Cell culture substrate and method of making thereof

Abstract

A cell culture substrate comprising a substrate having a coating of a plurality of amine functionalized nanoparticles is disclosed. In one embodiment, the amine functionalized nanoparticle is a polymer of an acrylamide monomer, a cross-linker and an amine monomer. There is also provided a method of making the cell culture substrate either by drying the amine functionalized nanoparticles when spread onto the said substrate or by covalent linkages of the substrate with thiol terminated nanoparticles. In addition, there is provided a method of culturing stem cells on the cell culture substrate having a coating of a plurality of the amine functionalized nanoparticles thereon.

Claims

1. A cell culture substrate comprising: a substrate having a coating of a plurality of amine functionalized nanoparticles thereon, wherein said amine functionalized nanoparticle is a polymer of an acrylamide monomer, a cross-linker and an amine monomer, and wherein said amine functionalized nanoparticles have a positive charge.

2. The cell culture substrate according to claim 1, wherein said amine functionalized nanoparticles have a particle size in the range of 50 to 200 nm.

3. The cell culture substrate according to claim 1, wherein said coating is hydrophilic.

4. The cell culture substrate according to claim 1, wherein said polymer has a molecular weight of 100 kDa to 300 kDa.

5. The cell culture substrate according to claim 1, wherein said coating has a thickness of 50 nm to 200 nm.

6. The cell culture substrate according to claim 1, wherein said amine functionalized nanoparticles have thiol terminal groups.

7. A method of making a cell culture substrate comprising a substrate having a coating of a plurality of positively charged amine functionalized nanoparticles thereon, said method comprising: an operation of spreading said plurality of positively charged amine functionalized nanoparticles onto said substrate, wherein said positively charged amine functionalized nanoparticle is a polymer of an acrylamide monomer, a cross-linker and an amine monomer.

8. The method according to claim 7, further comprising an operation of providing said plurality of positively charged amine functionalized nanoparticles in a fluid medium, thereby forming a suspension of said positively charged amine functionalized nanoparticles.

9. The method according to claim 8, wherein said suspended positively charged amine functionalized nanoparticles are present at a concentration from 2 mg/ml to 20 mg/ml.

10. The method according to claim 7, further comprising an operation of drying said positively charged amine functionalized nanoparticles after the nanoparticles are spread onto said substrate.

11. The method according to claim 7, wherein said positively charged amine functionalized nanoparticles have thiol terminal groups.

12. The method according to claim 11, further comprising an operation of functionalizing said substrate with a silane containing compound to form terminal functional groups on the surface of said substrate, wherein said terminal functional groups are capable of forming covalent linkages with said thiol-terminated positively charged nanoparticles.

13. The method according to claim 11, wherein said spreading step comprises an operation of reacting said functionalized substrate with said thiol-terminated positively charged nanoparticles.

14. The method according to claim 7, wherein said cross-linker is present at a concentration of 2.5 mol % to 15 mol %.

15. The method according to claim 7, wherein said amine monomer is present at a concentration of 2.5 mol % to 15 mol %.

16. The method according to claim 7, further comprising an operation of sterilizing said cell culture substrate.

17. The method according to claim 7, further comprising an operation of obtaining positively charged amine functionalized nanoparticles by reacting a plurality of amine functionalized nanoparticles with an acid.

18. A method of culturing stem cells, comprising: culturing stem cells in the presence of a culture medium, the stem cells cultured on a cell culture substrate, wherein said cell culture substrate comprises a substrate having a coating of a plurality of amine functionalized nanoparticles thereon, wherein said amine functionalized nanoparticle is a polymer of an acrylamide monomer, a cross-linker and an amine monomer, and wherein said amine functionalized nanoparticles have a positive charge.

19. The method according to claim 18, further comprising passaging said cultured stem cells.

20. The method according to claim 18, wherein said amine functionalized nanoparticles have thiol terminal groups.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) 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.

(2) FIG. 1 is a, a schematic diagram showing the synthesis of the amine functionalized nanoparticles.

(3) FIG. 2 shows the characterization of the cell culture substrate (the nanoparticle coated cover slips made in accordance with Example 1 below). a, Field Emission Scanning Electron Microscopy images of a cell culture substrate (i) and (ii) after coating. b, Atomic force microscopy images of (i, iv) uncoated (before coating), (ii, v) NP1- and (iii, vi) NP2-coated cover slips, where (ii, iii) were taken immediately after coating and (iii, vi) were taken after washing the coated covers three times with water. c, X-ray photoelectron spectroscopy analysis of (i) blank, (ii) NP1- and (iii) NP2-coated cover slips. Presence of nitrogen peaks confirms the presence of NP1 and NP2 on the surface.

