WO2013012397A1

WO2013012397A1 - Method for encapsulating living cells in a keratin-containing hydrogel

Info

Publication number: WO2013012397A1
Application number: PCT/SG2012/000265
Authority: WO
Inventors: Kee Woei NG; Shuai WANG; Francesca TARABALLI
Original assignee: Nanyang Technological University
Priority date: 2011-07-21
Filing date: 2012-07-23
Publication date: 2013-01-24

Abstract

The present invention relates to a method for encapsulating living cells in a keratin-containing hydrogel, comprising (i) providing a solution of keratin; (ii) adjusting the pH of the keratin solution by adding a pH buffer with a pH of about 2 to 5; (iii) incubating the keratin solution to form a keratin precipitate phase and a supernatant phase; (iv) removing the supernatant phase; (v) adjusting the pH of the keratin solution by covering the keratin precipitate phase with a liquid medium of a desired pH, wherein the desired pH is in the range of about 6 to 8; (vi) optionally repeating steps (iv) and (v) until the desired pH is achieved; (vii) adding living cells suspended in a liquid medium to the keratin precipitate phase and mixing both to form a homogeneous solution; and (viii) incubating the homogeneous solution to form a keratin-containing hydrogel encapsulating the living cells. Use of the living cells encapsulated keratin-containing hydrogel in cell delivery, drug delivery, wound dressing, or tissue regeneration is also disclosed.

Description

METHOD FOR ENCAPSULATING LIVING CELLS IN A KERATIN- CONTAINING HYDROGEL

Cross-Reference to Related Application

[0001] This application claims the benefit of priority of United States of America Provisional Patent Application No. 61/510,312, filed 21 July 201 1, the contents of which being hereby incorporated by reference in its entirety for all purposes.

Technical Field

[0002] The invention relates to a method of encapsulating living cells in a keratin- containing hydrogel and the use thereof.

Background

[0003] Cells in the human body typically experience a 3D environment. They are completely surrounded by other cellsand extracellular matrix components, an environment that is essential for proper cell behavior. In order to emulate the in vivo scenario more accurately, scientists have devoted much attention to the creation of 3D models in vitro to study cellular behaviours or engineer transplantable tissues. Among these, the use of hydrogels is one of the most established because of the close resemblance to the in vivo histo-architecture of natural extracellular matrix (ECM).

[0004] Hydrogels are crosslinked 3D network of polymers. Both natural and synthetic polymers have been explored for this purpose. Hydrogels based on synthetic polymers have more controllable physical properties and are more reproducible. However, there are problems such as poor biocompatibility, lack of bioactivity and poor resemblance to the natural environment. Vascular smooth muscle cells grown in these hydrogels demonstrated increased proliferation and migration. A more physiological material in the form of self-assembling oligopeptides has also been shown to form hydrogels effectively. Hepatocytes cultured in this hydrogel showed higher viability and better preservation of their metabolic machinery compared to those cultured in 2D collagen matrices.

[0005] The feasibility of using natural materials including polysaccharides and proteins has been well-demonstrated. The most common polysaccharide used as a hydrogel is probably alginate, which readily absorbs water and has been found to be biocompatible and mechanically stable. Among the most common proteins used as a hydrogel are collagen and fibrin. These are natural proteins found in the human body and therefore hold great promise in clinical applications. However, the supply of such natural proteins is limited, resulting in their high cost. Autologous sources are impossible except for limited amounts of fibrin. Animal sources are available, but these raise concerns of pathogen transfers and immunological reactions. Therefore, the development of an alternative, potentially autologous source of natural protein as a hydrogel could be immensely beneficial for clinical applications.

[0006] Human hair exists in great abundance and is readily available. Despite its availability, there has been surprisingly little work done to make good use of it. Besides its obvious use as a raw material for making wigs, only a few applications of human hair in its native state has been found: as hair mats for gardening and absorbing oil spills. Given the society's push for "greener" approaches and waste recycling today, hair as a largely untapped and free resource is likely to be readily accepted as a raw material of choice for any application that is proven feasible.

