AU2013204780A1 - Formulation and Method - Google Patents

Abstract

H:\SXD\Intemoven\NRPortbl\DCC\SXD\5072760-I.doc-12/04/2013 5 Formulations suitable for the suspension of viable cells to form a bio-ink for the controlled deposition of such cells as well as to bio-ink formulations that include viable cells. The invention is also directed to methods of producing such formulations and to methods of printing such bio-ink formulations to deposit viable cells in a controlled manner.

Description

H:\SXD\Interwoven\NRPortbl\DCC\SXD\5072760_.doc-12/04/203 Formulation and Method Field of the invention The present invention is directed to formulations suitable for the suspension of viable cells 5 to form a bio-ink for the controlled deposition of such cells as well as to bio-ink formulations that include viable cells. The invention is also directed to methods of producing such formulations and to methods of printing such bio-ink formulations to deposit viable cells in a controlled manner. 10 Background of the invention The field of biofabrication, wherein living cells are used to produce cellular constructs or complex cell containing structures such as human, animal or plant tissues or organs or components of devices such as assay arrays, diagnostic and prognostic kits and prosthetic or implantable devices and the like, is rapidly emerging. In such contexts it is desirable to 15 be able to deposit living and viable cells in a controlled manner. Similarly there are other contexts involving cell processing, such as cell testing and analysis and drug and toxicology screening, where it is also desirable to be able to deposit viable cells in a controlled manner. Cell printing, which allows computer controlled deposition of viable cells to produce a prescribed two dimensional or three dimensional arrangement, offers 20 great promise in this field and utilises existing printing technology (such as microvalve deposition and piezoelectric inkjet printing) to deposit drops of "bio-ink" comprising viable cells in a controlled manner and at a desired locus. A bio-ink suitable for cell printing should allow the stable suspension of cells without significant cytotoxic effects. 25 The suite of bioprinting techniques that allow the controlled deposition of living cells has expanded to include extrusion printing and laser printing,3'4 as well as drop-on-demand approaches like microvalve printing ' and inkjet printing. Drop-on-demand cell printing techniques are attractive due to their relative simplicity and capability for precise non contact deposition, yet have been hindered by some critical limitations. Cell settling and 30 aggregation within printer reservoirs obstructs nozzles and leads to non-uniform cell distribution so that cell output significantly decreases or fails when printing over extended H:\SXD\Interwoven\NRPortbl\DCC\SXD\5072760_.doc-12/04/203 -2 time periods." Gentle agitation of inkjet print heads and microvalves can reduce cell settling 12

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13 and addition of ethylenediaminetetraacetic acid (EDTA) limits aggregation, 1 4 but these strategies are only partly effective and can be detrimental to cell viability. Printing cells in high viscosity collagen solutions can retard settling, although this 5 approach is limited to specialized printing systems.

5 Inkjet printing presents additional challenges as the ink must meet stringent fluid property requirements (e.g. viscosity and surface tension) for efficient deposition." Currently, non-ideal ink formulations have been printed using single- or few-nozzle devices, ''','1 or using outdated thermal inkjet heads. 8,10,1-20 Piezoelectric inkjet print-heads with multiple nozzles are the current 10 standard for high-end printing applications, and offer promise for higher throughput and fabrication of larger cellular constructs. Rather than developing bio-inks that are suitable for use in these conventional printing systems bio-ink design has up to now focused on two-component fast-gelling reactive 15 approaches. Cells have been mixed with alginate and printed into cross-linking Ca 2 + solutions, 16,21 or mixed with Ca2+ and printed into either alginate or alginate/collagen solutions. Similar approaches have utilized the fibrin/thrombin reaction 8'19 or photopolymerisable inks.20 However, these printed environments are not suitable for all cell types and applications. 20 Current bio-inks that are based on salt solutions allow cell settling and aggregation and tend to clog nozzles and fluid pathways. Other known bio-inks that consist of viscous biopolymer solutions or hydrogels limit cell settling and aggregation, confine the cells to the biopolymer matrix after processing and require precise temperature control during 25 processing, both of which may be detrimental to cell viability. To deliver on the initial promise of drop-on-demand cell printing, it is desirable to develop bio-inks that are tailored to satisfy the seemingly disparate demands of printability and cell function, and that are preferably amenable to printing using standard hardware. It is with 30 this background in mind that the present invention has been conceived.

H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_.doc-12/04/20 3 -3 Summary of the invention According to one embodiment of the present invention there is provided a formulation suitable for stable suspension therein of viable cells to form a bio-ink for controlled deposition of said cells, comprising an aqueous microgel suspension of one or more gelling 5 biopolymer/s and one or more suitable salts. According to another embodiment of the present invention there is provided a bio-ink formulation for controlled deposition of viable cells, said formulation comprising a stable aqueous microgel suspension of one or more gelling biopolymer/s, one or more suitable 10 salts and viable cells of one or more types. In a further embodiment of the present invention there is provided a method of preparation of a formulation suitable for stable suspension therein of viable cells to form a bio-ink for controlled deposition of said cells, said method comprising dissolving one or more gelling 15 biopolymer/s in hot aqueous medium that comprises one or more suitable salts to form a solution that is cooled under shear conditions to produce a microgel suspension. In another embodiment of the present invention there is provided a method of preparation of a bio-ink formulation for controlled deposition of viable cells, said method comprising 20 dissolving one or more gelling biopolymer/s in hot aqueous medium that comprises one or more suitable salts, to form a solution that is cooled under shear conditions to produce a microgel suspension, to which viable cells of one or more types are added. In a still further embodiment of the present invention there is provided a method of 25 controlled deposition of viable cells to a desired locus comprising loading a bio-ink formulation as described above to a compatible computer controlled printer and directing controlled release of the bio-ink to said desired locus by computer operation. Further aspects of the present invention will become apparent from the following detailed 30 description thereof.

H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 -4 Brief description of the figures Fig. 1: Bio-ink structure and cell settling. (a) Structure of the bio-ink visualized by staining with Derivan ink and imaged by phase-contrast microscopy. Scale bar 200 [m. (b) Cell 5 settling (percentage of cells on the base of a 96 well plate) as a function of time for C2C12 cells suspended at 1 x 106 cells per mL in DMEM (open squares) or bio-ink (filled circles). Error bars represent one standard deviation from the mean. Insets show the base of well plates at indicated time points (scale bars 100 [m) and cartoons depicting the ability of the microgel suspension to keep cells in suspension. (c) Spiral patterns of C2C12 cells 10 suspended in bio-ink and deposited on a glass slide by microvalve printing. Scale bar 500 [m. (d) Average number of cells per drop over time, normalized to the number of cells in initial drops, for C2C12 cells suspended at 2 x 10 5 cells per mL in DMEM (open squares) or bio-ink (filled circles) and deposited by microvalve printing. Error bars represent one standard error of the mean (n = 10). A statistically significant difference (compared to t = 0 15 min) was assessed by unpaired Student's t-test and reported with 99% (**) or 99.9% confidence (***). Fig. 2: Printing cells from one inkjet print head. (a) Printed cell number across print head width was analyzed by counting cells printed in squares of 10 x 10 droplets (utilizing 10 20 nozzles each). Each sample contained 18 replicate squares as illustrated, printed in a single pass. (b) Cell number in the six squares positioned across the print head width, averaged for the three vertical replicates in three samples printed sequentially. Error bars represent one standard error of the mean (n = 3). One-way ANOVA indicated no statistically significant difference between the number of printed cells in each of the six positions. (c) 25 Cells-per drop distribution was analyzed by counting cells in individual drops printed in 10 x 10 arrays. Each sample contained 9 replicate arrays as illustrated, printed in a single pass. (d) Frequency distribution (bars) of the number of cells within individual printed droplets. Values were obtained by averaging the distributions in 3 arrays across the print head for two samples printed sequentially. Error bars represent one standard error of the 30 mean (n = 3). The line graph represents a Poisson distribution, calculated using the total average of cells per drop in the analysed arrays. Inset: single printed droplets on glass H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 -5 containing C2C12 cells (black arrows). Scale bar 200 [m. Fig. 3: Printed cell viability, proliferation and differentiation. (a) Viability (assessed by live/dead staining after 2 h in culture) of C2C12 and PC12 cells from typical experiments 5 where cells were either suspended in the bio-ink for 2 h and pipetted into culture wells ('exposure' conditions), or suspended in the bio-ink and printed into cell culture media by inkjet or microvalve printing. The control cells were suspended in DMEM for 2 h and pipetted into the culture wells. (b) MTS assay indicating proliferation of printed C2C12 (microvalve and inkjet printed) and PC12 (inkjet printed) cells in comparison to non 10 printed controls over 48 h in culture. MTS absorbance was normalized to the 2 h time point to account for the differences in initial cell numbers. (c) Differentiated C2C12 and PC12 cells on tissue culture polystyrene, comparing inkjet printed and control cells. Cells were stained for desmin (C2C12) or F-actin (PC12), as described in the ESIt (scale bars 100 [m for C2C12, or 50 [m for PC 12). (d) Viability (after 2 h in culture) of C2C12 cells printed 15 from bio-ink containing 0.1% v/v P188 (P188+), or with this surfactant omitted (P188-). (e) Comparison of C2C12 cells inkjet printed immediately and 1 h after loading the cells into inkjet print head. Top left - printed cell viability at both time points assessed by live/dead staining after 2 h in culture. Bottom left - average number of cells/drop at both time points. Right - Representative live/dead images of cells at both time points (scale bars 20 200 pm). (a, b, d, e) Error bars represent one standard error of the mean (n = 3), and the statistical significance was assessed by an unpaired Student's t-test and reported with either 99.9% (***) or 95% (*) confidence. Fig. 4: Patterning of two cell types printed simultaneously from two separate inkjet print 25 heads onto collagen substrates. (a) Schematic representation of multiple head printing. (b, c) C2C12 (red) and PC12 (green) cells pre-stained with CellTracker T M dyes and printed in various patterns. Images were taken 1 h after printing, following the addition of the culture medium. (d, e) Printed patterns of C2C12 and PC12 cells after 8 days under differentiation conditions. Cells were immunostained for desmin (C2C12, green) and p-III tubulin (PC12, 30 red). The dotted lines represent the outline of the printing pattern. Scale bars represent 500 [m (B-D) and 200 [m (E).

