PROCESS FOR MAKING AN ARRAY

Abstract

There is disclosed a method of making an array for cell assays comprising the step of providing an array of structures on a substrate, each of said structures having a pre-defined topography thereon, and wherein at least one structure has a different topography from at least one other structure.

Claims

1.-57. (canceled)

58. A method of making an array for cell assays comprising the step of providing an array of structures that each extend from a substrate and having a pre-defined topography thereon, wherein at least one structure (A) has a different topography from at least one other structure (B), wherein structure (A) is imprinted separately to structure (B), and wherein structure (A) is capable of being imprinted at an imprinting condition (A.sub.C) that is different to imprinting condition (B.sub.C) of structure (B).

59. The method as claimed in claim 58, wherein the structures are generally elongate and have a proximal end that extends from the substrate and a distal end that is opposite to said proximal end.

60. The method as claimed in claim 59, wherein the topography of said structures is provided on said proximal end.

61. The method as claimed in claim 60, wherein the elongate structures have a longitudinal axis extending from their proximal end to their distal end and wherein said longitudinal axis is generally normal to a planar surface of said substrate.

62. The method as claimed in claim 58, wherein the providing step comprises the step of adhering said structures to said substrate.

63. The method as claimed in claim 62, wherein said adhering step comprises the step of providing an adhesive layer on said substrate before said adhering step.

64. The method as claimed in claim 63, wherein said providing an adhesive layer on said substrate comprises the step of spin-coating a polymer solution onto said substrate.

65. The method as claimed in claim 64, removing the polymer solution while said array of structures are disposed in said polymer solution to form a polymer layer that adheres said structures to said substrate.

66. The method as claimed in claim 58, wherein said topography is formed by formations in the microscale or the nanoscale size range.

67. The method as claimed in claim 66, comprising, before said providing step, the step of forming said topographic formations on a sacrificial substrate.

68. The method as claimed in claim 67, wherein the step of forming said topographic formations on a substrate comprises an imprint lithography step.

69. The method as claimed in claim 68, wherein the step of forming said topographic formations comprises the step of providing the sacrificial substrate with an array of the topographic structures in the microscale or nanoscale size range.

70. The method as claimed in claim 69, wherein the array of microscale or nanoscale topographic structures on said sacrificial substrate are selected from the group consisting of trenches, pillars, gratings, dimples, wells, and other 3-dimensional structures.

71. The method as claimed in claim 69, comprising the step of removing a portion of said microscale or nanoscale topographic structures while attached to said sacrificial substrate.

72. The method as claimed in claim 58, wherein said providing step comprises providing an ordered array of structures on said substrate.

73. The method as claimed in claim 58, wherein said structures are formed from a thermoplastic polymer.

74. The method as claimed in claim 73, wherein said thermoplastic polymer comprises monomers selected from the group consisting of acrylates, phthalamides, acrylonitriles, cellulosics, styrenes, alkyls, alkyls methacrylates, alkenes, halogenated alkenes, amides, imides, aryletherketones, butadienes, ketones, esters, acetals, carbonates and co-monomers thereof.

75. The method as claimed in claim 70, wherein the gratings and dimples are imprinted under an imprinting condition of a temperature of 180 C. at 60 bars for 600 seconds.

76. The method as claimed in claim 70, wherein the pillars are preheated, prior to imprinting, at 180 C. for 120 seconds and imprinted under an imprinting condition of a temperature of 180 C. at 40 bars for 300 seconds.

77. The method as claimed in claim 70, wherein the topographic structures are hierarchically imprinted under a first imprinting condition of a temperature of 180 C. at 60 bars for 600 seconds and a second imprinting condition of a temperature of 140 C. at 40 bars for 300 seconds, wherein the first imprinting condition and the second imprinting condition are performed sequentially.

Description

BRIEF DESCRIPTION OF DRAWINGS

[0085] The accompanying drawings illustrate a disclosed embodiment and serves to explain the principles of the disclosed embodiment. It is to be understood, however, that the drawings are designed for purposes of illustration only, and not as a definition of the limits of the invention.

[0086] FIG. 1A-FIG. 1C are schematic diagrams showing a process for producing an array as described herein.

[0087] FIG. 2 is a photograph showing a working prototype of the array made according to an embodiment of the present disclosure.

