WO2007017653A1

WO2007017653A1 - Electrohydrodynamic jet processing for deposition of cells

Info

Publication number: WO2007017653A1
Application number: PCT/GB2006/002931
Authority: WO
Inventors: Peter A. M. Eagles; Suwan N. Jayasinghe
Original assignee: Eagles Peter A M; Jayasinghe Suwan N
Priority date: 2005-08-05
Filing date: 2006-08-04
Publication date: 2007-02-15
Also published as: GB0516155D0

Abstract

A method is provided for delivering a quantity of a concentrated biological suspension to a targeted location in which the quantity of the concentrated biological suspension is delivered in the form of a droplet by an electrohydrodynamic jet connected to a high voltage power supply. The invention also provides methods for treating a tissue lesion, implanting cells in an animal and preparing a tissue graft. A device suitable for use in such methods is also provided.

Description

ELECTROHYDRODYNAMIC JET PROCESSING FOR DEPOSITION OF CELLS

The present invention relates to a process for delivery of biological material including cells to defined target locations using microfluidics technology.

Ink-jet printing (UP) (Ago et al (2003), Siπinghaus et al (1998), Mott & Evans (1999)) is a jet-based technology which uses an ink reservoir with a series of miniature electrically heated chambers. Printing is achieved by pulsating a current through a heating element. A steam type explosion in the reservoir generates a bubble, forcing the formation of a droplet. However, current ink-jets use piezoelectric crystals within needles. The crystal is flexed by applying a current influencing droplet generation. Such printing technology utilises electrostatics or piezoelectricity to direct streams of ink droplets. Ultrasound-assisted means have also been employed for creating waves from which droplets can be generated. Ink-jet printing technology has been used to deliver biological material (see for example EP-A- 1459782, or R. M. Rao Digit, 27-30 (2005)).

Despite rapid advances in ink-jet printing employed for processing a variety of biological samples (Xu et al (2005), Turcu et al (2003), Varghese et al (2005)), it has been difficult exploring the processing of concentrated suspensions (e.g. having a material loading of ~20vol.%) for producing droplets in a size range of hundreds of nanometers. This is due to this technology predominantly driven around the jetting needle size, where the droplet diameter is approximately double the diameter of the internal orifice of the needle. If droplets in the size range of tens of micrometers are required from the processing of a concentrated suspension reducing the needle diameter to < 30jU,m, for producing droplets of -60 μm, can promote serious needle blockages. In practice after spreading of these generated droplet deposits this technology is limited to a resolution >100/.tm.

Although considerable potential problems are faced when using ink-jet printing, this technology has previously been employed for the processing of living cells (Roth et Ia (2004), Wilson & Boland (2003), Tsang & Bhatia (2004), Mourlas et al (2002)) moreover, various two-dimensional and three-dimensional tissue structures have been produced (Sanjana & Fuller (2004), Wood (2004), Sachlos et al (2003) Ma (2004)). Unfortunately due to the inherent problems encountered in IJP the processing of concentrated bio-suspensions have been severely limited by its cellular loading, and this has resulted in the formation of rather coarse structures.

According to a first aspect of the invention, there is provided a method for delivering a quantity of a concentrated biological suspension to a targeted location in which the quantity of the concentrated biological suspension is delivered in the form of a droplet by an electron ydrodynamic jet connected to a high voltage power supply.

The delivery of a suspension according to a method of the present invention may be as a series of discrete droplets, a spray, a mist, or other collection of droplets or drops.

The quantity of the suspension to be delivered is formed as a droplet. Therefore the amount of material deposited will depend in part on the dimensions of the needle and the strength of the electrical field applied.

The concentrated suspension may have a material (e.g. powder, particulate matter) loading of >20vol%, up to about 40vol% or 45vol%, or 50% or 70% of sub-micron sized particles of average diameter 0.00 lμm (lnm) to 40μm, suitably 0.5μm or less

(although percentage lower loadings are entirely feasible). Suitable ranges may therefore be from 1% to 75%, 10% to 50%, 15% to 45%, 20% to 40%. The suspension may comprise a suspension of biological material such as viruses, cells, cellular material, or other biological material or polymers, including biopolymers.

The cells may be prokaryotic cells (i.e. bacteria) and/or eukaryotic cells. Suitable sources of eukaryotic cells include, animal, plant, yeast or fungal cells. Cells may have average diameters of the order of about 5μm, in the range of from around 3μm to around 40μm. The concentration of cells in a the suspension may be up to 10²⁰ cells/ml, suitably in a range of from 10¹⁰ to 10¹⁴ cells/ml, for example 10¹² cells/ml. The methods of the present invention may find particular use in the delivery of animal cells, for example mammalian cells or avian cells. The term animal includes, mammals and non-human mammals (for example primate species, including Homo sapiens (or human) and non-human primates, and non-primate species, including ungulates, such as porcine, caprine, equine, bovine, ovine, or including rodents such as the genus Rattus or rat, or the genus Mus or mice, e.g. Mus musculus), birds (for example Gallus gallus or chicken), fishes (for example Brachydanio rerio or Danio rerio, the Zebra Danio), amphibians and insects (for example Drosophila).