H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 -6 - 6 Fig. 5: Image reproduction by deposition of C2C12 cells, suspended at 1x106 cells/mL in bio-ink, onto standard glass slides. (A,B) Bitmap images of spirals (A) or a lattice (B) were transformed to a plate-map and printed using a single layer of 50 nL droplets with a 5 spacing of 500 pm. (C,D) Sections of the printed patterns imaged by optical microscopy, showing a uniform cell distribution. Scale bars 500 pm. Fig. 6: Printing cellular arrays for analysis by LESA-MS. (A) Schematic representing the deposition from a Xaar-126 print head of a single cell spot into a micro-array on a glass 10 slide. (B) Either 100 droplets (50-100 cells) or a single droplet (average 1 cell) were deposited into array regions. (C) Image showing the LESA probe depositing solvent on a printed cell spot. (D,E) Comparison of droplet containing a single L929 cell before (D) and after (E) LESA analysis. Scale bars represent 200 pm. 15 Fig. 7: Precursor ion scans (PIS) on inkjet printed cells. (A-C) Typical m/z 184.1 PIS obtained from printed cell microarray spots (50-100 cells per spot) of L929 (A), PC12 (B), and C2C12 (C) cells. Spectra were produced from 100 summed PIS. (D-F) Typical m/z 184.1 PIS on printed cell microarray spots (1 cell per spot) of L929 (D), PC12 (E), and C2C12 (F) cells. Spectra were produced from 200 summed PIS. Values in the top right of 20 each spectrum correspond to the average intensity in counts per second (cps) per scan of the base peak. Fig. 8: Principal component analysis (PCA) on inkjet printed cells. PCA score plots using PC/SM lipid profiles acquired from 5 microarray spots for each cell type (L929, C2C12, or 25 PC12 cells) containing (A) up to 100 printed cells and (B) a single cell. Triangles indicate the data point obtained by direct infusion of lipid extract from non-printed L929 cells. Fig. 9: Printing a single cell type - C2C12 cells printed onto collagen hydrogel surfaces and live/dead stained after 2 hrs in culture. (A) Cells printed on an uneven hydrogel 30 surface. Dehydration in the centre of well led to cell death (stained red). (B) Cells printed in a simple linear pattern on a flat hydrogel surface. Dotted lines indicate the outline of the H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 -7 printing pattern, and the white arrow highlights a cell outside the pattern region. Scale bars represent 500 pm. Fig. 10: Patterns of two cell types printed simultaneously from two separate print heads 5 onto collagen hydrogel substrates. (A) Schematic representation of multiple head printing. (B,C) C2C12 (red) and PC12 (green) cells pre-stained with CellTracker TM dyes and printed in various patterns. Images were taken 1 hr after printing, following the addition of culture media. (D,E) Printed cell patterns after 8 days under differentiation conditions. Cells were immunostained for desmin (C2C12, green) and p-III tubulin (PC12, red). Dotted lines 10 represent the outline of the printing pattern. Scale bars represent 500 Pm (B-D) and 200 pm (E). Detailed description of the invention Throughout this specification, unless the context requires otherwise, the word "comprise", 15 or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. The reference to any prior art in this specification is not, and should not be taken as, an 20 acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia. As used herein, the singular forms "a", "and" and "the" are intended to include plural aspects unless the context clearly dictates otherwise. Thus, for example, reference to "a 25 cell" includes a single cell as well as two or more cells; reference to "an agent" includes one agent, as well as two or more agents; and so forth. The full disclosures of publications referred to within this specification are taken to be included herein in their entirety by way of reference, unless it is clear from the context in 30 which the publication is referred to that this is not the intention.

H:\SXD\Interwoven\NRPortbl\DCC\SXD\5072760_.doc-12/04/203 In its broadest sense the present invention relates to formulations suitable for stable suspension therein of viable cells to form a bio-ink for controlled deposition of said cells, comprising an aqueous microgel suspension of one or more gelling biopolymer/s and one or more suitable salts. Such formulations can be considered as precursors to bio-ink 5 formulations themselves, which will generally, although not necessarily, have living cells added to them shortly before the intended use of the bio-ink. In other contexts, however, and especially with particularly robust cells types (that for example exhibit a low level of metabolic activity and/or slow proliferation), cells may be maintained within the bio-ink formulation for some time prior to use. It will therefore be understood in the context of the 10 present invention that the phrase "suitable for stable suspension therein of viable cells to form a bio-ink" is intended to convey that the bio-ink precursor formulations are such that upon addition of viable, living cells the cells will not only be maintained in a viable condition but that the bio-ink formulation so produced will then be compatible for use in a cell printing process. That is, the bio-ink will have the appropriate physical characteristics, 15 such as viscosity, surface tension and size of microgel particles, that will enable it to be used as a bio-ink in conventional printing apparatus. Of course, a key aspect of the present invention is the bio-ink formulations themselves, which allow for controlled deposition of viable cells, and which comprise a stable aqueous 20 microgel suspension of one or more gelling biopolymer/s, one or more suitable salts and viable cells of one or more types. In this context, therefore the term "bio-ink" is intended to convey a formulation that can be used, for example in conventional printing apparatus, for the controlled deposition of viable cells. By this it is meant that the volume of bio-ink (and therefore cells) and/or the two dimensional or three dimensional location of deposition or 25 release of the cell containing ink can be controlled, for example by way of a computer operated printing software tool that controls a printer apparatus. Release or deposition of the bio-ink will in most cases be onto some form of substrate and in most cases the substrate will be hydrated to ensure continued cell viability, although depending upon the nature of the cells concerned and their intended longevity at the locus of deposition there 30 will be some circumstances where the substrate is dry.

