WO2020254650A1

WO2020254650A1 - In-flow acoustophoretic alignment of cells in a hydrogel filament

Info

Publication number: WO2020254650A1
Application number: PCT/EP2020/067234
Authority: WO
Inventors: Peter Reichert; Dhananjay DESHMUKH; Mark TIBBITT; Jürg Dual
Original assignee: ETH Zürich
Priority date: 2019-06-20
Filing date: 2020-06-19
Publication date: 2020-12-24

Abstract

A process of continuous production of a filament of solidified hydrogel, having cells aligned therein along the longitudinal axis of the filament according to a predetermined and preferably continuously tunable pattern, from an essentially tubular cavity comprising an inlet section, a patterning section, a solidification section, and an outlet section, whereby a growing plug of solidified material, as It is formed in the solidification section, is displaced out of the solidification section and discharged from the outlet section as a result of the continuous injection of precursor material into the Inlet section of the tubular cavity such as to form the filament of solidified hydrogel.

Description

TITLE

IN FLOW ACOUSTOPHORETIC ALIGNMENT OF CELLS IN A HYDROGEL

FILAMENT

TECHNICAL FIELD

The present invention relates to a process of producing an endless hydrogel filament, in which cells are embedded in a hydrogel matrix and aligned within the hydrogel filament according to a predetermined and/or tunable pattern via in-flow acoustophoresis.

PRIOR ART

Hydrogels have emerged as a major class of biomaterial in multiple applications, such as for the culture of mammalian cells outside of the body. Owing to their high content of water and tunable modulus, hydrogels are an attractive mimic of soft tissue extracellular matrix (ECM) that surrounds the cells in vivo. Within this field of extracorporeal culture, a large emphasis has been on the fabrication of functional micro- and macroscale tissues for regenerative medicine, ex vivo disease modeling, as well as the production of soft robotics.

However, it remains difficult to produce aligned cellular structures within a three dimensional environment on large scale, such as for example engineered musculoskeletal tissues. Other methods to arrange cells are limited by their rigid designs or by the lack of accuracy in cell positioning.

P. Lata, James & Guo, Feng & Guo, Jinshan & Huang, Po-Hsun & Yang, Jian & ,/un Huang. Tony, Surface Acoustic Waves Grant Superior Spatial Control of Cells Embedded in Hydrogel Fibers. Advanced Materials. 28. 10.1002 discloses a device and a method for controlling the spatial orientation of cells in a fiber of hydrogel matrix, where the cells are oriented via Surface Acoustic Waves (SAWs). A hydrogel precursor is introduced into a tube where in a predefined pattern SAWs are imparted on the cells by controlling a pair of interdigital transducers and after turning off the interdigital transducers, the hydrogel is cross-linked with UV light. Subsequently, the cross-linked fiber is extracted from the tube by pushing it out with a syringe. A poloxamer is added to the hydrogel in order to aid in extraction of the fiber from the tube, as this also aids with cell viability in the fiber in contrast to the coating of the tube lumen. Lata thus discloses a batch-wise production of individual fibers having a certain length, limited to the length of the tube is disclosed. However, this limitation in length is both undesirable for production cost reasons and for practical reasons. It is thus desirable to provide a device or method allowing the continuous production of fiber of arbitrary length, or which can be rolled up on a bobbin for later use. In addition, the use of SAWs restricts the depth at which particles in a liquid may be manipulated and thus the tube geometry is limited to diameters of less than 280 pm. It is thus further desirable to provide a device or method allowing the continuous production of fiber using larger tube geometries.

Kitagawa Y, Nagamima Y, Yajima Y, Yamada M, Seki M, Patterned hydrogel microfibers prepared using multilayered microfluidic devices for guiding network formation of neural cells. Biofabrication. 2014 Sep;6(3):035011 discloses a device and a method of using micronozzle arrays to obtain microfibers of hydrogel in which cells are aligned according to a pattern reflected in the arrangement of the micronozzles. However, a change in pattern in the fiber is tied to a change in the micronozzle array, which is not practical.

