Stem-cell differentiation on polymer fibres

Abstract

The present disclosure broadly relates to methods for stem-cell differentiation on polycaprolactone fibres to which growth factors have been immobilised. The disclosure also relates to growth factor-immobilised polycaprolactone fibres and methods for their preparation.

Description

Stem-cell differentiation on polymer fibresTechnical field

[0001] The present disclosure broadly relates to methods for stem-cell differentiation on polymer fibres.Background

[0002] Any discussion of the prior art throughout this specification should in no way be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Over the last decade tissue engineering has evolved alongside regenerative medicine as a promising approach for the treatment and modelling of currently incurable or poorly treatable diseases and conditions. Since the discovery of mouse embryonic stem-cells (mouse ES cells) in 1981 significant advances have been made to our understanding of how cells communicate with each other and their environment to build, repair and regenerate tissue. However, much of what has been learnt in vitro has been studied in either a two- dimensional or a three-dimensional spheroid environment. Neither of these environments ideally recapitulate the complex anatomical and histological geometry observed in many organs in vivo. Importantly, the ability to understand how biophysical cues (for example, stiffness, porosity, topography, surface geometry, and stress) control cell growth, proliferation and differentiation is undermined in both environments.

[0004] A key tenet of tissue engineering and regenerative medicine is the establishment of a three-dimensional biomaterial complex that recapitulates in vivo tissue structure and function. The rapid generation and characterisation of suitable biomaterials as scaffolds has hastened implementation and integration with candidate cell types. However, producing the desired cell types in sufficient numbers, purity and location in a manner that mimics the relevant tissue structure at the histological and geometric level remains a challenge.

[0005] Many biological structures such as peripheral nerves, blood and lymphatic vessels, skeletal muscle fibres and the tubes of the gastrointestinal and urogenitary tracts exhibit longitudinal geometries with distinct cell types extending along both the length and width of internal linear axes. Modelling these three-dimensional structures in vitro is challenging: the best defined stem-cell differentiation systems are monolayer cultures or organoids generally established using a source of pluripotent stem-cells such as mouse ES cells or inducedpluripotent stem-cells (iPSCs). Organoids are typically spheroidal in structure. However, few organs display this architecture at the level of their multicellular clusters. As such, there is a need for in vitro methods for producing biological structures having longitudinal complexity that recapitulate the anatomical and histological geometry observed in vivo.Summary

[0006] In a first aspect of the disclosure, there is provided a method for preparing differentiated cells from stem-cells, the method comprising:(a) providing a plasma-treated polycaprolactone (PCL) fibre;(b) immobilising a growth factor on the plasma-treated PCL fibre by direct covalent attachment of the growth factor to the plasma-treated PCL fibre so as to provide a growth factor-immobilised plasma-treated PCL fibre;(c) attaching stem-cells to the growth factor-immobilised plasma-treated PCL fibre prepared in step (b); and(d) culturing and differentiating the stem-cells on the growth factor-immobilised plasma- treated PCL fibre in order to provide differentiated cells.

[0007] The following options may be used in conjunction with the first aspect of the disclosure, either individually or in any combination.

[0008] The plasma-treated PCL fibre may be a plasma immersion ion implantation-treated PCL fibre, whereby the growth factor-immobilised plasma-treated PCL fibre is a growth factor- immobilised plasma-immersion ion implantation-treated PCL fibre.

[0009] The plasma-treated PCL fibre may be an individual fibre.

[0010] The plasma-treated PCL fibre may have a three-leaf cross-section, a tubular crosssection or a circular cross-section.

[0011] The plasma-treated PCL fibre may be fabricated by drawing.

[0012] Step (b) may be performed by submerging the plasma-treated PCL fibre into a solution or suspension or emulsion or microemulsion comprising the growth factor.

[0013] In step (b), a plurality of growth factors may be immobilised on the plasma-treated PCL fibre.

[0014] In step (b), the growth factor or growth factors may be immobilised along the entire length of the fibre.

[0015] Step (c) may be performed by seeding the stem-cells on the growth factor-immobilised plasma-treated PCL fibre.

[0016] In step (c), the stem-cells may be attached along the entire length of the fibre.

[0017] The stem-cells may be pluripotent stem-cells.

[0018] The growth factor may be GDNF, BDNF, CTNF, BMP4, VEGF, FGF2, BMP4, SCF, IL3, M-CSF, RANKL, SCF, IL3, G-CSF, SCF, IL2, IL7, dexamethasone, IBMX, insulin, rosiglitazone, SCF, EPO, TPO, VEGF, EPO, FGF2 or IL6, or a mixture or combination of any two or more of these.

[0019] The differentiated cells may be T- and / or B-cells, neurons, vascular endothelial cells, osteoclasts, adipocytes and / or cells of the erythroid, myeloid and lymphoid lineages, or muscle cells, or may be mixtures or combinations of any two or more of these.

[0020] The differentiated cells may form a three-dimensional tissue construct.

[0021] The three-dimensional tissue construct may recapitulate anatomical and histological geometry observed in a biological structure such as nerves, blood and lymphatic vessels, bones, and organs of the gastrointestinal, urinary, reproductive or respiratory tracts.

[0022] In a second aspect of the disclosure, there is provided a PCL fibre to which a growth factor is immobilised by direct covalent attachment.

[0023] The following options may be used in conjunction with the second aspect of the disclosure, either individually or in any combination.

[0024] The PCL fibre may be a plasma treated PCL fibre. It may be a plasma-immersion ion implantation-treated PCL fibre.

[0025] The PCL fibre may be an individual fibre.

[0026] The PCL fibre may have a three-leaf cross-section, a tubular cross-section or a circular cross-section.

[0027] A plurality of growth factors may be immobilised on the PCL fibre.

[0028] The growth factor or growth factors may be immobilised along the entire length of the fibre.

[0029] The growth factor may be GDNF, BDNF, CTNF, BMP4, VEGF, FGF2, BMP4, SCF, IL3, M-CSF, RANKL, SCF, IL3, G-CSF, SCF, IL2, IL7, dexamethasone, IBMX, insulin, rosiglitazone, SCF, EPO, TPO, VEGF, EPO, FGF2 or IL6. Any two or more of these may be immobilised on the fibre.

[0030] In a third aspect of the disclosure, there is provided a method for preparing a PCL fibre to which a growth factor is immobilised by direct covalent attachment, the method comprising reacting a growth factor with a plasma-treated PCL fibre.

[0031] The following options may be used in conjunction with the third aspect of the disclosure, either individually or in any combination.

[0032] The plasma-treated PCL fibre may be a plasma-immersion ion implantation-treated PCL fibre.

[0033] The PCL fibre may be an individual fibre.

[0034] The plasma-treated PCL fibre may have a three-leaf cross-section, a tubular crosssection or a circular cross-section.

[0035] The plasma-treated PCL fibre may be fabricated by drawing.

[0036] The growth factor may be immobilised along the entire length of the fibre.

[0037] The growth factor may be GDNF, BDNF, CTNF, BMP4, VEGF, FGF2, BMP4, SCF, IL3, M-CSF, RANKL, SCF, IL3, G-CSF, SCF, IL2, IL7, Dexamethasone, IBMX, insulin, rosiglitazone, SCF, EPO, TPO, VEGF, EPO, FGF2 or IL6, or a mixture or combination of any two or more of these.

[0038] In a fourth aspect of the disclosure, there is provided a three-dimensional tissue construct comprising differentiated cells located on the fibre, or on an assembly of fibres, of the second aspect.

[0039] The following options may be used in conjunction with the fourth aspect of the disclosure, either individually or in any combination.

[0040] The differentiated cells may comprise more than one type of cell.

[0041] The differentiated cells may be T- and / or B-cells, neurons, vascular endothelial cells, osteoclasts, adipocytes, and / or cells of the erythroid, myeloid and lymphoid lineages, or muscle cells or a mixture or combination of any two or more of these.

[0042] The three-dimensional tissue construct may be a longitudinal structure.

[0043] The three-dimensional tissue construct may be a tubular structure.