(4) FIG. 3 shows BG01V/hOG hESCs attachment to nanoparticle-coated coverslips according to Example 2. Fluorescent and light images of BG01V/hOG cells grown on a, NP1 and b, NP2 on day 1 and day 7; the nanoparticles were coated onto the cover slip by deposition via evaporation. On day 7, the cells maintained the green fluorescence and formed cell colonies. Scale bar represents 200 μm. c, Cell attachment measured by plate reader for nanoparticle-coated coverslips. NP1 and NP2 NPs were introduced at the concentrations specified, and coated onto the coverslips via deposition by evaporation or via covalent conjugation.

(5) FIG. 4 shows immunostaining of BG01V/hOG cells cultured for 7 days on cover slip coated with NP1 according to Example 2. NP1 nanoparticles were introduced at the concentrations specified and coated via deposition by evaporation. Antibodies against pluripotent markers Nanog, Oct3 and SSEA-4 were used, and DAPI was employed to stain the nucleus of the cells. Scale bar represents 200 μm.

(6) FIG. 5 shows immunostaining of BG01V/hOG cells cultured for 7 days on cover slip coated with NP2 according to Example 2. NP2 nanoparticles were introduced at the concentrations specified and coated via deposition by evaporation. Antibodies against pluripotent markers Nanog, Oct3 and SSEA-4 were used, and DAPI was employed to stain the nucleus of the cells. Scale bar represents 200 μm.

(7) FIG. 6 shows the characterization of HUES-7 cells grown on nanoparticle surface according to Example 2. Light microscopy images of colonies grown on a, NP1 and b, NP2 coatings. The boxed region of the colony in the left image was shown under higher magnification in the right image to show the details of cell attachment on the NP coatings. Immunostaining of the colonies grown on c, NP1 and d, NP2 coatings at passage 10. The colonies were stained with DAPI (D), and with antibodies against Nanog (N), Oct3 (0), Sox2 (SO), SSEA-4 (SS) and TRA 1-60 (T). The boxed region in SS was shown under higher magnification in SS' to illustrate the membranous location of SSEA-4. Scale bar represents 50 μm, except for SS' (5 μm).

(8) FIG. 7 shows Field Emission Scanning Electron Microscopy images of HUES-7 cells grown on a-b, NP1 and c-d, NP2 coatings according to Example 2. The boxed regions in a and c were shown in higher magnification in b and d, respectively. Scale bar represents 50 μm (a, c) and 10 μm (b, d).

(9) FIG. 8 shows pluripotent gene expression analysis of HUES-7 cells grown for 10 passages on a, NP1 and b, NP2 substrates according to Example 2. The fold difference was calculated with the expression value of cells cultured on Matrigel set as 1 fold. The expression values were normalized with respect to GAPDH. Karyotyping by G-banding of HUES-7 cells grown for 10 passages on c, NP1 and d, NP2 substrates. These cells did not show any chromosomal abnormalities after long-term culture on NP1 and NP2 substrates.

(10) FIG. 9 shows pluripotent gene expression analysis of DF 6-99 cells grown for 10 passages on a, NP1 and b, NP2 substrates according to Example 2. The fold difference was calculated with the expression value of cells cultured on Matrigel set as 1 fold. The expression values were normalized with respect to GAPDH.

(11) FIG. 10 shows karyotyping by G-banding of a-b, H1 and c-d, DF 6-99 cells grown for a b 10, c, 12 and d, 13 passages on a, c, NP1 and b, d, NP2 substrates according to Example 2. These cells did not show any chromosomal abnormalities after long-term culture on NP1 and NP2 substrates.

