MATRICES FOR CELL CULTURE

Abstract

There is provided a cell culture matrix comprising a fungal derived protein. Also provided is a composition comprising the cell culture matrix as described herein, a cell culture system comprising the cell culture matrix as described herein, and a method of forming a cell culture matrix thereof.

Claims

1. A cell culture matrix comprising a fungal derived protein.

2. The cell culture matrix of claim 1 wherein the fungal derived protein comprises a cell-adhesive sequence, optionally, the fungal derived protein retains its primary sequence.

3. The cell culture matrix of claim 1, wherein the fungal derived protein is free of native functions.

4. The cell culture matrix of claim 1, wherein the fungal derived protein forms particles having an average diameter of about 500 nm to about 1600 nm.

5. The cell culture matrix claim 1, wherein the fungal derived protein forms particles having a zeta potential of about −10 mV to about −50 mV.

6. The cell culture matrix of claim 1, wherein the fungal derived protein extract forms a gel and/or a coating, optionally the fungal derived protein extract forms a hydrogel.

7. The cell culture matrix of claim 1, wherein the fungi are non-pathogenic fungi.

8. The cell culture matrix of claim 1, wherein the fungi are from the order of Agaricales, optionally from the division Basidiomycota.

9. The cell culture matrix of claim 1, wherein the fungi are species selected from the group consisting of Flammulina velutipes, Lentinus edodes, and Hypsizigus mannoreus.

10. The cell culture matrix of claim 1, wherein the cell culture matrix further comprises a biomaterial component, optionally the cell culture matrix further comprises one or more selected from the group consisting of alginate, chitosan, chitin, and cellulose fibres.

11. The cell culture matrix of claim 1, where in the cell culture matrix further comprises crosslinkers, optionally the crosslinker is calcium.

12. The cell culture matrix of claim 1, wherein the cell culture matrix forms a coating comprising alginate that is calcium crosslinked.

13. A composition comprising the cell culture matrix of claim 1.

14. A cell culture system comprising a cell culture matrix comprising a fungal derived protein.

15. (canceled)

16. A method of forming a cell culture matrix from a fungus, comprising extracting a fungal derived protein from the fungi.

17. The method of claim 16, wherein the extracting comprises aggregating the fungal derived protein to forma self-aggregating precipitate.

18. The method of claim 16, wherein the extracting comprises aggregating the fungal derived protein by boiling the fungal derived protein and/or incubating the fungal derived protein in a condition that allows self-aggregating precipitate.

19. The method of claim 16, wherein the extracting comprises a fungal derived protein denaturation step that causes aggregation to occur, optionally wherein the denaturation step retains the primary sequence structure of the fungal derived protein, optionally the denaturation step retains the cell-adhesive sequence of the fungal derived protein.

20. The method of claim 16, wherein the extracting comprises an aggregation step comprising heat treating the fungal derived protein for about 1 min to about 30 mins, optionally the aggregation step comprises heating the fungal derived protein from about 50° C. to about 350° C.

Description

DETAILED DESCRIPTION OF FIGURES

[0067] Example embodiments of the disclosure will be better understood and readily apparent to one of ordinary skill in the art from the following discussions and if applicable, in conjunction with the figures. It should be appreciated that other modifications related to structural and/or functional changes may be made without deviating from the scope of the invention. Example embodiments are not necessarily mutually exclusive as some may be combined with one or more embodiments to form new exemplary embodiments. The example embodiments should not be construed as limiting the scope of the disclosure.

[0068] FIG. 1 shows the comparison of fresh extract from F. velutipes and extract after standing at room temperature (25° C.) for several days. Spontaneous precipitation had occurred to give a suspension of white particulates.

[0069] FIG. 2 shows photos of boiling of extracts from mushroom extracts yield particulate suspensions.

[0070] FIG. 3 shows photos of extracts from mushroom extracts. (A) Particles can be dispersed easily in water to give a particulate suspension. Light microscope images of (B) clusters of particulates in boiled solution; (C) uniformly dispersed particles after isolation by centrifugation and brief vortexing.

[0071] FIG. 4 shows microscopic images of cells in F. velutipes particles. (A) C2C12 cells entrapped in a matrix of F. velutipes particles upon mixing 25 μL of a cell suspension in DMEM media (with serum) and 10 μL of particle suspension. Cells appear in different focal planes within the gel; Live-dead assay after 4 hours showing good viability of the entrapped cells: (B) bright field image; (C) Calcein staining for live cells, shows the spreading of cells within the gel matrix; (D) Ethidium homodimer staining for dead cell nuclei.