[0044] The tubular structure may recapitulate the anatomical and histological geometry observed in a biological structure such as nerves, blood and lymphatic vessels, bones, and organs of the gastrointestinal, urinary, reproductive or respiratory tracts.Description of the Drawings

[0045] Figure 1 : Overall methodology for targeted pluripotent stem-cell differentiation on plasma-treated, growth factor-immobilised PCL fibres. Schematics of the fibre drawing process (Figure 1A) and plasma treatment, growth factor-immobilisation attachment of pluripotent stem-cells and their subsequent growth and differentiation (Figure 1 B). A. Three- leaf cross-sectional shaped PCL fibres were fabricated using a fibre draw tower set to 90 °C. The preform was fed downwards into a furnace at a constant rate of 2 mm / min and fibres were drawn with a capstan wheel at rates of 0.5-1 .5 m / min. PCL fibres were subsequently cut into 5 cm pieces and left in ethanol overnight for surface decontamination. B. Step 1 : Plasma treatment occurred in a custom-built plasma reactor and loaded into a silica tube subjected to pulsed high voltage for 40 min (-6 kV at 500 Hz and 20 ps pulse length). Step 2: To immobilise growth factors onto the surface of the plasma-treated fibre, fibres were submerged into a custom mould containing a gelatin solution with the desired growth factors and left for 1 h. Step 3: Fibres were then placed into a 12-well plate, irradiated with UV light prior to seeding cells ES. Step 4: Medium was changed every other day for 2-3 weeks until end-stage differentiation was reached.

[0046] Figure 2: Results of plasma treatment of PCL fibres. A. Mouse ES cells were seeded on untreated (UT) and plasma-treated (PT) PCL fibres and cultured for 1 week. Phase contrast images were taken 3 weeks later using a Zeiss Axiovert microscope with a 5x objective. B. Cultured mouse ES cells on UT and PT PCL fibres were fixed with 4% PFA, stained with 4',6-diamino-2-phenyl indole (DAPI) and imaged using a Zeiss LSM800 confocal microscope. DAPI (pseudo-coloured yellow) was excited using the 405 nm laser. The number of DAPI+cells were compared between the UT and PT PCL fibres of the same length. A paired Student’s t-test was used to determine statistical significance. ****p <0.0001. C. UT and PT PCL fibres were submerged in a solution containing interleukin 5 (IL5) and incubatedat 37 °C in culture medium for 1 week. Fibres were then stained for IL5 and imaged using a Zeiss LSM800 confocal microscope. Anti-I L5 (pseudo-coloured magenta) was excited using the 565 nm laser. Scale bars for B and C = 1 mm. Data represent mean ± SEM.

[0047] Figure 3: Haematopoietic-promoting growth factors immobilised on plasma-treated fibres induces loss of stem-cell markers and increases haematopoietic marker expression. Mouse ES cells were seeded onto UT and PT, growth factor-immobilised PCL fibres and left to grow for 16 days. Cells were harvested at multiple time points and single-cell suspensions co-stained for pluripotency markers EpCAM and c-kit or co-stained for haematopoietic markers CD45 and CD2. Representative flow cytometric plot of unstained (black), day 0 mouse ES cells (orange) and day 16 differentiated mouse ES cells (blue) stained with A. EpCAM and c-kit or C. CD2 and CD45. A two-way ANOVA was used to compare B. pluripotency and D. haematopoietic markers between UT+ growth factor (GF) and PT+GF fibres across several time points. Data represent mean ± SEM. *** P <0.005, **** P <0.0001.

[0048] Figure 4: Generation of erythroid- and myeloid-derived cell types from mouse ES cells on GF-immobilised, PT PCL fibres. Mouse ES cells were seeded on PT or UT PCL fibres with or without previous immobilisation of SCF (stem-cell factor), EPO (erythropoietin) and TPO (thyroid peroxidase) and were cultured for 3 weeks. This is summarised below:A. After21 days cells were fixed with 4% PFA and stained for erythroid-lineage marker Teri 19 (green), pan-haematopoietic marker CD45 (magenta) and nuclei with DAPI (blue) and imaged using a Zeiss LSM800 confocal microscope. A tile-scan image with a z-stack of at least 200 pm was taken to image the entire PCL fibre. B. Comparisons of CD45+cells and Ter119+cells between groups. Undifferentiated mouse ES cells were seeded on PT or UT PCL fibres with or without previous immobilisation of SCF, IL3 and G-CSF and were cultured for 3 weeks. This is summarised below:C. After 21 days cells were fixed with 4% PFA and stained for CD11b (macrophages, yellow, arrowhead), Gr-1 (granulocytes, magenta, arrow), F-actin stain phalloidin (green) and nuclei stain DAPI (blue) and imaged using a Zeiss LSM800 confocal microscope. A tile-scan image with a z-stack of at least 200 pm was taken to image the entire PCL fibre. D. Comparisons of the percentage of CD11b+cells and Gr-1+cells between groups. Data in B. and D. were analysed using a one-way ANOVA with Tukey’s post hoc analysis. Data represent mean ± SEM. * P <0.05, ** P <0.01 , *** P < 0.005, **** P <0.0001.

[0049] Figure 5: ES cell-derived vascular endothelium and adipocytes generated on GF- immobilised, PT PCL fibres. Mouse ES cells were seeded on PT or UT PCL fibres with or without previous immobilisation of IL6, VEGF, FGF2 and EPO and were cultured for 3 weeks. This is summarised below:A. After 21 days, cells were fixed with 4% PFA and stained for vascular endothelial cadherin (VE-Cadherin; magenta); F-actin, with phalloidin (green) and nuclei, with DAPI (blue) and imaged using a Zeiss LSM800 confocal microscope. A tile-scan image with a z-stack of at least 200 pm was taken to image the entire PCL fibre. Insets show areas of characteristic vascular endothelial cell morphology.Aand # correspond to areas on the fibre that have been enlarged. B. Quantification of staining of VE-Cadherin+ cells directed toward vascular endothelium. Mouse ES cells were seeded on PT or UT PCL fibres with or without previous immobilisation of 3-isobutyl-1 -methylxanthine (IBMX), dexamethasone, insulin, and rosiglitazone and were cultured for 3 weeks. This is summarised below:C. After 21 days cells were fixed with 4% PFA and stained with lipid droplet probe Nile Red (magenta); unsaturated free fatty acid dye Nile Blue (blue), and nuclei, with DAPI (yellow) and imaged using a Zeiss LSM800 confocal microscope. A tile-scan image with a z-stack of at least 200 pm was taken to image the entire PCL fibre. D. Quantification of staining of Nile Red+in cells directed towards adipocytes. Data in B. and D. were analysed using a one-way ANOVA with Tukey’s post hoc analysis. Data represent mean ± SEM. * P <0.05, *** P <0.005, **** p <0.0001.

[0050] Figure 6: ES cell-derived T- and B-cells generated on GF-immobilised, PT PCL fibres. Mouse ES cells were seeded on PT and UT PCL fibres with or without immobilisation of SCF, IL2 and IL7 and were cultured for 3 weeks. This is summarised below:A. and B.: After 21 days cells were fixed with 4% PFA and stained for T-cells, with CD2 (magenta), B-cells, with CD19 (yellow), F-actin with phalloidin (green) and nuclei with DAPI (blue) and imaged using a Zeiss LSM800 confocal microscope. A tile-scan image with a z- stack of at least 200 pm was taken to image the entire PCL fibre. Insets show clusters of A. CD2+cells and B. cells that encapsulated the fibre (arrow). C. Statistical significance of CD19+cells and CD2+cells between groups was tested using a one-way ANOVA with Tukey’s post hoc analysis. Data represent mean ± SEM.* P <0.05, ** P<0.01.