(12) FIG. 11 shows the differentiation potential of the HUES-7 cells cultured on the synthetic surfaces for 10 passages according to Example 2. a, The differentiation potential of the cells cultured on NP1 surfaces were tested by embryoid body formation assay using a ultra-low attachment plates. PCR analysis indicated that the genes associated with the three germ layers were not detected in the undifferentiated cells (lane 1), while these markers were expressed in the differentiated cells (lane 2). b, The pluripotent stem cells cultured on NP1 were implanted subcutaneously into SCID mice. Formation of teratoma was evident with the histology of the explants. Various tissues such as (i) blood vessels, (ii) cartilage, (iii) neurons, (iv) alveolar epithelium, (v) glandular epithelium, (vi) neuronal rosettes, (vii) skin epithelium, (viii) muscle, and (ix) adipocytes have been identified. Scale bar represents 100 μm.

(13) FIG. 12 shows HUES-7 cells cultured on the cover glasses coated with NP1 and NP2 nanoparticles according to Example 2. Cells grown at a confluent stage were photographed with a Nikon digital camera. Cover glasses with a, b, NP1 coating and c, d, NP2 coating a, c, before seeding the HUES-7 cells, and b, d, after culturing to a confluent stage were shown. Scale bar represents 5 mm.

(14) FIG. 13 shows compatibility of the synthetic nanoparticle surfaces with xeno-free media according to Example 3. Three different commercially available xeno-free media were used in the study, and compared with mTesR1 media. HUES-7 cells were seeded onto the NP1 and NP2 surfaces, and images were taken after 2 days of culture in the respective media. Scale bar represents 200 μm.

(15) FIG. 14 is a comparison of HUES-7 and DF 6-99 proliferation on various surfaces according to Example 3. Pluripotent stem cells were cultured on Matrigel (MG), Synthemax (SYN), NP1 and NP2 surfaces a, with mTesR1 media for 6 days, and b, with mTesR2 media for 4 days. The fold proliferation was obtained from cell counting using a hemocytometer, and the number of cells attached after 24 hours of seeding was taken as 1 fold. c, Doubling time for cell proliferation on different substrates under mTesR1 and mTesR2.

(16) FIG. 15 shows extracellular matrix and integrin subunit gene expression analysis according to Example 4. a, Real-time PCR analysis of the extracellular matrix genes associated with the pluripotency of the stem cells were examined for HUES-7 cells cultured on Matrigel, Synthemax, NP1 and NP2 surfaces. b, Expression profile of various integrin subunits was studied for HUES-7 cells grown on Matrigel, Synthemax, NP1 and NP2 surfaces. The expression levels were normalized against GAPDH and expressed as absolute expression levels.

(17) FIG. 16 shows extracellular matrix and integrin subunit gene expression analyses performed on DF 6-99 cells grown on various substrates according to Example 4. a, Real-time PCR analysis of the ECM genes associated with the pluripotency of the stem cells were examined for DF 6-99 cells cultured on Matrigel, Synthemax, NP1 and NP2 surfaces. b, Expression profile of various integrin subunits was studied for DF 6-99 cells grown on Matrigel, Synthemax, NP1 and NP2 surfaces. The expression levels were normalized against GAPDH and expressed as absolute expression levels.

EXAMPLES

(18) Non-limiting examples of the invention 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.

(19) All chemicals, surfactants, reagents and solvents were purchased from Sigma-Aldrich (Missouri of the United States of America) or Polysciences Inc (from Pennsylvania of the United States of America) and used without further purification.

Example 1

(20) General Synthesis of Nanoparticles

(21) Nanoparticles were synthesized by a water-in-oil reverse microemulsion method based on FIG. 1a. Typically, the monomer (16 mmol), crosslinker (1.6 mmol, 10 mol % crosslinking) and the amine-terminated monomer (1.6 mmol, 10 mol %) as mentioned above were dissolved in phosphate buffer (4 mL, 10 mM, pH 7.2) by sonication for 2 minutes to obtain a clear solution. The resulting monomer solution was added to a 250-mL round-bottom flask containing an argon-purged, well-stirred solution of dioctyl sulfosuccinate (AOT or Aerosol AT (3.2 g) and Brij 30 (6.4 mL) in hexanes (100 mL). The mixture was stirred under an argon blanket at room temperature for 10 minutes. The reaction mixture was treated with freshly prepared aqueous ammonium persulfate (65 μL, 10%) and N,N,N′,N′-tetramethylethylenediamine (TEMED) (85 μL) to initiate polymerization, and stirred further at room temperature (or about 20° C.) overnight under argon to ensure complete polymerization. The hexane solvent was evaporated under reduced pressure to obtain a thick residue, which was suspended in absolute ethanol (100 mL) by sonication. The precipitated particles were filtered and thoroughly washed with ethanol (10×100 mL) in an Amicon stirred cell equipped with a Millipore cellulose filter membrane (100 kDa, filtration pressure=1.5 bar nitrogen). The solid material was gently crushed into a fine powder and subjected to air drying. The product was suspended in water (20 mg/mL) and sonicated to obtain a homogeneous solution, filtered through 0.45-mM filter, and purified by ultrafiltration using Millipore cellulose filter (100 kDa) and water (10×50 mL). The concentrated sample was made into an aqueous solution (20 mg/mL) and stored at 4° C. until further use.

