than fluid in the second input syringe 110. For example, a gear train ratio of 3: 1, as shown in Figure IE, will equate to fluid in the first input syringe 108 being ejected at three times the flow rate of fluid in the second input syringe 110, if the first and second input syringes 108, 110 are substantially identical in dimension. It is noted that the first input syringe may include an aqueous cell suspension and the second input syringe may include a biomaterial solution, and that the gear ratios may be adapted accordingly to achieve the desired mixing ratio of the two solutions.

[0060] As noted previously, the size of the input syringe 108, 110 is selected based on the volume of the first and second solutions to be mixed. In some embodiments, for a 10:1 ratio, a 5-mL syringe may be used for the first input syringe (e.g. holding biomaterial ink) and a 1-mL syringe may be used for the second input syringe (e.g. holding a cell suspension). In this way, it is appreciated that the predetermined flow rates for both input syringes can be modified with factors independent to the gear train ratio, such as by syringe size or dimensions. In someembodiments for a 4:1 and 3:1 ratio, the first and second input syringes may have identical dimensions, such as sizes of 1-, 3-, or 5-mL.

[0061] In this embodiment, the secondary gear 154 and tertiary gear 156 (i.e. the compound gear) are coupled to the base 106 via a post 158. Further, the primary gear 152 is coupled to the base via a post 158. In some embodiments, each gear is fixed to the post, and the post is configured to rotate within the base. In other embodiments, like the embodiment of Figures 1 and 2, each gear is rotatable relative to the post, and the post is fixed within the base. In this embodiment, each post comprises a stainless-steel dowel pin with a 4 mm diameter and a 30 mm length, although other materials and dimensions are envisaged in other embodiments. In some embodiments, the gear train comprises bearings configured to aid rotation of the primary gear, secondary gear and tertiary gear, or to aid rotation of the posts.

[0062] In some embodiments, the primary gear, secondary gear and / or tertiary gear are configured to be removably retained on the base. In this way, the system can be modified, as desired, to employ a gear train with a different gear ratio. Preferably, the second gear rack is removably retained on the base. In this way, the second gear rack, secondary gear and tertiary gear (i.e. compound gear) may be removed and replaced by an alternative unit providing a different gear train ratio, as illustrated in Figures 1D-1F.

[0063] A gear ratio of 10:1 is illustrated in Figure ID. Figure ID illustrates an entire gear assembly 120’. Similarly, each of Figures IE and IF illustrate respective alternative embodiments of gear assembly 120”, 120’” which may be interchanged with the gear assembly 120 of Figure 1. Namely, the diameter of the secondary gear 154’ is ten times that of the diameter of the primary gear 152’, and the diameter of the tertiary gear 156’ is greater than the diameter of the primary and secondary gears 152’, 154’. The plurality of teeth 142’ of the second gear rack 126’ are also illustrated. Similarly, gear train ratios of 3:1 and 4:1, respectively, are illustrated in Figures IE and IF. Namely, in Figure IE, the diameter of the secondary gear 154” is three times that of the diameter of the primary gear 152” and the diameter of the tertiary gear 156” is greater than the diameter of the primary and secondary gears 152”, 154”. In FigureIF, the diameter of the secondary gear 154”’ is four times that of the diameter of the primary gear 152’”, and the diameter of the tertiary gear 156’” is greater than the diameter of the primary and secondary gears 152’”, 154’”. The respective first gear racks 124’, 124”, 124”’ and second gear racks 126’, 126”, 126’” are also illustrated. In the embodiment of Figure ID, the secondary gear rack 126’ comprises a stepped portion to accommodate the size of the secondary gear 154’.

[0064] In these non-limiting embodiments, the second gear rack recess 150 is configured to house a plurality of second gear racks 126’, 126”, 126’” with a clearance fit, as illustrated in Figure 1 A. The configuration of the second gear rack may be altered to better suit the diameter of the secondary gear, as shown in Figure ID compared to Figures IE and IF. For example, the elongate member of the gear rack may be stepped out to accommodate a larger gear while retaining the positioning of the plunger abutting head relative to the plunger of the syringe. It is appreciated that the elongate member may be any suitable shape to accommodate the gear train, provided that the shape does not adversely affect function.

[0065] In some embodiments, the base of the syringe proportioner and the primary gear, secondary gear and / or tertiary gear may be fabricated using the same material. In some embodiments, the base, primary gear, secondary gear and tertiary gear are formed using additive manufacturing methods, such as three-dimensional (3D) printing. In some embodiments, the base, primary gear, secondary gear and tertiary gear are made of polylactic acid (PLA) , acrylonitrile butadiene styrene (ABS), polyethylene terephthalate glycol (PETG), polycarbonate (PC), or mixtures of these polymers with reinforcing materials like carbon nanofibers or glass fibers.

[0066] The system further comprises a channel 160 comprising a dual inlet and at least one outlet 164. The dual inlet comprises a first inlet 162A and a second inlet 162B. The channel 160 is configured to be fluidly coupled, at the dual inlet, to first and second fluid expelling ends 166 A, 166B of each of the first and second input syringes 108, 110, respectfully. In this way, the channel 160 is configured to receive solution from the first and second syringes 108, 110 via the dual inlet 162A and 162B.

[0067] The channel 160 is further configured to be fluidly coupled at the at least one outlet 164 to a static mixer 168. It is to be noted that the coupling of the channel 160 to the first and second inlets 162A / B, and / or to the static mixer 168 at the fluid receiving end 170 thereof, may be fixed, or preferably, may be removably coupled to readily permit cleaning and / or replacement of the channel 160 and / or the static mixer 168.

[0068] The static mixer 168 comprises a fluid receiving end 170 and a fluid discharging end 172, wherein the fluid receiving end 170 is adapted to fluidly couple with the at least one outlet 164 of the channel 160 and receive solution therefrom. In some embodiments, the fluid receiving end 170 of the static mixer 168 is connected directly to the outlet 164 of the channel 160.

[0069] The fluid discharging end 172 of the static mixer 168 is adapted to discharge mixed fluid from the static mixer. The mixed fluid may be discharged into any suitable receiving container or vessel for future use. In one embodiment, the fluid discharging end is adapted to fluidly couple with an output syringe 174 as illustrated. In this embodiment, the static mixer 168 is configured to be coupled to the input syringes 108, 110 via the channel 160 and connected directly to the output syringe 174, for example at the hub of the syringe. In this embodiment, the first and second fluid expelling ends 166A, 166B of each of the first and second input syringes 108, 110 are configured to be connected to the channel 160 via Luer Lock fittings, such that the connection between the input syringes 108, 110 and the channel 160 inhibits the escape of fluid. Similarly, in this embodiment, the output syringe 174 is configured to be connected to the fluid discharging end 172 of the static mixer 168 via a Luer Lock fitting, such that the connection between the output syringe 174 and the mixer 168 inhibits the escape of fluid. In other embodiments, alternative screw coupling or, for example, a ‘Slip Tip’ coupling (i.e. slip or push-on connection) may be used. In other embodiments, the fluid discharging end 172 is adapted to discharge fluid into a container or vessel and adaptation to fluidly connect to a receiving vessel is not required. It is appreciated that this list is non-exhaustive and any suitable fitting may be used. The connection used may be the same for each syringe or different. As illustrated in Figure 1A, the channel 160 of this embodiment is substantially Y-shaped.The arrangement of the channel may be different in other embodiments. It is envisaged that the channel may be substantially any suitable shape, for example, T-shaped.

[0070] The static mixer 168 comprises an internal geometry within a tube 176 which provides mixing of a first solution 178 from the first input syringe 108 and a second solution 180 from the second input syringe 110. In some embodiments, the internal geometry of the static mixer comprises a convoluted pathway. In some embodiments, the internal geometry of the static mixer comprises at least one obstacle. In some embodiments, the obstacle comprises one or more of the following: a chainlike structure; a helical or ribbon structure; a spiral structure; a grid-like structure; a lattice-like structure; and / or a fin structure. It is understood that each of these structures may be formed from one or more baffles or blades arranged to provide a convoluted pathway for fluid to traverse within the static mixer and promote mixing of the two fluids. It is appreciated that the internal mixer geometry may comprise multiple obstacles within the tube of the static mixer, which may be the same or different. For example a non limiting embodiment may include a static mixer comprising one or more spiral structures and one or more lattice structures. It is appreciated that any suitable combination of two or more obstacles may be incorporated in the static mixer.

[0071] Further, the static mixer may comprise two or more static mixers arranged in series. Namely, a first static mixer may be fluidly connected to a second static mixer such that the output of the first static mixer is the input of the second static mixer. In this way, the fluid may encounter multiple internal geometries during mixing. It is appreciated that dead volume and pressure loss within the static mixer may be detrimental, as discussed further herein, and so there may be limitations on the types and number of individual static mixers that may be effectively arranged in series.