Groschl M, Coakley WT, McLoughlin AJ, A new immobilisation method to arrange particles in a gel matrix by ultrasound standing waves, Ultrasound Med Biol. 2005 Feb;31 (2):261 -72 discloses a setup in which an alginate gel is guided through a glass capillary lodged in a tubular transducer coupled to a vibration source to orient latex particles in the alginate. The alginate comprising the oriented latex particles is then introduced into a curing solution of calcium ions immediately upon leaving the capillary, whose outlet is submerged in the curing solution. At relatively low concentrations, alginate has a high viscosity in excess of 200 mPa s that hinders the dissipation of the latex particles in the bulk of the hydrogel precursor due to the flow field and drag forces within. This "freeze" of the latex particles in high viscosity alginate allows for the slower curing afforded by immersion in calcium ion solutions, but makes it impossible to process hydrogels precursors having lower viscosities in a similar manner.

US2015/010845 A1 discloses a method for the acoustic manipulation of particles in a How of particle-containing fluid along a pathway, where acoustic standing waves are used to orient the particles in the particle-containing fluid while flowing along the pathway. However, hydrogels are not mentioned as matrix for the particles and no cross-linking of the particle-containing fluid takes place.

WO2016/025518 A1 likewise discloses a method for the separation of low abundance cells from a fluid using SAWs. However, hydrogels are not mentioned as matrix for the particles and no cross-linking of the particle-containing fluid takes place.

WO2016161 109 A 1 discloses a method of ordering rod-shaped mineral particles suspended in a solution, wherein the anisotropic particles suspended in a solution are put to a channel, wherein the anisotropic particles are unordered when entering the channel; and applying sound waves to the channel, wherein the frequency of the sound wave is tuned to create one or more columns of anisotropic particles oriented in the same direction. However, it is not disclosed that cells, which correspond essentially spherical particles and therefore rather isotropic, can be used in such method. Furthermore, a lubricating fluid is side-fed to the cured hydrogel comprising the anisotropic particles as it exits the print head. The addition of lubricating fluids is impractical and depending on the chemical nature of the lubricant fluid can interfere with medical applications in the case cells would be incorporated. A frequency of 20 kHz was used.

SUMMARY OF THE INVENTION

It has been found that the above shortcomings of the prior art can be avoided or at least diminished by producing filaments of solidified hydrogel according to the process of the present invention and the device for carrying out the process, i.e., that a filament of solidified hydrogel can be obtained in a simple manner that allows to freely and independently choose the pattern of cells, the chemical nature of photopolymerisable or thermocurable hydrogel, the length of the filament, the size of the tube and ensuing filament as well as the spatial resolution of the cells.

The process of the present invention is characterized in that it is a process in which both acoustophoretic patterning of cells in a hydrogel precursor and gelation of the hydrogel precursor are carried out in- flow, thereby allowing a continuous filament formation.

It is an object of the present invention to provide a process of continuous production of a filament of solidified hydrogel, having cells aligned therein along the longitudinal axis of the filament according to a predetermined and/or tunable pattern, from an essentially tubular cavity comprising an inlet section, a patterning section, a solidification section, and an outlet section, the process comprising the steps of, preferably in this order:

a. establishing a continuous flow of precursor material, in which cells are suspended in a hydrogel precursor, across the tubular cavity from the inlet section of the tubular cavity towards the outlet section of the tubular cavity by continuous injection of precursor material into the inlet section of the tubular cavity,

b. patterning the cells in the precursor material flowing across the patterning section of the tubular cavity into the predetermined and tunable pattern by acoustophoretic patterning, c. solidifying the precursor material, in which cells have been brought into the predetermined and preferably tunable pattern, flowing across the solidification section of the tubular cavity such as to form a growing plug of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament,

characterized in that the growing plug of solidified material, as it is formed in the solidification section, is displaced out of the solidification section and discharged from the outlet section by the continuous injection of precursor material into the inlet section of the tubular cavity such as to form the filament of solidified hydrogel, which filament may be of an arbitrary length.

The continuous flow or injection of precursor material in the essentially tubular cavity can be provided by for example a pump, such as a syringe pump.

Without wishing to be held to a certain theory, it is believed that the in-flow formation of a plug in the solidification section of the essentially tubular cavity leads to a near plug-type flow of the upstream precursor material that is yet to be solidified near the plug, and in particular in the patterning section of the essentially tubular cavity. The plug-type flow allows patterning the cells within the precursor material more reliably because the cells transit the patterning section at a velocity that is independent of the radial position of the cells with respect to the longitudinal axis of the essentially tubular cavity.