H:\SXD\Interwoven\NRPortbl\DCC\SXD\5072760_.doc-12/04/203 -9 Examples of types of substrates onto or into which bio-ink formulations of the present invention can be deposited include fluids such as, salt solution, cell culture media and broth, cell colonies, tissues or organs, gels such as agar, collagen and the like, polymers and plastics material, metals, glass, ceramics and composite materials. Specific substrates 5 for bio-ink deposition can, for example, take the form of in vivo plant or animal tissues or organs, artificial plant or animal tissues or organs, scaffolds for artificial tissue or organ production, assay or high throughput plates, plastic, polymer, glass, metal, ceramic or composite surfaces, medical, prosthetic, surgical or implantable devices or components thereof, such as stents, nails, screws, plates, pins, staples, artificial vessels, sutures, 10 artificial limbs, joints, bone replacement material, dental implants, ocular implants including lenses and cornea, artificial blood, serum or the like, pacemakers, cochlear implants, neuro- stimulatory probes etc. By the phrase "stable suspension" it is intended to convey that viable cells are maintained 15 evenly distributed within the bio-ink formulations of the invention without significant cell aggregation or cell settling, under conditions encountered when the bio-inks are stored prior to use or are in use. For example, it is desirable that the bio-ink formulations of the invention maintain stability, without detectable or significant cellular aggregation or settling that would hinder the function of the bio-ink, for a period of at least 30 minutes, at 20 least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours or at least 24 hours. A major component of the formulations of the present invention is an aqueous microgel suspension of one or more gelling biopolymers. For example, gelling biopolymers that can 25 be used, alone or in combination, in preparation of the aqueous microgel suspension include gellan gum, alginate, fibrin, thrombin, fibronectin, laminin, elastin, proteoglycan chitosan, gelatine, chitin, xanthan gum, guar gum, pectin, collagen, cornstarch, locust bean gum, agar, carageenan, beta-glucan, gum arabic, gum tragacanth, karaya gum, mastic gum, psyllium gum, spruce gum, ghatti gum and glucomannan. Although extensive work has 30 been conducted by the present inventors in preparation of bio-ink formulations comprising aqueous microgel suspensions of gellan gum the inventors expect that other biopolymers, H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 -10 such as those mentioned above, that have the ability to form a semi-solid cross-linked dispersion in aqueous medium, can equally be used in the formulations of the invention. For example, gelling biopolymer can be included within formulations of the invention at a 5 concentration of from about 0.01% w/v to about 0.5% w/v, such as from about 0.02% w/v to about 0.4% w/v or from about 0.08% w/v to about 0.25% w/v, depending upon the specific biopolymer selected and the desired characteristics and end use of the bio-ink. In one aspect of the invention gellan gum (GG) is adopted wherein the GG concentration is from about 0.02% w/v to about 0.5% w/v, such as from about 0.05% w/v to about 0.25% 10 w/v. In the context of ink-jet printing a GG concentration of about 0.05% (w/v) is suitable in certain contexts as at this concentration a GG microgel suspension that can pass through the 50 pm nozzles of conventional inkjet print heads can be produced. The dispersion medium used to form the microgel is water or a substantially aqueous 15 liquid mixture or solution. When the liquid is water (or a substantially aqueous solution), the gel formed is a hydrogel. The formulations of the invention additionally comprise one or more suitable salts. Such salts are suitable in the sense that they are compatible with cell viability in respect of the 20 particular cell types to be included in the bio-ink. Preferably the salt solution will be an isotonic salt solution such that the cells included in the bio-ink will not be subject to damaging osmotic stress. However, this will not be required in all cases, depending upon the particular cells to be included in the bio-ink and the intended function and longevity of the cells after printing. Suitable salts include salts of physiologically compatible cations 25 such as sodium, potassium, lithium, calcium, magnesium, ammonium and alkylammonium; acid addition salts of pharmaceutically acceptable inorganic acids such as hydrochloric, orthophosphoric, sulphuric, phosphoric, nitric, carbonic, boric, sulfamic and hydrobromic acids; or salts of physiologically compatible organic acids such as acetic, propionic, butyric, tartaric, maleic, hydroxymaleic, fumaric, citric, lactic, mucic, gluconic, 30 benzoic, succinic, oxalic, phenylacetic, methanesulphonic, trihalomethanesulphonic, toluenesulphonic, benzenesulphonic, salicyclic, sulphanilic, aspartic, glutamic, edetic, H:\SXD\Interwoven\NRPortbl\DCC\SXD\5072760_.doc-12/04/203 - 11 stearic, palmitic, oleic, lauric, pantothenic, tannic, ascorbic and valeric acids. Suitable associated anions include sulphate, tartrate, citrate, chloride, nitrate, nitrite, phosphate, perchlorate, halosulfonate or trihalomethylsulfonate. Particularly preferred salts include calcium chloride, potassium chloride, sodium chloride, monosodium phosphate and 5 magnesium sulfate. The aqueous liquid mixture or solution adopted in preparation of the formulations of the invention can conveniently include ionic buffer solutions (e.g., phosphate buffer solution, citrate buffer solution, etc.), or cell culture medium (e.g., modified Eagle's medium 10 ("MEM"), Dulbecco's modified Eagle's medium (DMEM), Hanks' Balanced Salts, etc.), and so forth. The aqueous medium used in the formulations can also include other components that are compatible with cell viability, such as solid particles or powders, such as of electrically conductive or resistive material, magnetically active material, miniature electrical components or devices, carbon nanotubes, quantum dots, nanowires or other fine 15 threads or fibres, ceramics, metals, glass beads, composite materials, polymers or may include colorants, pharmaceutically or veterinarily active agents, proteins, nucleic acids, nucleotides, amino acids, antibodies, fatty acids, lipids, carbohydrates (including sugars such as glucose), enzymes, liposomes, nutritional supplements, vitamins, pH buffers, preservatives, anti-oxidants, contrast agents, fluorophores, phosphorescent agents, 20 radioactive isotopes, surfactants or the like. In some aspects of the invention it is desirable, for example to optimise the bio-ink for application in inkjet or other forms of printing, to reduce the surface tension. In this context a suitable surface tension is, for example, in the order of from about 20 mN/m to 25 about 50 mN/m, such as from about 25 mN/m to about 50 mN/m, or from about 28 mN/m to about 33 mN/m, such as about 30 mN/m, which is the optimal range specified for Xaar 126 piezoelectric inkjet print heads. To optimise bio-ink surface tension for printing applications where this is desirable it is advantageous to include one or more surfactant/s that are compatible with cell viability within the formulation. A particular category of 30 suitable surfactants is the non-ionic polymeric surfactants and particularly useful surfactants within that category include the Poloxamer surfactants, which are a family of H:\SXDUterwovenNRPrtbl\CC\SXD\5072760_Ldoc-2/34/2013 - 12 non-ionic triblock copolymers comprising a central hydrophobic poly(propylene oxide) (PPO) core flanked by two hydrophilic chains of poly(ethylene oxide) (PEO), as shown in Compound I. They are also known by the trade names Pluronic* and Lutrol®. The number nomenclature for Poloxamers defines their structure; the first two numbers (i.e. 188) 5 multiplied by 100 give the molecular weight of the PPO core (i.e. 18x100 = 1,800 gmolI) while the last number (i.e. 188) multiplied by 10 gives the percentage of PEO content by CH3 H{O OHO OH x y z mass (i.e. 8x10 = 80% w/w PEO). A particularly suitable Poloxamer surfactant is Poloxamer P188 (P188). 10 Compound I; Structure of the Poloxamer family of non-ionic block copolymers, PEO PPO-PEO. For Poloxamer 188, y = 31 and x+z = 164. It is recognised that P188 can protect cells from fluid-mechanical damage in agitated and 15 aerated cell cultures, and it has been proposed that this protective effect could be due to either biological (enhanced membrane integrity) or physical (reduced shear forces and bubble break-up) factors. More recent work has elucidated mechanisms by which P188 was shown to insert into perforated cell membranes, restore membrane integrity, and then itself be excluded from the membrane once repair is completed. This makes it suitable to 20 protect and repair cells exposed to high shear forces. P188 is therefore of great interest in cell printing applications, not only for its surfactant properties and biocompatibility, but also its ability to protect cells from injury. Parsa and co-workers2 used P188 as an additive to improve the droplet formation in inkjet cell printing, but while they showed that P188 did not decrease the viability or proliferation of cells in culture, they did not demonstrate a 25 protective effect of P188 on printed cells.

H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 13 Another category of surfactant that can usefully be included within the formulations of the invention is the non-ionic polymeric fluorinated surfactants (fluorosurfactants). A specific example of a useful fluorosurfactant is sold under the trade name NovecTM FC4430 (3MTM). The exact structure of this surfactant is proprietary, but it is known that it is a 5 fluoroaliphatic ester28,29 . The fluorosurfactants possess both high surface activity and low cytotoxicity. The high surface activity is attributed to strong hydrophobic interactions and low van der Waals interactions as a result of the fluorinated chains. There is a growing body of evidence that increasing the degree of surfactant fluorination decreases cytotoxicity in comparison with hydrocarbon analogues, and this could be due to a number 10 of factors including a decreased propensity for the removal of membrane proteins. Surfactants such as those referred to above can, if adopted at all, be used within the formulations of the invention either alone or in combination with others. In one aspect of the invention a target surface tension for the bio-ink (approximately 30 mN/m) for ink-jet 15 printing was reached by addition of the Novec FC-4430 fluorosurfactant at a concentration of 0.05% v/v. Although P188 did not exhibit significant surface activity alone, it remained of interest as a cell-protective additive to the bio-ink formulation. We therefore also investigated the surface tension when both surfactants were added to DMEM together. Novec FC-4430 was added at a range of concentrations to DMEM containing a fixed 20 concentration of 0.01% (v/v) P188. At a Novec FC-4430 concentration of 0.005% (v/v), the surface tension was lower than for either surfactant alone; this apparent synergistic effect could be due to the ability of surfactants to co-exist at the liquid-air interface. At higher concentrations of Novec FC-4430, the surface tension matched closely with this surfactant alone, indicating that it replaced the less surface active P188 at the surface. This 25 suggested that blends of the two surfactants could be utilised without compromising the surface tension reduction afforded by Novec FC-4430. For example, surfactant can be included, depending upon the cells adopted, the nature of the intended printing application and the specific cells adopted, at concentrations of from 30 about 0.001 % (v/v) to about 0.5% (v/v), such as from about 0.005 % (v/v) to about 0.1 % (v/v) or 0.01% (v/v) to about 0.05% (v/v). In one aspect of the invention P188 is included H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 14 in the formulations at a concentration of about 0.1% (v/v). In another aspect of the invention a fluoroaliphatic ester surfactant such as Novec FC-4430 is included in the formulation at a concentration of about 0.05% (v/v). In a further aspect of the invention P188 is included in the formulations (for example at a concentration of about 0.1% (v/v)) 5 and a fluoroaliphatic ester surfactant such as Novec FC-4430 is included in the formulation (for example at a concentration of about 0.05% (v/v)). A wide variety of cell types can be included within the bio-inks of the invention. Depending upon the intended use of the bio-ink it may be appropriate to include within the 10 bio-ink cells of more than one type. For example, either prokaryotic and/or eukaryotic cells can be used. Prokaryotes are cells having no nucleus or only a primitive nucleus, including both bacterial and cyanobacteria, such as, E. coli, algae, and so forth. On the other hand, eukaryotes are cells having a defined nucleus, such as fungi, protozoa, plant cells, mammalian cells (e.g., bovine aortal endothelial cells ("BAEC"), Chinese hamster 15 ovary cells ("CHO"), smooth muscle cells ("SMC"), etc.), and so forth. Prokaryotes typically have a cell diameter of from about 2 to about 10 micrometres, while eukaryotes typically have a cell diameter of from about 10 to about 200 micrometres. Most bacterial cells, for instance, have a cell diameter of about 2 micrometres, while most mammalian cells have a cell diameter of from about 15 to about 200 micrometres. 20 In general, the choice of cell type will vary depending on the type of construct to be printed. For example, if the bio-ink is to be used to print a blood vessel type three dimensional structure, the bio-ink will advantageously comprise a cell type or types typically found in vascular tissue (e.g., endothelial cells, smooth muscle cells, etc.). In 25 contrast, the composition of the bio-ink will vary if a different type of construct is to be printed (e.g., intestine, liver, kidney, etc.). One skilled in the art will thus readily be able to choose appropriate cell type(s), based on the intended end use of the bio-ink. Non-limiting examples of suitable cell types include contractile or muscle cells (e.g., striated muscle cells and smooth muscle cells), neural cells, connective tissue (including bone, cartilage, 30 cells differentiating into bone forming cells and chondrocytes, and lymph tissues), parenchymal cells, epithelial cells (including endothelial cells that form linings in cavities H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 15 and vessels or channels, exocrine secretory epithelial cells, epithelial absorptive cells, keratinizing epithelial cells, and extracellular matrix secretion cells), and undifferentiated cells (such as embryonic cells, stem cells, including pluripotent stem cells, induced pluripotent stem cells and totipotent stem cells, and other precursor cells), among others. 5 Cells adopted in the invention can be derived from microorganisms such as bacteria, fungi, nemotode, protozoa, helminth, etc., plants, algae, yeast, animals such as mammals, avians, reptiles, fish and humans. Cells utilized in the bio-inks may suitably be derived from mammals such as from domestic animals (e.g cats, dogs), laboratory animals (e.g. rats, 10 mice, guinea pigs, rabbits), agricultural animals (e.g. cattle, sheep, goats, horses etc.) captive wild animals and particularly from humans. Examples of specific cell types that can be incorporated in bio-inks of the invention include C2C12 murine skeletal muscle, PC12 murine pheochromocytoma, L929 murine 15 fibroblast, Rosa murine primary myoblast and RenCX human neural progenitor cells. Cells incorporated within bio-inks according to the invention will be included at varying concentrations depending upon the end use of the bio-ink. In some contexts it will be desirable to include an average of a single cell per printed drop while in other contexts it 20 will be desirable to print at higher or lower cell densities. A skilled person can readily determine the appropriate cell concentration depending upon the intended use of the bio ink. For example cells can be suspended in the bio-ink formulation at a concentration of from about 1 x 102 to about 1 x 108 cells/mL, such as from about 1 x 104 to about 5 x 106 cells/mL. 25 The distribution of cell number within bio-ink droplet is relevant, in particular, to the fabrication of cell-based microarrays where the number of cells contained in individual droplets is important, not just the density across a population of droplets. The cells/drop distribution can be fitted to a Poisson distribution according to Equation 1, as follows: 30 H:\SXD\Intrwoven\NRPortbl\DCC\SXD\5072760_1.de-12/04/2013 - 16 n! , Equation 1 where P is the probability of dispensing a given number (n) of cells in a given aliquot of 5 liquid, and ), is the average number of cells in the aliquot volume calculated by taking the overall average of cells/drop across all printed droplets. The term "viable" in the context of cells means that a substantial proportion of the cells are living and that such preferably maintain normal metabolic function, as demonstrated by 10 exhibiting normal levels of cell proliferation, differentiation and function. Standard assays are available to test cell viability such as by determining intracellular potassium to sodium ratio (as a test of cell membrane function or sodium-potassium pump function), cytolysis or membrane leakage, mitochondrial activity, cell proliferation, cell differentiation, production of normal cellular products and the like. Preferably at least 90%, at least 95%, 15 at least 98%, at least 99%, at least 99.5% or at least 99.9% of the cells within the bio-ink are maintained in a viable state for a period of at least 30 minutes, at least 1 hour, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours or at least 2 days after printing of the bio-ink using a conventional printing apparatus. 20 For example, cell viability can be assessed using live/dead staining against a suitable cell culture medium control (e.g. DMEM) after 2 hr in culture post inclusion in the test formulation. Cell morphology can readily be determined by standard microscopy techniques utilising dyes for specific cell features. 25 An example technique that can be utilised for analysis of cell proliferation is the MTS metabolic assay. This colorimetric method employs a reagent combining the tetrazolium compound MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4 sulfophenyl)-2H-tetrazolium] and an electron coupling reagent PES (phenazine ethosulfate) in a stable solution. The MTS compound is converted by metabolically active 30 cells into a coloured formazan product that is soluble is culture media, and can be quantified by measuring the absorbance at 490 nm. This absorbance level is directly H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 17 proportional to the number of metabolically active cells. Cell populations can for example be analysed 2, 24, and 48 hr after inclusion in test formulation and printing. A set of wells with cell-free culture media can also included in each plate as a method blank. At each time point, 30 pL of warmed CellTiter 96* AQueous One Solution reagent is added to each 5 well and incubated under culture conditions in the dark for 90 min before reading absorbance on a plate reader (SpectraMax 190, Molecular Devices) at 490 nm (formazan product) and 650 nm (background). The absorbance at 650 nm is subtracted from that at 490 nm for each individual well, and then the average net absorbance for blank wells is subtracted from each cell-containing well. The final absorbance at the 24 and 48 hr time 10 points is normalised to the 2 hr time point in order to account for differences in initial cell numbers between conditions. For example, cell differentiation can be analysed by culturing cells under differentiation conditions for 5 or 6 days before fixing and immunostaining against desmin or before 15 fixing and staining F-actin filaments with phalloidin. Differentiated cells are fixed with 3.7% (w/v) paraformaldehyde (PF, Fluka) in PBS for 10 min at room temperature, washed twice with phosphate buffered saline (PBS) and, if required, stored in PBS at 4'C prior to staining. For immunostaining against desmin, cells are permeabilised with 50:50 (v:v) methanol:acetone on ice for 5 min and washed with PBS before blocking in 10% (v/v) 20 donkey serum (DS, Chemicon) with 0.05% (v/v) Tween-20 (Sigma) for 1 hr at room temperature (RT). Mouse monoclonal anti-desmin primary antibody (Novocastra, 45 mg/L) is diluted 1:100 in blocking solution and incubated at 4'C overnight. After two 10 min washes in PBS, cells are incubated for 1 hr at RT in the dark with the secondary antibody Alexa-546 donkey anti-mouse (Invitrogen, 2 mg/mL) diluted 1:1000 in blocking 25 solution. After two 10 min washes in PBS, 4',6-diamidino-2-phenylindole (DAPI, 1 mg/mL) is added at 1:1000 in PBS for 5 min at RT, before replacing with PBS. For phalloidin staining, fixed cells are permeabilised with 0.1% Triton X-100 (Sigma) in PBS for 5 min at RT. After washing twice with PBS, cells are incubated for 20 min at RT in the dark with Alexa-488 phalloidin (Molecular Probes) at 165 mM in 1% (v/v) bovine serum 30 albumin (BSA) in PBS. After two washes in PBS, cells are stained with DAPI as above.