[0007] Human hair is made up of filamentous keratins (80%) and non-filamentous matrix proteins. Human hair keratins contain high proportions (14%) of cysteine-rich regions compared to other proteins, resulting in high proportions of crosslinks through disulphide bonds which account for much of the strength of hair. A major reason for the lack of successful, widespread uses of hair is the difficulty to effectively dissolve hair to extract its constituents. However, work in this respect in recent years has shown that it is possible to extract keratins from various keratinous tissues including human and animal hair, feathers and horns.

[0008] Keratins are intermediate filament proteins, which are a major component of the cytoskeleton of nearly every cell type in vertebrates. They are also the major components that make up epithelial appendages such as hair and nails. Intermediate filaments are intracellular and mostly cytoplasmic, forming long filaments of about lOnm in diameter. They are divided into 6 classes (Type I to VI) on the basis of their protein sequences. Of these, keratins make up the type I (acidic) and type II (basic) members, which together account for about three quarters of all known intermediate filament proteins in the human being. As with other intermediate filament proteins, the keratin protein is made up of a long central domain of mostly a-helix structure flanked by non-helical head and tail domains. Keratins will only polymerise into filaments when a type I member is present together with its type II member partner in the right environment. These heterodimers further interact in anti-parallel arrangement to form tetramers and, subsequently, non-polar protofilaments of 2-3nm in diameter. The final lOnm diameter filament is believed to compose of 16 dimeric strands.

[0009] Keratins play important structural roles and are also implicated as vital members in mechotransduction signalling pathways. The structural integrity of keratin filaments can be directly correlated to its amino acid composition. Keratins contain a high proportion of the two smallest amino acids: glycine and alanine. This allows sterically- unhindered hydrogen bonding between the amino and carboxyl groups of peptide bonds on adjacent protein chains, facilitating their close alignment and strong binding. In addition, keratins have large amounts of the sulphur-containing amino acid cysteine, resulting in the formation of disulfide bridges that act as permanent, thermal-stable crosslinks to confer additional strength and rigidity to the filaments. The amino acid sequence of keratins contains a cell adhesion motif, LDV (leucine - aspartic acid - valine). This motif is also found in ECM proteins such as fibronectin and is recognised by the cell adhesion molecule o¾/3i-integrin. Therefore, the presence of the LDV motif suggests that keratins may function as effectively as other ECM proteins in supporting cell attachment and growth. Indeed, keratins have been proven to be promising as 2D substrates for several biomedical applications. Attempts have also been made to construct 3D keratin sponges via lyophilisation as a tissue regeneration template.

However, uneven cell distribution in such a sponge is the major drawback.

[0010] Keratins are capable of self-polymerising in vitro to form hydrogels. Keratin- based hydrogels have been designed to serve as physical barriers to prevent the infiltration of inflammatory cells following the transplantation of neural stem/progenitor cells for nerve regeneration. In another study, keratin hydrogels have been shown to enhance nerve regeneration in vivo. It has further been demonstrated the potential of using keratin hydrogels as an internal haemostat and shown that reduction in size of these gels was observed only after 3 months implantation in vivo. Although various conditions for keratin to gel have been described, the mechanism for gelling is not well understood.

[0011] Thus, there remains a need to provide for a method of encapsulating living cells in a keratin-containing hydrogel wherein the gelling process is controlled and physiological conditions are maintained during the encapsulation process.

Summary

[0012] According to a first aspect of the disclosure, there is provided a method for encapsulating living cells in a keratin-containing hydrogel. The method may include: (i) providing a solution of keratin;

(ii) adjusting the pH of the keratin solution by adding a pH buffer with a pH of about 2 to 5;

(iii) incubating the keratin solution to form a keratin precipitate phase and a supernatant phase;

(iv) removing the supernatant phase;

(v) adjusting the pH of the keratin solution by covering the keratin

precipitate phase with a liquid medium of a desired pH, wherein the desired pH is in the range of about 6 to 8;

(vi) optionally repeating steps (iv) and (v) until the desired pH is achieved;

(vii) adding living cells suspended in a liquid medium to the keratin

precipitate phase and mixing both to form a homogeneous solution; and

(viii) incubating the homogeneous solution to form a keratin-containing hydrogel encapsulating the living cells.