The present invention allows to align cells along the longitudinal axis of the filament according to a predetermined and/or preferably continuously tunable pattern. In this context, the term "continuously tunable pattern" is to be interpreted such that the pattern can be tuned without interruption of process, i.e., on-line or in-flow, by appropriately adjusting the vibration settings of the bulk piezoelectric transducer.

In a preferred embodiment of the process of continuous production of a filament o solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, at least in the solidification section, and preferably at least in the solidification section and the outlet section of the essentially tubular cavity, the surface of the inner or luminal wall of the tubular cavity, which surface of the inner wall is in contact with the growing plug of solidified hydrogel, is formed of or coated with a solid material exhibiting a low coefficient of friction and/or is hydrophobic, thereby lowering the drag encountered by the sliding plug when compared to the uncoated surface of the inner wall.

In the context of the present invention, the term " solid material exhibiting a low coefficient of friction " refers to a material having a coefficient of friction lower than 0.1 and preferably of from 0.01 to 0.1 when measured according to ASTM D1894 standard. For example, PTFE having a coefficient of friction of from 0.04 to 0.1 may be used as a solid material exhibiting a low coefficient of friction.

Without wishing to be held to certain theory, it is believed that by providing at least the solidification section, and preferably the outlet section of the essentially tubular cavity as well, with a solid material exhibiting a low coefficient of friction and/or being hydrophobic, the plug being formed is less likely and/or prevented from clinging or adhering to the surface of the inner or luminal wall of the tubular cavity, which surface of the inner wall is in contact with the growing plug of solidified hydrogel. This prevents the plug from first clinging or adhering to the tube, i.e., slowing down the flow across the essentially tubular cavity and from speeding forward after pressure buildup from the continuously injected precursor material and thereby enables a more constant linear velocity of precursor material with respect to the longitudinal axis of the essentially tubular cavity. The low coefficient of friction also allows for higher cell viability during the filament production process, because the pressure needed to displace the plug towards the outlet is inferior, i.e. lower, compared with an essentially tubular cavity in which the luminal wall is not provided with a solid material exhibiting a low coefficient of friction and/or being hydrophobic.

It will be understood that the solidification section of the essentially tubular cavity may in some cases be made integrally from a suitable solid material exhibiting a low coefficient of friction and/or being hydrophobic or in some cases may be formed by another material coated on the luminal side with a suitable solid material exhibiting a low coefficient of friction and/or being hydrophobic.

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the solid material exhibiting a low coefficient of friction and/or being hydrophobic is a synthetic solid polymer such as for example fluoropolymer or a polysiloxane. A suitable fluoropolymer may for example be PTFE. In the case where at least the solidification section is made integrally from a suitable solid material exhibiting a low coefficient of friction and/or being hydrophobic, the solidification section or the essentially tubular cavity may be made from a fluoropolymer such as PTFE, and in particular may be in the form of a tube of fluoropolymer of PTFE.

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the acoustophoretic patterning is carried out via bulk acoustic wave (BAW) patterning, preferably at frequencies of between 0.5 MHz to 10 MHz. In contrast, surface acoustic wave (SAW) patterning typically is carried out at higher frequencies exceeding 20 MHz, which implies smaller wavelengths and hence limits the maximum distance between pressure nodes. This also implies that the channels used in SAW devices are smaller and hence the surface to volume ratio is higher. This results in higher surface of contact between the gel and the channel which hinders the continuous production of hydrogel fibre.

In the context of the present invention, the acoustic field inside the tubular cavity can be induced by a bulk acoustic wave (BAW) or a coupled fluid-structure resonance. A standing acoustic wave in the channel is generated by the vibration of a bulk piezoelectric transducer, which excites bulk waves and a resonance in channels within an acoustically hard material, mostly glass, rather than waves confined to the transducer’s surface. The standing waves inside the channel cause the manipulation of particles. The choice of frequency allows standing waves of one- or multiple half-wavelengths and different cellular arrangements with the same device. For example cells can be arranged in lines or clumps and produce a cell structure in a 3D environment.

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the step of solidifying the precursor material is earned out by irradiation with radiation, preferably using electromagnetic radiation such as, e.g., photopolymerisation, such as UV or 1R radiation.