[0072] With reference to Figures IB and 2B, the internal geometry comprising a lattice-like structure obstacle is illustrated. In particular, three lattice structures 184, spaced apart within the mixer 168, are present. Figure 2A illustrates an example mixer 168’ comprising a spiral structure obstacle 186, where non-uniform spiral ribbon shaped blades are located within the tube 170’. Figures 2C and 2D illustrate example chain-like structure obstacles 188, 188’. In the embodiment of Figure 2C, the chain-like structure obstacle 188A of the mixer 168” comprises equidistant nodes connected by four cylindrical connecting posts. In the embodiment of Figure 2D, the chain-like structure obstacle 188B of the mixer 168”’ is substantially serpentine-esque, with irregular, curved chain links. It is appreciated that the illustrated examples are non-exhaustive.

[0073] The internal geometry is configured to combine fluid received in the fluid receiving end 170 of the mixer 168 prior to outputting the combined fluid at the fluid discharging end 172 of the mixer 168. In particular, the first input syringe 108 may comprise fluid in the form of a first solution 178, for example, a biomaterial solution, and a second input syringe 110 may comprise fluid in the form of a second solution 180, for example, a cell suspension. When the first input syringe 108 is retained on the first input syringe retainer 112, the plunger 108 A of the first input syringe 108 may be driven (i.e. pushed to expel fluid 178 from the syringe 108) by the first input syringe driver 128 such that fluid 178 from the first input syringe 108 enters the first inlet 162A of the dual inlet of channel 160. Similarly, the second input syringe 110 is retained on the second input syringe retainer 114, the plunger 110A of the syringe 110 may be driven by the second input syringe driver 130 such that fluid 180 in the second input syringe 110 enters the second inlet 162B of the dual inlet of channel 160.

[0074] The two fluids 178, 180 are partially combined and may be partially mixed within the channel 160, although mixing in the channel 160 is not essential, before passing out of the channel 160 via the outlet 164. The fluids 178, 180 then enter the static mixer 168 via its fluid receiving end 170. The internal geometry of the mixer 168 promotes mixing of the two fluids 178, 180 together within tube 176 of the mixer 168, such as by promoting turbulent flow of the fluids. In this way, the fluid discharged at the fluid discharging end 172 into the output syringe 174 is substantially combined compared to the two discrete fluids 178, 180 entering the channel 160 at the dual first and second inlets 162A / B.

[0075] It is understood that actuation of the gear assembly 120, by either directly rotating an exposed portion of the tertiary gear 156, or by an actuator coupled to the compound gear, expels fluid from the input syringes 108, 110 into the channel160 via the dual first and second inlets 162A / B, into the mixer 168 and then into the output syringe 174. In particular, rotation of the compound gear causes the plungers 108A, 110A of the input syringes 108, 110 to mechanically activate, thereby driving fluid 178, 180 from the syringes 108, 110 due to pressure being generated within the barrel of the syringe 108, 110 expelling the fluid 178, 180. The user may rotate a portion of the compound gear until the desired quantity of mixed fluid is driven into the output syringe 174.

[0076] In some embodiments, the mixer is formed from a single integral member, while in other embodiments, the mixer is formed from a plurality of members. As illustrated in Figure 1A, the mixer 168 and the channel 160 are configured to connect in a manner which inhibits the escape of fluid (e.g. a watertight manner). Further, the mixer 168 may be removed or otherwise separated from the channel 160. In this way, the mixer 168 with internal geometry may be swapped out or otherwise replaced with an alternative mixer comprising alternative internal geometry, depending on the application. Alternatively, the mixer and channel may be provided as a single integral member.

[0077] As noted, the present system 100 is for use to mix a first solution 178 with a second solution 180, and is particularly useful to mix solutions differing in viscosity, for example, a viscous biomaterial with a less viscous aqueous solution. The first solution may be a biomaterial solution such as a biomaterial ink or bioink. Suitable such biomaterials for use as a bioink include biocompatible synthetic, natural or hybrid polymers. Examples of suitable natural polymers include alginate, agarose, gellan gum, chitosan, collagen and gelatin. Suitable synthetic polymers include carbomers, poloxamers and polyethylene glycol. For example, the first solution may comprise polyethylene glycol diacrylate (PEGDA)-based biomaterials or gelatin methacrylate (GelMA)-based biomaterials. It is understood that the term “biomaterial ink” may comprise any suitable composition for three-dimensional bioprinting of, for example, tissue structures.

[0078] In some embodiments, the second solution is an aqueous cell suspension, in some embodiments, a live cell suspension. In some embodiments, thecell suspension may comprise one or more of the following: endothelial cells; fibroblast cells; bone marrow stem cells; and induced pluripotent stem cells. In other embodiments, the second solution may be a microparticle solution.

[0079] Preferably, the viscosity of the aqueous second solution is less than the viscosity of the first biomaterial solution. In some embodiments, the viscosity of the second solution is no greater than about 1000 Ns / m2In other embodiments, the viscosity of the second solution is no greater than about 500 Ns / m2, 400 Ns / m2or 300 Ns / m2. In some embodiments, the viscosity of the first solution is no less than about IxlO'4Ns / m2. In other embodiments, the viscosity of the first solution is no less than about 5 x 1 O'4Ns / m2or 1 x 10'3Ns / m2. In this way, a system for mixing a high viscosity fluid with a low viscosity fluid is provided. In some embodiments, especially when the first solution is PEGDA, the viscosity of the first solution is no less than about 1 Ns / m2. In some embodiments, especially when the first solution is PEGDA, the viscosity of the first solution is no greater than 900 Ns / m2. In some embodiments, especially when the first solution is GelMA, the viscosity of the first solution is no less than about 0.5 Ns / m2. In some embodiments, especially when the first solution is GelMA, the viscosity of the first solution is no greater than about 250 Ns / m2. In some embodiments, especially when the first solution is a cell suspension in water at 37°C, the viscosity of the first solution is no less than about 0.5x10'3Ns / m2. In some embodiments, especially when the first solution is a cell suspension in a cell culture medium at 37°C, the viscosity of the first solution is no greater than about IxlO'3Ns / m2. The present invention therefore provides an improved system 100 for effective and accurate mixing of two fluids 178, 180. Dispensing the two fluids 178, 180 at predetermined flow rates, based on a selected gear train ratio, ensures that a desired ratio of the two fluids 178, 180 enter the static mixer 168 of the system for mixing and subsequent dispensing.

[0080] In particular, when mixing fluids comprising cells with viscous biomaterial fluids, there is a risk of damage to the cells within the fluid. Overmixing or otherwise mixing with excessive force can cause cell damage due to shearing under these mechanical strains. As such, care must be taken to ensure adequate mixing of the fluids, without damage to the cells. Under-mixing is also undesirable as the two fluids will not be uniformly combined. The present system provides a means for optimizingor otherwise improving the balance between homogeneity of the dispensed fluid and limiting cell damage. In particular, the present system achieves homogeneous mixing of solutions with minimal sheer stress, e.g. with a sheer stress of less than lOkPa, such as a sheer stress of less than 5 kPa, less than 4 kPa, less than 3kPa, less than 2 kPa or 1.5 kPa or less.

[0081] As a result, the system is particularly useful for mixing cell-containing solutions with viscous biomaterial solutions with minimal damage to the cells. In this regard, the present system exhibits effective mixing of cell -containing solutions while significantly maintaining cell viability, for example, maintaining at least 80%, 85%, 90%, 95% or greater of cell viability within the mixed cell-containing solution.

[0082] Turning now to Figures 8 and 9 and a second embodiment of a system 200 for mixing first and second solutions 278, 280 according to the first aspect of the invention is illustrated. Similar reference numerals are used to describe like features of the first and second embodiments.

[0083] The system 200 comprises a syringe proportioner 202 and a static mixer 268, the syringe proportioner 202 comprising a base 206. In this embodiment, the base 206 comprises a substantially rectangular casing 204, having a removable back panel 290. In this embodiment, the back panel 290 may be removed from the base 206 by unscrewing the four screws affixing it to the base 206. It is envisaged that any suitable fixing mechanism may be used to removably retain the back panel to the base.

[0084] In this non-limiting embodiment, the base 206 is adapted to receive first and second input syringes 208, 210 and comprises a first input syringe retainer 212 and a second input syringe retainer 214. The first input syringe retainer 212 is configured to retain the first input syringe 208 in position on the base 206, illustrated in situ in Figures 8A-8C, and the second input syringe retainer 214 is configured to retain a second input syringe 210 in position on the base 206, also shown in situ in Figures 8A-8C.