Suitable animal cells include any cell of the body at all stages of development, or from a cell line in culture. The cells may be from an adult, foetus or an embryo. The cells may be somatic cells or non-somatic cells (germ cells), including embryonic stem (ES) cells and/or embryonic germ (EG) cells . Embryonic cells may be cells of the endoderm, ectoderm and/or mesoderm. Adult or foetal cells may be of any tissue, organ or type. The cells may isolated from an adult (i.e. a non-embryo source) and cultured in vitro prior to use. Cell lines may be derived for any convenient source of cells, including timour derived cell lines. Examples of suitable cell lines include, but are not limited to Jurkat cells.

In vertebrate embryos, endodermal cells include the gastrointestinal tract, respiratory tract, and endocrine glands. The ectoderm can be distinguished as three parts, each giving rise to different tissues: the external ectoderm, cells of the neural crest and neural tube. The external ectoderm includes skin (along with glands, hair, nails), mouth and nasal cavity epithelium, lens and cornea of the eye. The cells of the neural crest include melanocytes, peripheral nervous system, facial cartilage and teeth dentine. The neural tube includes brain (rhomencephalon, mesencephalon and prosencephalon), spinal cord and motor neurons, the retina and pituitary. The mesoderm gives rise to tissues including connective tissue, muscles and the circulatory system. The mesoderm is also believed to be responsible for the formation of the central nrevous system, for example the notochord is responsible for releasing certain factors which induce the ectoderm to become neural tissue. Suitable cells from an adult may include differentiated somatic cells, or undifferentiated (or relatively undifferenttiated) adult stem cells of the mesenchymal, neural or haematopoietic cell lineages. Other adult stem cells are also included.

Differentiated adult somatic cells include, but are not limited to, fibroblasts, epithelial cells, hepatocytes, chondrocytes, osteoblasts, osteoclasts, osteocytes, chondrocytes, myocytes, cardiomyocytes, neurons, leucocytes, erthyrocytes, lymphocytes, monocytes, Islets of Langerhans, or a cell-line created from such cells

Suitable cell lines include Jurkat cells.

In addition to cells, the suspension may also comprise cellular material, such as internal cellular material including organelles, membranes, vesicles, or other structures, or structures formed by cells.

Biological polymers (biopolymers) in the suspension may include collagen, nucleic acid, such as DNA or RNA, polypeptides (which includes the term protein unless the context specifies otherwise, as well as glycoproteins and non- glycoproteins). Such polymers may be isolated from cells or synthesised in a biological or chemical system according to standard techniques as appropriate.

The targeted location may be any suitable substrate material. The use of biological or non-biological substrate materials is equally contemplated but it may desirable for the material to be compatible with the biological suspension. Potential biological materials include, for example, collagen. The substrate material may be an inert polymer material, such as cellulose or nitrocellulose, plastics material, or non-metal, e.g., silicon, or a metal, e.g. tungsten. A non-metal, e.g. silicon, based material may used as the basis of a "chip" on which individual cells are placed in a desired arrangement on the surface of the material.

Alternatively, the substrate may be a biological material such as collagen (for example a collagen matrix in the form of a collagen scaffold), dentine, epithelial skin tissue, endothelial vascular tissue, bone, hydroxyapatite, muscle, connective tissue, bone substitute, or corneal tissue. The biological material may have been cultured in vitro and may have been prepared in situ or it may be biological material from another animal. Such biological substrates may be used as supports for individually placed cells on the surface of the material in a desired arrangement.

The droplet delivered according to such a method may have a diameter less than about 50μm, suitably less than about lOμm or more preferably of a diameter in a range of from about lOμm to 50μm.

The methods of the present invention utilise an electrohydrodynamic jet. The physical process of applying a potential difference between an aperture, having within it a flow of liquid medium, with respect to a grounded electrode placed centrally below, gives rise to the phenomenon referred to as electrohydrodynamic jetting (EHDJ) also known as electrospraying (ES) (Rayleigh (1878)). The charged medium exiting the aperture enters a high intensity electric field where it forms numerous liquid geometries from which jet(s) evolve. These jet(s) later undergo several nonlinear effects that promote fragmentation and initiate the formation of droplets. The size and distribution of these droplets can be controlled, based on the applied potential difference, flow rate to the aperture and liquid properties (Taylor (1917)).

The electrohydrodynamic jet suitably comprises a cylindrically shaped tube having an internal lumen, which may be in the form of a hollow needle or nozzle. However, the jet may also comprise any shaped article capable of delivering a jet spray. Suitable geometrically shaped articles or polygons therefore also include a cube, trapezium, pyramid, as well as a cylinder. The jet may therefore have any generally suitable cross-section, which may be circular, ellipsoid, square, triangular, or even irregular provided that a jet is created by the passage of medium through the aperture..