It will be understood that in the case where solidifying the precursor material is carried out by irradiation with radiation, the solidifying section of the essentially tubular cavity is formed of one or more materials essentially transparent to the radiation such as to allow the radiation to penetrate the walls of the essentially tubular cavity forming the solidifying section and allow sufficient irradiation of the precursor material. A suitable material essentially transparent to the radiation, in particular UV radiation, are fluoropolymers such as PTFE.

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the precursor material is free of a synthetic lubricating agent such as for example poloxamers since the solid material exhibiting a low coefficient of friction and/or being hydrophobic provides for an eased displacement by the precursor material being injected. In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the steps b. and c. are carried out in a joint patterning and solidification section in which the patterning of the cells in the precursor material flowing across the joint patterning and solidification of the precursor material flowing across the joint patterning are carried out simultaneously in the joint patterning and solidification section such as to form a growing plug of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament. Stated alternatively, a preferred embodiment lies in a process of continuous production of a filament of solidified hydrogel, having cells aligned therein along the longitudinal axis of the filament according to a predetermined and/or tunable pattern, from an essentially tubular cavity comprising an inlet section, a joint patterning and solidification section, and an outlet section, the process comprising the steps of, preferably in this order:

b. simultaneously patterning the cells in the precursor material flowing across the joint patterning and solidification section of the tubular cavity into the predetermined and tunable pattern by acoustophoretic patterning and solidifying the precursor material flowing across the joint patterning and solidification section of the tubular cavity, in which cells have been brought into the predetermined and preferably tunable pattern such as to form a growing plug of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament,

characterized in that the growing plug of solidified material, as it is formed in the joint patterning and solidification section, is displaced out of the joint patterning and solidification section and discharged from the outlet section by the continuous injection of precursor material into the inlet section of the tubular cavity such as to form the filament of solidified hydrogel, which filament may be of an arbitrary length.

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the essentially tubular cavity has a constant diameter along its longitudinal axis and/or has a constant cross- sectional shape along its longitudinal axis.

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the resonance frequencies of / e [1.75, 2.16, 2.81 , 6.81] MHz enabled the formation of multiple l/2 wavelength standing waves leading to a spatial resolution ranging between 100-600 mih.

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the inner diameter of the essentially tubular is in excess of 300 pm, more preferably of between 300 pm and 5 mm and most preferably of 1 mm to 3 mm, and is preferably constant.

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern is continuously extruded at a flow rate of about 5 pl/min.

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the pressure required to set the initial hydrogel plug into motion per unit area of contact between the initial hydrogel plug and the solid material exhibiting a low coefficient of friction and/or being hydrophobic equals 8.625 kN/m².

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the spatial resolution of the pattern ranges between 100 and 600 pm.

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the cells are eukaryotic cells of a multicellular organism such as for example human cells and in particular are muscle cells or cardiovascular cells of a multicellular organism such as for example human muscle cells or human cardiovascular cells.

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the essentially tubular cavity being formed of an outer casing such as for example an outer glass casing that is vibrationally coupled to a wave generating device and of an inner fluoropolymer cavity, that is lodged inside the outer casing and that is vibrationally coupled to the outer casing. In a much preferred embodiment, the inner fluoropolymer cavity is a tube of fluoropolymer that is removably lodged in the outer casing. The tube of fluoropolymer may for example be vibrationally coupled to the outer casing by a body of coupling liquid such as water located between the outer casing and the tube of fluoropolymer. In a much preferred embodiment the glass is quartz glass.

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the inner wall of the essentially tubular cavity is formed of a fluoropolymer or PTFE tube having a round cross-sectional shape, which is preferably lodged inside an outer casing, the inner wall of the essentially tubular activity being directly or indirectly vibrationally coupled to a wave generating device, and where preferably the outer casing is a tubular glass casing having a square cross-sectional shape and where the interstices between the fluoropolymer or PTFE tube and the glass capillary are filled with an acoustic coupling medium, which may for example be water.