H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 18 Formulations suitable for stable suspension therein of viable cells to form a bio-ink for controlled deposition of said cells, can be prepared by dissolving one or more gelling biopolymer/s in hot aqueous medium that comprises one or more suitable salts to form a 5 solution that is cooled under shear conditions to produce a microgel suspension. For example, the aqueous medium and biopolymer can be heated to a temperature of from about 40'C to about 90'C, such as from about 50'C to about 85'C or about 60'C to about 80'C, depending upon the solubility of biopolymer adopted and the intended microgel features of the bio-ink. The solution can then conveniently be cooled, for example to a 10 temperature of from about 5'C to about 60'C, such as from about 10 0 C to about 50'C, from about 15'C to about 45'C or from about 20'C to about 40'C, again depending upon the specific biopolymer and the desired characteristics of the bio-ink. Shear can, for example, be applied using an overhead laboratory homogeniser or magnetic 15 stifrer, the use of a large volume (from example -30 mL) cup and vane-rotor assembly within a rheometer, and the use of a standard laboratory vortex mixer. Characterisation of the microgel suspension produced can be achieved by confocal scanning laser microscopy (CSLM). Microgel particles can also be stained with the 20 common cell nuclear stain DAPI, and imaged by fluorescence microscopy. In another example, microgel particles can be visualised by addition of a pigmented ink (such as Derivan ink) and imaged by phase contrast microscopy. Vortex mixing produces a homogenous network of relatively small microgel particles. Microgel particles can take a wide variety of forms depending upon the biopolymer adopted and microgel production 25 conditions. Particles can be spherical, elliptical, elongate such as in the form of rods or filaments or may be of irregular shape. In the case of GG microgels, and although high density of the microgel network makes accurate characterisation of particle size difficult, particles may be irregular and elongated in shape with dimensions on the scale of from about 5 prm to about 50 pm. This non-spherical shape can be significant in the 30 development of viscoelastic properties of the microgel suspensions even at low polymer concentration. Importantly, the particle dimensions should be such that they should be able H:\SXD\Interwoven\NRPortbl\DCC\SXD\5072760_.doc-12/04/203 - 19 to pass unimpeded through the nozzles of microvalve and inkjet printers, or other printer nozzles depending upon the intended use of the bio-ink. Cells can conveniently be added to the bio-ink precursor formulation after the microgel 5 suspension has been formed. Other components of the bio-ink can also be added at this stage. In some aspects addition of cells will take place just prior to use of the bio-ink, while in other cases cells can be added at an earlier stage such as several hours or even a day or several days prior to use. 10 In a still further embodiment of the present invention there is provided a method of controlled deposition of viable cells to a desired locus comprising loading a bio-ink formulation as described above to a compatible computer controlled printer and directing controlled release of the bio-ink to said desired locus by computer operation. 15 Printability characteristics of the bio-ink can be optimised depending upon the intended use of the formulation. However, in some contexts, such as for ink-jet printing it will be desirable for the bio-ink to exhibit a viscosity of less than about 12 mPa.s and a surface tension of from about 28 to about 33 mN/m. Printing of the bio-inks of the invention can be conducted using a wide variety of conventional printing devices such as laser printers, 20 microvalve printers and ink-jet printers. An example of a microvalve printer is the DeeracEquator T M GX1 liquid handling system. In this system volume is simply selected in the DeeracTM software. This variation in dispensed volume is one key advantage of microvalve printing over, for example, inkjet printing, where each inkjet nozzle dispenses a fixed droplet volume. The deposition mechanism is also relatively simple in comparison 25 to inkjet printing and the outlet dimensions are larger. Consequently, microvalve printing can deposit liquids with a much larger range of fluid properties. For example, the DeeracTM control software has preset 'liquid classes' for fluids with a wide range of viscosities, from water to 40% glycerol. The system therefore easily handles the deposition of surfactant free bio-ink. 30 The DeeracTM system operates by aspirating solutions from assignable reservoirs prior to H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 20 deposition; a feature that can be exploited to print either the same cells at different concentrations, or different cell types. The integrated tip-washing programs in the system ensure minimal cross-contamination between the two different ink reservoirs. 5 The printing techniques of the present invention may be utilized to form various types of arrays on a substrate. Two-dimensional arrays may be formed by printing a single layer of a cell composition onto a substrate. The single layer can contain cells optionally mixed with a support compound, such as a thermogelling polymer. Two-dimensional arrays are particularly useful when depositing prokaryotic cells (e.g., bacteria) onto a substrate. 10 Besides two-dimensional arrays, three-dimensional arrays may also be formed. Three dimensional cell arrays are commonly used in tissue engineering and biotechnology for in vitro and in-vivo cell culturing. In general, a three-dimensional array is one which includes two or more layers separately applied to a substrate, with subsequent layers applied to the top surface of previous layers. The layers can, in one embodiment, fuse or otherwise 15 combine following application or, alternatively, remain substantially separate and divided following application to the substrate. Three-dimensional arrays can be formed in a variety of ways in accordance with the present invention. For example, in one embodiment, three dimensional arrays may be formed by printing multiple layers onto a substrate. 20 Bio-inks of the invention can also be used in inkjet printing applications, such as using Xaar-126 piezoelectric inkjet print heads. An attractive feature of inkjet printing for cell deposition is its capacity for high resolution deposition. For example, the average diameter of droplets once on the substrate can be approximately 170 Pm or less. This does not necessarily equate to the minimum feature size achievable, as the diameter is dependent on 25 droplet volume as well as the properties of the fluid and substrate that control droplet spreading. Deposition onto a substrate where instantaneous gelation takes place, or altering the printing waveform to achieve droplet volumes closer to a nominal volume of 80 pL, may enable smaller features to be printed. Importantly, inkjet printing provides the capability to deposit structures smaller than the diffusion limit of oxygen in tissues (-200 30 pm), which is a key advantage of inkjet printing over microvalve printing H:\SXD\Interwoven\NRPortbl\DCC\SXD\5072760_.doc-12/04/203 - 21 Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in 5 this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. The invention will now be described with reference to the following Examples. These Examples are not to be construed as limiting the invention in any way 10 Examples Example 1 - Formulation and testing of Bio-ink for on-demand printing of living cells 15 In this example we report on the development of a general purpose bio-ink that addresses challenges of prior art formulations to allow facile cell deposition by drop-on-demand printing using both a commercial microvalve deposition system, and many-nozzle piezoelectric inkjet print heads. 20 Experimental Bio-ink Endotoxin-free low-acyl gellan gum (Gelzan CM, a gift from CP Kelco) was dissolved in hot (80 C) Milli-Q water (resistivity 18.2 MQ cm) at 1% (w/v) by stirring for 1-2 h. This 25 hot solution was combined with heated (80 C) Milli-Q and 2x concentrated Dulbecco's Modified Eagles Medium (DMEM, Invitrogen) to produce a range of gellan gum concentrations in 1x DMEM. The mixture was sheared using a vortex mixer while cooling to 25 'C to create a microgel suspension, i.e. the bio-ink. The surfactant-containing bio inks were prepared through addition of Poloxamer 188 surfactant (Lutrol@ F68, Sigma) 30 and/or fluorosurfactant (Novec@ FC-4430, 3M) solutions to the microgel suspension. All bio-inks were prepared under aseptic conditions.