[0013] According to a second aspect, there is provided a living cells encapsulated keratin-containing hydrogel obtained from the method of the first aspect.

[0014] A third aspect relates to the use of the living cells encapsulated keratin- containing hydrogel of the second aspect in cell delivery, drug delivery, wound dressing, or tissue regeneration.

Brief Description of the Drawings

[0015] In the drawings, like reference characters generally refer to the same parts throughout the different views. The drawings are not necessarily drawn to scale, emphasis instead generally being placed upon illustrating the principles of various embodiments. In the following description, various embodiments of the invention are described with reference to the following drawings. [0016] Fig. 1 shows a process flow chart for carrying out the method in accordance with a first aspect.

[0017] Fig. 2 shows the evaluation of extracted human hair protein samples by SDS- PAGE and Western blotting. Le t. Coomassie-stained gel showing presence of dominant double bands of sizes 40-60 kDA expected of basic and acidic keratins. Right:

Immunoblotting using antibody against hair cortex keratins (AE 13) proved presence of basic and acidic keratin fractions.

[0018] Fig.3 shows A) mixing of L929 fibroblasts in human hair extracted keratin. Cells are homogenously suspended in physiological conditions; B) keratin hydrogel with encapsulated L929 fibroblasts in 6-well plate. Keratin hydrogel reforms in 1 minute, trapping viable cells within the gel matrix.

[0019] Fig.4 shows the distribution and proliferation of L929 fibroblasts within keratin hydrogels. Live cells were labeled green by Calcein AM and observed under a fluorescent microscope. At A) day 1 of culture, cells were evenly distributed within the hydrogels;by B) day 3 of culture, single cells have proliferated to small multi-cell colonies, indicating their proliferative capacity within the keratin hydrogels. Out-of- focus colonies could be observed, demonstrating the distribution of colonies throughout the depth of the hydrogels. Scale bars represent 100 um.

[0020] Fig.5 shows the relative proliferation of L929 fibroblasts in keratin hydrogels measured using PicoGreen DNA quantification assay. Data represent relative double- stranded DNA (dsDNA) levels over 6 days of culture, normalized to controls at day 1 , presented as means ± standard deviation of quadruplicate samples. This result suggests that the cells proliferated normally, indicating that the keratin hydrogels were biocompatible. [0021] Fig. 6 shows the formation of keratin floes (or precipitates) with increasing keratin concentration. The ratio of PBS: 1 M pH 3 citrate buffer is 10:0.5 (v/v).

[0022] Fig. 7 shows the effect of citrate buffer concentration on keratin hydrogel formation. Visible light penetration (transparency) was used to measure gel formation. pH7.2 PBS buffer was mixed with 1M pH3 citrate buffer at the volume ratio of 10:0 (control), 10:0.3, 10:0.5, 10: 1 and 10:2. Equal volumes of lOmg/ml keratin solution was then added into individual buffer. At ratios of 10:0.5 and above, keratin solution reaches flocculation point.

[0023] Fig. 8 shows that almost 96.7% of the keratin in the solution was flocculated under this condition. Only 3.89% remained inside supernatant.

[0024] Fig. 9 shows temperature dependent crosslinking of keratin floes. Suspensions of keratin floes prepared with 10:0.5 PBS to citric buffer (v/v) were tested for their stability (transparency). At every time point indicated , either water or PBS was added to the opaque keratin floes (equal volumes as keratin floes) at 4 °C or 37 °C. Results show that at 37 °C, adding water has no effect on the stability of the floes. Adding PBS (increasing pH) destabilizes the floes resulting in increased transparency (low absorbance). However, with time, adding PBS gradually stops destabilizing the keratin floes. If the incubation was carried out at 4 °C, keratin floes will always destabilize the floes, suggesting that crosslinking is temperature dependent.

[0025] Fig. 10 shows microarchitecture of keratin gels made with different keratin concentrations: a) 25 mg/ml; b) 40 mg/ml; c) 50 mg/ml.

[0026] Fig. 11 shows the storage modulus measurement of keratin gels made with different keratin concentrations.