In a preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the inner wall of the essentially tubular cavity is formed of a fluoropolymer or PTFE tube having a cross- sectional shape having an outer diameter of 2 mm and/or an inner diameter of 1.7 mm, which is preferably lodged inside an outer casing, the inner wall of the essentially tubular activity being directly or indirectly vibrationally coupled to a wave generating device, and where preferably the outer casing is a tubular glass casing having a square cross-sectional shape and having an outer dimensions of 2.4 x 2.4 mm an inner dimensions of 2 x 2 mm, where the interstices between the fluoropolymer or PTFE tube and the glass capillary are filled with an acoustic coupling medium, which may for example be water.

In a preferred preferred embodiment of the process of continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament in a predetermined and/or tunable pattern according to the present invention, the wave generating device is a bulk piezoelectric transducer.

It is an object of the present invention to provide a device for the continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament according to a predetermined and/or tunable pattern, comprising an essentially tubular cavity comprising an inlet section, a patterning section, a solidification section, and an outlet section, wherein

a. the inlet section for accepting a flow of precursor material in which cells are suspended in a hydrogel precursor,

b. the pattering section for acoustophoretically bringing the cells in the precursor material into the predetermined and/or tunable pattern, as the flow of precursor material passes through the patterning section, c. the solidification section for solidifying the precursor material, in which cells have been brought into the predetermined and preferably tunable pattern, such as to form a growing plug of solidified hydrogel having cells aligned therein along the longitudinal axis, as the flow of precursor material passes through the solidification section, and

d. the outlet section for discharging the growing plug of solidified hydrogel such as to continuously form the filament of solidified hydrogel formed having cells aligned therein along the longitudinal axis of the filament according to the predetermined and preferably tunable pattern.

It is another object of the present invention to provide a device for the continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament according to a predetermined and/or tunable pattern, comprising an essentially tubular cavity comprising an inlet section, a joint patterning and solidification section, and an outlet section,

a. the inlet section accepting a flow of precursor material in which cells are suspended in a hydrogel precursor,

b. the joint patterning and solidification section acoustophoretically bringing the cells in the precursor material into the predetermined and/or tunable pattern, as the flow of precursor material passes through the joint patterning and solidification section and solidifying the precursor material, in which cells have been brought into the predetermined and preferably tunable pattern, such as to form a growing plug of solidified hydrogel having cells aligned therein along the longitudinal axis, as the flow of precursor material passes through the joint patterning and solidification section, and

c. the outlet section for discharging the growing plug of solidified hydrogel such as to continuously form the filament of solidified hydrogel formed having cells aligned therein along the longitudinal axis of the filament according to the predetermined and preferably tunable pattern.

In a preferred embodiment of the device for the continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament according to a predetermined and/or tunable pattern according to the present invention, at least in the solidification section, and preferably at least in the solidification section and the outlet section of the essentially tubular cavity, the surface of the inner or luminal wall of the tubular cavity, which surface of the inner wall is in contact with the growing plug of solidified hydrogel, is formed of or coated with a solid material exhibiting a low coefficient of friction and/or is hydrophobic, thereby lowering when compared to the uncoated surface of the inner wall.

In a preferred embodiment of the device for the continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament according to a predetermined and/or tunable pattern according to the present invention, the solid material exhibiting a low coefficient of friction and/or being hydrophobic is a synthetic solid polymer such as for example fluoropolymer or a polysiloxane. A suitable fluoropolymer may for example be PTFE. In the case where at least the solidification section is made integrally from a suitable solid material exhibiting a low coefficient of friction and/or being hydrophobic, the solidification section or the essentially tubular cavity may be made from a fluoropolymer such as PTFE, and in particular may be in the form of a tube of fluoropolymer of PTFE.

In a preferred embodiment of the device for the continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament according to a predetermined and/or tunable pattern according to the present invention, the patterning section is equipped with an wave generator device such as a bulk piezoelectric transducer to carry out acoustophoretic patterning, preferably via bulk acoustic wave (BAW) patterning.

In a preferred embodiment of the device for the continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament according to a predetermined and/or tunable pattern according to the present invention, the solidification section is equipped with a radiation or heat source for solidifying the precursor material either by radiation such as UV, VIS or IR radiation or by heating to a solidification temperature.

In a preferred embodiment of the device for the continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament according to a predetermined and/or tunable pattern according to the present invention, the essentially tubular cavity has a constant diameter along its longitudinal axis and/or has a constant cross-sectional shape along its longitudinal axis.