[0072] FIG. 5 shows microscopic images of cells in L. edodes particles. (A) C2C12 cells entrapped in a matrix of L. edodes particles upon mixing 10 μL of particle suspension and 50 μL of a cell suspension in DMEM media (with serum). Live-dead assay after 7 hours showing good viability of the entrapped cells: (B) bright field image; (C) Calcein staining for live cells; (D) Ethidium homodimer staining for dead cell nuclei. The gel matrix picks up some of the red stain.

[0073] FIG. 6 shows light microscope images of MCF7 cells seeded on F. velutipes particle-coated and non-coated plates after 20 h of culture.

[0074] FIG. 7 shows photos of various fibres drawn from F. velutipes particle. (A) Fibre drawn from the interface between chitosan and alginate-F. velutipes particle solutions. Note the distinct interface boundary between the two solutions; (B) Fibre being washed in deionized water; (C) Fibre exhibits a uniform appearance and dispersion of particles.

[0075] FIG. 8 shows microscopic behaviours of cell lines on F. velutipes particles. Good attachment of C2C12 mouse myoblast cell line on chitosan-alginate fibre incorporated with F. velutipes protein particles. Panel A: At lower magnification; Panel B: At higher magnification, showing cells packed closely on fibre surface; Panel C: At higher magnification, showing good cell spreading with formation of cellular processes.

[0076] FIG. 9 shows light microscope images of MCF7 cells seeded on mushroom extract-coated and non-coated plates at 18 h.

[0077] FIG. 10 shows light microscope images of primary human skeletal muscle cells seeded on mushroom extract-coated (A, C) and non-coated (B, D) plates after 3 h and 18 h of culture.

[0078] FIG. 11 shows ultraviolet subtraction spectrum of extract from F. velutipes before and after spontaneous precipitation. A.sub.max at 276 nM indicates protein being precipitated out of the solution.

[0079] FIG. 12 shows the addition of phosphate buffered saline (PBS) to L. edodes particulate suspension leads to particle aggregation and formation of a particulate gel. (A,B) Prior to adding PBS; (C,D) Immediately after addition of PBS.

[0080] FIG. 13 shows the formation of a gel upon addition of 11 mg/mL F. velutipes particles to C2C12 cell suspension in DMEM media, after 10 mins.

[0081] FIG. 14 shows C2C12 cells cultured in F. velutipes particulate gel over 2 days. (a) bright field image; (b) Calcein staining for live cells; (c) Ethidium homodimer staining for dead cell nuclei.

[0082] FIG. 15 shows the FT-IR spectra of cell-adhesive particles precipitated from F. velutipes (A, B) and L. Edodes (C). The y-axis and x-axis are % absorbance and wavelength (cm-1) respectively.

[0083] FIG. 16 shows particulate gel from F. velutipes promotes muscle cell alignment.

[0084] FIG. 17 shows water extract of F. velutipes (A) before, and (B) after boiling; (C) Isolated precipitate; (D) Precipitate dispersed uniformly in water forming a white, milky suspension; (E) Addition of media to particle suspension results in a particulate gel.

[0085] FIG. 18 shows photographs of (A) alginate spheres incorporating FV particles; and (B) attachment of C2C12 mouse myoblasts to FV-alginate incorporated spheres.

[0086] FIG. 19 shows photographs of (A) FV-alginate-coated chitosan microspheres; and (B) attachment of C2C12 cells to the microspheres.

[0087] FIG. 20 shows photographs of (A) C2C12 cells attached to FV particle incorporated water-soluble chitin (WSC)-alginate fibers after 1 h; and (B) cells observed to have spread on the fiber surface at 24 h.

[0088] FIG. 21 shows photographs of (A) attachment of C2C12 cells on FV-alginate coated cellulose fibers; and (B) cells had spread and elongated along the axis of the fibers after 2 days.

[0089] FIG. 22 shows photographs of (A) planar constructs from chitosan and sodium alginate; (B) immersion of the constructs in FV-alginate suspension; (C) coagulation of FV-alginate suspension to form films; and C2C12 cells seeded on the films that showed (D) good attachment after 1 h, and (E) spreading after 1 day.