[0027] Fig. 12 shows subcutaneous implantation of keratin hydrogels for acute toxicity assessment. Keratin hydrogels were implanted subcutaneously into 2 black 6 mice over 7 days (*). Masson Trichrome staining shows insignificant fibrotic encapsulation. No acute toxicity was observed.

[0028] Fig. 13 shows subcutaneous implantation of keratin hydrogels for acute toxicity assessment.H&E staining shows remodeling at gel/tissue interface after 7 days (*). Significant cell infiltration was evident.

Description

[0029] The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the invention may be practised. These embodiments are described in sufficient detail to enable those skilled in the art to practise the invention. Other embodiments may be utilized and changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

[0030] There are currently no suitable methods available to physiologically encapsulate living cells homogenously in a three-dimensional (3D) keratin-containing hydrogel. This is technically challenging because of the chemicals and extreme pH used in the keratin extraction and gelling processes. A physiological keratin-containing hydrogel system will be advantageous for the purpose of clinically relevant cell and/or drug delivery, for example.

[0031] Thus, a first aspect of the disclosure relates to a method for encapsulating living cells in a keratin-containing hydrogel. By employing the present method, living cells can concurrently beencapsulated or loaded in a keratin-containing hydrogel during the gelling process of a keratin-containing hydrogel precursor solution.The present method further allows mixing of living cells in suspension with a keratin-containing hydrogel precursor solution under physiological conditions followed by the formation of the keratin-containing hydrogel in a controlled environment, thereby promoting uniform cell distribution, maximum viability and reproducible hydrogel micro-architecture. In this manner, the living cells are subjected to only physiological conditions and environments which enable or maximize cell viability and phenotypic profile.

[0032] In the present context, a hydrogel is commonly known in the art to be formed of a 3D network or matrix of a hydrophilic polymeric material, which can swell in water and hold a significant amount of water while maintaining its general network structure. The network structure may be linked together through chemical or physical links. A 3D network can be formed by crosslinking hydrophilic polymers via covalent bonds, hydrogen bonding, van der Waals interactions, or physical entanglement.

[0033] Accordingly, in the present context, the term "keratin-containing hydrogel", or simply "keratin hydrogel", both terms interchangeably used herein, refers to a hydrogel whose network structure comprises or consists essentially of or contains keratin. As described earlier, keratins are intermediate filament proteins, which are a major component of the cytoskeleton of nearly every cell type in vertebrates. They are also the major components that make up epithelial appendages such as hair and nails.

Intermediate filaments are intracellular and mostly cytoplasmic, forming long filaments of about 1 Onm in diameter. They are divided into 6 classes (Type I to VI) on the basis of their protein sequences. Of these, keratins make up the type I (acidic) and type II (basic) members, which together account for about three quarters of all known intermediate filament proteins in the human being. As with other intermediate filament proteins, the keratin protein is made up of a long central domain of mostly a-helix structure flanked by non-helical head and tail domains. Keratins will only polymerise into filaments when a type I member is present together with its type II member partner in the right environment. These heterodimers further interact in anti-parallel arrangement to form tetramers and, subsequently, non-polar protofilaments of 2-3nm in diameter. The final lOnm diameter filament is believed to compose of 16 dimeric strands.

[0034] Keratins play important structural roles and are also implicated as vital members in mechotransduction signalling pathways. The structural integrity of keratin filaments can be directly correlated to its amino acid composition. Keratins contain a high proportion of the two smallest amino acids: glycine and alanine. This allows sterically- unhindered hydrogen bonding between the amino and carboxyl groups of peptide bonds on adjacent protein chains, facilitating their close alignment and strong binding. In addition, keratins have large amounts of the sulphur-containing amino acid cysteine, resulting in the formation of disulfide bridges that act as permanent, thermal-stable crosslinks to confer additional strength and rigidity to the filaments. The amino acid sequence of keratins contains a cell adhesion motif, LDV (leucine - aspartic acid - valine). This motif is also found in ECM proteins such as fibronectin and is recognised by the cell adhesion molecule o¾/3i-integrin. Therefore, the presence of the LDV motif suggests that keratins may function as effectively as other ECM proteins in supporting cell attachment and growth.