[0088] FIG. 3A-FIG. 3J show Scanning Electron Microscopy (SEM) images of the different topographies of an array of the present disclosure.

[0089] FIG. 4A-FIG. 4C shows representative Atomic Force Microscopy (AFM) section analyses of different designs on the array, demonstrating some of the different co-existing height topographies achievable on the array.

[0090] FIG. 5 is a photograph showing a Poly(dimethylsiloxane) (PDMS) replica of the design from an array according to the present disclosure.

[0091] FIG. 6 is a graph showing the percentage of Tull positive vs GFAP positive cells attached on various topographies of an array of the present disclosure.

[0092] FIG. 7 is a graph showing the percentage of BrdU incorporation for Human Mesenchymal Stem cells attached on various topographies of an array of the present disclosure.

[0093] FIG. 8A and FIG. 8B are graphs showing the amount of IL-2 secretion for T cells attached on various topographies of an array of the present disclosure.

[0094] FIG. 9A and FIG. 9B are graphs showing the percentage of CD44.sup.+Cd24.sup.lowESA.sup.+ cancer cells attached on various topographies.

DETAILED DESCRIPTION OF DRAWINGS

[0095] FIG. 1A shows a schematic diagram of a method 201 for making a topographical structure in the form of a grating pattern onto a sacrificial substrate polymer film (steps (A) to (C)).

[0096] In Step (A) of FIG. 1A, there is shown a Nano-Imprint Lithography (NIL) step to form a substrate having a topographical formation in the form of imprints as shown in the inset. NIL is a known technique to persons skilled in the art. In the NIL technique, the mold 1 is pressed into the surface of the substrate in the form of substrate 2, at a temperature of 180 C. and pressure of 60 Bars for 600 s to form a topographical structure on the substrate 2.

[0097] In Step (B) of FIG. 1A, the substrate 2 is cooled before demolding the substrate 2 from the mold 1.

[0098] In Step (C) of FIG. 1A, a portion of the topographical formation is removed from the substrate 2 using a tissue punch 3 to form a structure 4 having a topographical formation thereon for subsequent use in making an array.

[0099] FIG. 1B shows a schematic diagram of a method 202 for fabricating a topographical formation in the form of a pillar pattern onto a sacrificial substrate polymer film (steps (A) to (C)).

[0100] In Step (A) there is shown a Nano-Imprint Lithography (NIL) step to form a substrate having a topographical formation in the form of an ordered array as shown in the inset. In the NIL method step A of FIG. 1B, a preheating step is first performed at 180 C. for 120 s. Then the mold 10 is pressed into the surface of the sacrificial substrate in the form of substrate 20, at a temperature of 180 C. and a pressure of 40 Bars for 300 s to form a topographical formation on the substrate 20.

[0101] In Step (B) of FIG. 1B, the substrate 20 is cooled before demolding the substrate 20 from the mold 10.

[0102] In Step (C) of FIG. 1B, a portion of the topographical formation is removed from the substrate 20 using a tissue punch 30 to form a structure 40 for subsequent use in making an array.

[0103] FIG. 1C shows a schematic diagram of a method 203 for making an array 103 on a substrate 100, a process whereby the structures produced by methods 201 and 202 are adhered to the substrate 100.

[0104] In step (A) of FIG. 1C, PDMS mixed in a ratio of 3:1 (elastomer:curing agent) is first degassed using a vacuum for 15 min to remove any bubbles in the mixture. The PDMS is then spin-coated onto the silicon substrate 100 at 3000 rpm for 30 seconds. The resultant layer of PDMS 101 acts as a bonding material to adhere the structures 4, 40 onto the substrate 100.

[0105] In step (B) of FIG. 1C, the structures 4, 40 having topographical formations (1# to N#) produced by method 201 and 202 are adhered to the PDMS layer 101 in a matrix arrangement. The PDMS is further degassed under vacuum conditions to remove any residual bubbles remaining in the PDMS layer 101. The PDMS layer 101 is then cured by heating the assembled substrate in a vacuum oven at 70 C. for 1 h. This adheres the structures 4, 40 to the substrate firmly 100, resulting in an array 102 according to the present disclosure.

EXAMPLES

[0106] Non-limiting examples of the invention and a comparative example will be further described in greater detail by reference to specific Examples, which should not be construed as in any way limiting the scope of the invention.