The hollow needle used in the methods of the invention may be in the form of a nozzle or a needle sufficient to channel the suspension out through an external aperture at one end of the needle under the influence of the high voltage electrical field such that the suspension forms a droplet and is delivered to the target location. The internal diameter of the needle may be in the range of from about lOμm to about 5000μm, suitably from about 50μm to about lOOOμm, suitably in the range of from about lOOμm to about 500μm. Preferred needle sizes may be lOOμm to 150μm, or 250μm to 500μm.

The aperture of the electrohydrodynamic jet may be connected to the high voltage supply by any suitable means. In some embodiments of the invention, the method may be performed using apparatus comprising the jet in a fixed location. Alternatively, the jet may be deployed more flexibly, in which case a flexible connection to the power supply may be required.

The high voltage power supply may be of the order of up to 45kV, suitably 3OkV, or in the range of from 5kV to 45kV, or 1OkV to 4OkV, suitably 15kV to 35kV, preferably 2OkV to 3OkV. Preferred values may be around 9kV, or lower for droplets containing individual cells. Suitably, the resolution of the supply is of the order of 0.IkV, or in the range of from about 0.IkV to 0.5kV. For example, EHDJ or ES processing may be carried out in an electrical field strength ranging from 0.5 to 4.5kV/mm, suitably from 0.5 to 0.9kV/mm.

The biological suspension may be syringed through the jet, e.g. in the form of a hollow needle, at a positive or at a negative potential difference with respect to a ground electrode that charged the medium containing the biological suspension. The current may be AC or DC.

In contrast to ink-jet technology, electrohydrodynamic jetting (EHDJ) or electrospraying (ES) suffers from none of the disadvantages referred to above and it can be used to process concentrated suspensions from needles sized a few hundred micrometers yet capable of generating droplet deposits in the few micrometers in size and below, for example less than 5μm, less than 2μm, or in a narrower range such as for example in the range of from 0.5μm to 1.5μm, preferably 0.8μm to 1.4μm. The following parameters show the inherent limitations of ink-jet technology and the potential of the new method described herein

The electrohydrodynamic jet processing or electrospraying phenomenon is a unique jet technology, which shows great utility in processing a wide range of structural, functional and biological materials. The exploitation of these jets as a processing route for generating droplets from the micrometer to the nanometer size range is based on their ability to handle the processing of concentrated suspensions (>20vol.%) using needles of diameter from lOμm to 5000μm, for example <100μm in diameter, exceptional features possessed by no other jet-based processing technology in this category. The present studies show here for the first time that this jet technology can be used to process and deposit living cells onto a surface and that the cells are intact, viable after jetting and that they continue to divide normally as expected.

In some embodiments of the invention, the concentrated suspension to be delivered according to a method as described above may be introduced into the aperture, e.g. a hollow needle, by means of a pump which operates under conditions of continuous flow. The flow rates of the pump may be in the range of from 10^~5 to 10^"25m³s^"1, suitably 10^"8mV. In order to control the formation and delivery of the droplet formed by EHDJ according to the methods described above, an electrode may be placed a short distance away from the external aperture, e.g. the aperture of the needle if the jet is formed from a needle. Suitably the electrode may be a ring-shaped electrode in the form of an annular disc. The diameter of the ring-shaped electrode may be variable or it may be fixed. For example, where the needle has an internal diameter of 500μm, the electrode may suitably have an internal and external diameter of 13 and 15mm respectively and may be held approximately 15mm centrally below the external aperture of the needle. Electrodes may be of any shape, for example: ring, plate or point or combinations of these configurations. The electrodes may be employed singly or multiply and may assist in focussing the droplets or the spray of the electrohydrodynamic jet.

The biological suspension may further contain other components as follows, such as pharmaceutically active substances, adjuvants and/or diluents. Pharmaceutically active substances may be added to assist in the maintenance of the viability of the biological material in the suspension, such as for example in the development and growth of cells in the suspension, i.e. growth factors, nutrients (including vitamins and/or minerals) etc. Other pharmaceutically active substances may be included such as antibiotics, immunosuppressants, cytokines, antibodies etc. In addition, other biologically active substances such as preservatives, including anti-oxidants, may be included.

Some preferred compositions of the biological suspension may therefore comprise, for example, osteocytes, collagen and at least one antibiotic.

Methods in accordance with the present invention may find application in a variety of fields. However, it is believed that such methods may find greatest use in the fields of human and veterinary medicine. For example, such methods may find use in preparing a tissue graft for implantation into a host, alternatively, a tissue lesion may be repaired in situ in a patient using these methods. According to a second aspect of the invention, there is provided a method of treating a tissue lesion on the surface of the body of an animal, comprising delivering a quantity of a concentrated biological suspension to a targeted location in said lesion in which the quantity of the concentrated biological suspension is delivered to the site of the tissue lesion in the form of a droplet by an electrohydrodynamic jet connected to a high voltage power supply.