[0051] Figure 7: Formation of osteoid-like structure from Mouse ES cells seeded on GF- immobilised PT PCL fibres. Mouse ES cells were seeded on PT or UT PCL fibres previously immobilised with BMP4, FGF2, SCF, IL3, M-CSF, RANKL and were cultured for 3 weeks. This is summarised below:A. and B. After 21 days cells were fixed with 4% PFA and stained with RANK (magenta); F- actin, with phalloidin (green) and nuclei, with DAPI (blue) and imaged using a Zeiss LSM800 confocal microscope. A tile-scan image with a z-stack of at least 200 pm was taken to image the entire PCL fibre. A. Dotted box shows regions of RANK+cells dispersed within a larger cluster of cells. B. Inset shows a large three-dimensional cluster of RANK+cells resembling an osteoid-like structure C. Comparisons of RANK-positive cells and Nile Red positive cells between groups was determined using a one-way ANOVA with Tukey’s post hoc analysis. Data represent mean ± SEM. **** P <0.0001.

[0052] Figure 8: Generation of neural cells from Mouse ES cells on GF-immobilised, PT PCL fibres. Mouse ES cells were seeded on PT or untreated UT PCL fibres with or without previous immobilisation of GDNF, BDNF, CTNF, BMP4 and VEGF. This is summarised below:After 3 weeks of differentiation, cells were fixed with 4% PFA and stained for A. F-actin, with phalloidin (green), lipid droplets, with Nile Red (magenta) and nuclei, with DAPI (blue) or B. Nestin (Type VI intermediate filaments; magenta) and nuclei with DAPI (blue). A. Top inset shows phal loidin+neuronal projections; bottom inset shows phalloidin* neural rosettes.Aand# correspond to areas on the fibre that have been enlarged. B. Inset and arrows shows networks of Nestin+neural progenitors. Cells were imaged by confocal microscopy using a Zeiss LSM800 confocal microscope. A tile-scan image with a z-stack of at least 200 pm was taken to image the entire PCL fibre. C. Differences in Nestin coverage was determined using a one-way ANOVA with Tukey’s post hoc analysis. Data represent mean ± SEM. ** P value <0.01 , *** P <0.005, **** P <0.0001.Description of embodiments

[0053] The present disclosure relates to an approach that is capable of generating longitudinal biological structures in vitro using stem-cells cultured on PCL fibres coupled to lineage-specific growth factors. The PCL fibres may be plasma-treated PCL fibres. They may be plasmaimmersion ion implantation-treated PCL fibres. The method of the disclosure is capable of effectively recapitulating the relevant in vivo tissue structure at the histological and geometric level. The method offers an efficient way of generating high yields of differentiated cells from stem-cells and provides an improved means of modelling longitudinal biological structures beyond conventional two-dimensional and three-dimensional cell culture.

[0054] In a first aspect of the disclosure there is provided a method for preparing differentiated cells from stem-cells, the method comprising:(a) providing a plasma-treated PCL fibre;(b) immobilising a growth factor on the plasma-treated PCL fibre by direct covalent attachment of the growth factor to the plasma-treated PCL fibre so as to provide a growth factor-immobilised plasma-treated PCL fibre;(c) attaching stem-cells to the growth factor-immobilised plasma-treated PCL fibre prepared in step (b); and(d) culturing and differentiating the stem-cells on the growth factor-immobilised plasma-treated PCL fibre in order to provide differentiated cells.

[0055] A stem-cell is an unspecialised cell that has the ability to self-renew and can give rise to differentiated cells, sometimes referred to as a progenitor cell. Examples include: embryonic stem cells, induced pluripotent stem cells and adult stem-cells such as haematopoietic stem and progenitor cells, neural stem and progenitor cells, mesenchymal stem cells etc.

[0056] The PCL fibre used in step (a) may be a plasma-treated PCL fibre. It may be a plasmaimmersion ion implantation-treated PCL fibre. Step (a) may comprise plasma-treating, e.g. plasma-immersion ion implantation treating, a raw or untreated PCL fibre.

[0057] The PCL fibre may have a three-leaf cross section (as depicted in Figure 1 A), a tubular cross-section or a circular cross-section. It may have one or more grooves extending along the length of the fibre. In the event that it has more than one groove, the grooves may be disposed symmetrically or asymmetrically around the circumference of the fibre. The three leaf cross-section represents a simple means of putting narrow longitudinal grooves on the surface. It is thought that neurons were encouraged to grow in long narrow structures. An advantage of the fibre drawing approach is that it lends itself to fine longitudinal structuring. Sample structures include: essentially circular or tubular structures, optionally with patterning on the surface (one or both surfaces for a tube). The patterning may be essentially longitudinal, and with a transverse dimension typically between 1 m and 100pm. It should be noted that the "V" in the clover leaf allows a variable width. Other suitable structures include a cross-section in the shape of a star or a cogwheel.

[0058] The fact that PCL is an FDA-approved, biocompatible and biodegradable material is an important factor for choosing it. It would be advantageous to increase the porosity of the fibre. The surface properties of the fibre may be important in its functioning. Nanoscale and / or microscale patterning may also improve cell adhesion.

[0059] The PCL fibre may be a drawn PCL fibre. Drawn PCL fibres are advantageous in that they are flexible, easy and inexpensive to fabricate, easy to handle and can be tailored to meet surface topographical requirements. The PCL fibre may, for example, be drawn as described in Farajikhah et al.''2In the method, the PCL fibre may be an individual fibre. It may be an isolated fibre. It may be a single fibre. In other words, the PCL fibre may not be part of an entity having multiple fibres, such as for example a fabric or a fibre bundle. In some embodiments however, there may be multiple fibres. The multiple fibres may be an ordered array of fibres, such as for example a parallel bundle or a side-by-side stack. The ordered array of fibres may be established by placing each fibre in a predetermined position based on the location of another fibre or fibres.

[0060] The PCL fibre is a plasma-treated, e.g. plasma-immersion ion implantation-treated PCL fibre. The plasma-immersion ion implantation may, for example, be performed as described in Zhang et al3and Tan et al.4Plasma-treatment, in particular plasma-immersion ion implantation treatment, of PCL fibres enables covalent attachment of lineage-directing growth factors. It is also thought that the plasma-treatment reduces the hydrophobicity of the fibre. This in turn may facilitate the attachment of growth factors and enhance its biocompatibility.Additionally, such treatment allows for covalent, multiprotein micropatterning, which in turn permits greater control of the cellular microenvironment. This multi-faceted instructional environment helps simulate the in vivo conditions that cells would normally experience as they differentiate into specific cell types, providing a controlled and predictable way to direct the differentiation of pluripotent stem-cells towards a specific lineage.

[0061] In step (b) a growth factor, or a combination of growth factors, is immobilised on the fibre of step (a) by direct covalent attachment. In other words, there is no linker or linkers present between the growth factor and the fibre. Rather, there are covalent bonds between the growth factor molecules and the fibre. Immobilisation may be performed by contacting the fibre with a solution or suspension or emulsion or microemulsion comprising the growth factor. In one embodiment, immobilisation may be performed by contacting the fibre with a solution or suspension or emulsion or microemulsion comprising the growth factor and incubating the fibre in a culture medium. The growth factor may be immobilised along the entire length of the fibre. The density of the immobilised growth factor on the fibre may be controlled by altering the concentration of the growth factor and the length and temperature of the incubation period. In some embodiments, different growth factors may be immobilised in different locations on the fibre. For example, in the case of a fibre with a tubular cross-section, a first growth factor may be immobilised on the outer surface of the fibre and a second growth factor may be immobilised on the inner surface of the fibre.

[0062] The growth factor may be any growth factor that is capable of being immobilised on the fibre. The growth factor may be selected depending on the differentiated cells that are desired to be prepared. Differentiated cells that may be prepared include, but are not limited to, T- and B-cells, neurons, vascular endothelial cells, osteoclasts, adipocytes and cells of the erythroid, myeloid and lymphoid lineages and muscle cells, e.g. smooth muscle cells. Exemplary growth factors include, but are not limited to, GDNF, BDNF, CTNF, BMP4, VEGF, FGF2, BMP4, SCF, IL3, M-CSF, RANKL, SCF, IL3, G-CSF, SCF, IL2, IL7, dexamethasone, IBMX, insulin, rosiglitazone, SCF, EPO, TPO, VEGF, EPO, FGF2 and IL6. Stem-cells that may be used in the method include pluripotent stem-cells, such as for example mouse ES cells of iPSCs or any type of stem-cell (including adult stem cells) or progenitor cells. These may be obtained from an in vivo source or may be grown in vitro. In one embodiment, the stem-cells are mouse ES cells or alternatively stem-cells from other species.