[0035] In various embodiments, the keratin-containing hydrogel does not comprise a significant amount of other structural proteins. For example, in some embodiments, the keratin-containing hydrogel does not comprise a significant amount of collagen (for example, less than about 5%, or 1%, or 0.5%, or 0.1% by weight of the total composition). In yet other embodiments, the keratin-containing hydrogel does not comprise a significant amount of chitosan (for example, less than about 5 %, or 1 %, or 0.5%, or 0.1% by weight of the total composition). In yet further embodiments, the keratin-containing hydrogel does not comprise a significant amount of

glycosaminoglycans (for example, less than about 5%, or 1%, or 0.5%, or 0.1% by weight of the total composition). In yet certain embodiments, the keratin-containing hydrogel does not comprise a significant amount of collagen and/or

glycosaminoglycans.

[0036] In the present context, by "living cells encapsulated keratin-containing hydrogel" is meant that the keratin-containing hydrogel is loaded or filled with living cells. The cells may be found at the surface, or in the interior (i.e. hydrogel matrix), or both locations of the hydrogel. The cell loading, i.e. the proportion of the cells based on the amount (such as volume or weight) of the hydrogel, may be adjusted in accordance with the target application of the keratin-containing hydrogel.

[0037] In the present context, the term "cell" as used herein refers to any type of prokaryotic or eukaryotic cell. In case of eurakyrotic cells, an assembly of cells which appear as one single cell (for example, eggs in early development) are also included in the meaning of the term "cell". Prokaryotic cells include any type of bacteria cell such as E. coli, or bacillus subtilis or any member of the microbacterium, streptococcus or streptomyces families of bacteria. Eukaryotic cells include any type of plant or animal cell, cells such as those found in plant xylem, phloem, or meristem; or cells such as those found in animal organs, such as osteoblasts, liver cells, lung cells, stomach cells, lymphatic cells, erythrocytes and leukocytes, including monocytes and lymphocytes. Eukaryotic cells also include yeast cells. Additionally, the term includes such cells obtained from a normally functioning organ as well as those that are pathological. Thus, in various embodiments, the living cells encapsulated in the keratin-containing hydrogel include, but are not limited to, smooth muscle cells, skeletal muscle cells, endothelial cells, stem cells, progenitor cells, myocytes, bone marrow cells, neurons, pericytes and fibroblasts. Living cells used in the present method may be of any source. They may for example be native cells, including cells isolated from tissue, or they may be cells of a cell line. Respective cells may also be modified, e.g. treated by an enzyme, exposed to radiation, transformed by the incorporation of heterologous matter (an organelle, genetic material, inorganic matter etc.), or they may be recombinant or transgenic. The cells may be seeded at any density, for example, as long as they are able to proliferate and as long as they are seeded below confluence. Depending on the cells used and the target applications of the keratin-containing hydrogel, they may be allowed to proliferate up to a density of about 80 % to about 100 %, such as about 85 %, 90 % or 95 %. The cells may be allowed to proliferate near to confluence. The term "confluence" is used herein - unless stated otherwise - in the regular meaning to describe a state in which cells have grown within a certain amount of space.

[0038] In certain embodiments, the living cells are encapsulated in the 3D hydrogel made from keratin extracted from human hair. Using keratin extracted from human hair offers the possibility of abundant supply of this hydrogel at a low cost. Furthermore, given the society's current push for "green" approaches, the strategy of using human hair (which is usually just discarded) as a raw material for making a product with potential medical benefits shows potential in wide acceptance and adoption.