In a preferred embodiment of the device for the continuous production of a filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament according to a predetermined and/or tunable pattern according to the present invention, the amount of precursor material per unit time introduced into the tubular cavity via the inlet is essentially equal to the amount of filament of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament per unit time.

Further embodiments of the invention are laid down in the dependent claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings,

Fig. 1 shows the device (1 ) according to the present invention, the essentially tubular cavity (2) formed of an inlet (3), a patterning section (4) and a solidification section (5), and outlet (6). As can be seen, in step 1 the essentially tubular cavity (2) is filled with the hydrogel precursor having cells suspended therein and a Poiseuille-type flow is established (arrows). In step 2, the cells (8) are patterned via the piezoelectric transducer (9) in the patterning section (4) in-flow as they flow across the patterning section (4) and the flow is stopped. In step 3, the precursor material, in which cells have been brought into the predetermined and preferably tunable pattern, is solidified to form an initial plug (10) at the height of the outlet (6). In step 4, the flow is re-instated and the flow rate is then adjusted such that the solidification is essentially complete as the filament exits the outlet (6) and plug-type flow (arrows) is established in the upstream vicinity of the plug (5), which plug (5) is continuously being formed on its upstream end in the solidification section (5). The plug-type flow allows to bring the cells into the predetermined and preferably tunable pattern in the hydrogel precursor to experience uniform UV radiation doses which would otherwise not be possible when having a Poiseuille-type flow. Fig. 2 shows an embodiment of the device (1) according to the present invention in cross- section along the x-/ y-plane (Fig. 2a) and in the y-/ z-plane (Fig. 2b). The PTFE tubular cavity (2) is formed of an inlet (3), a patterning section (4) and a solidification section (5), and outlet (6). The essentially tubular cavity (2) having an inner diameter (r) and an outer diameter (D) is lodged in an glass outer casing of square having a side of length (D) and of wall thickness (t) in which water as a vibrational coupling fluid is comprised between the essentially tubular cavity (2) and the outer casing, which transmits vibrations from the piezoelectric transducer (9) glued to the outer casing to the essentially tubular cavity (2) and hydrogel precursor therein. The essentially tubular cavity (2) is filled with the hydrogel precursor having cells (8) suspended therein, which cells (8) are patterned in the patterning section (4) in-flow as they flow across the patterning section (4) after which the hydrogel precursor is solidified to form an plug (5) that continuously grows upstream at essentially the same spreed as the downstream end is pushed downstream and exits the outlet (6) as filament (6).

Fig. 3 A/B shows micrographs in which MDCKII cells (8) are aligned within a solidified hydrogel filament along the longitudinal axis of the filament. In Figure 3A, cells aligned within the filament were patterned using a frequency of 6.82 MHz, In Figure 3B stained cells (8) are visible within the filament.

DESCRIPTION OF PREFERRED EMBODIMENTS

In a preferred embodiment of the device according to the present invention, the device is mounted to a holder and is connected to a syringe pump, and comprises a function wave generator wired to a piezoelectric transducer as well as a UV light source for curing the hydrogel precursor, an objective, a cavity, a camera, and a light source. The syringe pump constantly provides one or more hydrogel precursors, via a tubing to the cavity that is a PTFE tube of OD = 2mm, ID = 1.7mm. The optical implements are used to localise the UV light to locally control polymerization of the hydrogel precursor. The camera helps with the monitoring of the process. The function wave generator generates the electrical signals transmitted via electrical wiring to the lead zirconate titanate piezoelectric transducer of 10 mm x 2 mm x 1 mm size. A trifurcation is used such that the PTFE tube passes through the trifurcation. The space between the square glass outer casing and the round PTFE tube is filled with water as coupling medium for the acoustic waves. The length between piezoelectric transducer and PTFE tube outlet is 3 mm. To generate an ultrasonic field within the fluidic domain within the cavity, the piezoelectric transducer vibrates at a tunable frequency f The piezoelectric substrate excites a vibration in the outer glass casing and thereby also in the coupling medium, the PTFE tube and the hydrogel precursor. Bounded by the mismatch of the characteristic acoustic impedance Z = p ^c c (density times speed of sound) of hydrogel precursor and glass, certain fluid resonance modes across the channel width w are feasible, when n l/2 = w fits between left and right channel wall with n = 1 , 2, 3 ... for the first, second and third harmonic and the acoustic wavelength l. If the transducer is tuned to a resonance frequency f = c n/(2w) with the speed of sound c in the precursor fluid, this leads to the formation of an ultrasonic standing wave in the fluid.