EXPERIMENTAL SECTION

[0090] Materials and Methods

[0091] Materials

[0092] The three fungal species, Flammulina velutipes, Lentinus edodes and Hypsizigus marmoreus are edible mushrooms which were procured from local retailers. 2-amino-2(hydroxymethyl)-1,3-propanediol hydrochloride (TRIS-HCl), magnesium chloride hexahydrate (MgCl.sub.2.6H.sub.2O) and 2-morpholinoethanesulfonic acid (MES) were obtained from Merck, Darmstadt, Germany. Breast cancer cell line, MCF7 and mouse myoblast cell line, C2C12 were obtained from American Type Culture Collection (ATCC), VA, USA. Primary human skeletal muscle cells were obtained from Promocell GmbH, Heidelberg, Germany.

[0093] Preparation of Fungal Extracts

[0094] The mushroom pileus and stipes were frozen at −80° C. for at least an hour, followed by grounding with mortar and pestle to achieve a slurry. 25 mL of buffer (0.01 M TRIS-HCl, 5 mM MgCl.sub.2) was added to 14 g of mushroom wet mass. The suspension was sonicated (Vibracell VCX130PB Ultrasonic Processor) for 30 min on ice, followed by centrifugation at 10,000 rpm for 15 min using an Eppendorf refrigerated centrifuge at 4° C. The supernatant was decanted into a fresh tube or blue cap bottle and designated as the mushroom extract.

[0095] Protein Particle Precipitation

[0096] Example 1: 28 g of ground F. velutipes was extracted using 25 mL of TRIS buffer. From the 35 mL of resulting extract, 15 mL was decanted into a 50 mL Blue Cap bottle and heated on a Heidolph hot-plate set at 150° C. Within 10 min, boiling of the solution had occurred, and a white precipitate had appeared in the solution, which was isolated by centrifugation at 7000 rpm for 5 min. The remaining 20 mL of extract was allowed to stand at room temperature (25° C.). Within 48 hours, a white turbidity had appeared in the solution, which could be isolated as the same precipitate.

[0097] Example 2: 14 g of ground L. edodes was extracted using 25 mL of TRIS buffer. The resulting extract was decanted into a 50 mL Blue Cap bottle and heated on a Heidolph hot-plate set at 200° C. Within 20 min, boiling of the solution had occurred, and a white precipitate had appeared in the solution.

[0098] Example 3: 20 g of ground F. velutipes or L. edodes was extracted using 25 mL of TRIS buffer. The resulting extract was decanted into a 50 mL Blue Cap bottle and heated on a Heidolph hot-plate set at 200° C. Within 10 min, boiling of the solution had occurred, and a white precipitate had appeared in the solution, which was centrifuged at 7000 rpm for 5 min. The yield of precipitate (particles) was measured to be 60-70 mg per 20 g of mushroom wet mass.

[0099] Characterization of Protein Particles

[0100] UV spectrophotometry. The uv-spectrum for a solution of F. velutipes extract and the supernatant of the same solution after spontaneous precipitation was measured in the uv wavelength range. The subtraction spectrum indicated that a protein compound had been precipitated from the solution, with maximum absorbance at 276 nm (FIG. 11).

[0101] Formation of Hydrogels by Particle Aggregation and 3D Mammalian Cell Culture

[0102] Phosphate buffered saline (PBS) was added to L. edodes or F. velutipes particle suspension on a coverslip. Aggregation of the particles was observed under the light microscope, leading to the formation of a particulate gel (FIG. 12).

[0103] 10 μL of F. velutipes or L. edodes particle suspension (8-15 mg/mL) was added to 25 μL or 50 μL of DMEM serum-containing media. A particulate gel was formed (FIG. 13).

[0104] For 3D cell culture, 25 μL or 50 μL of C2C12 cell suspension in serum-containing DMEM media was added to a 96-well plate. 10 μL F. velutipes or L. edodes particle suspension (8-15 mg/mL) was then added to the cell suspension and immediately mixed by tituration. Formation of a slightly turbid particulate gel was observed, entrapping the cells (FIG. 14). The C2C12 cell suspension could also be added to the particle suspension in the well, followed by mixing.

[0105] Results

[0106] Isolation and Characterization of Particles

[0107] A typical protein extraction procedure under slightly basic conditions (0.01 M TRIS-HCl, 5 mM MgCl.sub.2) was employed. However, it was found that the particles could also be isolated using a pH 6 2-morpholinoethanesulfonic acid (MES) buffer.