H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 22 Cell culture C2C12 (CRL-1772), PC12 (CRL-1721) and L929 (CCL-1) murine cell lines were obtained from ATCC. C2C12 and L929 cells were maintained in DMEM (Invitrogen) supplemented 5 with 10% fetal bovine serum (FBS, Invitrogen), while PC12 cells were maintained in DMEM with 10% fetal bovine serum and 5% horse serum (HS, Sigma). Cells were cultured at 37 'C in a humidified incubator with 5% CO 2 and passaged every 2-3 days. Bio-ink characterization 10 Rheology of the bio-ink was characterized using a controlled stress ARG2 rheometer (TA Instruments), using a sandblasted 40 mm parallel plate geometry with a measurement gap of 0.5 mm and Peltier plate thermal control. A solvent trap was used to prevent evaporation of water during measurements. After loading, samples were subjected to 30 s pre-shear at 500 s-1 followed by 1 min equilibration before measurement. Shear-dependent viscosity 15 was measured by a stepped ramp of shear rate from 1-1000 s- . Each shear rate (10 points per decade) was held for 20 s, and the viscosity over the last 10 s was averaged. Apparent yield stress was measured by a continuous ramp of shear stress from 0-2 Pa over 5 min. Constitutive modelling was facilitated by Rheology Advantage data analysis software (TA 20 Instruments). Silicone oil standards (Scientific Polymer Products) were used to validate experimental conditions. Surface tension was measured using a Dataphysics OCA contact angle system with SCA 20 software. The structure of the bio-ink was visualized by negative staining with a pigmented ink 25 (Derivan Ink, black) that was excluded from microgel particles. Derivan Ink (1 : 5) was added to the bio-ink, 20 [L, and was immediately placed on a glass slide and cover-slipped prior to imaging. The ability of the bio-ink to maintain cells in suspension was determined by suspending 30 cells at 1-6 x 106 cells ml in the ink or in serum-free DMEM as the control. 100 [L aliquots of both suspensions were added to 96-well plates, and the base of each well was H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 23 imaged over time. Image J software was used to count the number of cells in a defined area of the wells at each time-point, allowing the number of settled cells to be plotted as a function of time. 5 Printer design Microvalve cell printing was facilitated through a DeeracTM GX1 liquid handling system (Labcyte Inc.), which dispenses droplets using a magnetic feedback-controlled microvalve. Cells were inkjet printed using a custom-built inkjet printing system with Xaar-126 piezoelectric inkjet print heads. Both printers were housed in a bio-safety cabinet and 10 sterilised regularly using 70% ethanol and UV light. Cell printing For microvalve printing, C2C12 cells were suspended in the bio-ink (without added surfactants) at 2 x 105-2 x 106 cells per mL and aspirated into the DeeracTM GX1 nozzle 15 reservoir. Patterns were designed using accompanying software (Spot Station/Plate Designer). For analysis of cell viability and proliferation, 50 drops were printed into 100 [L of the cell culture media supplemented with 100 units per mL penicillin and 100 [g mUL streptomycin (Pen/Strep, Gibco). 20 For inkjet printing, cells (C2C12 or PC12) were suspended in the surfactant-containing bio-ink at 1-6 x 106 cells per mL, and loaded into the print heads by aspirating through the nozzle plate. Patterns were designed in Microsoft Paint and loaded into Xaar XUSB software. 25 For analysis of cell viability, proliferation and differentiation, rectangular patterns (25 x 50 drops) were printed into supplemented media as above. This media was contained within thin (1 mm) PDMS wells, and subsequently transferred to a 96-well plate for further culture and analysis. 30 For analysis of the cell/drop distribution, cells were printed directly onto glass slides and allowed to dry. The number of cells in each drop, or the number of cells in a printed H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 24 pattern, was then counted manually or imaged using a Zeiss Axiovert 40 CFL inverted fluorescence microscope (Carl Zeis AG) and counted using Image Pro software. For patterning experiments, cells were inkjet printed onto collagen bio-paper. Collagen I 5 (rat tail, 5 mg mU 1 , Invitrogen) was sonicated for 5 min on ice, combined with cold 5x concentrated DMEM to a final concentration of 4 mg mU 1 and neutralised with 0.1 M NaOH. The cold collagen solution was pipetted into 0.5 mm thick PDMS wells and polymerized for 2 h at 37 'C. 1 mm thick PDMS wells were then placed on top of the existing PDMS to create a reservoir. Collagen bio-papers were soaked in cell culture media 10 supplemented with Pen/Strep for 1-2 h, and excess medium was removed prior to cell printing. Cell patterns were printed onto collagen bio-papers, and incubated at 37 'C for 1 h to allow cells to attach prior to further addition of the culture medium. In dual cell printing experiments, cells were stained prior to printing with CellTrackerTM Probes (Molecular Probes, Invitrogen). C2C12 cells were stained with CellTrackerTM Red 15 CMPTX (20 [M) and PC12 cells were stained with CellTracker T M Green CMFDA (20 PM), following the manufacturer's protocols. Cell viability Cell viability was assessed by fluorescent live/dead staining using Calcein AM and 20 propidium iodide (both from Molecular Probes). Calcein AM was added at 5 pg/mL and incubated at 37 0 C for 15 mins in the dark, followed by addition of propidium iodide at 1 pg/mL. Cells were imaged immediately using fluorescence microscopy. Viability was assessed by manual counting live and dead cells using Image J software, or by automated counting with Image Pro software (MediaCybernetics). Comparison of manual and 25 automated counts showed good agreement. Cell proliferation and differentiation Cell proliferation was assessed using the MTS colorimetric assay (Promega). Printed and control cells were seeded into 96 well plates at -5xlO3cells/well in 100 PL cell culture 30 media. Enzymatic activity was measured after 2, 24 and 48 hrs by adding 20 PL MTS reagent (Promega) to each well, and incubating at 37 0 C for 90 mins before reading H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 25 absorbance on a plate reader (SpectraMax 190, Molecular Devices) at 490 nm (formazan product) and 650 nm (background). Absorbance at 24 and 48 hr time points was normalized to the 2 hr time point in order to account for differences in initial cell numbers. Calibration curves ensured that cell numbers were within the linear range of the assay. 5 Printed and control cells were seeded in 96-well plates at ~1x10 4 cells/well for differentiation experiments. For C2C12 cells, media was changed gradually to differentiation conditions: the serum content was reduced to 5% FBS after 24 hrs, 2% FBS after 48 hrs and finally to 2% horse serum (HS) after 72 hrs. For PC12 cells media was changed to differentiation conditions, DMEM with 1% HS and nerve growth factor (NGF, 10 50ng/mL, Invitrogen), after 24 hrs. Differentiation media was changed every 48 hrs. PC12 cells were fixed after 5 days in differentiation media, and C2C12 cells after 4 days in final differentiation media, with 3.7% paraformaldehyde for 10 mins at room temperature (RT, 21'C). For co-cultures of printed C2C12 and PC12 cells on collagen gels, cells were maintained in C2C12 proliferation media (DMEM with 10% FBS) for 24 hrs before being 15 changed to PC12 differentiation media. Cells were fixed as previously described after 8 days in differentiation conditions. Immunostaining C2C12 cells were permeabilised with 50:50 methanol:acetone on ice for 5 mins, and 20 washed with phosphate buffered saline (PBS, Sigma) before blocking solution in 10% donkey serum (DS, Chemicon) with 0.1% v/v Tween-20 (Sigma) for 1 hr at RT. Mouse monoclonal antidesmin primary antibody (Novocastra) was diluted 1:100 in blocking solution and incubated at 4'C overnight. After two 10 min washes with PBS, cells were incubated for 1 hr at room temperature in the dark with the secondary antibody Alexa-546 25 donkey anti-mouse (Invitrogen) diluted 1:1000 in blocking solution. After two further 10 min washes in PBS, DAPI was added at 1:1000 in PBS for 5 mins at room temperature, before replacing with PBS. For differentiated PC12 and C2C12 co-cultures on collagen, the above protocol was used with cells incubated in a mixture of primary antibodies in blocking solution. C2C12 cells were labelled with Cell Signalling rabbit monoclonal anti 30 desmin (GeneSearch) diluted 1:100, while PC12 cells were labelled with mouse anti neuronal p-III tubulin (Covance) diluted 1:1000. Secondary antibodies were Alexa-488 H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_.doc-12/04/20 3 - 26 donkey anti-rabbit (Invitrogen) and Alexa-546 donkey anti-mouse (Invitrogen). For differentiated PC12 cells on tissue culture polystyrene, cells were stained with a phalloidin dye to avoid lengthy immunostaining protocols that could have detached cells from the substrate. Fixed cells were permeabilised with 0.1% v/v Triton X-100 (Sigma) in PBS for 5 5 mins at room temperature. After washing twice with PBS, cells were incubated for 20 mins at RT in the dark in Alexa-488 phalloidin (Molecular Probes) at 165 nM in 1% bovine serum albumin in PBS. After two washes, cells were stained with DAPI as above and imaged. 10 Results and discussion Bio-inks were prepared by producing microgels (a dispersed phase of discrete polymeric gel particles) in standard cell culture media (DMEM) using the biopolymer gellan gum. This linear anionic polysaccharide has found widespread use in the food and cosmetic industries as a gelling and stabilizing agent and more recently as a material for tissue 15 engineering applications. The choice of gellan gum is justified as follows. Gellan gum is a linear anionic polysaccharide. Gelation of gellan gum is preceded by a conformational transition from coil to double helix, and association of these helices in junction zones is facilitated through 20 either monovalent or divalent cations. Consequently, gellan gum hydrogels may be formed at low concentrations of divalent cations. Gellan gum can even form gels in the presence of monovalent cations alone. Gellan gum is particularly attractive for its ability to form microgels at low concentrations, 25 which allows the mass content of the bio-ink to be kept at low levels. Furthermore, the concentration window to form microgels is quite broad for gellan gum. A range of gellan gum concentrations was investigated and 0.05% (w/v) was found to be the lowest concentration at which microgels form. Imaging of the bio-ink structure at this concentration clearly revealed an associated network of elongated microgel particles (Fig. 30 la). This tenuous network structure imparted pseudo-plastic properties that we elucidated by rheological measurements of both the apparent yield stress and the apparent viscosity as H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 27 a function of shear rate. The bio-ink exhibited an apparent yield stress of -30 mPa followed by shear-thinning flow behaviour that showed good agreement with constitutive modeling. 5 Importantly, these properties are suitable to satisfy the dual aims of cell-suspending ability and printability. Cell settling in a fluid can be described by Stoke's law, which defines a minimum yield stress of -5 mPa for zero settling velocity. Thus the yield stress of the bio ink is, theoretically, sufficient to keep the cells suspended. Additionally, the shear-thinning behaviour presents a high viscosity to settling cells (shear rates <10 s-1) to maintain 10 suspensions, and a low viscosity during droplet ejection (shear rates >103 s-1) to aid printability. To confirm this we performed cell settling tests and found that cells in the bio ink remained suspended with no sign of aggregation, whereas cells suspended in DMEM alone completely settled to the base of a 96-well plate within 15 min (Fig. 1b). The consequences of this for drop-on-demand cell printing were directly demonstrated by 15 analyzing cell output over time by microvalve deposition. With DMEM alone, cell output showed significant variation with a sharp peak due to cell settling, followed by a steady decrease during the deposition of cell-depleted media, whilst cell output was steady over 1 h of printing with the bio-ink (Fig. ld). This allowed the deposition of relatively large scale patterns with uniform cell distribution (Fig. Ic). Previous work has shown that 20 printing cells from bio-inks consisting of cell culture media alone leads to inconsistent cell output from both microvalve and inkjet" printing systems. This was attributed to cell settling and aggregation. The present bio-ink addresses these challenges to achieve consistent cell output. 25 Efficient deposition of the bio-ink by inkjet printing was improved with the addition of surfactants that reduced the surface tension to low (-30 mN m-I) levels without cytotoxicity. The non-ionic polymeric surfactant Poloxamer 188 (P188) is an established medium additive which has been well documented for protecting cells from fluid mechanical damage. However, P188 alone did not reduce surface tension to a significant 30 extent.