Example 1

[0107] This example illustrates a process of the present disclosure for fabricating an array in accordance with the present disclosure using methods 201-203 as depicted in FIG. 1A-FIG. 1C.

[0108] To demonstrate the wide range of different topographies which could be incorporated on a single substrate, a mixture of designs was used. The various topographies include, but are not limited to: gratings, pillars and dimples, curved lens structures as well as complex three-dimensional hierarchical structures, with different resolutions and aspect ratios.

[0109] FIG. 1A and FIG. 1B respectively illustrate a process for producing a topographical formation with a grating pattern and a pillar pattern.

[0110] Thermal nanoimprint lithography was first used to fabricate various topographical formations separately onto individual substrates 2, 20 (for example, Polycarbonate (PC) or Poly (methyl methacrylate film) (PMMA). Referring to FIG. 1A for example, molds 1, 10 bearing the desired topography were pressed into the surface of the substrates 2, 20 at various optimized temperatures, pressures and durations depending on the desired topography, in order to imprint the desired pattern onto the sacrificial substrates 2, 20. For gratings, dimples and curved lens structures, a single imprinting condition was used: 180 C., 60 Bars, 600 s. For delicate structures such as pillar structures, a preheating step was performed at 180 C. for 120 s before the imprinting at 180 C., 40 Bars, 300 s to allow high yield imprinting of such structures. For hierarchical structures, a sequential imprinting process was employed: 180 C., 60 Bars, 600 s for the primary imprinting process; followed by 140 C., 40 Bars, 300 s for the secondary imprinting process.

[0111] Next, the substrates 2, 20 were cooled before they were demolded from their respective molds 1, 10. A Tissue puncher 3, 30 was then used to cut out a uniform 2 mm diameter sized portion of the substrate 2, 20 resulting in the production of structures 4, 40 bearing the desired topographical formations.

[0112] The above process was repeated for different topographical formations, thereby producing N number of topographical formations (indicated as #1-#N, in FIG. 1C).

[0113] FIG. 1C illustrates the process of making an array in accordance with the disclosure. Firstly, PDMS (Sylgard184 mixed in a ratio of 3:1 (elastomer:curing agent)) was degassed in a vacuum for 15 minutes to remove any bubbles in the mixture. The PDMS was then spin-coated onto a silicon substrate 100 at 3000 rpm for 30 s. The resultant layer of PDMS 101 acts as a bonding material for securing the structures to the substrate 100.

[0114] Next, the structures with various topographical formations were assembled manually using tweezers onto the PDMS layer 101 in a matrix arrangement The PDMS layer acts as a bonding layer to adhere the structures onto the substrate. Further degassing was then performed using under vacuum conditions to remove residual bubbles remaining beneath the PDMS layer 101. The PDMS layer 101 was then cured by heating the assembled substrate in a vacuum oven at 70 C. for 1 h. This bonded the topographical structures (#1-#N) firmly onto the substrate 100, thereby producing an array 102 of the current invention.

[0115] FIG. 2 shows a working prototype 300 of an array fabricated using the above process. The arrow points to an example of a 2 mm topographical structure 301. The prototype 300 consists of a matrix of 66 structures 301 having different topographical formations thereon.

[0116] SEM images of some of the different topographical formations that were fabricated are shown in FIG. 3A-FIG. 3J. The topographies shown are: FIG. 3A 2 m line, 2 m space; FIG. 3B 1 m line, 2 m space; FIG. 3C 2 m line, 1 m space; FIG. 3D 250 nm line 250 nm space; FIG. 3E 1 m dimple, 9 m pitch; FIG. 3F 2 m pillars, 12 m pitch; FIG. 3G 500 nm pillars, 10 m pitch; FIG. 3H 250 nm pillars, 500 nm pitch; FIG. 3I 1 m pitch microlens; FIG. 3J 2 m line 2 m space 250 nm line 250 nm space.

[0117] FIG. 4A-FIG. 4C shows a range of topographical formations with different heights, clearly demonstrating the possibility of incorporating topographical formations of different aspect ratios onto a single array. The different topographies shown are: FIG. 4A 250 nm line 250 nm space gratings with 120 nm height; FIG. 4B 250 nm pillars, 500 nm pitch with 250 nm height; FIG. 4C 1 m pitch microlens with 90 nm sag.