In a preferred embodiment of the present invention, a standing acoustic wave in the tubular cavity is generated by a bulk piezoelectric transducer. The piezoelectric transducer excites bulk waves and resonance in the tubular cavity within an acoustically hard material such as for example glass. The standing waves inside the tubular cavity cause the manipulation of particles. The choice of frequency allows standing waves of one- or multiple halfwavelengths and different cellular arrangements with the same device. For example cells can be arranged in lines or clumps and produce a cell structure in a 3D environment. Unlike other methods, acoustophoresis works on a broad range of cell types with few physical requirements, as long as cells differ from the hydrogel precursor in terms of density and/or speed of sound. Dependent on these hydrogel precursor and cell properties, the acoustic waves exert an acoustic force on cells suspended in said hydrogel precursor towards the pressure nodal line of the standing wave or towards the pressure antinode line. This contact- free and controllable external force field acts selectively and on demand on the cells. Furthermore, the biocompatibility of acoustic methods with regard to cells in solution is well documented. Bulk acoustic wave acoustophoresis has formerly focused on particle handling in water, but in the context of the present invention it is applied in combination with a hydrogel precursor having cells suspended therein and the subsequent fluid-solid transition of said precursor solution. In a preferred embodiment of the present invention, the cells that are suspended in a hydrogel precursor can be patterned using frequencies of about 0.5 to 7 MHz.

In a preferred embodiment of the present invention, the precursor material has a viscosity of about 1 mPa s to about 200 mPa s, more preferably of about 1 mPa s to about 175 mPa s. when measured at a shear rate of 1 Hz using an Anton Paar MCR 502 rheometer. Suitable low viscosity precursor solutions may be based on poly(ethylene glycol) (PEG)-based hydrogel precursors such as poly(ethylene glycol) norbornene or polyethylene glycol) diacrylate, which in addition also exhibit good cytocompatibility. For reference, the viscosity of 10 wt% poly( ethylene glycol) diacrylate is 4.6 mPa s at a shear rate of 1 Hz using an Anton Paar MCR 502 rheometer.

It is understood that the precursor material may comprise additives suitable for aiding with photopolymerization such as photoinitiator additives and crosslinker additives. In the case of poly(ethylene glycol) norbornene based hydrogel precursors, a suitable photoinitiator may be lithium acyl phosphate (LAP) and a suitable crosslinker additive may be dithiothreitol (DTT).

At least one electromechanical transducer is attached to the channel adapted to excite BAW standing waves of a predetemiined harmonic resonance mode between said channel walls. In other embodiments, the glass tube may be replaced by other acoustically hard materials. The fluid channel can be of any shape connected by an inlet.

In an embodiment, the piezoelectric transducer is attached to a rectangular glass tube. The electromechanical transducers for cell acoustophoresis are preferably bulk piezoelectric transducers. The glass tube has a rectangular cross section to maximize the attachment surface to the transducer for an efficient coupling of piezoelectric vibration into the fluid channel. The glass tube side walls provide the reflective surfaces for the acoustic waves and the PTFE tube defines the volume of the cell suspension. The PTFE tube has a round cross section and spaces to the rectangular tube are filled with water. The length of the glass tube is usually 20 to 30 times longer than the width. The PTFE tube has a density and speed of sound close to the ones of water; therefore, the PTFE tube is almost transparent for the acoustic waves. The PTFE tube is not physically attached to the glass walls and can be easily replaced for single use. Such single-use system is important for bioprocesses with cells to avoid cross or microbial contamination. In another embodiment, the acoustically hard channel may be coated directly with a low-friction material like PTFE or with a slippery lubricant infused porous surface (SLIPS).

The filament obtained by the process of the present invention may be used as a tissue model for high throughput drug testing especially for tissues with defined cellular structure like skeletal muscles and cardiovascular tissues. In another embodiment, the filament obtained by the process of the present invention may be used to create autologous tissues such as skeletal muscles, which would avoid transplant rejection for in muscle repair.