(22) Coating of Nanoparticles on the Cover Slips by Evaporation

(23) The purified nanoparticles were suspended in deionized water in appropriate concentrations, and the desired volume (100 L) was spotted on the cover slips placed in a 24-well tissue culture plate. The solution was spread uniformly over the cover slips by gentle shaking, and was dried under air or at 50° C. until a uniform, dry layer was obtained. The coated cover slips were directly used for stem cell culture after sterilization with UV irradiation.

(24) Coating of Nanoparticles on the Cover Slips by Covalent Linkage

(25) The cover slips were washed thoroughly with hexane and ethanol and dried. The surface of cover slips was functionalized with silanization using the appropriate functionalized trimethoxysilyl reagent in dry methanol or heptane to obtain the reactive surface groups such as amine, polyamine, PEGylated amine, carboxyl, thiol, isocyanate, isothiocyanate, urea, etc. For example, amine-functionalized cover slips were reacted with sulfo-succinimidyl-4-[N-maleimidomethyl]cyclohexane-1-carboxylate (sulfo-SMCC) to obtain terminal maleimide groups. In parallel, the amine-functionalized NPs were reacted with iminothiolane hydrochloride to obtain a thiol derivative. The thiol-terminated nanoparticles were reacted with maleimide-functionalized cover slips to obtain the covalently linked nanoparticles on the surface via maleimide chemistry. These cover slips were sterilized under UV irradiation and directly used for stem cell culture.

(26) In terms of supporting the self-renewal of stem cells, the coating of cover slips by nanoparticles achieved via evaporation was as excellent as that obtained via covalent conjugation. The former obviously presented a major advantage of simplicity and practicality giving its ease of application with a suspension of nanoparticles. In addition, the polymeric nanoparticulate suspension would be much less expensive than commercially available materials (such as Matrigel and Synthemax), can be easily and stably stored at room temperature, and is free of immunogenicity issues.

(27) Tables 1 to 3 below show the synthesis of nanoparticles of various compositions and with varying amounts of components.

(28) TABLE-US-00001 TABLE 1 Synthesis of nanoparticles of various compositions and different methods showing particle size, zeta potential and contact angle Evapo- Functional Zeta Contact ration Group Size Potential Angle Conju- Attach- Monomer Cross-Linker (10%) (nm) (eV) (°) gation ment — 102 −2  52 Evapora- tion No —(CH.sub.2).sub.3NH.sub.2  88 +88 38 Both Yes (NP1) —(CH.sub.2).sub.3NH.sub.3Cl 106 +96 24 Both Yes (NP2) —(CH.sub.2).sub.3NH.sub.2 150 +65 41 Both Yes 0 —(CH.sub.2).sub.3NH.sub.2 104 −4  43 Both No —(CH.sub.2).sub.3NH.sub.2  91 +38 51 Both Partial, after 2 days —(CH.sub.2).sub.3NH.sub.2  79 +88 21 Both Partial, after 2 days —(CH.sub.2).sub.3NH.sub.2 102 +44 54 Both Partial —(CH.sub.2).sub.3NH.sub.2 109 −5  34 Both No 0 —(CH.sub.2).sub.3NH.sub.2 137 +46 36 Both Partial

(29) Dynamic light scattering (DLS) showed that NP1 and NP2 have an average size of 88 nm and 150 nm, respectively, and a zeta potential of +88 and +65 mV, respectively (Table 1). The purified nanoparticles (20 mg/mL) were suspended in deionized water and coated on the cover slips by evaporation. The NP1 and NP2 coatings were hydrophilic, with a contact angle of 38° and 24° respectively (Table 1). Nanoparticles NP1 and NP2 were then selected for further experiments.