The lesion may be in the skin, or other external exposed tissues of the body, such as the eye, including the cornea, mouth, mucus membranes, teeth etc. The lesion may be in an internal tissue structure that is exposed (whether as a result of an injury or for the purposes of therapy and/or surgery). Internal tissue structures include endothelial vascular tissue, bone, teeth, nerves (including nervous tissue), muscle, connective tissue, including tendon.

According to a third aspect of the invention, there is provided a method of implanting a quantity of cells into an animal, comprising delivering a quantity of a concentrated biological suspension of cells to a targeted location in the body of the animal in which the quantity of the concentrated biological suspension of cells is delivered in the form of a droplet by an electrohydrodynamic jet connected to a high voltage power supply

The cells to be delivered may be as described herein, but it is envisaged that particular cells of interest for implantation according to this aspect of the invention may include, skin fibroblasts, corneal cells, Islets of Langerhans, nerve cells (neurons, including dopaminergic neurons), hepatocytes, kidney cells, myocytes (including cardiomyocytes), osteoblasts, osteoclasts, osteocytes, chondrocytes. For example, such methods may be used for forming rapid healing dense and porous thin bio-films.

According to a fourth aspect of the invention, there is provided a method of preparing a tissue graft for implantation into an animal body, comprising delivering a quantity of a concentrated biological suspension of cells to a targeted location in which the quantity of the concentrated biological suspension of cells is delivered in the form of a droplet by an electrohydrodynamic jet connected to a high voltage power supply The construction of a tissue graft suitable for implantation may comprise the construction of either a two-dimensional or a three-dimensional structure. For example, a monolayer of cells or a biological polymer may be regarded as being essentially two-dimensional, whereas a multi-layer of cells, a hydroxyapatite structure, a bone structure, a bone-substitute structure or a collagen scaffold may be regard as being a three-dimensional structure. Such structures may be formed using the methods of the present invention. The principle of constructing a three- dimensional structure using electrohydrodynamic jet spray technology has been shown in Wang et al Review of Scientific Instruments, vol. 76, 075105-1, (2005). Suitable structures, such as for example a collagen "scaffold" structure composed of a network of overlaid collagen threads, may be seeded with desired cells also according to the methods of the present invention prior to implantation.

For example, such methods may be used in "printing" nerve cells on collagen substrates for enhanced regeneration of nerve growth "printing" 2-dimensional/3- dimensional composite structures (containing sandwich-like structures having layers of collagen/desired cells/collagen/chitosan, or as tissue/cells/muscle, or in any desired order or in gradation, building composite structures from bone through to skin, with concentric needle systems for deposition of encapsulated cells in collagen, in forming a novel combinatorial cell line for building cell libraries (using any suitable cellular material), in 2-dimenasional/3-dimensional building of tissue structures in any composition with arrays of ES needles for patterning in both in vitro and in vivo, in microfabrication of closed/open cell foams and emulsions, and as a robust cellular coating technique. It is contemplated that structures may be prepared for implantation as implants to repair or replace skin, teeth, bone, muscle, cornea, amongst others.

Such methods of tissue reconstruction in vivo or in vitro may be achieved using a suitable electrospray device. According to a fifth aspect of the invention, there is provided a device comprising a chamber having an inlet and an outlet, in which the chamber is connected to a high voltage supply and in which the outlet forms an aperture having a ground electrode connected thereto within the lumen of the chamber, wherein in use a quantity of a concentrated biological suspension is delivered to a targeted location through the device in which the quantity of the concentrated biological suspension of cells is delivered in the form of a droplet by an electrohydrodynamic jet connected to the high voltage power supply.

For example, a device may be constructed in the form of a "pen" into which a suspension of cells is delivered via a suitable needle under the influence of an appropriate electrical field. The device or "pen" may be cylindrical in shape having an inlet for introduction of the cell suspension at one end. After introduction of the suspension into the cylinder, the suspension is passed through the needle under the influence of the electrical field prior to being sprayed out of an aperture at the other end of the cylinder. The aperture may be of variable dimensions sufficient to permit the exit of the droplets and the device may also comprise gulley traps, for examples at the aperture for collecting stray particles and the like. In another embodiment, it is envisaged that a high voltage deflection plate could be arranged at the aperture to influence the exit of the droplets as they are sprayed out of the aperture. Preparation of a tissue graft may be at the level of a two-dimensional layer of cells, or a three- dimensional construct of cells and a matrix.

Such methods may also find use in segregating different cell types from a heterogeneous population, such as for example from a biological fluid such as blood. It is often advantageous to be able to separate out the different cells found in circulating blood, such as erythrocytes, leucocytes, megakaryocytes, macrophages, monocytes etc. Alternatively, the technique could be used to segregate a population of cells from a suspension of biological material, for example a tissue or an organ from an animal.