[0108] Upon standing for two days at room temperature, spontaneous precipitation of solid particulates from the extract from F. velutipes occurred (FIG. 1). Precipitation took a substantially longer time (in the order of weeks) for the extracts from the other two mushroom species, and to lesser degree. After the F. velutipes suspension was centrifuged to isolate the precipitate, and the supernatant allowed to stand further, more precipitate formed in the supernatant with time.

[0109] It was found that precipitation could be induced to obtain the same particulate suspension by boiling the F. velutipes and L. edodes extracts for 5-20 min (FIG. 2a). However, the H. Marmoreus extract could not be induced to precipitate by boiling alone (evaporation to concentrate the solution was required). For the extracts from F. velutipes and L. edodes, boiling is postulated to result in denaturation and unfolding of the RGD-containing protein, leading to its self-aggregation to form particles. In this case, protein denaturation is desirable as the cell-adhesive function of the RGD motif, being in the primary sequence, would not be lost, while the protein would lose other native functions that depend on its higher level structures (The latter functions may not be desirable for a general matrix for 3D cell culture). After being centrifuged to obtain a pellet, the particles could be dispersed easily by brief vortexing (˜1 min) in deionized water (FIG. 3). To further purify the particles for characterization, they were dialyzed against several changes of deionized water overnight.

[0110] By zetasizer analysis, the particles from F. velutipes were found to possess an average diameter of 1050 nm and zeta potential of −28 mV, while the particles from L. edodes measured 960 nm with a zeta potential of −20 mV. The relatively high negative surface charge of the particles leads them to repel each other and prevents aggregation, explaining the easy dispersibility and stability of the particles in aqueous solution.

[0111] Particulate Gels and Coatings

[0112] When the suspension of F. velutipes particles (8-15 mg/mL) was mixed with PBS or media (typically at a 1:2.5 or 1:5 ratio), aggregation of the particles occurred, resulting in a particulate gel. When a cell suspension was used in place of media, the cells were entrapped in the 3D particulate matrix (FIG. 4). Additional medium could be dispensed on top of the hydrogel, with regular changes as necessary for continuous cell culture. It was observed that cultured cells could spread out and extend processes in the hydrogel matrix, attributed to the presence of the RGD motif in the protein of the particles (FIG. 4c). In the same way, cells could be cultured in gels formed from L. edodes particle suspensions (8-15 mg/mL) (FIG. 5). For both gel types, cells remained viable when cultured continuously over the longer term.

[0113] The particulate gels could be redispersed in water, an advantage for the passaging of cells and their retrieval for analysis, e.g. for RT-PCR.

[0114] The particles from the mushroom extracts could also be used for coating of cell culture surfaces. MCF7 cells adhered on F. velutipes particle-coated plates and demonstrated good viability (FIG. 6).

[0115] Incorporation of Particles into Chitosan-Alginate Fibres and Cell Adhesion on the Fibers

[0116] 1 mL of a 12 mg/mL suspension of F. velutipes particles in deionized water was centrifuged at 7000 rpm for 5 min in an Eppendorf vial. 700 μL of supernatant above the pellet was withdrawn using a pipette. The pellet was redispersed in the remaining 300 μL of water by vortexing for several minutes. 3 mg of sodium alginate (medium molecular weight, Sigma-Aldrich) was then added to this particulate suspension and vortexed further for at least 10 min to completely dissolve the alginate.

[0117] By the process of interfacial polyelectrolyte complexation, the resulting mixture (40 mg/mL particles and 1% alginate) was then used to draw fibre against 1% chitosan (high molecular weight, Sigma-Aldrich) in 0.15M HOAc. A linear motor with an attached pipette tip at a vertical upward speed of 40 mm/min was employed to draw fibre at the interface between the two solutions (FIG. 7A).

[0118] The fibres were washed with water in a petri dish (FIG. 7B), after which the samples were transferred to wells of a 96-well tissue culture plate. Light microscope observation indicated that the particles were well-distributed in the fibres (FIG. 7C). A C2C12 mouse myoblast cell line was then seeded on these fibres. A LIVE/DEAD Viability/Cytotoxicity Kit (Thermo Fisher Scientific) was used to visualize cell attachment to the fibres after 72 h of culture. Excellent adhesion of cells onto the fibres was observed (FIG. 8).