Since the filament obtained by the process of the present invention can be produced continuously, the filament may be spooled and collected, and may further be woven to provide more complex textile structures.

Claims

1. A process of continuous production of a filament of solidified hydrogel, having cells aligned therein along the longitudinal axis of the filament according to a predetermined and preferably continuously tunable pattern, from an essentially tubular cavity comprising an inlet section, a patterning section, a solidification section, and an outlet section, the process comprising the steps of:

b. patterning the cells in the precursor material flowing across the patterning section of the tubular cavity into the predetermined and preferably continuously tunable pattern by acoustophoretic patterning,

c. solidifying the precursor material, in which cells have been brought into the predetermined and preferably continuously tunable pattern, flowing across the solidification section of the tubular cavity such as to form a growing plug of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament,

characterized in that the growing plug of solidified hydrogel, as it is formed in the solidification section, is displaced out of the solidification section and discharged from the outlet section as a result of the continuous injection of precursor material into the inlet section of the tubular cavity such as to form the filament of solidified hydrogel.

2. The process of continuous production of a filament of solidified hydrogel according to claim 1, characterized in that at least in the solidification section, and preferably at least in the solidification section and the outlet section, the surface of the inner wall of the tubular cavity, which surface of the inner wall is in contact with the growing plug of solidified hydrogel, is formed of a solid material exhibiting a low coefficient of friction and/or being hydrophobic.

3. The process of continuous production of a filament of solidified hydrogel according to claim 1 or 2, wherein the acoustophoretic patterning is carried out via bulk acoustic wave (BAW) patterning.

4. The process of continuous production of a filament of solidified hydrogel according to any preceding claim, wherein the solid material exhibiting a low coefficient of friction and/or being hydrophobic is a synthetic solid polymer, preferably a fluoropolymer or a polysiloxane.

5. The process of continuous production of a filament of solidified hydrogel according to any preceding claim, wherein solidifying the precursor material is carried out by irradiation with radiation, such as UV, VIS or IR radiation.

6. The process of continuous production of a filament of solidified hydrogel according to any preceding claim, wherein the precursor material is tree of a lubricating agent.

7. The process of continuous production of a filament of solidified hydrogel according to any preceding claim, wherein the cells are human cells, in particular human muscle cells.

8. The process of continuous production of a filament of solidified hydrogel according to any preceding claim, wherein the inner wall of the essentially tubular cavity is formed of a fluoropolymer or PTFE tube, which is preferably lodged inside an outer casing, the inner wall of the essentially tubular activity being directly or indirectly vibrationally coupled to a wave generating device.

9. The process of continuous production of a filament of solidified hydrogel according to any preceding claim, wherein the steps b. and c. are carried out simultaneously in a joint patterning and solidification section, in which the patterning of the cells in the precursor material flowing across the joint patterning and solidification section and the solidification of the precursor material flowing across the joint patterning and solidification section are carried out simultaneously in the joint patterning and solidification section such as to form a growing plug of solidified hydrogel having cells aligned therein along the longitudinal axis of the filament.

10. A device for the continuous production of a filament of solidified hydrogel, having cells aligned therein along the longitudinal axis of the filament according to a predetermined and preferably continuously tunable pattern, comprising an essentially tubular cavity comprising an inlet section, a patterning section, a solidification section, and an outlet section, wherein

a. the inlet section serves the puipose of accepting a flow of precursor material in which cells are suspended in a hydrogel precursor,

b. the pattering section serves the purpose of acoustophoretically bringing the cells in the precursor material into the predetermined and preferably continuously tunable pattern, as the flow of precursor material passes through the patterning section,

c. the solidification section serves the purpose of solidifying the precursor material, in which cells have been brought into the predetermined and preferably tunable pattern, such as to form a growing plug of solidified hydrogel having cells aligned therein along the longitudinal axis, as the flow of precursor material passes through the solidification section, and d. the outlet section serves the purpose of discharging the growing plug of solidified hydrogel such as to form the filament of solidified hydrogel formed having cells aligned therein along the longitudinal axis of the filament according to the predetermined and preferably tunable pattern.

11. The device for the continuous production of a filament of solidified hydrogel according to claim 10, wherein the patterning section is equipped with a wave generator device to carry out acoustophoretic patterning via bulk acoustic wave (BAW) patterning.