[0085] In this embodiment, the first input syringe retainer 212 comprises a bracket 216A configured to be upstanding from the base 206. Similarly, the second input syringe retainer 214 comprises a bracket 216B configured to be upstanding fromthe base 206. A further central bracket 216C is common to both the first input syringe retainer 212 and the second input syringe retainer 214. Each of the three brackets 216A, 216B, 216C extend planar to one another such that a channel 292 is formed between brackets 216A and 216C, and 216B and 216C. The channel 292 is configured to house a portion of the barrel 218 (also called the barrel flange) of each input syringe 208, 210 with a clearance fit. In this way, a syringe 208, 210 retained on the first input syringe retainer 212 or second input syringe retainer 214 can be refilled or otherwise replaced. In other embodiments, the first and second input syringe retainers may each comprise any suitable fixing mechanism or retaining means for retaining a syringe on the base, preferably for retaining the flange or barrel of the input syringe. A non-exhaustive list of suitable mechanisms includes: dowels or posts, a clip, a strap, one or more screws, hook and loop fastener and an adhesive or tape.

[0086] It is envisaged that the system 200 may be compatible with any suitable syringe size and shape. In particular, it is envisaged that the first and second input syringe may have a size of 1 mL, 3 mL or 5 mL, although embodiments of the device may be compatible with other syringe sizes. The input syringe retainers are designed accordingly to retain the particular sized input syringes selected for use with the system.

[0087] In the non-limiting embodiment of Figures 8 and 9, the first input syringe retainer 212 is parallel to and spaced apart on the base 206 from the second input syringe retainer 214. In this way, the first input syringe 208 is configured to be retained on the first input syringe retainer 212 parallel to, and facing the same direction as, the second input syringe 210 retained on the second input syringe retainer 214. The upstanding brackets 216A, 216B, 216C inhibit motion of the first and second input syringes 208, 210 in the direction of force applied to each plunger 208 A, 210A of the respective input syringes 208, 210 when the plunger 208A, 210A is activated. In other embodiments, the first and second input syringe retainers may be configured differently, e.g. arranged in a non-parallel manner provided that the use of the input syringes, including their insertion into and removal, within the retainer is not adversely impacted.

[0088] The syringe proportioner 202 further comprises a gear assembly 220 comprising a gear train 222 and first and second gear racks 224, 226. The first gear rack 224 comprises a first input syringe driver 228 configured to drive a plunger 208A of the first input syringe 208 retained in the first input syringe retainer 212. Similarly, the second gear rack 226 comprises a second input syringe driver 230 configured to drive the plunger 210A of the second input syringe 210 retained in the second input syringe retainer 214. The first gear rack 224 comprises an elongate member 232 comprising a plurality of teeth 234 along a gear facing surface 236. The elongate member 232 further comprises a plunger abutting head 238 at an end of the elongate member 232 configured to abut the plunger 208A of the first input syringe 208 retained in the first input syringe retainer 212.

[0089] Similarly, the second gear rack 226 comprises an elongate member 240 comprising a plurality of teeth 242 along a gear facing surface 244. The elongate member 240 further comprises a plunger abutting head 246 at an end thereof configured to abut the plunger 210A of the second input syringe 210 retained in the second input syringe retainer 214.

[0090] The base 206 comprises a first gear rack recess 248 configured to house the first gear rack 224 with a clearance fit and a second gear rack recess 250 configured to house the second gear rack 226 with a clearance fit. In this way, the primary and second gear racks 224, 226 can slide within their respective gear rack recesses.

[0091] The gear assembly 220 is configured to drive the plunger 208A, 210A of each of the input syringes 208, 210 to empty solution from each of the input syringes 208, 210 at a predetermined flow rate. The gear assembly 220 comprises the gear train 222 comprising, in this embodiment, a primary gear 252, a secondary gear 254, a tertiary gear 256 and a pair of intermediate gears 294A, 294B. The secondary gear 254 and tertiary gear 256 form a compound gear. In this embodiment, all gears are spur gears. The primary gear 252 has a first diameter and is configured to engage with the plurality of teeth 234 of the gear facing surface 236 of the first gear rack 224. Similarly, the secondary gear 254 has a second diameter and is configured to engage with the plurality of teeth 242 of the gear facing surface 244 of the second gear rack 226. Thetertiary 256 gear has a third diameter and is not configured to engage directly with the teeth 234, 242 of either the first or second gear racks 224, 226.

[0092] In this embodiment, and differing from the embodiment of Figures 1 and 2, a plurality of teeth 252 A of the primary gear 252 are not configured to engage directly with a plurality of teeth 256A of the tertiary gear 256. Instead, the two intermediate gears 294A, 294B couple the primary gear 252 to the tertiary gear 256, such that rotation of the tertiary gear 256 causes rotation of both intermediate gears 294A, 294B that in turn cause rotation of the primary gear 252. Further, the secondary gear 254 is configured to be coupled concentrically to the tertiary gear 256 to form a compound gear. In Figure 9A, the secondary gear 254 is in direct contact with the tertiary gear 256. In other embodiments, the secondary gear and tertiary gear may be coupled via, for example, a post so as to be spaced from one another. As the secondary gear 254 is coupled to the tertiary gear 256, rotation of the tertiary gear 256 causes rotation of the secondary gear 254.

[0093] In this embodiment, the tertiary gear 256 and intermediate gears 294A, 294B are set in the underside of the casing, and the primary gear 252 and secondary gear 254 extend from the underside of the casing into the casing 204 in order to couple with the first and second input syringe drivers 228, 230, respectively. Thus, the primary and secondary gears 252, 254 possess an elongated cylindrical configuration in order to permit coupling with the syringe drivers. Thus, the depth of the primary and / or secondary gears 252, 254 may be greater than the depth of the tertiary gear 256. Access to the gear train 222 is granted by removing the back panel 290 of the base 206.

[0094] The first gear rack 224 and second gear rack 226 are arranged within the casing 204 such that the respective gear facing surfaces 236, 244 of each gear rack 224, 226 are facing away from one another. In this way, the configuration of the first gear rack 224 and second gear rack 226 of this embodiment differ from that of the embodiment of Figures 1 and 2.

[0095] In the embodiment of Figures 8 and 9, the primary gear 252 is coupled to the plurality of teeth 234 of the first input syringe driver 228 and the secondary gear 254 is coupled to the plurality of teeth 242 of the second input syringe driver 230. Theprimary and secondary gears 252, 254 are configured to directly contact the first and second input syringe drivers 228, 230, respectively, although embodiments wherein further gears couple but separate the primary and secondary gears from the first input syringe driver and the second input syringe driver, respectively, are envisaged.

[0096] Rotation of the tertiary gear 256 triggers rotation of the primary gear 252. A plurality of teeth 256A of the tertiary gear 256 engage with teeth of the first intermediate gear 294A; the teeth of the first intermediate gear 294A engage with teeth of the second intermediate gear 294B; and the teeth of the second intermediate gear 294B engage with the teeth 252A of the primary gear 252, causing rotation of the primary gear 252 in the opposite direction to the direction of rotation of the tertiary gear 256. In this way, the rotation motion of the primary gear 252 is translated into linear motion of the first gear rack 224, actuating the first input syringe driver 228. The plunger abutting head 238 of the first gear rack 224 is drawn closer to the first input syringe retainer 212, driving the plunger 208 A of the first input syringe 208 retained in the first input syringe retainer 212 and causing fluid to be ejected from the first input syringe 208. Rotation of the tertiary gear 256 therefore causes fluid to be driven from the first input syringe 208 retained in the first input syringe retainer 212.

[0097] In this embodiment, a portion 296 of the tertiary gear 256 protrudes from the casing 204 such that the user may rotate the tertiary gear 256 in the activation direction to drive the plungers 208 A, 210A of the input syringes 208, 210.

[0098] Rotation of the tertiary gear 256 simultaneously triggers rotation of the secondary gear 254 in the same direction as the gears 254, 256 are coupled to one another forming a compound gear. In this way, the rotation motion of the secondary gear 254 is translated into linear motion of the second gear rack 226, actuating the second input syringe driver 230. The plunger abutting head 246 of the second gear rack 226 is drawn closer to the second input syringe retainer 214, driving the plunger 210A of the second input syringe 210 retained in the second input syringe retainer 214 and causing fluid to be ejected from the second input syringe 210. Rotation of the tertiary gear 256 of the compound gear therefore causes fluid to be driven from the second input syringe 210 retained in the second input syringe retainer 214 simultaneously to fluidbeing driven from the first input syringe 208 retained in the first input syringe retainer 212.