[0119] Cell Adhesive Properties of the Fungal Extracts

[0120] It was found that all three fungal extracts (from L. edodes, F. velutipes and H. marmoreus), prior to precipitation, could mediate cell attachment. Cells attached and spread out to a higher degree on extract-coated PS microtiter wells, compared to the non-coated control wells, for both MCF7 cells (FIG. 9), and primary human skeletal muscle cells (FIG. 10).

[0121] In view of the above, the ability to formulate hydrogels from protein particles induced to precipitate from fungal extracts introduces a spectrum of new materials for 3D cell culture. Furthermore, the use of fungal, as opposed to animal-derived materials as cell-adhesive extracellular matrices for mammalian cells, may be environmentally more sustainable in the long term, and holds particular promise for Clean Meat applications. The three species of mushroom presented as examples in this work are all edible mushroom species from the order Agaricales (gilled mushrooms). Given that there are an estimated 13,000 species of fungi in this order alone, there is a high possibility that other candidates exist which may yield cell-adhesive mushroom extracts for biomaterials and cell culture applications.

Example A1

[0122] To further confirm the proteinaceous nature of the cell-adhesive particles extracted from the fungi, Fourier Transform-Infra-Red (FT-IR) spectra of F. Velutipes particles obtained by TRIS buffer extraction (A) and water extraction (B) were obtained, in addition to that of L. Edodes particles obtained by TRIS buffer extraction (C).

[0123] All three FT-IR spectra were similar (see FIG. 15), and showed characteristics of protein samples, with NH stretching vibrations at 3300 cm.sup.−1 and 3070 cm.sup.−1, amide I vibration at 1650 cm.sup.−1, amide II vibration at 1550 cm.sup.−1, and amide III vibrations between 1200 and 1400 cm.sup.−1. A relative strong absorption band was observed at 1100 cm.sup.−1, especially for the TRIS buffer-extracted F. Velutipes particles, which is believed to be due to a relatively high content of the amino acid, tryptophan in the proteins.

Example A2

[0124] It was demonstrated that the fungal-derived cell adhesive matrices were able to promote alignment of C2C12 mouse myoblast cells.

[0125] 15 μL of F. Velutipes particle suspension in water (concentration of approx. 10-15 mg/mL) was mixed with 25 μL C2C12 suspension (˜1.8×10.sup.5 cells/mL) in a 96 well-plate, followed by an additional 25 μL of DMEM media (10% FBS) after 50 min. Control wells where 15 μL of sterile water was used in place of the particles were also prepared (n=4). At the end of 1 day, wells were topped up with 25 μL DMEM (without FBS).

[0126] After 1 day in culture, cells in the wells containing particles had begun to align themselves (FIG. 16A). These aligned cells that appeared to connect clusters of myoblasts persisted after 3 days of culture, as demonstrated by a Live Dead assay, which also indicated excellent cell viability (FIGS. 16 B, and C). In contrast, cells in the control wells were viable but not aligned (FIG. 16 D).

[0127] Alignment of muscle cells is an important step that precedes myotube formation (Zhao Y et al. Biotechnol Bioeng. 102(2) (2009) 10.1002/bit.22080). Thus, promotion of muscle cell alignment by the fungal-derived protein particles may be advantageous for muscle cell differentiation and subsequent maturation.

Example A3

[0128] Extraction of F. velutipes particles could be carried out using deionized water, i.e. TRIS or other buffer is not required.

[0129] 20 g of ground F. velutipes was extracted using 25 mL of deionized water. The resulting extract was decanted into a 100 mL media storage bottle and heated on a Heidolph hot-plate set at 200° C. Within 10 min, boiling of the solution had occurred, and a white precipitate had appeared in the solution, which was isolated by centrifugation at 7000 rpm for 5 min (see FIG. 17).

[0130] As for the TRIS buffer-extracted protein particles, particulate gels could be prepared in the same way using the water-extracted protein particles.

Example A4

[0131] The same protein extraction and precipitation procedure, as presented in this TD, was applied on another edible fungal species, Grifola Frondosa. As for the case of Hypsizygus Marmoreus, boiling of the extract alone did not yield any protein particles. It is believed that this is an argument in favour of an inventive step, as the boiling procedure is not expected to yield precipitate (protein particles) for any fungal extract in general.

[0132] The inventors also found that protein extraction and precipitation used heat that is much higher than what is typically used. In particular, general knowledge in the art holds that low temperatures (such as 30° C.) should be used to obtain protein from fungal species, and the fungus should not be heated to temperatures of 100° C. or higher, as the latter would result in protein denaturation. However, in contrast to general knowledge, the inventors found that heating the protein fungal extract results in particles that retain cell-adhesive function, which suggests that heating advantageously removes higher structures but still retains primary structures and cell-adhesive sequences such as the RGD motif.