[0039] Existing hydrogels used clinically are either allogeneic (e.g. human fibrin) or xenogenic (e.g. bovine collagen, rat collagen). The present disclosure makes it possible to offer an autologous hydrogel system that is derived from the patient's own hair, thereby circumventing the shortcomings of excessive immunological reactions, risks of pathogen transfers and high cost. A patient needing a non-emergency hydrogel source can donate his/her own hair and return to the hospital a few days later after the keratin is extracted and ready for use, to get the required procedure done. Such a strategy will eliminate the shortcomings of using other natural and synthetic materials as hydrogels. Such a strategy will revolutionize clinical practice where hydrogels are being used. This is done by applying a pH-induced precipitation method to accelerate the in vitro keratin self-assembly process.At neutral pH, keratin molecules carry net negative charges which cause electrostatic repulsion between them (for instance, Isoelectric points of hair keratins are mostly below 7). By lowering the pH, keratin molecules become protonated, thus becoming less charged. This reduces the solubility of keratin molecules and promotes interactions between keratin molecules, thus resulting in gelling.

[0040] Thus, a further aspect of the disclosure relates to use of the living cells encapsulated keratin-containing hydrogel in cell delivery, drug delivery, wound dressing, or tissue regeneration.

[0041] The method of the first aspect includes:

(i) providing a solution of keratin;

(iv) removing the supernatant phase;

(v) adjusting the pH of the keratin solution by covering the keratin

(vi) optionally repeating steps (iv) and (v) until the desired pH is achieved;

(vii) adding living cells suspended in a liquid medium to the keratin

precipitate phase and mixing both to form a homogeneous solution; and

(viii) incubating the homogeneous solution to form a keratin-containing

hydrogel encapsulating the living cells. [0042] In various embodiments such as the one illustrated in Fig. 1, a solution of keratin is first provided. In certain embodiments, the keratin solution is prepared by extracting keratin as taught in U.S. Patent No. 7,169,896 B2. For example, keratin may be extracted from human hair, animal hair, animal feathers, or animal horns. For example, a 5-10 mg/ml keratin solution may be used.In one embodiment, keratin from human hair is extracted. For example, human hair samples are first washed with soap, followed by 70% ethanol, rinsed extensively with water and air-dried. The cleaned hair was delipidized by soaking in a mixture of chloroform and methanol (2:1 v/v) for 24 h. The delipidized hair was subsequently air-dried and cut into 1 -cm-long fragments for keratin extraction. Delipidized hair fragments (50 g) were immersed in 1 1 0.125 M Na₂S solution and incubated at 40 °C for 4 h. The resulting mixture is then filtered and exhaustively dialyzed against 2 1 deionized water, by using cellulose tubing. The dialysis step is repeated six times. Keratin concentration was thereafter quantified by using the 660-nm protein assay kit. The extracted keratin solution is stored at 4 °C until use.

[0043] The pH of the keratin solution is then adjusted by adding a pH buffer with a pH of about 2 to 5 to the keratin solution.The pH buffer serves to maintain the pH of the keratin solution at physiological conditions at¹ all times in subsequent steps. In various embodiments, the pH buffer may have a pH of about 2, or 2.5, or 3, or 3.5, or 4, or 4.5, or 5. In certain embodiments, the pH buffer has a pH of about 3.

[0044] Suitable pH buffers include buffer systems with acidic pH. Exemplary pH buffer systems include, but are not limited to, citric acid and acetic acid based buffers such as citric acid-sodium citrate, citric acid-disodium hydrogen phosphate [Na₂HP0₄], or acetic acid-sodium acetate. In various embodiments, the pH buffer is selected from the group consisting of citrate buffer, phosphate buffered saline (PBS), acetic acid, citric acid-disodium hydrogen phosphate [Na₂HP0₄], acetic acid-sodium acetate, and a mixture thereof. In certain embodiments, the pH buffer is a citrate buffer.

[0045] In one embodiment, the pH buffer is a pH 3 citrate buffer. For example, the pH buffer may be 0.1 M pH 3 citrate buffer.

[0046] After adding the pH buffer to the keratin solution, the keratin solution is incubated to form a keratin precipitate phase and a supernatant phase. In various embodiments, precipitates of keratin are obtained by a phase-separation of the keratin solution. The keratin solution may be incubated in suitable containers such as a petri dish. After the phase-separation has completed, a keratin precipitate phase and a supernatant phase are formed. The keratin precipitate phase comprises or consists essentially of precipitates or floes of keratin.