[0063] In step (c) stem-cells are attached to the PCL fibre prepared in step (b). Attachment may be achieved by contacting the stem-cells with the fibre, such as for example by seeding or by contacting a solution or suspension comprising the stem-cells with the fibre. In some embodiments the fibre may be irradiated with UV light prior to attachment of the stem-cells.Following step (c), in step (d) the stem-cells are cultured and differentiated on the fibre in order to provide differentiated cells. Advantageously, the method does not require the use of feeder layers, transgenic cell lines, cell sorters or Matrigel® and hence in an embodiment the method does not use these. The method allows for the generation of differentiated cells from multiple lineages including, for example, neurons, vascular endothelial cells, osteoclasts, adipocytes and cells of the erythroid, myeloid, and lymphoid lineages, and muscle cells, e.g. smooth muscle cells.

[0064] The disclosure also relates to growth factor immobilised PCL fibres. Accordingly, in a second aspect of the disclosure there is provided a PCL fibre to which a growth factor is immobilised by direct covalent attachment. The fibre and growth factors may be as defined and described in connection with the first aspect. The fibre may be a plasma-treated fibre. It may be a plasma-immersion ion implantation-treated fibre. The fibre may be fabricated by drawing.

[0065] The disclosure further relates to a method for preparing growth factor immobilised PCL fibres. Accordingly, in a third aspect of the disclosure there is provided a method for preparing a PCL fibre to which a growth factor is immobilised by direct covalent attachment, the method comprising reacting a growth factor with a plasma-treated, optionally plasma-immersion ion implantation-treated, PCL fibre. Immobilisation may be performed as described above in connection with step (b) of the first aspect.

[0066] The longitudinal geometry cell culture provided by the method of the first aspect allows for the generation of longitudinal three-dimensional tissue constructs that are more closely related to the anatomical and histological geometry observed in vivo as compared to conventional two-dimensional and three-dimensional cell cultures. Accordingly, in a fourth aspect of the disclosure there is provided a three-dimensional tissue construct comprising differentiated cells located on the fibre, or on an assembly of fibres, of the second aspect. The three-dimensional tissue construct may comprise an assembly of fibres in the form an ordered array, such as for example a parallel bundle or a side-by-side stack. The fibres can be assembled and oriented in such a way, depending on the three-dimensional tissue construct that is desired to be prepared, that the resulting three-dimensional tissue construct more closely mimics the anatomical and histological geometry observed in vivo. The cells may be selected from the group consisting of T- and B-cells, neurons, vascular endothelial cells, osteoclasts, adipocytes and cells of the erythroid, myeloid or lymphoid lineages, and muscle cells. In some embodiments the three-dimensional tissue construct is a tubular structure, such as for example a mimic of a biological structure such as a nerve, blood or lymphatic vessel,bone, or organ of the gastrointestinal, urinary, reproductive and respiratory tract or it may recapitulate anatomical and histological geometry observed in such a biological structure.Definitions

[0067] The following are some definitions that may be helpful in understanding the description of the present disclosure. These are intended as general definitions and should in no way limit the scope of the present disclosure to those terms alone, but are put forth for a better understanding of the following description.

[0068] Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

[0069] The terms "a" and "an" are used herein to refer to one or to more than one (i.e. to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element, unless the context requires otherwise.

[0070] In the context of this specification the term "about" is understood to refer to a range of numbers that a person of skill in the art would consider equivalent to the recited value in the context of achieving the same function or result. The term "about" may refer to ±10% of the recited value.

[0071] Any numerical range recited herein is intended to include all sub-ranges of the same numerical precision subsumed within the recited range. For example, a range of 1.0 to 5.0 is intended to include all sub-ranges between (and including) the recited minimum value of 1.0 and the recited maximum value of 5.0, that is, having a minimum value equal to or greater than 1.0 and a maximum value equal to or less than 5.0, such as 2.1 to 4.5. Any maximum numerical limitation recited herein is intended to include all lower numerical limitations subsumed therein and any minimum numerical limitation recited herein is intended to include all higher numerical limitations subsumed therein.

[0072] The term "may" is intended to encompass both positive and negative recitations, and can therefore be interpreted as "may or may not", unless the context indicates otherwise. Thus, for example, the statement "A may be B" should be taken to encompass both "A is B" and "A is not B".

[0073] The term "polycaprolactone" as used herein may be taken to encompass polymers and copolymers comprising monomer units derived from caprolactone. Thus it encompasses bothpolycaprolactone homopolymer and copolymers of caprolactone. In such copolymers, monomeric units derived from caprolactone may comprise at least about 50% of monomer units by number, or at least about 60, 70, 80 or 90% by number. The caprolactone copolymers may be resorbable copolymers.

[0074] Any description of prior art documents herein, or statements herein derived from or based on those documents, is not an admission that the documents or derived statements are part of the common general knowledge of the relevant art.

[0075] For the purposes of description, all documents referred to herein are hereby incorporated by reference in their entirety unless otherwise statedExamples

[0076] The present disclosure is further described below by reference to the following nonlimiting example.

[0077] In this example ES are directed to differentiate to specific cell types on the surface of PCL fibres treated by plasma-immersion ion implantation to which lineage-specifying molecules have been directly, covalently immobilised. The example demonstrates the utility of the method of the disclosure for efficiently generating high yields of the following cell types: neurons, vascular endothelial cells, osteoclasts, adipocytes and cells of the erythroid, myeloid, and lymphoid lineages.Materials and methodsSummary of methodology

[0078] The methodology employed for targeted pluripotent stem-cell differentiation on plasma- treated, growth factor-immobilised PCL fibres is that summarised in Figure 1 . Mouse ES cells were used together with a range of growth factors (see Table 2). A range of cell types were produced (see Table 3).Fabrication of PCL fibres

[0079] PCL fibres with a three-leaf cross sectional shape were fabricated from PCL preforms as previously described.1 2Briefly, three-leaf cross-sectional shaped PCL fibres were fabricated by drawing using a fibre draw tower as shown in Figure 1 . The drop-off temperature was set to 90 °C (the actual hot zone temperature inside the furnace was approximately 52 °C) while the subsequent drawing was performed at 85 °C (hot zone temperature about 48 °C). The preform was fed downwards into a furnace at a constant rate of 2 mm / minute, andfibres were drawn with a capstan wheel at rates of 0.5-1.5 m / minute. The PCL fibres were subsequently cut into 5 cm pieces and left in ethanol overnight for surface decontamination.Plasma-treatment of PCL fibres

[0080] PCL fibres were plasma treated using plasma-immersion ion implantation inside a custom-built plasma reactor as described previously.34PCL fibres were cut into 5 cm pieces and left in ethanol overnight to remove surface contamination. Fibres were then removed from ethanol and air dried before being loaded into an 11 mm inner diameter silica tube that was subjected to pulsed high voltages (-6 kV at 500 Hz frequency and 20 ps pulse length) delivered by a RUP 6-25 generator (GBS Electronics, Germany). Prior to plasma treatment, the silica tube was evacuated to 0.10 Torr. Nitrogen gas was then injected into the silica tube to maintain pressure at 0.70 Torr at upstream and 0.30 Torr at downstream, respectively, with a flow rate of 2.0 standard cm3 / minute (seem). This pressure was maintained for 10 minutes before applying high voltages. All plasma treatments were carried out for 40 minutes.