[0118] The array of the present disclosure can also be used to form replica copies of the design by using a conventional PDMS casting method for example. A PDMS replica copy 500 of the design from an array is shown in FIG. 5. The replica array 500 has an array of structures 501 having different topographical formations thereon.

Example 2

[0119] In this example, an array as described herein was used to illustrate an application to facilitate fast microscopy based screening to investigate neural precursor cell differentiation enhancement with respect to various topographies.

[0120] Neural progenitor cells were isolated from the hippocampal region of a postnatal day 5 mouse brain. They were expanded on the tissue culture plate and coated with 8 g/ml of natural mouse laminin (Invitrogen Pte. Ltd.) in a proliferation medium composed of DMEM/F12 (Biological Industries), N2 supplements (GIBCO, Invitrogen Pte Ltd) and Penicillin/Streptomycin. Growth factors, basic fibroblast growth factor, bFGF, (GIBCO, Invitrogen Pte. Ltd.), and epidermal growth factor, EGF, (Invitrogen Pte. Ltd.), were added freshly at a final concentration of 20 ng/ml to the expansion medium.

[0121] Polydimethyl siloxane (PDMS) substrates were replicated from an array master mold using a 10:1 ratio of elastomer and curing agent (SYLGARD184 Elastomer Kit, Dow Corning). The elastomer and the curing agent were mixed thoroughly at a ratio of 10:1 and the mixture was degassed before pouring onto the microarray chip. Degassing was repeated subsequently for 45 minutes until there were no residual visible bubbles. The curing was completed by keeping the sample inside a curing oven at a temperature of 65 C. overnight. The replica array was peeled off from the PDMS substrate.

[0122] For neural differentiation, the PDMS replica substrates were plasma treated and coated consecutively with Poly-L-ornithine (Sigma Aldrich) and laminin at 20 g/ml overnight. The cells (Passage 12-17) were detached from the tissue culture plate and seeded on the PDMS substrates at a density of 7,500 cells/cm.sup.2. The differentiation medium during the first phase consisted of DMEM/F12, N2 supplements, Penicillin/Streptomycin and B27 supplements (GIBCO, Invitrogen Pte. Ltd.) with bFGF at a concentration of 5 ng/ml. After 7 days, the differentiation medium was changed to the second phase medium made up of neurobasal medium (Invitrogen Pte. Ltd.) supplemented with B27 and 0.25 N2 and mixed at a ratio of 1:1 with normal Neural Precursor Cell (NPC) expansion medium without N2 supplements and bFGF.

[0123] At the end of the time point, the cells were fixed in 4% paraformaldehyde and immunocytochemical staining was performed with the following primary antibodies with their respective optimized concentrations: anti-rabbit -tubulin III or Tuj1 (Tuj1=neuron-specific class III beta tubulin) antibody (1:600, Sigma Aldrich); anti-mouse glial fibrillary acidic protein, GFAP, antibody (1:600, Chemicon, Millipore Pte Ltd). The secondary antibodies were purchased from Invitrogen Pte Ltd: Alexor Fluor 546 Goat anti-mouse IgG (H+L) and Alexor Fluor 488 Goat anti-rabbit IgG (H+L) both at the concentration of 1:750. Nuclei were counterstained with 4,6-diamidino-2-phenylindole, DAPI, (Invitrogen).

[0124] Imaging was performed with a Leica fluorescent microscope equipped with Q imaging camera (Canada) and with the software, Q capture Pro. From each topographical formation, 7-10 images were captured and image analysis was done using the software, Image J (NIH). Statistical analysis with one way ANOVA was done using Microsoft Excel after compiling the raw data. The percentage of neural precursor cells which differentiated into immature neurons (positive for Tuj1 staining) and astrocytes (positive for GFAP staining) was compared with respect to different topographical formations as well as coverslip control samples (CS). FIG. 6 shows the proportion of immature neurons vs astrocytes attached to different topographical formations. The different topographies compared were: CS: control; I: 2 m line, 2 m space; II: 1 m line, 2 m space; III: 2 m line, 1 m space; IV: 250 nm space, 250 nm space; V: 1 m diameter holes, 9 m pitch; VI: 2 m diameter pillars, 12 m pitch; VII: 500 nm diameter pillars, 10 m pitch; VIII: 130 nm diameter pillars, 400 nm pitch; IX: 2 m line, 2 m space 250 nm line, 250 nm space (hierarchy). The error bars in FIG. 6 represent standard deviations from the mean.