Additional Examples

[0133] The inventors of the present disclosure further found that a suitable and effective approach to imbue cell-adhesive factors into the biomaterial forms was by bulk-incorporation or coating of a suspension of particles from F. Velutipes in alginate solution (FV-alginate), followed by crosslinking with calcium.

[0134] In the following, examples are presented of bulk-incorporation and coating of FV-alginate fibers in/on fiber, sphere and film forms of various biomaterials. In each case, the precipitate that had been obtained by boiling the F. Velutipes extract and centrifuging at 7000 rpm for 5 min (see Examples 1-3 above) was used as a starting material. The precipitate could be further washed with water by centrifugation and stored either as the wet precipitate or a suspension in water. These are referred to as ‘FV precipitate’ or ‘ FV particle suspension’ respectively.

Example A5

[0135] FV-Alginate Incorporated Spheres

[0136] The FV particle suspension was centrifuged down at 7000 rpm for 5 min. The supernatant was decanted and 500 μL of deionized water was used to disperse the resulting pellet. 2 mL of 3% alginate (low molecular weight, Sigma-Aldrich) was added to this suspension and titurated/vortexed till the particles were uniformly dispersed (FV-alginate suspension). The suspension was drawn into a 3 mL syringe using a 21½ G needle. By gently depressing the plunger to produce tiny droplets while immersing and rapidly withdrawing the syringe needle in and out of 10 mL 5% (w/v) CaCl.sub.2) solution contained in a 50 mL beaker, alginate spheres incorporating FV particles were obtained. The particles were washed and sterilized in 70% ethanol in a 24-well plate for 3 h, washed with phosphate buffered saline (PBS) and transferred to fresh wells. A suspension of C2C12 mouse myoblasts in DMEM/10% FBS (>10.sup.6 cells/mL) were seeded into these wells.

[0137] Results

[0138] Spheres were formed with diameters ranging from ˜1-2 mm in diameter (FIG. 18A). No observable leaching of FV particles occurred from the spheres. 2.5 h after cell seeding, C2C12 mouse myoblast attachment was observed on the spheres (FIG. 18B).

Example A6

[0139] FV-Alginate Coated Chitosan Microspheres

[0140] Chitosan microspheres (˜100 μm in diameter) were prepared according to a water-in-oil microemulsion method. 500 μL of 3% FV-alginate suspension was added to 500 μL of a microsphere suspension in a 1.5 mL centrifuge tube. After 10 min of standing, the microspheres had sedimented to the bottom of the tube. 500 μL of the supernatant was removed and replaced with deionised water. The above step was repeated 4 times, allowing at least 2 min for microsphere deposition between each repeat. At the end of the last repeat, the supernatant was clear, indicating complete removal of the FV-alginate particles. The supernatant was removed until approx. 50 μL remained over the deposited microspheres, 500 μL 5% (w/v) CaCl.sub.2) solution was added and the suspension was titurated several times. The coated microspheres were allowed to deposit, then the CaCl.sub.2) solution was removed and replaced with 75% ethanol. The microspheres were transferred to a 24-well plate and allowed to stand for 1 h, after which they were washed with phosphate-buffered saline. The appearance of the FV-alginate-coated chitosan microspheres is shown in FIG. 19A. A suspension of C2C12 mouse myoblasts in DMEM/10% FBS (>10.sup.6 cells/mL) were then seeded onto the microspheres. After 2 h, the microspheres were transferred into fresh wells for observation.

[0141] Results

[0142] Within 2 h of cell seeding, attachment of C2C12 cells to the microspheres had occurred. Large numbers of attached cells could be observed on the periphery of the microspheres. After overnight (16 h) culture, the cells had spread out onto the microspheres, whose surfaces had transited from a more granular appearance where individual cells could be discerned, to a smoother, homogenous surface with bumps. (FIG. 19B).