[0099] In this way, the gear train 222 is coupled to the first input syringe driver 228 and the second input syringe driver 230, wherein rotation of the compound gear of the gear train 222 in an actuation direction actuates the first input syringe driver 228 and the second input syringe driver 230 simultaneously, driving fluid from the first and second input syringes 208, 210 at the same time and at a predetermined flow rate. Rotation of the gears in their respective actuation directions is therefore configured to engage the first and second gear racks 224, 226 and actuate the first and second input syringe drivers 228, 230 to drive fluid from the first and second input syringes 208, 210, respectively.

[0100] The predetermined flow rate is based, in part, on the ratio of the diameters of the primary gear 252, secondary gear 254 and tertiary gear 256. In particular, the relationship between the first diameter of the primary gear 252 and the second diameter of the secondary gear 254 acts to proportion or deposit fluid from each input syringe 208, 210 at the predetermined rate. The ratio may be calculated by second diameter: first diameter (i.e. the ratio of the diameter of the secondary gear to the diameter of the primary gear). As will be appreciated in the art, when the first diameter equals the second diameter, the flow rate will be equal. A decrease in the first diameter relative to the second diameter (or an increase in the second diameter relative to the first diameter) will result in an increase in the flow rate of fluid expelled from the first input syringe. The greater the decrease in the first diameter relative to the second diameter, the greater the flow rate of fluid expelled from the first input syringe.

[0101] In some embodiments, the first diameter and the second diameter are equal i.e. when the predetermined ratio is 1:1. In other embodiments, the second diameter is greater than the first diameter. In select embodiments, the first diameter is greater than the second diameter. Preferably, the third diameter is greater than both the first diameter and second diameter, although embodiments where this is not the case are envisaged. A non-exhaustive list of ratios (i.e. the ratio of the diameter of thesecondary gear to the diameter of the primary gear) includes 3 : 1 , 4 : 1 and 10:1, although it is appreciated that any suitable ratio may be used.

[0102] The first input syringe 208 may contain a first solution 278, such as a biomaterial and the second input syringe 210 may contain a second solution 280, such as a cell suspension. In this way, the first input syringe driver 228 may be configured to be driven at a faster rate than the second input syringe driver 230, such that fluid 278 in the first input syringe 208 is ejected at a faster rate than fluid 280 in the second input syringe 210. For example, a gear train ratio of 3: 1 will equate to fluid in the first input syringe being ejected at three times the flow rate of fluid in the second input syringe, if the first and second input syringes are substantially identical in dimension.

[0103] As noted previously, the size of the input syringe is selected based on the volume of the first and second solutions to be mixed. In some embodiments, for a 10:1 ratio, a 5-mL syringe may be used for the first input syringe (e.g. holding biomaterial ink) and a 1-mL syringe may be used for the second input syringe (e.g. holding a cell suspension). In this way, it is appreciated that the predetermined flow rates for both input syringes can be modified with factors independent to the gear train ratio, such as by syringe size or dimensions. In some embodiments for a 4:1 and 3:1 ratio, the first and second input syringes may have identical dimensions, such as sizes of 1-, 3-, or 5-mL.

[0104] In this embodiment, the secondary gear 254 and tertiary gear 256 (i.e. the compound gear) are coupled to the base via a post 258. Further, the primary gear 252 is coupled to the base via a post 258. In some embodiments, each gear is fixed to the post, and the post is configured to rotate within the base. In other embodiments, like the embodiment of Figures 8 and 9, each gear 252, 254, 256 is rotatable relative to their respective post 258, and the post 258 is fixed within the base 206. In this embodiment, each post 258 comprises a stainless-steel dowel pin with a 4 mm diameter and a 30 mm length, although other materials and dimensions are envisaged in other embodiments. In some embodiments, the gear train comprises bearings configured to aid rotation of the primary gear, secondary gear and tertiary gear, or to aid rotation of the posts.

[0105] In this embodiment, the primary gear 252 and compound gear (the secondary gear 254 and tertiary gear 256) are configured to be removably retained within the casing 204 of the base 206. In this way, the system 200 can be modified, as desired, to employ a gear train with a different gear ratio, as illustrated in Figure 9B. A gear ratio of 4: 1 is illustrated in Figures 8 and 9. Namely, the diameter of the secondary gear is four times that of the diameter of the primary gear.

[0106] The system further comprises a channel 260 comprising a dual inlet and at least one outlet 264. The dual inlet comprises a first inlet 262A and a second inlet 262B. The channel 260 is configured to be fluidly coupled, at the dual inlet, to first and second fluid expelling ends 266 A, 266B of each of the first and second input syringes 208, 210, respectfully. In this way, the channel 260 is configured to receive solution 278, 280 from the first and second syringes 208, 210 via the dual first and second inlets 262A / B. The channel 260 is further configured to be fluidly coupled at the at least one outlet 264 to the static mixer 268. The static mixer 268 comprises a fluid receiving end 270 and a fluid discharging end 272, wherein the fluid receiving end 270 is adapted to fluidly couple with the at least one outlet 264 of the channel 260 and receive solution therefrom. In some embodiments, the fluid receiving end is connected directly to the outlet of the channel.

[0107] The fluid discharging end 272 of the static mixer 268 is adapted to discharge mixed fluid from the static mixer. The mixed fluid may be discharged into any suitable receiving container or vessel for future use. In one embodiment, the fluid discharging end is adapted to fluidly couple with an output syringe 274 as illustrated. In this embodiment, the first and second fluid expelling ends 266A, 266B of each of the first and second input syringes 208, 210 are configured to be connected to the channel 260 via Luer Lock fittings, such that the connection between the syringes 208, 210 and the channel 260 inhibits the escape of fluid. In other embodiments, alternative screw coupling or, for example, a ‘Slip Tip’ coupling (i.e. slip or push-on connection) may be used. It is appreciated that this list is non-exhaustive and any suitable fitting may be used.

[0108] As illustrated in Figure 8A, 8B and 8C, the channel 260 of this embodiment is substantially Y-shaped. The arrangement of the channel may be different in other embodiments. It is envisaged that the channel may be substantially any suitable shape, for example T-shaped.

[0109] The static mixer 268 comprises an internal geometry within tube 276 which provides mixing of the first solution 278 from the first input syringe 208 and the second solution 280 from the second input syringe 210. The internal geometry of mixer 268 is as described previously, comprising lattice-like structures 284.

[0110] The embodiment of Figs. 8 and 9 functions similar to that of the embodiment of Fig. 1. In particular, a first input syringe 208 comprising a fluid 278, for example a biomaterial, and a second input syringe 210 comprising fluid 280, for example a cell suspension are prepared. When the first input syringe 208 is retained on the first input syringe retainer 212, the plunger 208A of the syringe 208 may be driven (i.e. pushed to expel fluid from the syringe) by the first input syringe driver 228 such that fluid 278 from the first input syringe 208 enters the first inlet 262A of dual inlet of the channel 260. Similarly, the second input syringe 210 is retained on the second input syringe retainer 214, the plunger 210A of the syringe 210 may be driven by the second input syringe diver 230 such that fluid 280 in the second input syringe 210 enters the second inlet 262B of the dual inlet of the channel 260.

[0111] The two fluids 278, 280 are partially combined and may be partially mixed within the channel 260, although mixing in the channel 260 is not essential, before passing out the of channel 260 via the outlet 264. The fluids then enter the static mixer via the fluid receiving end. The internal geometry 276 promotes mixing of the two fluids 278, 280 together within the mixer 268, such as by promoting turbulent flow of the fluid. In this way, the fluid discharged at the fluid discharging end 272 into the output syringe 274 is substantially combined compared to the two discrete fluids 278, 280 entering the channel 260 at the dual first and second inlets 262A / B.

[0112] It is understood that actuation of the gear assembly 222, either directly rotating the exposed portion 296 of the tertiary gear 256, or by an actuator coupled to the compound gear, expels fluid from the input syringes 208, 210 into the channel 260via the dual first and second inlets 262A / B, into the mixer 268 and then into the output syringe 264. In this embodiment, the user may rotate the exposed portion of the compound gear with, for example, their thumb or finger. In particular, rotation of the compound gear causes the plungers of the input syringes to mechanically activate, thereby driving fluid from the syringes due to pressure being generated within the barrel of the syringe expelling the fluid. The user may rotate the compound gear until the desired quantity of mixed fluid is driven into the output syringe.

[0113] In some embodiments, and as illustrated in Figures 8 and 9, the mixer 268 is formed from an integral member. In other embodiments, the mixer may be formed from a plurality of members. In other embodiments, the mixer and channel may be integrally formed. It is appreciated that any suitable internal geometry may be used in the mixer of the embodiment of Figures 8 and 9, such as any of the example obstacles illustrated in Figures 2A to 2D.Definitions

[0114] Unless otherwise indicated, the definitions and embodiments described in this and other sections are intended to be applicable to all embodiments and aspects of the present disclosure herein described for which they are suitable as would be understood by a person skilled in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only and is not intended to be limiting.