[0125] As can be seen from FIG. 6, a significant difference in the proportion of immature neurons vs astrocytes was observed for topographies I and IX.

Example 3

[0126] In this example, an array as described herein was used to illustrate an application to facilitate fast microscopy based screening to investigate the regulation of Human Mesenchymal Stem Cell proliferation with respect to various topographical formations.

[0127] BrdU incorporation was used as an indicator of DNA synthesis and therefore a proxy for measuring the extent of cell proliferation.

[0128] Polydimethyl siloxane (PDMS) substrates were replicated from an array in the form of a master mold using a 10:1 ratio of elastomer and curing agent (SYLGARD184 Elastomer Kit, Dow Corning). The elastomer and the curing agent were mixed thoroughly at a ratio of 10:1 and the mixture was degassed before pouring onto the array chip. Degassing was repeated subsequently for 45 minutes until there was no residual visible bubble. The curing was completed by keeping the sample inside a curing oven at a temperature of 65 C. overnight. The array was peeled off from the PDMS substrate. The PDMS replica array was then used in the cell assay. Human mesenchymal stem cells were cultured for 7 days on a PDMS stitch array replica and serum was withdrawn from serum-depleted cells for 6 hours on day 5.

[0129] On day 7, BrdU was added into culture medium (1:1000) and the samples were incubated for 4 hours. The cells were then fixed with 4% paraformaldehyde, permeabilized with 0.1% Triton-X100, further incubated in 4N HCl for 10 minutes and then blocked with 10% goat serum and 1% BSA in PBS. BrdU was labeled with mouse anti-human BrdU diluted 1:50 as primary antibody, Alexa-Fluor 546 conjugated goat anti-mouse antibody diluted 1:1000 as secondary antibody, and the nuclei were counterstained with 4,6-Diamidino-2-phenylindole. Samples were then imaged with fluorescence microscope. Acquired fluorescence images were analyzed by Image J software. BrdU incorporation percentage was calculated as the percentage of BrdU positive-stained cells to the total number of cells on each of the different topographical formations of the replicated PDMS substrate.

[0130] FIG. 7 shows the results obtained. The topographies compared were: A: 2 m line, 2 m space; B: 2 m line, 1 m space; C: 1 m line, 2 m space; D: 250 nm line, 250 nm space, 125 nm height; E: 1 m diameter pillars, 9 m pitch; F: 2 m diameter holes, 12 m pitch; G: 500 nm diameter holes, 10 m pitch; H: 130 nm diameter holes, 400 nm pitch; I: 2 m line, 2 m space 250 nm line, 250 nm space (hierarchy); J: 2 m line, 2 m space//250 nm line 250 nm space (hierarchy); K: 2 m line and 2 m space+250 nm diameter holes (hierarchy); L: 250 nm diameter holes, 500 nm pitch; M: 250 nm line, 250 nm space, 250 nm height; N: 1 m diameter concave lens, 1 m pitch; O: 1 m diameter convex lens, 1 m pitch; P: 1.8 m diameter concave lens, 2 m pitch; Q: 1.8 m diameter convex lens, 2 m pitch. The percentage of BrdU incorporation indicates the extent of proliferation. In particular, the higher the percentage of BrdU incorporation, the greater the extent of Human Mesenchymal Stem Cell proliferation.

[0131] From the results it can be seen that narrower line widths of the topographical formation result in decreased stem cell proliferation. Secondly, topographies comprising pillars appear to significantly enhance proliferation. Lastly, the preliminary studies suggested that the line width may exert a more significant effect on the proliferation of Human Mesenchymal Stem Cells compared to the spacing between the lines.

Example 4

[0132] In this example, an array as described herein was used to illustrate an application to facilitate fast microscopy based screening to investigate T-cell activation with respect to various topographical formations.