According to a sixth aspect of the invention, there is provided a method for segregating different cell types from a heterogeneous population, comprising delivering a quantity of a concentrated biological suspension of cells to a targeted location in which the quantity of the concentrated biological suspension of cells is delivered in the form of a droplet by an electrohydrodynamic jet connected to a high voltage power supply, wherein said droplets are passed through an annular earth electrode and subsequently between high voltage deflection plates.

Such methods may be achieved by preparing electrospray droplets as described previously and passing the droplets through an annular earth electrode after which the droplets are passed between high voltage deflection plates. Separation of the cells can then suitably take place depending on the charge of the droplets. Collection vessels can be deployed as necessary to collect cells separated by such methods.

According to a seventh aspect of the invention, there is provided a method for the preparation of a substrate material containing cells, comprising delivering a quantity of a concentrated biological suspension of cells to a targeted location on a substrate in which the quantity of the concentrated biological suspension of cells is delivered in the form of a droplet by an electrohydrodynamic jet connected to a high voltage power supply. Suitably, the substrate may comprise a plurality of wells in the surface of the substrate. The substrate material may be as described above.

One embodiment of the invention provides for the production of a "chip" composed of living cells as described above. Such devices or "chips" may be prepared as follows. A substrate graticule may be formed that contains wells sufficient to hold the cells deposited. The material may be suitably compatible with the cells and may be composed of a suitable biological or non-biological material as described above. The device may be of any suitable dimensions, such as for example lmm by lmm, with wells of around 20-50μm square thus permitting 400 to 2500 wells to be constructed. Each well may contain one or a few cells that may be deposited by a method of the invention. The cells may be from any convenient source as described above. After preparation the device may be open, or it may be covered with a suitable porous or semi-porous membrane, or an impermeable membrane.

The present investigations show that the electrohydrodynamic or electrospray jet, driven by high intensity electric fields, can be exploited for processing living cells, and this technology is not hampered by the disadvantages encountered with ink-jets. Furthermore no adverse effects on living cells have been observed when processed via this technique.

Preferred features of the second a subsequent aspects are as for the first aspect mutatis mutandis.

The present invention will now be further described by way of example with reference to the following Examples which are present for the purposes of illustration only and are not to be construed as being limiting on the invention. Reference is also made in the Examples to a number of Figures in which:

FIGURE 1 shows a schematic representation of the electrohydrodynamic or electrospray (ES) equipment used.

FIGURE 2 shows 3 plume configurations Figure 2(a) ring, Figure 2(b) plate and Figure 2(c) point, as follows, enlarged picture of the needle and ground electrode with Petri dish below as set up in sterile hood. Sparking at an applied voltage of ~8.9kV seen at a flow rate of 10^~8 mV¹

FIGURE 3 shows a series of optical micrographs of control and electrosprayed cells taken using 4OX objective. The bar represents 20microns. The time at which the samples were observed is indicated. A): Control cells not taken through the process of electrohydrodynamic atomisation (electrospraying), B) to F) : A sample of cells that were electrosprayed at 11:30am and observed at the following times B) 15:30, C) 15:35, D) 15:40, E) 15:45, F) 15:50

FIGURE 4 shows characteristic droplet size distribution for the cells undergoing electrohydrodynamic jetting in the unstable mode

FIGURE 5 shows enlarged picture of the needle and ground electrode with

Petri dish below as set up in sterile hood. Sparking at an applied voltage of ~8.9kV seen at a flow rate of 10^"8 mV¹. FIGURE 6 shows a typical high-speed sequence ((a-f) depicting the jetting of the Jurkat cell suspension. The scale bar denotes 810μm.

FIGURE 7 shows aspects of the uses of the methods of the invention in creating tissue structures. Figure 7a shows Electrodeposition in 3D for tissue development in both in-vitro and in-vivo; Figure 7b shows illustrates a 3D patterning device which can be used for creating 3D structures (Figure 7c) from a host of cells; Figure 7(c) shows a 3D structure formed of zirconia.

FIGURE 8 shows droplets of Jurkat cells labelled with Rhodamine G and the medium labelled with FITC.

FIGURE 9(a) to (f) shows formation of droplets by electrospray in the formation of a chip of living cells.

FIGURE 10 shows a schematic representation of a portable electrospray device ("E-spray pen") in which shows a device comprising motor and battery compartment (1), screw thread (2), gas tight plunger (3), medium tank (4), skirting (5), needle/nozzle (6), cone-jet spray mode (7), stable jet (8), 3-D spray area (9), substrate (10).

FIGURE 11 shows a schematic representation of a fixed electrospray device ("E-spray pen") in which shows a device comprising motor and battery compartment (1), screw thread (2), gas tight plunger (3), medium tank (4), skirting (5), needle/nozzle (6), cone-jet spray mode (7), stable jet (8), 3-D spray area (9), substrate (10), power cable (11).