[0115] In understanding the scope of the present disclosure, the term “comprising” and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, “including”, “having” and their derivatives. The term “consisting” and its derivatives, as used herein, are intended to be closed terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of’, as used herein, is intended to specify the presence ofthe stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0116] Terms of degree such as “substantially”, “about” and “approximately” as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies. In addition, all ranges given herein include the end of the ranges and also any intermediate range points, whether explicitly stated or not.

[0117] As used in this disclosure, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise.

[0118] In embodiments comprising an “additional” or “second” component, the second component as used herein is chemically different from the other components or first component. A “third” component is different from the other, first, and second components, and further enumerated or “additional” components are similarly different.

[0119] The term “and / or” as used herein means that the listed items are present, or used, individually or in combination. In effect, this term means that “at least one of’ or “one or more” of the listed items is used or present.

[0120] The abbreviation, “e.g.” is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Thus, the abbreviation “e.g.” is synonymous with the term “for example.” The word “or” is intended to include “and” unless the context clearly indicates otherwise.

[0121] It will be understood that any component defined herein as being included may be explicitly excluded by way of proviso or negative limitation, such as any specific compounds or method steps, whether implicitly or explicitly defined herein.

[0122] Embodiments of the invention are described by reference to the following specific examples which are not to be construed as limiting.Example 1

[0123] This work evaluates the mixing performance, shear stress, and cell viability using four different types of static mixers intended for high viscosity mixing. Three static mixers were based on the Sulzer SMX, Ross ISG, and serpentine mixers and fabricated using resin 3D printing. CELLMIXER, a Kenics-style static mixer commercially available through CELLINK, was used as a comparator. Two biomaterial inks based on PEGDA and methacrylated gelatin were used to characterize each mixer’s performance. Shear stress was estimated via fluid dynamics simulations using shearthinning attributes measured experimentally through rheology. Mixing effectiveness was evaluated using fluorescent beads, from which the most effective design was chosen for live cell mixing experiments. Viability of cell lines (A549 and NIH-3T3) and primary human lung fibroblasts was evaluated post-mixing. A demonstration of extrusion bioprinting was performed using the mixed bioinks.Methods

[0124] Mixer design and fabrication - Three static mixer geometries intended for mixing in laminar flow regimes were designed using computer-aided design software (Autodesk Fusion) and compared with the Kenics-style CELLMIXER purchased from CELLINK (SKU: KT0000000000, CELLINK, Gothenburg, Vastergotland, Sweden). The lattice-style geometry was designed based on the Sulzer SMX plus mixer. The interfacial surface generator (ISG) style geometry was designed based on the Ross ISG mixer. The serpentine geometry was designed based on a microfluidic mixer intended for viscous laminar micromixing via stream splitting and chaotic advection. Each mixer was designed to have a dead volume equal to that of the CELLMIXER system, which was manually estimated as 310 pL using pipettes and an analytical balance (AB265-S, Mettler Toledo, Columbus, OH, USA). To maximize the number of mixing units occupying this 310 pL volume constraint, each mixer was designed with the minimum printable channel size of a Form 3B stereolithography 3D printer (700 pm channel diameter). The resulting number of mixing units for the lattice, ISG, and serpentinemixers were 3, 7, and 7 units, respectively. A Y-channel connector with a dead volume of 80 pL was also designed in Autodesk Fusion to combine the input materials into a single stream going into the mixer. The designed mixers and y-channel were fabricated using a stereolithography resin printer (Form 3B, Formlabs, Somerville, MA, USA) with BioMed Clear resin (RS-CFG-BMCL-O, Formlabs). After printing, the parts were rinsed thoroughly with 99% isopropanol using a wash bottle and automated washing machine (FH-WA-01, Formlabs) to remove uncured resin before performing the final cure in an automated curing machine (FH-CU-01, Formlabs) for 60 minutes at 60°C. The mixers were autoclaved before use in experiments with cells.

[0125] Syringe proportioner fabrication - A 3D printed syringe proportioner was designed for combining the two input materials, a biomaterial ink and cell suspension, at defined ink: suspension ratios of 3: 1, 4: 1, and 10: 1, respectively. The syringe proportioner base and interchangeable gears were designed using computer-aided design software (Autodesk Fusion) and fabricated with polylactic acid (PLA) filament using a fused-deposition 3D printer (Ultimaker S3, Ultimaker, Utrecht, Netherlands). Stainless steel dowel pins (4 mm diameter, 30 mm length; McMaster Carr, 91585A457) were heated using a piezo torch and inserted into the holes of the base for the gear axes. The proportioner is compatible with BD syringes of volumes 1 mL (14-823-30, Fisher Scientific, Waltham, MA, USA), 3 mL (B309657, Fisher Scientific, Waltham, MA, USA), and 5 mL (B309646, Fisher Scientific, Waltham, MA, USA). Syringes from different manufacturers may also be compatible with the system.

[0126] Biomaterial inks and rheological characterization - Polyethylene glycol diacrylate (PEGDA)-based biomaterials (PEGDA ink; Tessella Biosciences, Dundas, ON, Canada) and gelatin methacrylate (GelMA)-based biomaterials (TissuGel Medium; Tessella Biosciences, Dundas, ON, Canada) were provided by Tessella Biosciences as pre-inks to be used in 3:1, 4:1, and 10:1 ink:cell suspension ratios. The rheological properties of each ink formulation were measured using a DHR Controlled Stress Single Head CMT torsional shear rheometer (HR20, TA instruments, New Castle, DE, USA) with 8 mm parallel plate geometry. The viscosity of the biomaterial inks was determined as a function of the shear rate (0.1 to 100 Hz). The shear thinning relationship of thebiomaterials was modelled from the rheological measurements via the Ostwald-de Waele power law:T)=KY(n-1)where i] is the effective viscosity, K is the flow consistency index, y is the shear rate, and n is the flow behaviour index. The values of K and n were determined via linear regression of the logarithmic plot of the shear rate vs. viscosity relationships:log( / j) = (n - 1) log(y) + log( / C)where (n-1) is the slope and log( / Q is the y-intercept.

[0127] Shear stress simulation - The measured rheological relationships of the PEGDA and TissuGel biomaterial inks were used to perform computational fluid dynamics to estimate the maximum shear stresses generated in fluid flowing within each mixer geometry. The CELLMIXER was 3D modelled in Autodesk Fusion 2024 with reference to caliper measurements. The mixer 3D models were exported from Autodesk Fusion 2024 as ACIS text files and imported to Autodesk CFD 2024. For each mixer, the Geometry Tools function was used to generate an internal fluid volume possessing the measured rheological properties of the 4: 1 ink: suspension formulation of PEGDA ink or TissuGel via the power law coefficients with density of 1 g / mL. CFD decisions followed best practices defined in a recent review on computational modeling of fluids. Static nonslip conditions were applied to all outer boundaries except the outlet, which was open to atmosphere, and the inlet, with flow rates of 0.12, 1.2, 6, or 12 mL / s. The chosen flow rates mimic a practical range of speeds at which a user will operate the syringe proportioner. Meshing was automatically generated with surface and gap refinement disabled. The meshes were inspected visually before computation to ensure regions of interest were adequately discretized. Fluid volumes were assumed as incompressible with a k-epsilon turbulence model. Solutions were computed until convergence with Intelligent Solution Control enabled and Automatic Convergence Assessment set to default. For each flow rate, the maximum shear stress within each mixer was recorded.

[0128] Mixing performance evaluation - PEGDA ink and TissuGel ink were supplied by Tessella Biosciences (Dundas, ON, Canada). Fluorescent polystyrene beadswith diameter 0.1 pm (FluoSpheres™ Carboxylate-Modified Microspheres, F8800, Invitrogen, Waltham, MA, USA) were diluted to a 0.04% w / v suspension. Both biomaterial inks were mixed with the fluorescent bead suspension at an ink: suspension ratio of 4: 1 using the CELLINK, lattice, ISG, and serpentine mixers. Mixing consisted of one pass through the mixer using the syringe proportioner. Each output mixture was collected in a 5 mL syringe and crosslinked with blue light (405 nm) for 5 minutes. The cured gel was then removed from the syringe and sliced axially using single edge industrial razor blades (55411-050, VWR International Co, Mississauga, ON, Canada) to generate 2 mm thick transverse cross-sectional cylinders. Slices were isolated at various locations along the output gel to investigate axial differences in mixing performance. The slices were placed on glass microscope slides (48312-004, VWR International Co, Mississauga, ON, Canada) and imaged with an inverted confocal microscope (Nikon AIR HD25, Nikon Canada, Mississauga, ON, Canada) using a 10x / 0.45NA objective at the Centre for Advanced Light Microscopy at McMaster University.