H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 28 After having investigated other alternatives we investigated fluorinated surfactants, which exhibit both greater surface activity24 and lower cytotoxicity 25 than their hydrocarbon analogues, to achieve further surface tension reduction. We established that addition of 0.05% (v/v) of the nonionic polymeric fluorosurfactant Novec FC-4430 (a fluoroaliphatic 5 ester surfactant) in combination with 0.1% (v/v) P188 reduced the surface tension of the bio-ink to -30 mN m-1. To the best of our knowledge, this is the first example where surfactants have been utilised to achieve considerable surface tension reduction in a bio ink, to within the optimal range for inkjet printing, 15 whilst maintaining the biocompatibility of the bio-ink. Importantly, this improved controlled deposition of three 10 different murine cell lines from commercially available Xaar-126 piezoelectric print heads. The use of these print heads represents a significant advance over currently employed piezoelectric print heads that have only a single nozzle. '' C2C12 (skeletal muscle), PC12 (neuronal model) and L929 (fibroblast) cells were reproducibly deposited from all 126 nozzles of the Xaar- 126 print heads during numerous print cycles. Analysis of printed 15 C2C12 patterns showed even cell density across the width of the print head (Fig. 2a and b), and by optimizing cell concentration in the bio-ink it was possible to print droplets that contained, on average, one cell per drop (Fig. 2c and d). The number of cells in each individual droplet followed the expected Poisson distribution (Fig. 2d), as previously observed by others using single-nozzle deposition methods.

1 7

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26 20 Exposure to the bio-inks (with and without surfactants) did not have an apparent cytotoxic effect on either the C2C12 or PC12 cells (Fig. 3a). In fact, the viability of the bio-ink exposed PC12 cells was significantly higher than the control cells exposed to DMEM alone. This is likely due to the maintenance of a single cell suspension in the bio-inks, as 25 opposed to cells in DMEM, which aggregated and settled and thus had to be resuspended intermittently. Inkjet printed PC12 cells, and both inkjet and microvalve printed C2C12 cells, retained >95% viability (Fig. 3a) and were shown to proliferate over 48 h at a rate comparable to non-printed controls (Fig. 3b). 30 A comparison of immunostained cells indicated that inkjet printed C2C12 and PC12 cells retained the ability to differentiate (Fig. 3c). Furthermore, omission of P188 from the H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 29 surfactant- containing bio-ink did decrease viability of inkjet printed C2C12 cells to some extent (Fig. 3d), indicating a direct protective effect of P188 during the inkjet printing process. To demonstrate the utility of the surfactant-containing bio-ink to prevent cell settling during inkjet printing, we compared C2C12 cells printed immediately and then 1 h 5 after loading into the print head. After a 1 h pause in printing, cell viability and density (average cells/drop) was no different to the initial values (Fig. 3e). Representative images of live/dead stained cells printed at these different time points (Fig. 3e) show cells with similar density, morphology and viability. Taken together, these results establish the present bio-inks as providing a unique combination of printability and cell-suspending 10 capability, whilst retaining the viability and function of printed cells. Printing multiple cell types from different print heads is a highly attractive feature of inkjet printing as a biofabrication tool, allowing the fabrication of more complex multi-cellular constructs. Fig. 4a and b show two cell types (C2C12 and PC12) printed simultaneously 15 from two different inkjet print heads in defined two-dimensional patterns onto collagen hydrogel substrates. Deposition of cells onto thin layers of collagen hydrogels ensured that the cells remained hydrated and viable for long enough to develop adhesions to the collagen, so that further addition of media did not disrupt the printed pattern. The cells were cultured under differentiation conditions and subsequently fixed and immunostained 20 to assess the retention of printed patterns and the establishment of post-printing cell-cell and cell-substrate interactions. The bio-ink did not impede cellular interactions with the collagen substrate and both neural (PC12) and skeletal muscle (C2C12) cells were unimpeded in their ability to express the respective neural (P-III tubulin) and skeletal muscle (desmin) markers and to differentiate normally, as evidenced by the extension of 25 dense neural networks from PC12 cells into surrounding areas populated by skeletal muscle cells (Fig. 4c and d). Conclusions The results reported in this example demonstrate key advances in addressing the major 30 challenges in the continuing evolution of drop-on-demand cell printing towards becoming a clinically relevant biofabrication tool. The present bio-inks display optimal fluid H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 30 properties whilst addressing the multiple complications that arise from cell settling and aggregation. As we have demonstrated, this means that cell-containing structures can be printed simultaneously from separate print heads, over extended time periods while maintaining printed cell density and viability. This capability is fundamental to the 5 fabrication of multi-cellular and/or larger structures. The printing of relatively simple dual-cell-type patterns in two dimensions would not have been possible had the issues of cell settling and aggregation not been addressed. That printing was reproducible across the width of these print heads is further evidence of the 10 utility of the present bio-inks. The present bio-inks will allow more facile cell deposition, and enhance the accessibility of the technique by enabling the use of standard commercially available print heads. Example 2 - 2D image printing 15 Drop-on-demand techniques, such as microvalve printing, have application in the fabrication of droplet-based arrays. For the fabrication of tissues and other cell constructs, droplets must be combined to form larger-scale patterns. The deposition of complex user defined patterns using the DeeracTM control software is limited. The two modes which enable patterning of droplets are the spotting and platemap dispense functions. In spotting 20 mode, a grid can be defined within a well region and droplet deposition at points of this grid can be toggled on/off. This is useful for the deposition of simple patterns, but is limited and tedious. Alternatively, the platemap dispense function allows the user to define a volume to be dispensed into each well in a given plate, and these values can be imported from a spreadsheet .csv file. Using simple plate designer software tool in conjunction with 25 the DeeracTM system, a 'plate' can be created where each 'well' represents a pixel of an image and a platemap dispense task can then be used to deposit a droplet of specified volume into these 'wells' to create an image. Simple bitmap images, or even gray-scale or colour images, can be deposited using this approach. However, to alleviate the need to manually enter volumes for each pixel of an image, we needed a method to easily convert 30 an image into meaningful deposition volumes in a .csv file that could be imported into a DeeracTM platemap dispense task.