FIGURE 12 shows a schematic representation of equipment set-up for a bio- patterning device, which comprises electrospray needle (1), stable cone (2), stable jet (3), patterned 2-D/3-D architecture (4), substrate (5), point-like ground electrode (6). FIGURE 13 shows a schematic representation of a device for bio-patterning and single-cell deposition (with following needle) in biochip manufacturing, which comprises electrospray needle (1), stable cone (2), stable jet (3), patterned 2-D/3-D architecture (4), substrate (5), point-like ground electrode

(6), following needle (7), stable cone (8), stable jet (9), detaching a single droplet containing the desired number of cells (10), point-like ground electrode for following needle (11).

FIGURE 14 shows the potential of exploiting this technique for segregating cell types from a concentrated mix of blood, for example segregating blood into red, white and platelets

Experimental The equipment used for electrohydrodynamic or electrospray (ES) jetting (Figure 1) consists of a stainless steel needle having an internal diameter of ~100μm which is connected to a high voltage power supply based upon (FP-30, Glassman Europe Ltd., Tadley, UK). The power supply has the capability to supply a voltage of up to 3OkV with a resolution of 0.IkV. The top end of this needle was connected via silicone tubing to a 10ml syringe placed on a precision syringe pump (PHD 4400, HARVARD Apparatus Ltd., Edenbridge, UK). The pump is capable of delivering flow rates in the

-5 -25 3 -1 range 10 to 10 m s . A ring-shaped ground electrode made of copper having an internal and external diameter of 13mm and 15mm respectively, was held approximately 15mm centrally below the needle. EHDJ processing was carried out in an electric field strength ranging from 0.5 to 4.5kV/mm for a flow rate regime of 10

8 3 -1 m s .

During the EHDJ of the cellular suspension a high-speed camera (Phantom V7,

Photo-sonics International Ltd, Oxford, UK) capable of capturing 150000fps in conjunction with a long distance microscope lens (Nikon 50 mm, Oxford Lasers Ltd,

Oxford, UK) was triggered simultaneously with a diode laser system (HSI5000, Oxford Lasers Ltd, Oxford, UK) allowing the jetting process to be recorded in real time from needle exit and beyond.

Example 1: Electrospray of Jurkat cells Jurkat cells (ATCC, USA) were grown for 48 hours, in 100 ml of RPMI 1640 growth medium (Invitrogen, UK) with 10% foetal calf serum (Invitrogen, UK), in an incubator (Heraeus BB 16, UK) at 37⁰C and 4% CO . Cell viability was assessed by staining with trypan-blue and viable cells were counted using a bright-field- haemocytometer (Sigma), with a phase microscope (Zeiss TD 02, Germany)

6 employing a 1OX objective. 2-5 mis of a cell suspension containing 1-2x10 cells/ ml were used for each electrospray experiment. For the control, similar volumes of the suspension were passed through the needle with no applied voltage and collected in a Petri dish. The cells were counted immediately after electrospraying and subsequently after a 24 hour period of incubation.

The cellular suspension was characterised for estimating its properties (electrical conductivity, viscosity, surface tension, relative permittivity and density) affecting the

Tm jet-processing by using a conductivity meter (HACH SensION 156 probe), Visco- Easy rotational viscometer, Kruss Tensiometer K9 (Du Novy's ring and plate method), a calibrated cell connected to a high precision multimeter and a standard density bottle, respectively.

Table 1. Measured and estimated properties of the cellular suspension and medium

The cells were syringed through a needle kept at a positive potential with respect to the ground electrode that charged the medium containing the cells. The medium was immediately dispersed on entering the external electric field. At an electric field strength of approximately 0.85kV/mm discharging was observed where sparks were found to cross between the needle and the ground electrode (Figure 5)

It is believed that these suspensions can be further loaded. It is estimated that this loading can be at least in the order of 10²⁰ ml^"1. However, a much greater loading is envisaged as experiments have shown the possibilities that it is possible to spray medium with liquids up to -lOOOOmPa s with a corresponding electrical conductivity of 10^"13Sm^"1 (S.N. Jayasinghe and MJ. Edirisinghe, Electrically Forced Jets and Microthreads of High Viscosity Dielectric Liquids, /. Aerosol ScL, 35(2004)233-243).