(30) TABLE-US-00002 TABLE 2 Effect of amine concentration in nanoparticle synthesis —(CH.sub.2).sub.3NH.sub.2 Cross-Linker Functional Evaporation/ Monomer (10%) Group (%) Conjugation Attachment 0 Evaporation No 2.5 Both Yes 5 Both Yes 10 Both Yes 0 25 Both Yes 50 Both Yes 75 Both Yes 100 Both Yes

(31) From Table 2, it can be seen that having the amine functional group is essential to obtaining attachment of the cells on the cell culture substrate.

(32) TABLE-US-00003 TABLE 3 Effect of cross-linkers in nanoparticle synthesis —(CH.sub.2)xNH.sub.2 Cross- (x = 2, 3) Linker Functional Evaporation/ Monomer Cross-linker (%) Group (%) Conjugation Attachment 10 0 Evaporation No 0 10 10 Both Yes 10 10 Both Yes 10 10 Both No 5 10 Both Yes 25 10 Both Yes 0 50 10 Both Yes 5 10 Both Yes, Gel Formation 5 25 Both Yes, Gel Formation

(33) From Table 3, it can be seen that having the amine functional group is essential to obtaining attachment of the cells on the cell culture substrate. In addition, where the cross-linker is negatively charged, having an excess of the amine functional group may aid in attachment.

(34) Characterizations of Nanoparticle Coated Cover Slips

(35) Nanoparticle coated cover slips were characterized as-coated and after they were washed three times with water.

(36) Field Emission Scanning Electron Microscope (FESEM) was conducted with JEOL JSM-7400F. The coverslips were frozen in liquid nitrogen prior to freeze drying to keep the sample's morphology intact. The coverslips were mounted on metal holders and vacuum coated with a platinum layer before FESEM studies. The FESEM images are shown in FIG. 2a where both (i) and (ii) are the images after coating, except that (i) was obtained at low resolution and (ii) was obtained at high resolution. The scale bar of (i) is at ×25 magnification while that for (ii) is at ×30,000 magnification.

(37) Atomic Force Microscopy (AFM) was performed using a tapping mode on a Veeco Multimode AFM AS-12V Scanner with a Bruker RTESPA tip. The coverslips were mounted on metal holders. The center of the coverslips was examined over a length and width of 1-10 μm at a scan rate of 1 Hz. The AFM images are shown in FIG. 2b whereby the surface morphology of the coated cover slips before and after washing with deionized water three times were illustrated. It can be seen that a thin, uniform layer of nanoparticles was achieved on the cover slips after washing.

(38) Theta Probe X-ray Photoelectron Spectroscopy (XPS) was used with monochromatic Al Kα X-rays (hu=1486.6 eV) at an incident angle of 30° with respect to surface normal. Photoelectronswere collected at a take-off angle of 50° with respect to surface normal. The analysis area was about 400 μm in diameter, and the maximum analysis depth was 4 to 10 nm. The XPS analysis is shown in FIG. 2c which confirmed the presence of nitrogen-containing surface species associated with the amine functionalized nanoparticles.

Example 2

(39) Cell Culture

(40) The tested cell culture substrates (such as the nanoparticle coated cover slips) were washed twice with Dulbecco's phosphate-buffered saline (DPBS, obtained from Invitrogen of California of the United States of America). The washed cover slips were placed in 24-well plates, and BG01V/hOG and HUES-7 cells were seeded and cultured for 48 hours in serum-free defined media mTesR1 (from Stem Cell Technologies of Singapore). BG01V/hOG was obtained from LifeTechnologies (California of the United States of America). HUES-7 cell line was obtained from Harvard University (Massachusetts of the United States of America). mTesR1 was prepared by mixing the supplements with basal media and the complete media was aliquoted in 50-mL tubes for regular use.

(41) Matrigel-coated 24-well plates (obtained from BD Biosciences, of New Jersey of the United States of America) and Synthemax plates (obtained from Corning of New York of the United States of America) were used as positive controls. Media was changed every 24 hours and monitored for unwanted differentiation of the cell colonies. The cells grown on the nanoparticle-coated glass cover slips became confluent in 6 to 7 days, and passaged subsequently onto similar nanoparticle-coated cover slips at 1:3 ratio using dispase. Here, the cells grown to confluence on the substrates were gently rinsed twice with knockout Dulbecco's medium (DMEM). Dispase was added to the cells, and incubated at 37° C. for 6 minutes. The cells were further rinsed twice with knockout DMEM. The pluripotent stem colonies were dislodged with a cell scraper, and seeded onto a new substrate at a dilution of 1:5.