[0081] Plasma treatment of PCL fibres promotes cell attachment and proliferation as well as the covalent attachment of growth factors.Maintenance of mouse ES cells

[0082] The mouse ES cell line used in this study was 129SVJ.5Mouse ES cells were plated on gelatin-coated 6-well plates, in the absence of a feeder layer, in maintenance medium (Table 1). Addition of 100 U mL'1leukaemia inhibitory factor (LIF) inhibited the differentiation of mouse ES cells and maintained an undifferentiated status. Mouse ES cells were passaged every two days.Table 1 : Media compositionsUsed in neuronal differentiation medium only. DMEM, Dulbecco’s Modified Eagle Medium; FBS, fetal bovine serum; IMDM; Iscove Modified Dulbecco’s Medium; KOSR, knockout serum replacer; LIF, leukemia inhibitory factor.Immobilisation of growth factors on plasma-treated PCL fibres

[0083] Growth factors (Table 2) were pipetted according to the desired cell type (Table 2 and 3) in gelatin and sterile filtered. 50 pL growth factor mix was pipetted into a custom mould in which the PT and UT PCL fibres were placed. Growth factors were left to immobilise for 1 h before fibre samples were transferred to a 24-well plate for addition of mouse ES cells.Table 2: List of growth factors usedBDNF, brain-derived neurotrophic factor; BMP4, bone morphogenetic protein 4; CTNF, ciliary neurotrophic factor; EPO, erythropoietin; FGF2, fibroblast growth factor 2; GCSF, granulocyte-colony stimulating factor; GDNF, glial cell-derived neurotrophic factor; IL 2; interleukin; LIF, leukaemia inhibitory factor; MCSF, macrophage colony-stimulating factor; RANKL, receptive activator of nuclear factor kappa beta ligand; SCF, stem-cell factor; TPO, thyroid peroxidase; VEGF, vascular endothelial growth factor.Table 3: Growth factors used for immobilisation to generate targeted cell types from mouse ES cells.BDNF, brain-derived neurotrophic factor; BMP4, bone morphogenetic protein 4; CTNF, ciliary neurotrophic factor; EPO, erythropoietin; FGF2, fibroblast growth factor 2; GCSF, granulocyte-colony stimulating factor; GDNF, glial cell-derived neurotrophic factor; IL 2; interleukin; LIF, leukaemia inhibitory factor; MCSF, macrophage colony-stimulating factor; RANKL, receptive activator of nuclear factor kappa beta ligand; SCF, stem-cell factor; TPO, thyroid peroxidase; VEGF, vascular endothelial growth factor.Culturing of mouse ES cells on PCL fibres

[0084] Cells were washed once with PBS and 500 pL TrypLE Express added into each well of a 6-well plate, which was placed into the incubator at 37 °C to initiate dissociation of the cells.1.5 mL maintenance medium (Table 1) was then added to quench the TrypLE Express and cells transferred into a 15 mL tube. The cells were centrifuged at 240 g for 5 minutes and the supernatant discarded. After centrifugation, cells were resuspended in 1 mL serum-free differentiation medium (Table 1) and 10 pL pipetted into a haemocytometer for cell counting. For all targeted cell types, 5 x 103cells were pipetted into the wells of a 24-well plate containing 0.5 cm length of the plasma-treated (PT) and untreated (UT) PCL fibres. An additional 1 mL serum-free differentiation medium was added. After 24-48 h, PCL fibres were transferred to a new well and cultured in 1 mL serum-free differentiation medium. Medium was changed every 2-3 days before cells were fixed at the desired time point.Confocal imaging

[0085] Images were taken using a Zeiss LSM 800 confocal microscope coupled with the Zen Blue software package. Antibodies conjugated to a fluorophore were excited according to the fluorophore attached; i.e., at 488 nm, 555 nm and 625 nm. No stain and secondary antibody only controls were included as negative controls. DAPI was excited at 405 nm. Tile-scan, zstack images were taken to visualise cell coverage and density along the entire PCL fibre.Flow cytometry

[0086] Mouse ES cells were seeded onto growth factor-immobilised PCL fibres and left to grow for 16 days. At days 4, 12 and 16 of differentiation, the fibres were transferred to a new dish and cells harvested using TrypLE Express, as described above. After incubation at 37 °C for 5 minutes, TrypLE Express was neutralised using PBS + 0.05% (w / v) BSA (FACS buffer) and centrifuged for 5 minutes at 240 g. Single-cell suspensions of differentiated and undifferentiated mouse ES cells were stained with fluorescently conjugated antibodies (Table 4) at a concentration of 6.5 pg mL1for 1 h on ice. Stained cells were then washed with FACS buffer and centrifuged for 5 minutes at 240 g. The cell pellet was resuspended in FACS buffer containing 0.1 % (v / v) propidium iodide for dead-cell exclusion. Flow cytometry was performed on a Beckman 8 Coulter Gallios flow cytometer and data collected using the Kaluza G software package. Data analysis was performed using FlowJo v10.1.Table 4: Markers used for cell type verificationData analysis

[0087] Data comparing the number of cells attached and the yield of cells differentiated from mouse ES cells are presented as mean ± SEM. GraphPad Prism 7 was used to generate graphs and determine statistical significance. A two-tailed Student’s t-test was used to compare the number of cells attached between PT and UT PCL fibres. A two-tailed one-way ANOVA with Tukey’s post hoc analysis was used to compare the number of cells attachedand the percentage yield of differentiated cells between the PT, UT, and growth factor- immobilised PCL fibres. A P <0.05 was deemed significant for all statistical tests used.ResultsPlasma treatment of PCL fibres promotes cell attachment and proliferation

[0088] To determine whether the PCL fibres are suitable for cell attachment and proliferation, 5 x 103mouse ES cells were seeded into 6-well plates containing UT and PT PCL fibres (Figure 2A). After three weeks of culture, phase contrast microscopy was used to determine cell attachment. Cells seeded on the PT PCL fibre had greater cell attachment, proliferation, and cluster formation compared to the UT PCL control (Figure 2A). In order to determine the number of cells present at the end of the culture period, fibres were fixed with 4% PFA, stained with DAPI and imaged by confocal microscopy (Figure 2B). There was a 3-fold greater attachment of cells on the PT PCL fibre compared to the UT PCL fibre control (7289 ± 752 cells vs 2414 ± 473 cells, P <0.0001).Growth factors covalently attach to plasma-treated PCL fibres

[0089] UT and PT PCL fibres were immersed in a solution containing anti-mouse interleukin- 5 (IL5) and incubated in culture medium for 1 week (Figure 2C). Confocal imaging showed several bright, punctate fluorescent particles indicative of IL5 staining on the PT fibre, while only a single particle was observed on the UT fibre (Figure 2C, dotted box). These findings show the attachment of IL5 one week after the initial immobilisation step.Mouse ES cells undergo spontaneous differentiation on PCL fibres

[0090] In the absence of LIF, mouse ES cells undergo spontaneous differentiation. Here, the inventors assessed whether the PCL fibres have any impact on their spontaneous differentiation. Epithelial cell adhesion molecule (EpCAM) and c-kit are cell-surface proteins used to detect epithelial cells and pluripotency. Mouse ES cells were seeded on UT and PT PCL fibres and cultured for 0, 4, 12, and 16 days. Four days after the differentiation process started, there was a >85% decrease in the expression of EpCAM and c-kit, decreasing further at day 12 (92%) and at day 16 (98%) of differentiation (Figure 3A,B). There were no differences between UT and PT PCL fibres, indicating that plasma treatment does not alter spontaneous differentiation on PCL fibres or influence pluripotency.Generation of haematopoietic cell types

[0091] UT and PT PCL fibres were exposed to a combination of haematopoietic cell-promoting growth factors including stem-cell factor (SCF), erythropoietin (EPO) and thrombopoietin (TPO). Mouse ES cells were subsequently seeded and cultured on these fibres for 16 days. At multiple time points, flow cytometry was used to quantify the number of cells expressingthe pan-haematopoietic marker (CD45) and T-cell marker (CD2) (Figure 3C,D). No surface protein expression of these two markers was seen at days 0 or 4 of culture. On days 12 and 16 of differentiation, cells on the PT PCL fibre with immobilised growth factors (PT+GF) exhibited 9 ± 1 % (SEM) and 18 ± 2% (SEM) surface-protein expression of both CD45 and CD2, respectively (Figure 3D). In contrast, cells grown on the UT PCL fibre with immobilised growth factors (LIT+GF) had significantly fewer cells double-stained for CD45+and CD2+at days 12 and 16 of differentiation; 2 and 6 ± 1% (SEM), respectively (P <0.0001). Taken together, differentiation to haematopoietic cell types was greatest on fibres that were plasma treated and immobilised with growth factors known to stimulate their formation from mouse ES cells.Generation of myeloid derivatives