[0133] An array in accordance with the disclosure was used as a master mold to form replica copies of the array using a material termed hard PDMS: 3.4 g of 7-8% vinylmethylsiloxane, 1 g of 25-30% methylhydrosiloxane, 18 microliters of Platinum(0)-2,4,6,8-tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane), and 10 microliters of 2,4,6,8-Tetramethyl-2,4,6,8-tetravinylcyclotetrasiloxane were mixed and spin coated onto the array. Following hardening, a thick layer of PDMS (Sylgard184, Dow Corning) with a mixture of (elastomer:curing agent of 10:1) was poured in order to allow easy handling of the replica.

[0134] The replica arrays were coated with antibodies to TCR and CD28 (receptors on the T cell surface that upon this binding provide activating and co-stimulatory signals, respectively). Nave CD4+ T cells were isolated from mice and seeded onto these surfaces in RPMI media supplemented with 10% fetal bovine serum. T-cell activation was measured on the basis of secretion of IL-2, a cytokine indicative of T cell activation, over the course of six hours. IL-2 secretion was assayed using a commercial, surface-capture method from Miltenyi Biotech (IL-2 secretion detection kit). Fluorescence associated with IL-2 secretion from this assay was measured on a cell-by-cell basis by microscopy, and compared using Kruskal-Wallis non-parametric approaches.

[0135] FIG. 8A and FIG. 8B illustrate the results obtained from the microscopy screening of a small subset of the topographical formations on the replica array. A-J in FIG. 8A are topographiesA: 2 m line, 2 m space; B: 2 m line, 1 m space; C: 1 m line, 2 m space; D: 250 nm line, 250 nm space, 250 nm height; E: 250 nm line, 250 nm space, 150 nm height; F: 460 nm line, 70 nm space, G: 2 m diameter holes, 12 m pitch; H: 500 nm diameter holes, 10 m pitch; I: 250 nm diameter holes, 500 nm pitch; J: 130 nm diameter holes, 400 nm pitch.

[0136] FIG. 8B illustrates the results obtained using topographies of lens geometry consisting of: 1 m diameter, 1 m pitch concave lens; 1.8 m diameter, 2 m pitch concave lens; 1 m diameter, 1 m pitch convex lens; 1.8 m diameter, 2 m pitch convex lens.

[0137] Comparison of IL-2 secretion across the replica array revealed that T cells are able to recognize and respond differently to different topographies. Most prominently, these T cells showed higher levels of IL-2 secretion on surfaces presenting arrays of 1 m diameter, 1 m pitch lenses than those with 1.8 m diameter, 2 m pitch lens features. In addition, IL-2 secretion was higher on convex lenses rather than concave ones (such as those illustrated in FIG. 8B).

[0138] A further potential extension of this application can be achieved by using this array for immunotherapy. T-cells can be extracted from patients, activated and expanded in cell culture for sufficient number of cells for therapeutic purposes with an array of specific topography. The activated T-cells are injected back to the patient for immunotherapy. As the substrate will not be transferred to the patients, there will be lower side-effects and toxicity. Thus this protocol/system will have limited regulatory barriers and can be brought to clinical trials very rapidly.

Example 5

[0139] This example was used to illustrate a potential application of using the microarray, as a unique, fast and low cost marker for cancer diagnostic applications, since different subpopulations of cancer cells display different adhesion responses to specific topographical formations.

[0140] This example shows that different topographical formations can influence the adhesion response of different sub-populations of cancer cells.

[0141] This example was not carried out on a replica array chip. This example has been included to illustrate that 1) different topographies can influence the response of cancer cells, and 2) since the microarray consists of a variety of different topographical formations, it can be potentially used as a cancer diagnostic tool.

[0142] FIG. 9A shows the results obtained for the breast cancer cell line MCF7 and FIG. 9B shows the results for human primary invasive ductal carcinoma (IDC) cells.

[0143] The results were analysed by ANOVA, which showed that there was a significant difference for all the topographies as compared to the control (flat surface/no pattern surface). For MCF7 cells, t-tests showed a preferential isolation of CD44.sup.+CD24.sup./lowESA.sup.+MCF7 cells on the nano-scale features (*p<0.05, meanSD, n=3) as compared to the micro-scale features (see FIG. 9A).

[0144] In the IDC cell study, significantly more primary cells adhered onto the 250 nm well (#p<0.001, meanSD, n=3) as compared to the control (see FIG. 9B).