(42) The free amine groups on the nanoparticles were made positively charged by treating with dilute hydrochloric acid (1 N) (NP2). Nanoparticles with free amine groups (NP1) and positive charge (NP2) showed similar behavior in cell adhesion and propagation (FIG. 3).

(43) Pluripotency

(44) Initial screening of the materials suitable for the stem cell attachment was conducted with BG01V/hOG cells, which express EmGFP (Emerald Green Fluroscent Protein) under the control of human Oct4 promoter. The expression of GFP is an indication that these cells maintain their pluripotency during culture. FIG. 3a shows that the cells cultured for 1 day and for 7 days on NP1 coating maintained the GFP expression. Moreover, the cells were observed to grow in colonies under light microscope. Similar results were observed for NP2 coating (FIG. 3b).

(45) The measured fluorescence intensity of the cells indicated that the cell attachment was not substantially affected by the nanoparticle concentration introduced or the method of nanoparticle coating (FIG. 3c) Immunostaining with antibodies also confirmed that the cells maintained their pluripotency at Day 7, regardless of the nanoparticle concentration used for both NP1 and NP2 coatings (FIG. 4 and FIG. 5 respectively). This suggested that a thin layer of nanoparticle coating on the glass surface was sufficient to support the attachment and proliferation of hESCs. Excess nanoparticles were found to disintegrate, and could be effectively washed away by water prior to cell seeding.

(46) Apart from the GFP-tagged hESCs, some of the common hESC lines (HUES-7, H1 and H7) and hiPSC lines (hFib2-iPS4 and DF 6-99) have also been examined. H1, H7 and DF 6-99 were purchased from WiCell Research Institute (Wisconsin of the United States of America). HUES-7 was investigated for long-term culture on the nanoparticle surface. The pluripotent stem cell attachment, cell doubling time, viability, colony morphology and pluripotency status were studied at the end of each passage. Light microscopy showed that the colonies adhered to the nanoparticle surfaces with a firm binding. Higher magnification images further illustrated the cellular processes for the cells that were present on the outer ring of the colony (FIG. 6a and FIG. 6b). This was further supported by FESEM images of the colonies (FIG. 7) Immunostaining with antibodies against Nanog, Oct3, Sox2, SSEA-4 and TRA 1-60 showed that the cells grown on NP1 and NP2 coatings for up to 10 passages maintained their pluripotency (FIG. 6c and FIG. 6d). Furthermore, flow cytometry results with pluripotent markers indicated that the cells cultured on NP1 and NP2 substrates were 98% positive for Nanog, Oct3, Sox2 and TRA 1-60 markers (Table 4). Fluorescence-activated cell sorting (FACS) with SSEA-4 antibody showed 71% and 70% positive cells for NP1 and NP2 substrates, respectively. It was remarkable that the percentage of cells that stained positive for the various pluripotent markers were either comparable to or higher than that achieved for Matrigel and Synthemax substrates under similar culture conditions.

(47) TABLE-US-00004 TABLE 4 Comparison of pluripotency by flow cytometry. The percentages of positively labeled cells are indicated in the table. Substrate Nanog Oct3 Sox2 TRA 1-60 SSEA-4 Synthemax 79 68 79 61 76 Matrigel 98 86 92 75 72 NP1 99 98 99 98 71 NP2 99 98 99 98 70

(48) Other cell lines (such as H1, H7 and DF 6-99 cells) cultured for 10 passages on NP1 and NP2 substrates showed similarly excellent pluripotency by FACS (Table 5).

(49) TABLE-US-00005 TABLE 5 H1, H7 and DF 6-99 cells were cultured for 10 passages on NP1 and NP2 surfaces. The percentage of cells detected positive for each antibody was tabulated. Cell Line Substrate Nanog Oct3 SSEA-4 H1 NP1 99 99 91 NP2 99 100 95 H7 NP1 100 100 93 NP2 100 100 93 DF 6-99 NP1 95 99 86 NP2 100 99 68

(50) The culture of HUES-7 cells on NP1 and NP2 were continued for up to 23 passages, and it was found that the pluripotency was not compromised on these nanoparticle substrates. At 23 passages, the cells grown on NP1 and NP2 were 94 to 98% positive for Oct3 and Nanog, and 73 to 83% positive for TRA 1-60 (Table 6). These results clearly illustrated that the stem cells maintained their pluripotency during the long-term culture on the NP surfaces.