H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 31 Using an image processing tool and the platemap dispense function, we deposited various C2C12 cell patterns onto glass slides (Fig. 5). C2C12 cells were suspended in bio-ink at 1xi0G cells/mL and 50 nL droplets deposited in 50x100 pixel spiral or lattice patterns using 5 a droplet (pixel) spacing of 500 pm. The individual droplets coalesced to form continuous patterns that accurately reflected the original images (Fig. 5A and B). Higher magnification images (Fig. 5C and D) showed that cells were homogeneously distributed throughout these patterns. 10 This demonstrated that the image processing tool could be utilised to print more complex patterns that would not otherwise be readily achieved using the DeeracTM Equator GX1 system. It also showed that the bio-ink could be used to print relatively large-scale patterns with uniform cell density throughout. Importantly, the principle of joining individual 'building blocks' to create a continuous structure was demonstrated. Printing gray-scale 15 (different droplet volumes) or colour (different cell types or concentrations) images was not investigated but it is expected that the software and system will be amenable to this approach. Example 3 - Mass spectrometry of single printed cells 20 Having demonstrated good control over cell patterning, even at the single-cell level, we endeavoured to show that printed cellular arrays could be utilised to subsequently detect and characterise cell-based analytes with single-cell resolution. The study of single cells is at the forefront of analytical chemistry; it provides insights into many important physiological processes that occur in individual cells, as well as the heterogeneity of cell 25 populations, and could aid in the detection and diagnosis of disease. Mass spectrometry (MS) is one powerful method that can provide an expanse of both qualitative and quantitative information about cellular components including proteins, peptides, glycoproteins, lipids and metabolites. Analysis on the single cell level has been facilitated by advances in the sensitivity and spatial resolution of MS techniques. Most commonly 30 these analyses have been implemented by sampling via extraction using a fine capillary, or the ablation of single cells using a focussed laser or ion beam.

H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 32 Liquid Extraction Surface Analysis (LESA) is a relatively new surface sampling technique where a liquid micro-junction is created between the substrate of interest and a conductive tip; extracted analytes are then injected into a nano-electrospray ionisation (nano-ESI) 5 source coupled to a mass spectrometer (MS). LESA-MS has been applied to biological systems previously, for example in the study of the distribution of drugs and their metabolites in tissue sections. We sought to couple this technique with inkjet printing of cell microarrays to enable analysis of individual cells by LESA-MS. In this proof-of principle study, we chose to detect and characterise the lipid profiles of these printed cells. 10 Lipids are ideal targets for direct analysis of single cells owing to their high concentrations near the surface (i.e. in cell membranes). Once considered spectators to biological interactions, lipids are now recognised to possess key roles in biochemical processes and have been implicated in numerous disease states. 15 Three different cell lines (C2C 12, PC12 and L929 cells) were suspended in the bio-ink and printed into micro-arrays on glass slides (Fig. 6A). The number of cells contained in each array region (Fig. 6B) was controlled by changing the number of deposited droplets. The printed droplets dried quickly on the glass slide, which resulted in dehydration of the cells. Techniques have been described to avoid this dehydration (i.e. covering the printed 20 droplets in a thin oil layer, but for the purposes of this experiment, analysis on dehydrated cells was sufficient. The number of cells in each array region was determined post-printing by microscopic visualisation. While this step was time consuming, it was unavoidable due to the random 25 distribution of cells within the bio-ink, and the resultant Poisson distribution of the number of cells in each printed droplet. Printed arrays were subsequently analysed by LESA-MS (Fig. 6C). Microscope images of the same array region before (Fig. 6D) and after (Fig. 6E) LESA sampling showed some remaining cell material, indicating that only a fraction of the cell content was analysed in this case. 30 Lipid profiling from printed cells was first demonstrated on array spots containing up to H:\SXD\Interwoven\NRPortbl\DCC\SXD\5072760_.doc-12/04/203 -33 100 cells (Fig. 7A-C). A variety of lipid classes including cholesterol esters, triacylglycerides, ceramides, glycosphingolipids, phosphatidylcholines (PC) and sphingomyelins (SM) could be detected in printed cells. PC and SM lipids were the most abundant species and were thus the focus of further characterisation. PC and SM lipid 5 profiles were also readily obtained from array spots containing single L929, C2C12 and PC12 cells (Fig. 7D-F). In each case, the spectra from single cells showed the same PC and SM lipids with similar relative abundances observed for array spots containing up to 100 cells. As expected, the peak signal was lower when analysing single cells (Fig. 7). These single cell spectra were reproducible for each cell type, and highlighted the excellent 10 sensitivity of this technique. The PC and SM lipid profiles from each printed cell type provided a characteristic "fingerprint" that allowed differentiation of cell type by principal component analysis. PCA plots produced using the spectral data from array spots containing up to 100 cells 15 (Fig. 8A), or single cells (Fig. 8B) showed clear groupings based on cell type, indicating that each cell type's lipid profile was different and characteristic. Cell culture conditions are known to affect cellular lipid compositions. While PC12 cells were cultured with a different serum composition to the other cell types, C2C12 and L929 cells were cultured under identical conditions and so the different lipid profiles observed for these cell types 20 can be attributed to inherent differences in membrane composition. Non-printed L929 cells analysed by lipid extraction and direct infusion nano-ESI analysis also showed tight PCA grouping with printed L929 cells analysed by LESA-MS (Fig. 8). This suggests that the printing and analysis process did not affect the lipid composition in cell membranes. This demonstrates that the components of the bio-ink formulation did not interfere with the 25 detection of cell-based analytes. The fact that the spectral data from single cell spots of the same cell type (Fig. 8B) were closely grouped suggests that there is little heterogeneity between individual cells for these cell lines. Example 4 - Patterning multiple cell types 30 The fabrication of constructs containing multiple cell types is an important advance in the development of both engineered tissues and cell-based assay devices. To achieve H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 34 functionality in engineered muscle tissues, for example, rapid integration with the host neuromuscular system is required. Several in vitro studies have shown that co-culturing muscle constructs with neural cells enhances muscle differentiation and force generation, and can lead to the formation of functional neuromuscular junctions. Spatial control over 5 these cell-cell interactions could further illuminate the role of innervation in muscle development and enable the fabrication of tissue constructs with enhanced functionality. Additionally, spatially-defined arrays of neuromuscular and other co-cultures can be implemented as tissue test systems for drug discovery and the study of cell behaviour. In this example, work on the inkjet printing of multiple cell types to fabricate patterned co 10 cultures is presented. For cells to remain alive after printing, they must be deposited into a hydrated environment. For experiments investigating the phenotypic response of printed cells, we printed cells directly into cell culture medium. Printing and retaining a defined pattern of 15 living cells, however, requires a different approach. To achieve this, we printed cells onto thin collagen hydrogels, as has been done previously. These hydrogels provided a hydrated environment along with cell attachment cues, so that cell dehydration was prevented during the development of cell-substrate adhesions. We initially formed collagen hydrogels (4 mg/mL) in thin custom-made PDMS wells that were plasma-bonded to cleaned glass 20 slides. This resulted in uneven collagen surfaces due to the meniscus effect, and consequently dehydration in the centre of wells (where there was very little collagen) led to cell death (Fig. 9A). This problem was solved by completely filling thin PDMS wells with collagen to achieve a flat hydrogel surface, and then attaching a replicate PDMS layer to act as a media reservoir. We pre-equilibrated the collagen hydrogels in CPM for ~1 hr, 25 removed all excess media prior to cell printing, and then allowed the printed cell patterns to attach to the collagen for ~1 hr in a humidified incubator prior to adding media on top of the patterns for continued culture. We found that this approach facilitated good retention of printed patterns with no clear reduction of cell viability (Fig. 9B). Some cells were found outside the pattern area (arrow in Fig. 9B), although it was not clear if this was due to 30 misfiring during printing or pattern disruption on media addition.

H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_.doc-12/04/20 3 - 35 Our custom printer housing was amenable to the inclusion of a second print head which allowed us to explore the simultaneous deposition of two different cell types onto collagen hydrogels to create patterned co-cultures (Fig. 10A). C2C12 and PC12 cells were pre stained with different CellTrackerTM dyes prior to printing to allow identification of each 5 cell type in the printed patterns. A relatively crude method was used to align the two print heads. Fig. 10B,C show examples of printed patterns 1 hr after cell printing, following the addition of excess media. Again, several cells were misplaced, either during printing or on media addition, although general pattern fidelity was good. Printed patterns were subsequently cultured under differentiation conditions for 8 days to assess cell-substrate 10 and cell-cell interactions. Cells were fixed and immunostained, using two different antibodies to simultaneously stain desmin (C2C12) and p-III tubulin (PC12) (Fig. 10D,E). The bio-ink did not impede cellular interactions with the collagen substrate and both neural (PC12) and skeletal muscle (C2C12) cells were unimpeded in their ability to express the respective neural (P-III tubulin) and skeletal muscle (desmin) markers and to differentiate 15 normally, as evidenced by the extension of dense neural networks from PC12 cells into surrounding areas populated by skeletal muscle cells (Fig. 10D,E). The results exhibit the utility of the bio-ink in printing multi-cellular structures and subsequent analysis of cell-cell and cell-substrate interactions. Multiple cell types have 20 been patterned previously by inkjet printing, however in previous work cell types were printed sequentially. The process of loading both print heads with cells and aligning the print heads in our work took, on average, 30 minutes. During this time, cells would normally settle significantly within the print heads, but with the cells suspended in the bio ink settling was avoided and cells could be printed at the desired density following 25 completion of pre-printing procedures.