Culturing of MCF7 Cells

[0145] The human breast cancer cell line, MCF7, was obtained from the American Type Culture Collection (ATCC) and maintained using Dulbecco's modified Eagle's medium (DMEM, Sigma Aldrich) supplemented with 10% fetal bovine serum (FBS), 0.01 mg/ml bovine insulin, 1% penicillin streptomycin and 1% non-essential amino acids solution. The medium was changed every three days.

Culturing of Invasive Ductal Carcinoma (IDC) Cells

[0146] IDC cells were obtained from the NUH-NUS Tissue Repository. These cells were derived from breast tumor tissue donated by patients under the approval of NUS Institutional Review Board. The cells were maintained in culture using mammary epithelial growth medium (MEGM, Lonza) supplemented with 10% FBS, amphotericin B and gentamicin.

Cell Seeding on Micro- or Nano-Topographical Substrates

[0147] Poly-L-lactic substrates with nanotopography patterns were cut to 1.0 cm by 1.0 cm dimensions. They were submerged in 70% ethanol solution and exposed to ultraviolet light simultaneously for 20 minutes for sterilization.

[0148] Either MCF7 or IDC cells were seeded onto the PLLA films with a seeding density of about 10,000 cells/cm.sup.2.

[0149] After 4 and 24 hours of culturing MCF7 cells on the PLLA films, the cells were fixed with 4% paraformaldehyde and immuno-fluorescence stain for the stem cell markers with standard protocol, using primary antibodies specific for human cell surface markers CD44, CD24 and epithelial cell adhesion molecule (EpCAM or ESA). The primary antibodies used were: rat anti-CD44 FITC (Abcam), mouse anti-CD24 PE (Abcam) and rabbit anti-ESA (Santa Cruz). The secondary antibodies used were: goat anti-mouse Alexa Fluor 546, goat anti-rabbit Alexa Fluor 647 and rabbit anti-rat FITC. The samples were then mounted on glass slides with fluoromount (Sigma) and imaged with a fluorescence microscope at 20, 40 and 63 magnification. Flow cytometry was also performed using the antibodies for CD44, CD24 and ESA. The breast cancer stem cells were identified as the CD44.sup.+/CD24.sup./ESA.sup.+ population.

APPLICATIONS

[0150] The disclosed process may be applied in numerous industrial applications, not least in therapeutic applications, pharmaceutical compositions. The fabrication of different topographies on a single substrate surface may be used for fast and efficient experimental studies investigating the effect of different surface topographies on a wide range of cell types. The array and its PDMS replica can also be used for fast microscopy screening for cell-topography interactions, for immunotherapy and cancer diagnostic applications and immunotherapy applications.

[0151] Advantageously, the disclosed process may be highly reproducible and may be used to simultaneously fabricate a large range of different topographies, such as different heights, dimensions and feature shapes onto a single substrate surface.

[0152] Advantageously, the method described herein provides a low cost fabrication process. In particular, nanoimprint technology, which is a cost-efficient, scalable, high throughput nanoimprinting technique, is used to create the different topographies which are otherwise prohibitively expensive if they are fabricated by conventional electron beam lithography.

[0153] It is a further advantage that the method as described herein provides a biocompatible array.

[0154] It is a further advantage of the method as described herein that the array may be replicated using known casting methods, for example PDMS casting. This provides a cost-effective way of producing replicas of the original array.

[0155] Advantageously, the method as described herein does not encounter problems of resolution limit in providing the different topographies, as nanoimprinting is used to form the topographies, where the limit of resolution is limited by the mold to be used for imprinting.

[0156] It is a further advantage of the method as described herein that the individual topographies of the array can be optimized as different topographies are imprinted separately.

[0157] It is a further advantage of the method as described herein that a dry/etch-free approach is used to form the different topographies on a single substrate.

[0158] Advantageously, the method as described herein permits the use of different types of polymer (for example, PMMA, PC, ETFE and the like) to form the structures having a pre-defined topography thereon.

[0159] Advantageously, the array of the invention can also be used to form replica copies of the array design by using various methods well known in the art, such as a conventional PDMS casting method.

[0160] It will be apparent that various other modifications and adaptations of the invention will be apparent to the person skilled in the art after reading the foregoing disclosure without departing from the spirit and scope of the invention and it is intended that all such modifications and adaptations come within the scope of the appended claims.