[0129] Confocal images were imported to ImageJ (v2.16.0, National Institutes of Health, Bethesda, MD, USA) and the mean and standard deviation of pixel intensity across the entire area of the slice were used to calculate the coefficient of variance ((Standard deviation) / Mean) of fluorescence intensity. A decision criterion was established a priori to select the mixer that produced the lowest mean coefficient of variance across three independent experiments for downstream evaluation of mixing performance with different ink: suspension ratios and live cell suspensions. The lattice geometry static mixer was the design that provided the optimal performance based on this quantitative assessment.

[0130] To evaluate mixing performance with different mixing ratios using the lattice mixer and syringe proportioner, the experiments described above for the mixer geometry selection were repeated using PEGDA ink and TissuGel ink for 3:1, 4:1, and 10:1 ink: suspension ratios.

[0131] Cell mixing viability - Cell viability was evaluated immediately postmixing without crosslinking the bioink using the following methods. A549, NIH-3T3, and primary human lung fibroblast cells were expanded in tissue culture flasks withDulbecco's Modified Eagle Medium (11965-092, Gibco, Waltham, MA, USA) with 10% FBS (090-110, Wisent BioProducts, Saint-Jean-Baptiste, QC, Canada) and antibiotic-antimycotic (100 U / mL penicillin / 100 pg / mL streptomycin, VWR International Co, Mississauga, ON, Canada). Cells were lifted from culture flask surfaces using 0.05% trypsin-EDTA (T3924, Sigma- Aldrich, St. Louis, MO, USA) and 25*106cell / mL suspensions were mixed with PEGDA ink at an ink: suspension ratio of 4:1 using the CELLINK mixer or the designed lattice mixer. Mixing consisted of one pass through the mixer using the syringe proportioner. Each output was collected in a 1 mL syringe which was not crosslinked to avoid irradiation-induced cell damage. The syringe graduations were used to dispense 200 pL samples into 48-well plates, which were immediately stained with LIVE / DEAD™ cell viability kit (LIVE / DEAD™ Cell Imaging Kit, 488 / 570, Life Technologies Corp., CA, USA). The stained samples were imaged using an inverted confocal microscope as described above. The images were imported to ImageJ, where the images were separated into green (live) and red (dead) channels and thresholded to generate binary masks of each channel before using the Analyze Particles function to count the number of cells. The viability was calculated by the number of cells in the green channel divided by the total number of cells in both channels. The samples for viability analysis were not crosslinked to minimize irradiation-induced cell death. Dispensing the samples into well plates likely impacted the cell distribution, so a separate method was used to evaluate cell distribution immediately post-mixing in crosslinked samples as discussed below.

[0132] Cell mixing distribution - To evaluate primary human lung fibroblast cell distribution post-mixing with the lattice and CELLMIXER geometries, the mixed outputs were collected in 1 mL syringes and crosslinked with 405 nm light for 5 minutes. The crosslinked gels were sliced axially using razor blades to generate 2 mm thick transverse cross-sectional slices. The slices were placed in a 48 well plate and washed 3 times with PBS for 30 minutes each before fixing and staining. Samples were fixed and permeabilized by incubating for 1 h with a solution of 4% paraformaldehyde (15712, Electron Microscopy Sciences, Hatfield, PA, USA) and 0.2 % Triton-X 100 (A14288SA, Invitrogen, Waltham, MA, USA) in PBS. Non-specific binding was blocked by incubating the samples overnight at 4°C with 3% bovine serum albumin (BSA; 800-095-EG, Wisent Bioproducts, Saint-Jean-Baptiste, QC, Canada) in PBS. The samples were then incubated overnight with DAPI (R36705, Invitrogen, Waltham, MA, USA) and phalloidin (T7471, Thermo Fisher, Mississauga, ON, Canada) diluted in a solution of 3% BSA and 0.02 % Triton-X 100 in PBS. The stained samples were stored in 3% BSA for up to 3 days before imaging with an inverted confocal microscope as described above. Images were imported into ImageJ and the phalloidin channel was thresholded to generate binary masks from which clustered cells were isolated using a watershed transform function. The entire slice was selected with a circular region of interest and the Biovoxxel plugin was used to run the Particle Distribution (2D) macro with minimum size of 10 pixels including holes with centroid nearest-neighbor distance calculation selected. The variance (o2) of measured nearest-neighbor distances in each image was normalized with respect to the minimum theoretical variance (o02):Area<JQ = 0.5nwhere n is the number of cells identified within the area of interest.

[0133] Extrusion bioprinting of mixed bioinks - A549 cells were mixed with sterile TissuGel ink in an ink: suspension ratio of 4: 1 using the lattice mixer, to achieve a final concentration of 5 / I06cell / mL. The output syringe was transferred to an Allevi 3 bioprinter (Allevi, Philadelphia, PA, USA) and kept at 37 °C throughout the bioprinting process. Mesh-like constructs of 0.6 cm x 0.6 cm width, 1 mm height, and 3 mm x 3 mm internal squares were bioprinted using a G25 needle covered in foil to protect biomaterial ink from premature cross-linking, 15% blue light (405 nm), and 10 mm / s speed. The final constructs were incubated at 37 °C and 5% CO2, with culture media changes every 3 days. The viability was measured on days 1, 3, and 7 post-bioprinting using the LIVE / DEAD™ kit as described above.Results

[0134] Mixing system design - Images and renderings of the purchased CELLMIXER and the designed lattice, ISG, and serpentine static mixers are displayedin Figure 2. The mixers connect via Luer Lock fittings to an output syringe and a Y-channel connecting two input syringes driven by the syringe proportioner, where the input syringes contain a biomaterial ink and cell suspension to be mixed (Figure 1 A). The total dead volume of the system is the sum of the internal volumes of the mixers (310 pL) and the Y-channel (80 pL), totaling 390 pL. The syringe proportioner consists of a base with interchangeable gears that allow mixing the components at three ink: suspension ratios of 10: 1, 4:1, and 3:1 (Figure IB). The mechanism contains a primary rack gear, a spur gear, a compound spur gear, and a secondary rack gear. The primary rack gear drives the secondary rack gear at the ratio defined by the gear diameter ratio. For the 10:1 configuration, 5-mL and 1-mL BD syringes must be used for the biomaterial ink and suspension, respectively. The 4:1 and 3:1 configurations must use identical syringes for both inputs, the size of which can be 1-, 3-, or 5-mL BD syringes.

[0135] Biomaterial rheology and shear stress simulation - The shear-thinning relationships of concentrated PEGDA and TissuGel inks prepared to be mixed at ink: suspension ratios of 3:1, 4:1, and 10:1 were interpolated from the measured rheological data with R2values of 0.9957, 0.9958, and 0.9957, respectively, for the PEGDA inks (Figure 3A), and 0.9946, 0.9930, and 0.9955, respectively, for the TissuGel inks (Figure 3C). As expected, the rheological relationships for both biomaterial inks were slightly different when prepared for the different ink: suspension mixing ratios. The flow simulations with interpolated rheological relationships of 4: 1 PEGDA and TissuGel yielded different maximum shear stresses for each mixer geometry at flow rates representing different speeds at which a user may push the syringe proportioner (Figure 3B / D). The lattice mixer yielded the lowest shear stress at all simulated flow rates with both biomaterial inks. The maximum shear stress was observed on the walls for all mixer geometries except for the lattice mixer in which the maximum shear stress was located at the acute vertices of the lattice intersections. With a high flow rate of 12 mL / s, the maximum shear stress in all mixer geometries did not exceed 1.5 kPa, which was well below the 5-10 kPa level that has been previously reported to decrease endothelial and fibroblast cell viability below 80%.

[0136] Mixing performance - Suspensions of fluorescent particles (0.1 pm diameter) were mixed into PEGDA and TissuGel inks using each mixer geometry at a4:1 ink: suspension ratio. These particles are much smaller than cells and were not intended to mimic cell mixing. Rather, their small size enables visualization with fine resolution of the mixing patterns for quantitative analysis of the mixing performance. In representative images of transverse cross sections of the mixed outputs, the lattice mixer provided the most homogenous distribution of fluorescent particles in both PEGDA and TissuGel inks with few saturated or black regions compared to the other mixers (Figure 4A). Quantification confirmed the qualitative assessment. The quantified coefficients of variation of pixel intensity of the lattice mixer yielded the lowest variation of fluorescence intensity when mixing both PEGDA (0.825 ± 0.062) and TissuGel (0.533 ± 0.044) inks with fluorescent particle suspensions (Figure 4B).