[0092] The inventors next sought to generate myeloid derivatives by immobilising SCF, IL3 and G-CSF on fibres to generate macrophages (CD11b+) and granulocytes (Gr-1+). Imaging of the PT+GF fibres showed a cluster of cells containing Gr-1+cells (Figure 4C, arrow) as well as cortical membrane staining of CD11b+(Figure 4C, arrowhead). The number of myeloid cells on the PT+GF PCL fibre was greater compared to the PT and UT PCL fibre controls (Figure 4D): at least 75% of cells differentiated on the PT+GF PCL fibre were Gr-1+compared to the PT and UT+GF PCL fibre controls which were only 13.5 ± 8.8% and 1 ± 0.5% Gr-1+, respectively (Figure 4D). In addition, at least 47% of cells that differentiated on the PT+GF PCL fibre were CD11b+compared to the PT and UT+GF PCL fibre controls which were 13.5 ± 8% and 9 ± 6% CD11b+, respectively (Figure 4D).Generation of vascular endothelial cells and adipocytes

[0093] Differentiation of mouse ES cells into vascular endothelial cells was achieved by immobilising IL6, VEGF, FGF2 and EPO on PT PCL fibres. After 22 days, cells were fixed and stained for vascular endothelial cadherin (VE-Cadherin) as well as phalloidin (to show cell morphology) and DAPI (nuclear stain) (Figure 5A). VE-Cadherin staining for vascular endothelium is indicated by the cortical membrane staining in these cells (Figure 5A, magenta). VE-Cadherin was detected on 37.5 ± 4% of all cells on PT+GF fibres (Figure 5B), while <10% of cells stained in the PT PCL fibre control, and <2% in the UT fibres (Figure 5B).

[0094] Similarly, differentiation of mouse ES cells to adipocytes was greatest on PCL fibres that had been previously immobilised with IBMX, dexamethasone, insulin, and rosiglitazone. Cells were stained with Nile Red for lipid droplets (a hallmark of adipogenesis), Nile Blue (a lipophilic dye for unsaturated free fatty acids), and DAPI (nuclear stain) (Figure 5C). There was a significant increase in Nile Red staining for the PT+GF PCL group (63 ± 9%) compared to all other groups, and a significant increase in Nile Red staining between the PT PCL group(33 ± 3%) and the UT PCL groups (8 ± 5% and 5 ± 5%) (Figure 5D). Thus, adipocyte differentiation may be spontaneous in the absence of growth factors. However, the use of growth factors results in a greater yield.Generation of T- and B-cells

[0095] Generation of immune cells from pluripotent stem-cells may provide an avenue for personalised medicine. Here, the inventors generated T- and B-cells from mouse ES cells on PT fibres. SCF, IL2 and IL7 were immobilised on PT PCL fibres prior to cell seeding to promote the differentiation of mouse ES cells to T- and B-cells (CD2 and CD19, respectively). Imaging of the PT+GF PCL fibre showed a cluster of CD2+cells (Figure 6A). CD19+cells were dispersed along the fibre (Figure 6A). However, at higher magnifications the signal was difficult to resolve due to the refractivity of the fibre (Figure 6A).

[0096] On another set of PT PCL fibres with the same growth factors immobilised, a very large cluster of cells formed which encapsulated the fibre (Figure 6B, arrow). CD2 and CD19 staining was observed in cells within the clump as well as cells dispersed along the fibre (Figure 6B).

[0097] The PT+GF PCL fibre had 50 ± 13% of CD2+cells compared to 8 ± 6% (P <0.01) in the PT PCL fibre and 5 ± 3% (P = 0.009) in the UT PCL fibre controls (Figure 6C). Similarly, the PT+GF PCL fibre had the greatest number of CD19+cells (Figure 6C). Differentiated cells on the PT+GF PCL fibre were 61 ± 13% CD19+cells compared to the PT (8 ± 6%, P <0.01) and the UT+GF (11 ± 7%, P <0.01) PCL fibre controls (Figure 6C).

[0098] Thus, differentiation of mouse ES cells to immune-cell derivatives on growth factor- immobilised, PT PCL fibres has been demonstrated.Generation of three-dimensional osteoid-like structure

[0099] To complete the generation of cell types from mesodermal lineages, the inventors generated osteoclasts from mouse ES cells on PT, growth factor immobilised fibres using a combination of BMP4, FGF2, SCF, IL3, M-CSF and RANKL.

[0100] Staining of RANK was used to quantify the percentage of osteoclasts. Confocal microscopy showed RANK+cells within large clusters of cells (Figure 7A, dotted box). On another set of PT+GF PCL fibres, a large, three-dimensional cluster of cells resembling an osteoid-like structure was observed (Figure 7B, inset, dotted region).

[0101] On the PT+GF PCL fibres, 33 ± 3 % cells were RANK+(Figure 7C). This yield was significantly greater than on PT PCL fibres (3 ± 0.3%, P <0.0001) and LIT+GF PCL fibres (0.7 ± 0.4%, P <0.0001) control fibres (Figure 7C).Generation of neurons

[0102] The cell types differentiated above all arise from the mesoderm germ layer. To determine whether mouse ES cells could differentiate to non-mesodermal cell types, such as neurons, growth factors promoting neuronal differentiation from mouse ES cells were immobilised on to PCL fibres. Mouse ES cells were seeded on UT and PT PCL fibres that had been immobilised with or without GDNF, BDNF, CTNF, BMP4 and VEGF. After three weeks of differentiation, cells were fixed and stained for F-actin with phal loidin , lipid droplets with Nile Red and nuclei with DAPI. Confocal images showed neuronal projections (Figure 8A top inset, arrow) and neural rosettes (Figure 8A bottom inset, arrow). Differentiated adipocytes were also present, as shown by Nile Red staining. While this could be indicative of the presence of myelin, further marker analysis is required.

[0103] Another set of fibres were stained for the neural stem-cell / progenitor marker, Nestin (Figure 8B, inset, arrows). There was a two-fold greater percentage of Nestin+coverage on the PT+GF PCL fibre (84 ± 8%) compared to the PT PCL control (40 ± 20%, P <0.005) and 5- fold compared to the UT PCL controls (17 ± 6%, P <0.005 and 10 ± 10%, P <0.0001) (Figure 8C).

[0104] A summary of the findings is shown in Table 5. Table 6 summarises published results and, where possible, the yield of cell types have been compared. All cell types, with the exception of osteoclasts, had similar or greater yields on PT+GF PCL fibres compared to results in two-dimensional culture obtained by other groups (Table 6). It is thought that a longer differentiation period may promote higher yields of osteoclasts (RANK+cells) on the fibres. Collectively, the inventors have demonstrated efficient differentiation of mouse ES cells to cell types derived from different germ layers and spanning multiple lineages.Table 5: Comparison of yields of cell type between plasma-treated and untreated ± growth factors immobilised on PCL fibres#Used in neuronal differentiation#Data presented is mean ± SEM (n = 3 individual experiments)Table 6: Comparison of yields of cell type achieved in this study against the published values'^AThe markers used to identify individual cell types differentiated from mouse ES cells on growth factor-immobilised, plasma-treated PCL fibres were compared (where possible) to published results mouse (m) or human (h) Mouse ES cells or induced pluripotent stem (IPS) cells. The markers used to identify the cell type of interest and the methodology used for differentiation are also shown.**Note: the markers used by Nishikawa et al.'13differ from those used in our study and may account for them differences in yield.*** Percentage is based on Nestin coverage.Discussion

[0105] Pluripotent stem-cells hold great promise in regenerative medicine because of their potential to become any cell type derived from the three germ layers and the possibility of their use in treating a range of currently poorly treatable human conditions, such as type I diabetes, Parkinson’s disease and spinal sever. Here, the inventors have shown that it is possible to generate a high yield of targeted cell types, simply and efficiently, from mouse ES cells seeded on growth factor-immobilised plasma-treated PCL fibres.