Example A7

[0143] FV Particle Incorporated Water-Soluble Chitin (WSC)-Alginate Fibers

[0144] FV precipitate was resuspended in 5 mL deionised water by vortexing for at least 1 min. 1 mL of the particle suspension was transferred to a 1.5 mL centrifuge tube. The suspension was centrifuged at 7000 rpm for 5 min and the resulting pellet was dispersed by vortexing in 200 μL 3% alginate (low MW, Sigma-Aldrich). Fiber was drawn by a process of interfacial polyelectrolyte complexation using 10 μL of the FV-alginate particle suspension and 0.5% WSC. The drawn fiber was immediately immersed into 5% (w/v) CaCl.sub.2) solution contained in a petri dish. The fiber was rinsed with deionised water and sterilized with 70% ethanol for at least 1 h. The ethanol solution was replaced with PBS. The PBS solution was removed and a suspension of C2C12 mouse myoblasts in DMEM/10% FBS (>10.sup.6 cells/mL) was transferred to the well containing the fiber. After 1 h, the fiber (with attached cells) was transferred to a fresh well.

[0145] Results

[0146] No leaching of FV particles from the fiber occurred during the washing process. At 1 h, a substantial number of cells was observed attached to the fiber (FIG. 20A). After 24 h, these cells appeared to have spread out on the fiber surface (FIG. 20B). A Live Dead assay showed that most of the attached cells retained good viability.

Example A8

[0147] FV-Alginate Coated Cellulose Fibers

[0148] Preparation of FV-alginate suspension: FV precipitate was resuspended in 5 mL deionised water by vortexing for at least 1 min. 1 mL of the particle suspension was transferred to a 1.5 mL centrifuge tube. The suspension was centrifuged at 7000 rpm for 5 min and the resulting pellet was dispersed by vortexing in 200 μL 3% alginate (low MW, Sigma-Aldrich).

[0149] Cellulose fibers were immersed in 25% NaOH solution in a petri dish overnight. The fibers were washed thrice with deionised water, soaked in water for 30 min, then immersed in 5% w/v CaCl.sub.2 solution for at least 10 min. By centrifugation at 3500 rpm for 5 min, the fibers were separated from the supernatant and washed with deionised water. Subsequently, they were immersed in the FV-alginate suspension, as prepared above. After at least 30 min of standing, the suspension was removed and deionised water added to wash the fibers. This process was repeated until the supernatant was clear. The fibers were transferred to a 24-well plate and immersed in 70% ethanol for at least half hour. The ethanol solution was then replaced with PBS, followed by a C2C12 mouse myoblast suspension in DMEM/10% FBS (>10.sup.6 cells/mL). After 2 h, the fibers (with attached cells) were transferred to fresh wells for observation.

[0150] Results

[0151] Good attachment of cells on the fibers was observed after 2 h (FIG. 21A). After 2 days of culture, the cells had spread and elongated along the axis of the fibers (indicated by arrows in FIG. 21B). At this time-point, a Live Dead assay indicated good viability of the cells.

Example A9

[0152] FV-Alginate Films

[0153] FV alginate films can be obtained in the following way. Planar constructs were made by an interfacial polyelectrolyte complexation process using chitosan and sodium alginate (FIG. 22A). As these constructs had been calcium crosslinked using 25 mM calcium chloride during the process, they contained calcium solution. FV-alginate suspension (as prepared in Example A8) was added to a petri dish containing the constructs to completely immerse them (FIG. 22B). After several minutes, the FV-alginate suspension directly on top of the constructs had coagulated to form films due to crosslinking by calcium leaching out from the constructs, and these could be easily be peeled off (FIG. 22C). C2C12 cells that were seeded onto these films showed good attachment after 1 h (FIG. 22D) and spreading after 1 d (FIG. 22E).

REFERENCES

[0154] 1. Yasuda T, Ishihara H, Amano H, Shishido K. Generation of basidiomycetous hyphal cell-aggregates by addition of the Arg-Gly-Asp motif-containing fragment of high-molecular-weight cell-adhesion protein MFBA derived from the basidiomycete Lentinus edodes, Biosci Biotechnol Biochem. 61(9) (1997) 1587-9 [0155] 2. Kondoh O, Muto A, Kajiwara S, Takagi J, Saito Y, Shishido K. A fruiting body-specific cDNA, mfbAc, from the mushroom Lentinus edodes encodes a high-molecular-weight cell-adhesion protein containing an Arg-Gly-Asp motif, Gene 154(1) (1995) 31-7 [0156] 3. Sakamoto Y, Azuma T, Ando A, Tamai Y and Miura K. Characterization of proteins expressed abundantly in the fruit-body of Flammulina velutipes, Mycoscience 41 (2000) 279-282