[0137] The lattice mixer geometry was chosen for further evaluation due to the superior mixing performance compared to the other mixer geometries with an ink: suspension ratio of 4:1. The utility of the syringe proportioner for mixing two alternate ink: suspension ratios of 3:1 and 10:1 was demonstrated by mixing fluorescent particle suspensions with PEGDA and TissuGel inks using the lattice mixer. Representative images of transverse cross sections of the mixed outputs revealed that the fluorescent particles were spread throughout the mixtures (Figure 4C). The coefficients of variation for the 3:1 ratio (PEGDA ink: 0.794 ± 0.090; TissuGel: 0.674 ± 0.056) were comparable to those of the 4 : 1 ratio with the lattice geometry (PEGDA ink: 0.813± 0.043 ; TissuGel: 0.533 ± 0.044). Despite having a much higher ratio of ink to fluorescent particles, the lattice mixer with a 10:1 ink: suspension ratio yielded coefficients of variation (PEGDA ink: 1.062 ± 0.240; TissuGel: 0.950 ± 0.030) comparable to those of the CELLMIXER with a 4:1 ratio (PEGDA ink: 1.088 ± 0.114; TissuGel: 1.169 ± 0.076).

[0138] Cell mixing viability and distribution - To evaluate the viability of cells immediately after mixing without crosslinking, A549 human alveolar epithelial cells, NIH-3T3 mouse fibroblast cells, or primary human lung fibroblasts were mixed with PEGDA ink at a 4:1 ink: suspension ratio using the CELLMIXER or the lattice mixer. For all cell types, the CELLMIXER maintained a viability higher than 92% and the lattice mixer maintained viability higher than 96% (Figure 5A / B).

[0139] The distribution of primary human lung fibroblasts within the PEGDA biomaterial ink was qualitatively more homogeneous with the lattice design than with the CELLMIXER system (Figure 6A). Quantitatively, the normalized variance of nearest neighbor distances between cells was significantly lower in bioinks prepared with the lattice mixer than the CELLMIXER (Figure 6B), demonstrating enhanced mixing efficiency.

[0140] Extrusion bioprinting of mixed bioinks - The mixing evaluations identified the lattice mixer as the optimal design for preparing bioinks among those investigated. We therefore set out to validate the optimized system by producing cellladen bioinks followed by 3D printing and assessment of cell viability over time. TissuGel bioinks with A549 cells mixed using our method with the lattice geometry were used to 3D bioprint 0.6 cm x 0.6 cm width, 1 mm height, and 3 mm x 3 mm internal square mesh-like constructs, and cell viability was assessed over 7 days (Figure 7A). The proportion of live cells increased from 73 ± 8% on day 1 post-printing to 89 ± 4% on day 3 and 86 ± 6% on day 7 (Figure 7B). The cell distribution was qualitatively similar throughout the printed construct on day 7 post-printing (Figure 7C). The visible cells at day 7 post-printing frequently formed multi-cell spheroids throughout the constructs (Figure 7D).Discussion

[0141] In the present work, we describe and validate an improved system for mixing aqueous cell suspensions gently and homogenously into viscous biomaterial inks. We designed and 3D printed three miniaturized static mixer geometries inspired by larger volume industrial viscous mixers, and compared them with the Kenics-style mixer used in the CELLMIXER product commercially available from CELLINK. Differences in the fluid dynamics within miniaturized static mixer geometries compared to industrial scale geometries may be because, with industrial scale geometries, inertial forces dominate over viscous forces, making turbulence more common. Whereas at microfluidic scales of the miniaturized geometries, viscous forces dominate inertial forces because there is less fluid mass, making laminar flow more common. There arose modifications in the dimensions of the obstacles, how they interact and cause turbulence within the fluid, aswell as on the arrangement of the lattices and the number of lattices used. These features were optimized for the particular use. Computational fluid dynamics simulating the measured shear-thinning behaviours of PEGDA ink and TissuGel biomaterials indicated that the lattice design, based on the Sulzer SMX mixer, yielded the lowest shear stress across a range of practical flow rates. The lattice design provided the most homogeneous mixing of fluorescent particles compared to the CELLMIXER and the other designed geometries. To enable the real-world implementation of the lattice static mixer design, we developed and demonstrated the utility of a custom-made 3D printed mixing platform that consisted of a syringe proportioner and Y -connector for mixing ink: suspension ratios of 3:1, 4:1, and 10:1. The viability of A549 and NIH-3T3 cell lines and primary human lung fibroblast cells was maintained post-mixing using both the lattice mixer and CELLMIXER, with a more uniform distribution of mixed cells using the lattice mixer. Finally, we demonstrated the utility of the static mixer and syringe proportioner system for 3D bioprinting of a cell-laden gelatin-based bioink with qualitatively uniform cell distribution.

[0142] An inherent limitation of static mixers is the dead volume that is difficult to recover from within the mixer and associated channels after mixing. We experimentally estimated the dead volume of the CELLMIXER as 310 pL. Additionally, the CELLMIXER application note reports that the first 500 pL of bioink is not completely mixed, yielding a total unusable volume of 810 pL for the CELLMIXER system. The lattice, ISG, and serpentine mixers modeled in this work were designed to match the 310 pL dead volume of the CELLMIXER. Combined with the dead volume of the Y-channel connecting the input syringes to the mixer, which is 80 pL, our designed system has an effective dead volume of 390 pL, a significant improvement over the CELLMIXER. Part of this dead volume can be recovered by detaching the input syringes from the Y-connector and pulling the output syringe plunger to draw in material from the mixer and Y-channel, but the exact volume that can be recovered has not been quantified.

[0143] Although the CELLMIXER and Vitrolnk cell mixing systems use static mixers, thorough evaluations of mixing performance and cell viability are not available in the literature. In this work, we performed such an evaluation of miniaturized static mixers mimicking the SMX, ISG, and serpentine mixers with dead volumes equal to theKenics-style CELLMIXER. Using two viscous shear-thinning biomaterials (Figure 3) we found that our lattice mixer, based on the SMX geometry, provided the most homogenous mixing of the viscous biomaterial inks with low viscosity particle suspensions (Figure 4), which was consistent with industrial scale static mixer evaluations.

[0144] A unique aspect of our system is its flexibility in accommodating several ink: suspension ratios. In bioprinting, the optimal ratio of bioink to cell suspension can vary depending on the specific application, the nature of the biomaterial ink, and the cell type involved. Whereas the CELLMIXER and Vitrolnk mixing systems only accommodate a single ink: suspension ratio of 10: 1 or 3: 1, respectively, our custom-made syringe proportioner enables reliable mixing with 3: 1, 4: 1, and 10: 1 ink: suspension ratios by simply interchanging two gears. This flexibility is useful as different tissue engineering applications may require fine adjustments to the bioink’s rheological properties to achieve the desired print fidelity and mechanical stability while ensuring a homogeneous cell distribution. Our designed syringe proportioner is useful for rapid production of small bioink batches (< 10 mL) with high reproducibility. For larger scale production, the syringe proportioner design can be scaled to accommodate 50 mL syringes. The input materials may also be mounted on syringe pumps and fed into the static mixer to automate the production of large batches.

[0145] Upon identifying the superior mixing performance of the lattice mixer design with fluorescent particles, we applied the system to live cells. Although a uniform distribution of cells in bioinks is desired, maintenance of cell viability post-mixing is necessary for the proper functioning of advanced tissue mimetics. A common source of cell death during the bioink preparation and bioprinting processes is wall shear stress. Endothelial and fibroblast cell viability have been found to decrease below 80% with 5-10 kPa of shear stress, although some cell types are more susceptible to mechanical damage like bone marrow stem cells and induced pluripotent stem cells. Using the measured rheological properties of the PEGDA and TissuGel inks, we simulated shear stresses that were well below this range for each mixer design at flow rates relevant to user operation (Figure 3). The simulations indicated that the lattice mixer was least likely to induce shear-related cell damage. Accordingly, high viability of A549, NIH-3T3, and primary human lung fibroblasts was maintained using both the lattice and CELLMIXERgeometries (Figure 5), with our designed lattice mixer providing a more homogenous cell distribution (Figure 6). Bioink mixing could be performed in a biosafety cabinet within one minute using our system. The mixer and Y-connector made with Formlab’s Biomed Clear resin are autoclavable, and the syringe proportioner made with PLA can be sprayed with ethanol into the biosafety cabinet to prevent contamination of the bioink.