Results and Discussion

Electrohydrodynamic or electrospray (ES) jetting of this living biological suspension was seen to taken place in the unstable mode (Figure 6a-f). A laser spectrometer was incorporated into the EHDJ set-up for measuring the droplet size distribution, below the ring shaped ground electrode as in our previous work (Jayasinghe et al (2004), Jayasinghe & Edirisinghe (2002)). The unstable mode of jetting was seen to give rise to a poly-disperse distribution of droplets (Figure 4). The droplet size distribution plot showed two significant peaks at -17 and ~37μm, which correlates well with the collected droplet relics sized at 43 and 97μm respectively, after taking into account spreading on substrate. During jetting the flow rate and applied voltage were varied over a rather large parametric space illustrating no regions for stable cone-jet mode of jetting. The non-existence of such a region is primarily due to the suspension properties: electrical conductivity and viscosity both having direct effects on the formation and stability of the mode of jetting (Jayasinghe et al (2004), Gana-Calvo (2004), Hartman et al (2000)). Nevertheless, the suspension was syringed through at a

-8 3 -1 flow rate of 10 m s and an applied voltage of 8.5kV where discharging was promoted forming sparks.

A sample of around 1.7xlO⁶ cells which were jetted and examined by means of a 4OX phase objective on a Zeiss Axiovert 200 inverted microscope. Characteristic micrographs were taken at 5 minute intervals which assisted in assessing cell survival. The cells appeared to have not incurred any cell damage or death (see Table 2). Table 2

A couple of hours after electrospraying, cells were observed using optical microscopy (Figure 3). Hence no signs of cellular damage were seen as a result of processing. Cells changed their morphology over time (Figure 3 B- F, cells X and Y) and in some cases underwent cytokinesis (Figure 3 B-F cell Q) as seen when processed via other routes. Four samples of electrosprayed cells were incubated for 24 hours, and then countered for cell survival Table 1. This points out that the process of electrospraying had no adverse affects on the rate of cell division, which continued as normal.

Samples of human peripheral blood monocytes and mouse CAD cells, and undifferentiated neuronal cell lines, were taken through a similar electrospraying protocol. Once again no evidence was seen that the process of EHDJ or ES had affected these cells structurally on their activity or their rate of cellular division.

Example 2: Results of Electrospraying experiment on Jurkat cells and varying the electrode potential Further work shown in Tables 3 to 6 also indicates that a range of cell types can be jetted over a range of conditions and after which the cells are still found to be viable over at least 96 hours. A potential difference of 6.8kV and 17kV was used and cells where examined at 24hrs and 96 hrs after electrospraying. Control cells were taken through the same procedure but not subjected to the potential field. It can be seen that electrospraying the cells at 6.8kV or 17k V did not affect their viability.

Table 3

Jurkat Initial counts xlO⁶ 24hours xlO⁶ 96hours xlO⁶

Control cells 4.8 .±0.22 5.35 ±0.22 6.8 ±0.25

ES at 6.8kV 4.8 » ±0.22 6.08 ±0.08 7.4 ±0.3

ES at 17kV 4.ϊ \ ±0.22 6.25 ±0.05 7.3 ±0.2

Example 4: Results of Electrospraying experiment at 17kV on Human Peripheral Blood Mononuclear Cells (PBMCs)

Cells were electrosprayed at 17kV and then incubated for 24 hrs and 96 hrs. Control cells were taken through the same procedure but not subjected to the potential difference. It can be seen that electrospraying the cells at 17kV did not affect their viability.

Table 4

Example 5: Results of EIectrospraying experiment at 17kV on CAD cells, a mouse neuronal cell line that can differentiate

Cells were electrosprayed at 17kV and then incubated for 24 hrs and 96 hrs. Control cells were taken through the same procedure but not subjected to the potential difference. It can be seen that electrospraying the cells at 17k V did not affect their viability. Cells were also induced to differentiate by removal of serum from the medium and they showed normal response.

Table 5

Example 6: Results of Electrospraving experiment at 9kV on Buffy coat residues that were obtained from human blood

Buffy coats contain a mixed population of cells but are enriched on PBMCs. The cellular suspension had a viscosity of 1200 mPa s, and a needle of internal diameter 400 um was necessary for electrospraying. Control cells were taken through the same procedure but without the application of the potential difference. The extent of lysis of red blood cells during the experiment was assessed by measuring haemoglobin levels, and comparing values obtained after electrospraying with samples that were completely lysed.

Table 6

Example 7: Experiment to show the distribution of droplet sizes and the distribution of cells within those droplets after electrospraving at kV

Fluorescent dyes have been used to label cells and media (see Figure 8) which demonstrates that the ES process can generate droplets containing single cells. We hope to explore this approach for direct writing of 2D to 3D pre-determined architectures for bio-patterning (Figures 7a to 7c).

Jurkat cells were labelled with a fluorescent dye, Rhodamine G, and the medium was labelled with another fluorescent dye, FITC. The sample was then electrosprayed and the droplets captured on nylon film. Fluorescence microscopy was used to visualise cells and the sizes of the droplets. In this photograph shown in Figure 8, five droplets are seen; the largest contains ten cells. Two droplets contain two cells each and two droplets contain one cell each.