(51) TABLE-US-00006 TABLE 6 HUES-7 cells were cultured for 23 passages on NP1 and NP2 surfaces. The percentage of cells detected positive for each antibody was tabulated. Substrate Nanog Oct3 TRA 1-60 NP1 94 98 73 NP2 97 96 83

(52) The gene expression profiles of pluripotent markers such as Oct3, SSEA-4 and Nanog were analyzed for HUES-7 cells grown on Matrigel and nanoparticle surfaces (FIG. 8a and FIG. 8b). For the nanoparticle surfaces, majority of the 14 genes showed an expression level similar to or higher than those for Matrigel. In particular, expression of Lin28 increased by about 3 folds on NP1 and NP2 substrates. Lin28 expression in hiPSC line DF 6-99 grown on nanoparticle surfaces remained similar to that on Matrigel. A consistent increase in the expressions of KLF4, REX1 and GDF3 in HUES7 and DF 6-99 cells on nanoparticle surfaces was also noticed at passage 10 (FIG. 8a, FIG. 8b and FIG. 9). On the other hand, expression of E-cadherin (E-cad) in HUES-7 and DF 6-99 cells grown on nanoparticle surfaces was noticed to be lower than that on Matrigel.

(53) Determination of Chromosomal Abnormalities

(54) Pluripotent stem cells are highly susceptible to chromosomal abnormalities depending on the culture conditions. To determine if any chromosomal abnormalities were introduced by the culture conditions, karyotyping of HUES-7, H1 and DF 6-99 cells that were cultured for ≥10 passages on nanoparticle surfaces was performed. These hESC and iPSC cell lines showed no alterations or modifications in their chromosomes after long-term culture on NP1 and NP2 substrates (FIG. 8c, FIG. 8d and FIG. 10). For long-term culture, the cells were continuously cultured and subcultured onto the specific substrate for at least 10 passages. Typically, each passage was done at day 7 of the culture.

(55) Differentiation Potential

(56) The differentiation potential of the pluripotent stem cells that were cultured for 10 passages on NP1 were tested via embryoid body (EB) formation assay. The differentiated EBs were examined for the expression of the three germ layers, i.e. endoderm, mesoderm and ectoderm specific markers by polymerase chain reaction (PCR) using gene-specific primers (FIG. 11a). Additionally, the differentiation potential was tested by in vivo teratoma assay. When subcutaneously implanted into immunocompromised mice, the passage-10 cells that were grown on NP1 developed into a teratoma that contained cell types of the three germ layers (FIG. 11b). Results obtained from the EB formation assay and teratoma assay illustrated that the cells maintained their potential to differentiate into multiple cell types of the three germ layers.

(57) Pluripotent stem cells cultured on Matrigel and Synthemax were observed to lose their stem cell like nature; this was the case especially for the cells at the edges of the colonies, and sometimes the entire colony. Routinely, researchers avoided these differentiated cells by a process called “colony picking”, in which only the undifferentiated colonies were picked up and expanded for downstream applications. When the pluripotent stem cells were cultured on NP1 and NP2, the differentiated cells would not attach to the substrates, leaving only the pluripotent colonies growing on the substrates. As result, “colony picking” was not required when NP1 and NP2 substrates were employed. This ease of culturing stem cells on the nanoparticulate surface would be a very beneficial feature in practical applications. The uniform growth of HUES-7 cells on NP1 and NP2 substrates could be clearly seen in the digital photographs (FIG. 12).

Example 3

(58) Effect of Culture Media

(59) The culture conditions stated above was mainly performed with mTesR1 media, which contained bovine serum albumin (BSA) as a supplement. To study if the nanoparticle coatings would be useful in translational applications whereby animal-derived components were avoided, the compatability of these synthetic substrates with xeno-free culture conditions were examined. Three xeno-free media for pluripotent stem cell culture were investigated, mTesR2 (from Stem Cell Technologies of Singapore), Nutristem (from Stemgent of Massachusetts of the United States of America) and Essential-8 (E8, from Invitrogen of California of the United States of America).