[0146] Having confirmed that cell viability was maintained for the cell types used, we 3D printed constructs using A549 cell-laden TissuGel bioinks prepared using the mixing system (Figure 7). The cell viability in the printed constructs was slightly lower than at the mixing stage. Our demonstration that the viability of different cell types was well maintained immediately following mixing, further indicated that the observed reduction in viability was a result of the extrusion process during printing and not due to mixing. Regardless of the cell viability, the distribution of cells across the constructs qualitatively demonstrated the effectiveness of the mixing process to prevent cell clumping or sedimentation, which could affect the functionality of the bioprinted constructs. The formation of spheroids within the bioprinted hydrogels highlighted the ability of the bioink to promote this behavior.Conclusion

[0147] In conclusion, our work provides a simple, reproducible, and flexible system for mixing cells into viscous biomaterial inks. By demonstrating mixing homogeneity, high cell viability, and the capacity to accommodate various ink: suspension ratios, our approach facilitates standardized fabrication of cell -laden tissue constructs to ensure consistency in the growing field of 3D bioprinting.Example 2

[0148] A system as shown in Fig. 8 and 9 comprising a static mixer and a fixed 4: 1 ratio syringe proportioner was combined with a y-channel that blends the input streams from two input syringes. The input syringes were mounted in a syringe proportioner base which may be adapted to accommodate syringes of different volumes, e.g. 3- or 5-mL input syringes. It is noted that the performance of the second embodiment is similar to that of the first embodiment.

[0149] The following listing of reference numerals is provided for convenience only, and no limitation is implied; the list is not necessarily exhaustive and may not be complete.100 System102 Syringe proportioner106 Base108 First input syringe108 A Plunger110 Second input syringe110A Plunger112 First input syringe retainer114 Second input syringe retainer116 Post118 Flange120 Gear assembly122 Geartrain124 First gear rack124’ First gear rack124” First gear rack124’” First gear rack126 Second gear rack126’ S econd gear rack126” S econd gear rack126’” Second gear rack128 First input syringe driver130 Second input syringe driver132 Elongate member134 Plurality of teeth136 Gear facing surface138 Plunger abutting head140 Elongate member142 Plurality of teeth’ Plurality of teethGear facing surface Plunger abutting head First gear rack recess Second gear rack recess Primary gear’ Primary gear” Primary gear’” Primary gearA Plurality of teeth Secondary gear’ Secondary gear” Secondary gear’” Secondary gearA Plurality of teeth Tertiary gear’ Tertiary gear” Tertiary gear’” Tertiary gearA Plurality of teethPostChannelA First inletB Second inletOutletA First fluid expelling end B Second fluid expelling end Static mixer’ Static mixer” Static mixer” Static mixerFluid receiving endFluid discharging end Output syringeTubeFirst solutionSecond solutionLattice-like structure Spiral structure obstacle A Chain-like structure obstacle B Chain-like structure obstacle SystemSyringe proportioner CasingBaseFirst input syringeA PlungerSecond input syringeA PlungerFirst input syringe retainer Second input syringe retainer A BracketB BracketC Central bracketFlangeGear assemblyGear trainFirst gear rackSecond gear rackFirst input syringe driver Second input syringe driver Elongate member Plurality of teethGear facing surface Plunger abutting headElongate member Plurality of teethGear facing surface Plunger abutting head First gear rack recess Second gear rack recess Primary gearA Plurality of teeth Secondary gear Tertiary gearA Plurality of teethPostChannelA First inletB Second inletOutletA First fluid expelling end B Second fluid expelling end Static mixerFluid receiving end Fluid discharging end Output syringeTubeFirst solutionSecond solution Lattice-like structure Back panelChannelA Intermediate gearB Intermediate gearPortion

Claims

CLAIMS1. A system for mixing first and second solutions, the system comprising:a syringe proportioner comprising a base adapted to receive first and second input syringes, and a gear assembly to drive a plunger of each of the input syringes to empty solution from each of the input syringes at a predetermined flow rate;a channel comprising first and second inlets and a single outlet, the first and second inlets being adapted to fluidly couple respectively with first and second fluid expelling ends of each of the first and second input syringes, to receive solution from the first and second syringes; anda static mixer comprising a fluid receiving end and a fluid discharging end, wherein the fluid receiving end is adapted to fluidly couple with the outlet of the channel and receive solution therefrom and the fluid discharging end is adapted to discharge fluid, said mixer comprising an internal geometry which provides mixing of a first solution from the first input syringe and a second solution from the second input syringe.

2. The system according to claim 1, wherein the gear assembly comprises a gear train and first and second gear racks respectively comprising first and second input syringe drivers, wherein rotation of the gear train in an actuation direction is configured to engage the first and second gear racks and actuate the first and second input syringe drivers to drive fluid from the first and second input syringes, respectively.

3. The system according to claim 2, wherein the gear train comprises primary, secondary and tertiary gears, wherein actuation of the tertiary gear engages with the primary and secondary gears to engage the first and second gear racks, respectively, and actuate the first and second input syringe drivers, wherein the primary gear has a first diameter and the secondary gear has a second diameter.

4. A system according to claim 3, wherein the ratio of the first and second diameters corresponds to the relative volumes of the first and second solutions to be dispensed by the syringe proportioner.

5. A system according to claim 3, wherein the first diameter is greater than the second diameter.

6. A system according to claim 5, wherein the ratio is selected from 3:1, 4:1 and 10:1.

7. A system according to any one of claims 3 to 6, wherein the depth of the primary and / or secondary gear is greater than the depth of the tertiary gear.

8. A system according to any one of claims 3 to 7, wherein the tertiary gear has a third diameter, and the third diameter is greater than the second diameter and the first diameter.

9. A system according to any one of claims 3 to 8, wherein the secondary gear is in direct contact with the tertiary gear.

10. A system according to any one of claims 3 to 8, wherein the secondary gear is in spaced apart from the tertiary gear, such that the secondary gear and tertiary gear are non-engaging.

11. A system according to any one of claims 3 to 10, wherein the gear train comprises at least one intermediate gear, the at least one intermediate gear coupling the primary gear to the tertiary gear.

12. A system according to any one of claims 1 to 11, wherein the internal geometry of the mixer comprises at least one obstacle that causes mixing of the first and second solutions.

13. A system according to claim 12, wherein the obstacle is selected from a chainlike structure; a helical or ribbon structure; a spiral structure; a grid-like structure; a lattice structure; and / or a fin structure.

14. The system according to any one of claims 1-13, wherein the static mixer exhibits a sheer stress of less than lOkPa during mixing of the first and second solutions.

15. The system according to claim 13, wherein the obstacle comprises at least two of a chain-like structure; a helical or ribbon structure; a spiral structure; a grid-like structure; a lattice structure; and / or a fin structure.

16. The system according to any one of claims 1 to 15, wherein the channel comprises a Y-shape or T-shape.

17. A system according to any one of claims 1 to 16, wherein the base of the syringe proportioner is adapted to removably retain first and second input syringes.

18. A system according to claim 17, wherein the base of the syringe proportioner is adapted to retain the first and second input syringes atop the base of the syringe proportioner.

19. A system according to claim 17, wherein the base of the syringe proportioner is adapted to retain the first and second input syringes within the base of the syringe proportioner.

20. A system according to any one of claims 2 to 19, wherein the gears of the gear train are removable.

21. A system according to any one of claims 1 to 20, wherein the first inlet of the channel is configured to receive a biomaterial solution and the second inlet of the channel is configured to receive an aqueous cell suspension.

22. A system according to any one of claims 1-21, wherein the channel is configured to removably connect to the first and second input syringes.

23. A system according to any one of claims 1-22, wherein the fluid discharging end of the static mixer is configured to fluidly couple to an output syringe.

24. A system according to any one of claims 1-21, wherein the channel is configured to removably connect to the static mixer.

25. A method of mixing first and second solutions having different viscosities, the method comprising: i) actuating the gear assembly of a system as defined in any one of claims 1-24 to drive solution contained within the first and second input syringes into the static mixer for mixing via the channel, and ii) collecting the mixed solution as it is discharged from fluid discharging end of the static mixer.

26. The method of claim 25, wherein the viscosity of the second solution is less than the viscosity of the first solution.

27. The method of claim 25, wherein the first solution comprises a biomaterial and the second solution is an aqueous cell-containing solution.

28. The method of claim 26 or 27, wherein the viscosity of the second solution is no greater than about 1000 Ns / m2.

29. The method of claim 28, wherein the viscosity of the second solution is no greater than about 500 Ns / m2.

30. The method of any one of claims 25 to 29, wherein the viscosity of the first solution is no less than about lxlO'4Ns / m2.

31. The method of any one of claims 25 to 30, wherein the static mixer exhibits a sheer stress of less than lOkPa during mixing of the first and second solutions.

32. The method of claim 27, wherein the static mixer maintains at least about 80% viability of cells in the mixed solution.

33. The method of any one of claims 25-32, wherein the gear assembly is selected to drive fluid from the first and second syringes at a predetermined flow rate.

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