[0106] The method of the disclosure overcomes several limitations of standard cell culture. For example, while conventional two-dimensional cell culture is relatively simple and allows for even distribution of nutrients and growth factors, and removal of waste products (resulting in controlled cell growth, differentiation, and proliferation), it fails to recapitulate the complex anatomical and histological geometry observed in vivo, particularly in organs that are longitudinally orientated.

[0107] Recently, organoids have taken centre stage as miniaturised systems for modelling disease and development. In these systems, stem-cells self-organise into three-dimensional structures which more closely resemble the in vivo environment, as measured by gene expression, function and architecture. To date, organoid systems have been developed for (but not limited to) the gut, stomach, kidney, liver, lungs, pancreas, prostate, thyroid, retina, and brain While organoids better recapitulate the heterogeneity and cell distribution found in an organ compared with cells cultured in adherent culture, they fail to recapitulate the gross anatomical geometry of many organs, especially those that are longitudinally orientated. In addition, cells within organoids inherently receive uneven exposure to growth factors over time, often resulting in quiescent and / or necrotic cores reducing their value as models for studying tissue regeneration. With these limitations in mind, the inventors have developed a way of reconstructing tubular structures such as nerves, blood and lymphatic vessels, bones, and organs of the gastrointestinal, urinary, reproductive and respiratory tracts, that does not rely on three-dimensional spheroid culture. The present disclosure combines the ability to: a)develop scaffolds that have a longitudinal geometry using an inexpensive, accessible, biocompatible and biodegradable polymer, and b) control the differentiation of pluripotent stem-cells by immobilising growth factors of interest, minimising the use of cell culture reagents and time.

[0108] In the present disclosure cultures grown on PCL fibres that were left to grow for extended periods of time resulted in the formation of three-dimensional structures without the need for Matrigel® or suspension culture. Specifically, the osteoclast-primed fibre and the lymphoid-primed fibre generated large three-dimensional structures that are likely indicative of bone and lymph node formation, respectively. The extended differentiation time may have allowed cells to self-organise into these structures more closely resembling tissue in vivo. In addition, the yield of cells achieved by the present disclosure is comparable to, or for some cell types greater than, those published using the aforementioned methods (see Table 6).

[0109] Another finding by the inventors was the long-term covalent attachment of biomolecules. The biomolecules could still be detected long after attachment and are presumably still capable of driving directed differentiation. For example, IL2 has a half-life of 3.5 minutes in culture, but it could be detected on the plasma-treated fibres one week after immobilisation.References1. Farajikhah, S., Rukhlenko, I., Stefani, A., Large, M., Chrzanowskic, W., and Fleming, S. (2019). Thermally Drawn Polycaprolactone Fibres with Customised Cross Sections (Paper 103). Australian and New Zealand Conference on Optics and Photonics (ANZCOP19). Proceedings of SPIE 11200.2. Farajikhah, S., Runge, A.F.J., Boumelhem, B.B., Rukhlenko, I.D., Stefani, A., Sayyar, S., Innis, P.C., Fraser, S.T., Fleming, S., and Large, M.C.J. (2021). Thermally drawn biodegradable fibers with tailored topography for biomedical applications. J Biomed Mater Res B Appi Biomater 109, 733-743. 10.1002 / jbm.b.34739.3. Zhang, A., Wong, J.K.U., Redzikultsava, K., Baldry, M., Alavi, S.K., Wang, Z., van Koten, E., Weiss, A., Bilek, M., Yeo, G.C., and Akhavan, B. (2023). A cost-effective and enhanced mesenchymal stem cell expansion platform with internal plasma-activated biofunctional interfaces. Mater Today Bio 22, 100727. 10.1016 / j.mtbio.2023.100727.4. Tan, R.P., Chan, A.H.P., Wei, S., Santos, M., Lee, B.S.L., Filipe, E.C., Akhavan, B., Bilek, M.M., Ng, M.K.C., Xiao, Y., and Wise, S.G. (2019). Bioactive Materials FacilitatingTargeted Local Modulation of Inflammation. JACC Basic Transl Sci 4, 56-71. 10.1016 / j.jacbts.2O18.10.004.5. Kuo, Y.M., Zhou, B., Cosco, D., and Gitschier, J. (2001). The copper transporter CTR1 provides an essential function in mammalian embryonic development. Proc Natl Acad Sci U S A 98, 6836-6841. 10.1073 / pnas.111057298.6. Nishikawa, S.I., Nishikawa, S., Hirashima, M., Matsuyoshi, N., and Kodama, H. (1998). Progressive lineage analysis by cell sorting and culture identifies FLK1+VEcadherin+cells at a diverging point of endothelial and hemopoietic lineages. Development 125, 1747-1757. 10.1242 / dev.125.9.1747.7. Cho, S.K., Webber, T.D., Carlyle, J.R., Nakano, T., Lewis, S.M., and Zuniga-Pflucker, J.C. (1999). Functional characterization of B lymphocytes generated in vitro from embryonic stem cells. Proc Natl Acad Sci U S A 96, 9797-9802. 10.1073 / pnas.96.17.9797.8. Carotta, S., Pilat, S., Mairhofer, A., Schmidt, U., Dolznig, H., Steinlein, P., and Beug, H. (2004). Directed differentiation and mass cultivation of pure erythroid progenitors from mouse embryonic stem cells. Blood 104, 1873-1880. 10.1182 / blood-2004-02-0570.9. Zhang, R., Liu, T.Y., Senju, S., Haruta, M., Hirosawa, N., Suzuki, M., Tatsumi, M., Ueda, N., Maki, H., Nakatsuka, R., et al. (2015). Generation of mouse pluripotent stem cell- derived proliferating myeloid cells as an unlimited source of functional antigen-presenting cells. Cancer Immunol Res 3, 668-677. 10.1158 / 2326-6066.CIR-14-0117.10. Kane, N.M., Meloni, M., Spencer, H.L., Craig, M.A., Strehl, R., Milligan, G., Houslay, M.D., Mountford, J.C., Emanueli, C., and Baker, A.H. (2010). Derivation of endothelial cells from human embryonic stem cells by directed differentiation: analysis of microRNA and angiogenesis in vitro and in vivo. Arterioscler Thromb Vase Biol 30, 1389-1397. 10.1161 / ATVBAHA.110.204800.11. Dani, C., Smith, A.G., Dessolin, S., Leroy, P., Staccini, L., Villageois, P., Darimont, C., and Ailhaud, G. (1997). Differentiation of embryonic stem cells into adipocytes in vitro. J Cell Sci 110 (Pt 11), 1279-1285. 10.1242 / jcs.110.11.1279.12. Cuaranta-Monroy, I., Simandi, Z., Kolostyak, Z., Doan-Xuan, Q.M., Poliska, S., Horvath, A., Nagy, G., Bacso, Z., and Nagy, L. (2014). Highly efficient differentiation of embryonic stem cells into adipocytes by ascorbic acid. Stem Cell Res 13, 88-97. 10.1016 / j.scr.2014.04.015.13. Nishikawa, K., Iwamoto, Y., and Ishii, M. (2014). Development of an in vitro culture method for stepwise differentiation of mouse embryonic stem cells and induced pluripotentstem cells into mature osteoclasts. J Bone Miner Metab 32, 331-336. 10.1007 / s00774-013- 0547-5.14. Kawasaki, H., Mizuseki, K., Nishikawa, S., Kaneko, S., Kuwana, Y., Nakanishi, S., Nishikawa, S.I., and Sasai, Y. (2000). Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 28, 31-40. 10.1016 / s0896- 6273(00)00083-0.15. Perrier, A.L., Tabar, V., Barberi, T., Rubio, M.E., Bruses, J., Topf, N., Harrison, N.L., and Studer, L. (2004). Derivation of midbrain dopamine neurons from human embryonic stem cells. Proc Natl Acad Sci U S A 101 , 12543-12548. 10.1073 / pnas.0404700101.16. Li, W., Sun, W., Zhang, Y., Wei, W., Ambasudhan, R., Xia, P., Talantova, M., Lin, T., Kim, J., Wang, X., et al. (2011). Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors. Proc Natl Acad Sci U S A 108, 8299-8304. 10.1073 / pnas.1014041108.Industrial applicability

[0110] The present disclosure is useful for generating differentiated cells from stem-cells and provides an improved means of modelling longitudinal biological structures.