H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 -36 References 1. V. Mironov, et al. Organ printing: tissue spheroids as building blocks, Biomaterials, 2009, 30, 2164-2174. 5 2. R. Gaetani, et al. Cardiac tissue engineering using tissue printing technology and human cardiac progenitor cells, Biomaterials, 2012, 33, 1782-1790. 3. N. R. Schiele, et al. Laser-based direct-write techniques for cell printing, Biofabrication, 2010, 2, 032001. 10 4. M. Gruene, et al. Laser printing of stem cells for biofabrication of scaffold-free autologous grafts, Tissue Eng., Part C, 2011, 17, 79-87. 5. S. J. Moon, et al. Layer by layer three-dimensional tissue epitaxy by cell-laden 15 hydrogel droplets, Tissue Eng., Part C, 2010, 16, 157-166. 6. F. Xu, et al. A droplet-based building block approach for bladder smooth muscle cell (SMC) proliferation, Biofabrication, 2010, 2, 014105. 20 7. W. C. Wilson and T. Boland, Cell and organ printing 1: protein and cell printers, Anat. Rec., 2003, 272, 491-496. 8. T. Xu, J. Jin, C. Gregory, J. J. J. J. Hickman and T. Boland, Inkjet printing of viable mammalian cells, Biomaterials,2005, 26, 93-99. 25 9. R. E. Saunders, J. E. Gough and B. Derby, Delivery of human fibroblast cells by piezoelectric drop-on-demand inkjet printing, Biomaterials, 2008, 29, 193-203. 10. T. C. Burg, C. a P. Cass, R. Groff, M. E. Pepper and K. J. L. Burg, Building off 30 the-shelf tissue-engineered composites, Philos. Trans. R. Soc. London, Ser. A, 2010, 368, 1839-1862.

H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 37 11. M. E. Pepper, V. Seshadri, T. C. Burg, K. J. L. Burg and R. E. Groff, Characterizing the effects of cell settling on bioprinter output, Biofabrication, 2012, 4, 5 011001. 12. S. Parsa, M. Gupta, F. Loizeau and K. C. Cheung, Effects of surfactant and gentle agitation on inkjet dispensing of living cells, Biofabrication, 2010, 2, 025003. 10 13. W. Lee, et al. Multi-layered culture of human skin fibroblasts and keratinocytes through three-dimensional freeform fabrication, Biomaterials, 2009, 30, 1587-1595. 14. C. A. Parzel, M. E. Pepper, T. C. Burg, R. E. Groff and K. J. L. Burg, EDTA enhances high-throughput two-dimensional bioprinting by inhibiting salt scaling and cell 15 aggregation at the nozzle surface, J. Tissue Eng. Regener. Med., 2009, 3, 260-268. 15. B. Derby, Inkjet printing of functional and structural materials: fluid property requirements, feature stability, and resolution, Annu. Rev. Mater. Res., 2010, 40, 395-414. 20 16. K. Arai, et al. Three-dimensional inkjet biofabrication based on designed images, Biofabrication, 2011, 3, 034113. 17. A. R. Liberski, J. T. Delaney and U. S. Schubert, "One cell one well": a new approach to inkjet printing single cell microarrays, ACS Comb. Sci., 2011, 13, 190-195. 25 18. T. Xu, et al. Viability and electrophysiology of neural cell structures generated by the inkjet printing method, Biomaterials, 2006, 27, 3580-3588. 19. 23 X. Cui and T. Boland, Human microvasculature fabrication using thermal inkjet 30 printing technology, Biomaterials, 2009, 30, 6221-6227.

H:\SXD\Interwovn\NRPortbl\DCC\SXD\5072760_ .doc-12/04/20 3 - 38 20. X. Cui, K. Breitenkamp, M. G. Finn, M. Lotz and D. D. D'Lima, Direct human cartilage repair using three dimensional bioprinting technology, Tissue Eng., Part A, 2012, 18, 1304-1312. 5 21. Y. Nishiyama, et al. Development of a three-dimensional bioprinter: construction of cell supporting structures using hydrogel and state-of-the-art inkjet technology, J. Biomech. Eng., 2009, 131, 035001. 22. T. Xu, et al. Characterization of cell constructs generated with inkjet printing 10 technology using in vivo magnetic resonance imaging, J. Manuf. Sci. Eng., 2008, 130, 021013. 23. E. T. Papoutsakis, Media additives for protecting freely suspended animal cells against agitation and aeration damage, Trends Biotechnol., 1991, 9, 316-324. 15 24. M. Krafft, Fluorocarbons and fluorinated amphiphiles in drug delivery and biomedical research, Adv. Drug Delivery Rev., 2001, 47, 209-228. 25. X. Li, et al. Hydrophobic tail length, degree of fluorination and headgroup 20 stereochemistry are determinants of the biocompatibility of (fluorinated) carbohydrate surfactants, Colloids Surf., B, 2009, 73, 65-74. 26. J. A. Barron, D. B. Krizman and B. R. Ringeisen, Laser printing of single cells: statistical analysis, cell viability, and stress, Ann. Biomed. Eng., 2005, 33, 121-130. 25 27. S. Parsa, M. Gupta, F. Loizeau, K. C. Cheung, Effects of surfactant and gentle agitation on inkjet dispensing of living cells, Biofabrication 2, 025003 (2010). 28. N. Quinete et al., Degradation studies of new substitutes for perfluorinated 30 surfactants., Archives of environmental contamination and toxicology 59, 20-30 (2010).

H:\SXD\Interwoven\NRPortbl\DCC\SXD\072760_.doc-12/04/203 - 39 29. R. G. A. Wills, M. J. Watt-Smith, F. C. Walsh, The Use of Fluorocarbon Surfactants to Improve the Manufacture of PEM Fuel Cell Electrodes, Fuel cells 9, 148 156 (2009). 5

Claims (22)

1. A formulation suitable for stable suspension therein of viable cells to form a bio ink for controlled deposition of said cells, comprising an aqueous microgel suspension of one or more gelling biopolymer/s and one or more suitable salts. 1. A bio-ink formulation for controlled deposition of viable cells, said formulation comprising a stable aqueous microgel suspension of one or more gelling biopolymer/s, one or more suitable salts and viable cells of one or more types.

2. The formulation of either claim 1 or claim 2 further comprising one or more suitable surfactant/s.

3. The formulation of claim 3 wherein said surfactant/s are non-ionic polymeric surfactants.

4. The formulation of claim 4 wherein said surfactants are selected from Poloxamer surfactants and fluorinated surfactants.

5. The formulation of claim 5 wherein said Poloxamer surfactant is Poloxamer 188.

6. The formulation of claim 5 wherein said fluorinated surfactant is a fluoroaliphatic ester.

7. The formulation of either claim 1 or claim 2 comprising Poloxamer 188 and fluoroaliphatic ester surfactant. H:\SXD\Interwoven\NRPortbl\DCC\SXD\5072760_ /doc-204/20l3 - 41

8. The formulation of any one of claims 1 to 8 wherein said gelling biopolymer/s are selected from one or more of gellan gum, alginate, fibrin, thrombin, fibronectin, laminin, elastin, proteoglycan chitosan, gelatine, chitin, xanthan gum, guar gum, pectin, collagen, cornstarch, locust bean gum, agar, carageenan, beta-glucan, gum arabic, gum tragacanth, karaya gum, mastic gum, psyllium gum, spruce gum, ghatti gum and glucomannan.

9. The formulation of any one of claims 1 to 8 wherein said gelling biopolymer comprises gellan gum.

10. The formulation of claim 10 wherein gellan gum concentration is from about 0.02 % w/v to about 0.5 % w/v.

11. The formulation of claim 10 wherein gellan gum concentration is about 0.05 % w/v.

12. The formulation of any one of claims 1 to 12 further comprising one or more of amino acids, glucose and vitamins.

13. The formulation of any one of claims 1 to 13 comprising cell culture medium.

14. The formulation of claim 14 comprising Dulbecco's Modified Eagle's Medium (DMEM). H:\SXD\Interwoven\NRPortbl\DCC\SXD\5072760_ /doc-204/20l3 - 42

15. The formulation of any one of claims 1 to 15 comprising one or more of calcium chloride, potassium chloride, sodium chloride, monosodium phosphate and magnesium sulfate.

16. The formulation of any one of claims 1 to 16 wherein microgel particles of said one or more gelling biopolymer/s have dimensions of from about 5prm to about 50pm.

17. The formulation of any one of claims 1 to 17 having viscosity of less than about 12 mPa-s.

18. The formulation of any one of claims 1 to 18 having surface tension of from about 25 mN/m to about 40 mN/m.

19. A method of preparation of a formulation suitable for stable suspension therein of viable cells to form a bio-ink for controlled deposition of said cells, said method comprising dissolving one or more gelling biopolymer/s in hot aqueous medium that comprises one or more suitable salts to form a solution that is cooled under shear conditions to produce a microgel suspension.

20. A method of preparation of a bio-ink formulation for controlled deposition of viable cells, said method comprising dissolving one or more gelling biopolymer/s in hot aqueous medium that comprises one or more suitable salts, to form a solution that is cooled under shear conditions to produce a microgel suspension, to which viable cells of one or more types are added. H:\SXD\Interwoven\NRPortbl\DCC\SXD\5072760_ /doc-204/20l3 - 43

21. A method of controlled deposition of viable cells to a desired locus comprising loading a bio-ink formulation of any one of claims 2 to 19 to a compatible computer controlled printer and directing controlled release of the bio-ink to said desired locus by computer operation.

22. The formulation of claim 1 or claim 2 or the method of any one of claims 20 to 22, substantially as hereinbefore described.