(60) Examination of initial cell attachment within 24 hours of seeding suggested that both NP1 and NP2 substrates were able to support the xeno-free culture of pluripotent stem cells (FIG. 13). Both nanoparticle coatings performed well, but NP2 performed better in terms of cell attachment compared to NP1 in all the media tested. It was also noted that cell attachment was better with the xeno-free media. A robust attachment of cells was observed within 2 hours of seeding under the xeno-free conditions (data not shown). The pluripotency of cells cultured under xeno-free conditions for 3 passages were examined by FACS (Table 7). E8 media was noticed to support the culture with the highest number of positive cells.

(61) TABLE-US-00007 TABLE 7 Expansion of HUES-7 cells was carried out for 5 passages with xeno-free media, mTesR2, Nutristem and Essential 8. At passage 5, the cells were analyzed for pluripotency using antibodies against Nanog, Oct3 and TRA 1-60 by FACS. The percentage of cells tested positive for each antibody was tabulated. mTesR2 Nutristem Essential 8 Nanog Oct3 TRA 1-60 Nanog Oct3 TRA 1-60 Nanog Oct3 TRA 1-60 NP1 89 98 85 99 89 61 96 90 97 NP2 89 91 81 92 97 77 93 92 85

(62) The proliferation of the pluripotent stem cells on nanoparticulate surfaces was compared with that on Matrigel and Synthemax substrates. Proliferation of pluripotent stem cells on NP1 and NP2 was observed to be slower than that on Matrigel and Synthemax in mTesR1 media (FIG. 14a). However, no significant difference in cell proliferation rate was observed on NP1, NP2, Matrigel and Synthemax in the xeno-free mTesR2 media (FIG. 14b). The doubling time for HUES-7 and DF 6-99 was calculated and tabulated in FIG. 14c. The doubling time was reduced significantly under xeno-free conditions for the NP1 and NP2 surfaces, as compared to that under mTesR1 media.

Example 4

(63) Gene Expression Profile

(64) In order to explore the mechanism involved in pluripotent stem cell culture on nanoparticulate substrates, the expression of the critical ECM genes and integrin subunits involved was analyzed. The expression profiles of ECM genes were similar for HUES-7 and DF 6-99 cell lines. The expression of CTGF (connective tissue growth factor) for cells cultured on NP1 and NP2 was lower compared to that on Matrigel and Synthemax (FIG. 15a and FIG. 16a). ECM proteins regulate the stem cell differentiation; the observed reduced expression of ECM genes could potentially protect the pluripotent stem cells against undergoing differentiation.

(65) Apart from this, expressions of ECM genes were observed to be mostly higher for cells cultured on NP1 surface than that on NP2 surface (FIG. 15a). The mechanism behind these observations still needs to be explored. The ability of the nanoparticulate surfaces to support stem cell's self-renewal would also involve regulation of gene expression from “outside-to-inside” of the cell, as mediated by integrins. To identify the integrins associated with attachment to NP1 and NP2 substrates and their possible role in the gene regulation leading to pluripotency maintenance, the various integrin subunits associated with cells cultured on Matrigel, Synthemax, NP1 and NP2 were profiled. Results indicated that subunits such as ITG-α5, ITG-α6, ITG-αV, ITG-β1 and ITG-β5 were highly expressed for cells cultured all the substrates, with varying levels of expression between the substrates (FIG. 15b). A similar observation was made with DF 6-99 cell line (FIG. 16b).

CONCLUSION

(66) The above examples demonstrated the development and application of synthetic, chemically defined nanoparticulate surfaces for the self-renewal of hESCs and hiPSCs. Cell lines of hESCs (H1 and HUES-7) and hiPSCs (hFib2-iPS4 and DF 6-99) were successfully maintained on amine and positively charged nanoparticulate surfaces for 25 serial passages in defined xeno-free medium. Pluripotent stem cells cultured on nanoparticulate surfaces retained stable doubling time, typical morphology of human pluripotent stem cells, stem cell marker expression, in vitro and in vivo pluripotency and normal karyotype.

INDUSTRIAL APPLICABILITY

(67) The disclosed cell culture substrates can be used for the propagation of stem cells of interest. The disclosed cell culture substrates can be used for large-scale production of human embryonic stem cells and induced pluripotent stem cells for regenerative medicine.

(68) The cell culture substrate can be used for tissue engineering, and where suitable, in drug delivery, implants, biosensors and microfluidic systems.

(69) 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.