Example 8: Production of a chip composed of living cells

A graticule can be produced to form the wells. This may be made on a suitable surface that is compatible with the cells and the walls of the well are made of a biological or non-biological material. The chip could be of any size but it might suitably be lmm x lmm and the wells might be 20-50 μm square, allowing 400-2500 wells to be formed. Each well may contain one or a few cells that would be deposited by electrospraying. The cells may be from established cell lines or primary cell lines and may be all of one type or of various types. The chip may be open or covered my a suitable membrane allowing the chip to moved from one medium to another without loss or disturbance of the cells. A diagram explaining this is shown in Figure 9.

Conclusions

This investigation has elucidated the ability to successfully jet-process living cells under high intensity electric fields without harming their integrity. Jurkat cells were used in this investigation because they provided a convenient source of cells that were easy to grow and to visualize. The study elucidated that electrospraying (ES) had no disruptive effect on the cell structure and neither on the basic activities of living cellular organisms. This technique used for processing living cells is not confined to just Jurkat cells as blood monocytes and a neuronal cell lines have also been electrosprayed. The investigation has shown this processing technique to be a robust route for processing cellular materials which will have a widespread importance in the field of bioscience and engineering. The potential of the present research findings has far reaching consequences in the processing and precise deposition of a range of living cellular materials and biologically related matrices, also for encouraging regeneration and fabrication of tissues at the micrometer and nanometer level. Thus in summary the electrohydrodynamic or electrospray (ES) jet-processing phenomenon with its potential for handling samples in the form of concentrated biological suspensions with needles of large bore looks to be strongly placed for competing with ink-jet technology in the race to develop biological tissues for repair

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Claims

1. A method for delivering a quantity of a concentrated biological suspension to a targeted location in which the quantity of the concentrated biological suspension is delivered in the form of a droplet by an electrohydrodynamic jet connected to a high voltage power supply,

2. A method as claimed in claim 1, in which the concentrated biological suspension has a material loading of >20vol% of particles of average diameter 0.001μm to 40μm.

3 A method as claimed in claim 1 or in claim 2, in which the biological suspension comprises a suspension of viruses, cells, cellular material, or other biological material or polymers.

4. A method as claimed in any one of claims 1 to 3, in which the targeted location is a substrate material.

5. A method as claimed in claim 4, in which the substrate material is a non- biological substrate material.

6. A method as claimed claim 5, in which the non-biological substrate material is selected from the group consisting of cellulose-based polymers, plastics materials, non-metals, and metals.

7. A method as claimed in claim 4, in which the substrate material is a biological substrate material.

8. A method as claimed in claim 7, in which the biological material is selected from the group consisting of collagen, dentine, epithelial skin tissue, endothelial vascular tissue, bone, hydroxyapatite, muscle, connective tissue, bone substitute, and corneal tissue.

9. A method as claimed in any preceding claim, in which the droplet delivered has a diameter less than about 50μm

10. A method as claimed in any preceding claim, in which the electrohydrodynamic jet comprises a hollow needle having an internal diameter in the range of from about lOμm to about 5000μm

11. A method of treating a tissue lesion on the surface of the body of an animal, comprising delivering a quantity of a concentrated biological suspension to a targeted location in said lesion in which the quantity of the concentrated biological suspension is delivered to the site of the tissue lesion in the form of a droplet by an electrohydrodynamic jet connected to a high voltage power supply.

12. A method of implanting a quantity of cells into an animal, comprising delivering a quantity of a concentrated biological suspension of cells to a targeted location in the body of the animal in which the quantity of the concentrated biological suspension of cells is delivered in the form of a droplet by an electrohydrodynamic jet connected to a high voltage power supply

13. A method of preparing a tissue graft for implantation into an animal body, comprising delivering a quantity of a concentrated biological suspension of cells to a targeted location in which the quantity of the concentrated biological suspension of cells is delivered in the form of a droplet by an electrohydrodynamic jet connected to a high voltage power supply

14. A device comprising a chamber having an inlet and an outlet, in which the chamber is connected to a high voltage supply and in which the outlet forms an aperture having a ground electrode connected thereto within the lumen of the chamber, wherein in use a quantity of a concentrated biological suspension is delivered to a targeted location through the device in which the quantity of the concentrated biological suspension of cells is delivered in the form of a droplet by an electrohydrodynamic jet connected to the high voltage power supply.

15. A method for segregating different cell types from a heterogeneous population, comprising delivering a quantity of a concentrated biological suspension of cells to a targeted location in which the quantity of the concentrated biological suspension of cells is delivered in the form of a droplet by an electrohydrodynamic jet connected to a high voltage power supply, wherein said droplets are passed through an annular earth electrode and subsequently between high voltage deflection plates. .

16. A method for the preparation of a substrate material containing cells, comprising delivering a quantity of a concentrated biological suspension of cells to a targeted location on a substrate in which the quantity of the concentrated biological suspension of cells is delivered in the form of a droplet by an electrohydrodynamic jet connected to a high voltage power supply.