[0111] Those skilled in the art will appreciate that the disclosure described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the disclosure includes all such variations and modifications. The disclosure also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps, features, compositions and compounds.

Claims

Claims:

1. A method for preparing differentiated cells from stem-cells, the method comprising:(a) providing a plasma-treated polycaprolactone fibre;(b) immobilising a growth factor on the plasma-treated polycaprolactone fibre by direct covalent attachment of the growth factor to the plasma-treated polycaprolactone fibre so as to provide a growth factor-immobilised plasma-treated polycaprolactone fibre;(c) attaching stem-cells to the growth factor-immobilised plasma-treated polycaprolactone fibre prepared in step (b); and(d) culturing and differentiating the stem-cells on the growth factor-immobilised plasma-treated polycaprolactone fibre in order to provide differentiated cells.

2. The method of claim 1 , wherein the plasma-treated polycaprolactone fibre is a plasma immersion ion implantation-treated polycaprolactone fibre, whereby the growth factor- immobilised plasma-treated polycaprolactone fibre is a growth factor-immobilised plasma-immersion ion implantation-treated polycaprolactone fibre.

3. The method of claim 1 or claim 2, wherein the plasma-treated polycaprolactone fibre is an individual fibre.

4. The method of any one of claims 1 to 3, wherein the plasma-treated polycaprolactone fibre has a three-leaf cross-section, a tubular cross-section or a circular cross-section.

5. The method of any one of claims 1 to 4, wherein the plasma-treated polycaprolactone fibre is fabricated by drawing.

6. The method of any one of claims 1 to 5, wherein step (b) is performed by submerging the plasma-treated polycaprolactone fibre into a solution, suspension, emulsion or microemulsion comprising the growth factor.

7. The method of any one of claims 1 to 6, wherein in step (b) a plurality of growth factors are immobilised on the plasma-treated polycaprolactone fibre.

8. The method of any one of claims 1 to 7, wherein in step (b) the growth factor or growth factors is / are immobilised along the entire length of the fibre.

9. The method of any one of claims 1 to 8, wherein step (c) is performed by seeding the stem-cells on the growth factor-immobilised plasma-treated polycaprolactone fibre.

10. The method of any one of claims 1 to 9, wherein in step (c) the stem-cells are attached along the entire length of the fibre.11 . The method of any one of claims 1 to 10, wherein the stem-cells are pluripotent stemcells.

12. The method of any one of claims 1 to 11 , wherein the growth factor is selected from the group consisting of: GDNF, BDNF, CTNF, BMP4, VEGF, FGF2, BMP4, SCF, IL3, M-CSF, RANKL, SCF, IL3, G-CSF, SCF, IL2, IL7, dexamethasone, IBMX, insulin, rosiglitazone, SCF, EPO, TPO, VEGF, EPO, FGF2 and IL6, or a mixture or combination of any two or more of these.

13. The method of any one of claims 1 to 12, wherein the differentiated cells are selected from the group consisting of: T- and / or B-cells, neurons, vascular endothelial cells, osteoclasts, adipocytes and / or cells of the erythroid, myeloid and lymphoid lineages, and muscle cells, or mixtures or combinations of any two or more of these.

14. The method of any one of claims 1 to 13, wherein the differentiated cells form a three- dimensional tissue construct.

15. The method of claim 14, wherein the three-dimensional tissue construct recapitulates anatomical and histological geometry observed in a biological structure selected from the group consisting of: nerves, blood and lymphatic vessels, bones, and organs of the gastrointestinal, urinary, reproductive and respiratory tracts.

16. A polycaprolactone fibre to which a growth factor is immobilised by direct covalent attachment.

17. The polycaprolactone fibre of claim 16, which is a plasma-immersion ion implantation treated polycaprolactone fibre.

18. The polycaprolactone fibre of claim 16 or claim 17, which is an individual fibre.

19. The polycaprolactone fibre of any one of claims 16 to 18, which has a three-leaf crosssection, a tubular cross-section or a circular cross-section.

20. The polycaprolactone fibre of any one of claims 16 to 19, wherein a plurality of growth factors is / are immobilised on the polycaprolactone fibre.21 . The polycaprolactone fibre of any one of claims 16 to 20, wherein the growth factor or growth factors is / are immobilised along the entire length of the fibre.

22. The polycaprolactone fibre of any one of claims 16 to 21 , wherein the growth factor is selected from the group consisting of: GDNF, BDNF, CTNF, BMP4, VEGF, FGF2, BMP4, SCF, IL3, M-CSF, RANKL, SCF, IL3, G-CSF, SCF, IL2, IL7, Dexamethasone, IBMX, insulin, rosiglitazone, SCF, EPO, TPO, VEGF, EPO, FGF2 and IL6, or a mixture or combination of any two or more of these.

23. A method for preparing a polycaprolactone fibre to which a growth factor is immobilised by direct covalent attachment, the method comprising reacting a growth factor with a plasma-treated polycaprolactone fibre.

24. The method of claim 23 wherein the plasma-treated polycaprolactone fibre is a plasmaimmersion ion implantation-treated polycaprolactone fibre.

25. The method of claim 23 or claim 24, wherein the polycaprolactone fibre is an individual fibre.

26. The method of any one of claims 23 to 25, wherein the plasma-treated polycaprolactone fibre has a three-leaf cross-section, a tubular cross-section or a circular cross-section.

27. The method of any one of claims 23 to 26, wherein the plasma-treated polycaprolactone fibre is fabricated by drawing.

28. The method of any one of claims 23 to 27, wherein the growth factor is immobilised along the entire length of the fibre.

29. The method of any one of claims 23 to 28, wherein the growth factor is selected from the group consisting of: GDNF, BDNF, CTNF, BMP4, VEGF, FGF2, BMP4, SCF, IL3, M-CSF, RANKL, SCF, IL3, G-CSF, SCF, IL2, IL7, Dexamethasone, IBMX, insulin, rosiglitazone, SCF, EPO, TPO, VEGF, EPO, FGF2 and IL6, or a mixture or combination of any two or more of these.

30. A three-dimensional tissue construct comprising differentiated cells located on the fibre, or on an assembly of fibres, of any one of claims 16 to 22.31 . The three-dimensional tissue construct of claim 30, wherein the assembly of fibres are in the form of an ordered array.

32. The three-dimensional tissue construct of claim 30 or claim 31, wherein the differentiated cells comprise more than one type of cell.

33. The three-dimensional tissue construct of any one of claims 30 to 32, wherein the cells are selected from the group consisting of: T- and / or B-cells, neurons, vascular endothelial cells, osteoclasts, adipocytes and / or cells of the erythroid, myeloid and lymphoid lineages, and muscle cells, or mixtures or combinations of any two or more of these.

34. The three-dimensional tissue construct of any one of claims 30 to 33, which is a longitudinal structure.

35. The three-dimensional tissue construct of any one of claims 30 to 34, which is a tubular structure.

36. The three-dimensional tissue construct of claim 35, wherein the tubular structure recapitulates anatomical and histological geometry observed in a biological structure selected from the group consisting of: nerves, blood and lymphatic vessels, bones, and organs of the gastrointestinal, urinary, reproductive and respiratory tracts.

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