[0047] In certain embodiments,incubating the keratin solution includes incubating for about 1 to 30 hours. For example, the mixture may be incubated for about 1 h, or 5 h, or 10 h,or 15 h,or 20 h,or 24 h,or 30 h. In an illustrative embodiment, the mixture is incubated for about 24 h.

[0048] In further embodiments, incubating the keratin solution also includes incubating at a temperature range of about 25 to 40 °C. For example, the incubation temperature may be about 25 °C, or 30 °C, or 32 °C, or 35 °C, or 37 °C, or 40 °C.In an illustrative embodiment, the mixture is incubated at about 37 °C.

[0049] Thus, in one embodiment, the keratin solution is incubated at 37 °C for 24 hours in a petri dish. Two aqueous phases may be seen to separate out: keratin precipitates in the bottom phase and a clear supernatant in the upper phase.

[0050] After the phase-separation has occurred, i.e. the keratin precipitate phase and the supernatant phase have formed, the supernatant phase is removed. In various embodiments, the supernatant phase is removed from the container using a manual or pump-assisted pipette or syringe, or simply by decanting.

[0051] After removal of the supernatant phase, the pH of the keratin solution is again adjusted by covering the keratin precipitate phase with a liquid medium of a desired pH, wherein the desired pH is in the range of about 6 to 8, such as about 6, or 6.5, or 7, or 7.2, or 7.5, or 8.

[0052] In the present context, by "covering" is meant carefully adding in the liquid medium over the keratin precipitate phase such that the keratin precipitate phase remains stable, i.e. without being disturbed, and the keratin precipitate phase remains at the bottom while the liquid medium phase is above the keratin precipitate phase, thereby covering the phase beneath it. In certain embodiments, adding the liquid medium includes adding a volume of the liquid medium about equivalent to the volume of supernatant phase removed.

[0053] In the present context, the term "liquid medium" refers to any type of pure liquid or liquid solution or mixture, including water, physiologically acceptable buffers such as saline, phosphate buffer saline (PBS) or Ringer's lactate, or any type of cell media or nutrient solution such as Hank's medium or Eagle's Medium. In one embodiment, the liquid medium is a cell culture medium, such as the Eagle's Medium.

[0054] In various embodiments, the liquid medium has a pH of about 7 to 7.5. For example, the liquid medium may have a pH of about 7.1, or 7.2, or 7.3, or 7.4, or 7.5. In certain embodiments, the liquid medium has a pH of about 7.2.

[0055] In various embodiments, the liquid medium is Dulbecco's modified Eagle's medium (DMEM).

[0056] In one embodiment, the liquid medium is DMEM with a pH of about 7.2. [0057] In additional embodiments, the step of adding the pH buffer to the keratin solution further includes adding a liquid medium to the keratin solution. In such embodiments, the above discussion on the liquid medium used for the adjustment of the pH of the keratin solution also applies to the liquid medium added in the above step.

[0058] The order of mixing the keratin solution, the pH buffer and the liquid medium is immaterial to the working of the present method. In some embodiments, the keratin solution, the pH buffer, and the liquid medium may be added simultaneously to a container for mixing. In other embodiments, the keratin solution may be added to a mixture of the pH buffer and the liquid medium. In yet alternative embodiments, the keratin solution is first added to the pH buffer, followed by the addition of the liquid medium.

[0059] In various embodiments, the keratin solution, the pH buffer, and the liquid medium are mixed in a ratio of about 5:1 :5 (v/v/v) to 20: 1 :20. For example, the ratio may be about 5: 1 :5, or 10: 1 : 10, or 15:1 : 15,or 20: 1 :20. In certain embodiments, the ratio is 10:1 : 10.

[0060] In one embodiment, about 5~10 mg/ml keratin solution, 0.1M pH 3 citrate buffer and pH 7.2 DMEM cell culture medium are mixed at the ratio 10: 1 : 10 (v/v/v).

[0061] In various embodiments, the step of removing the supernatant phase from the keratin solution and/or the step of adjusting the pH of the keratin solution by covering the keratin precipitate phase with a liquid medium of a desired pHis optionally repeated until the desired pH is achieved. By repeating the above steps at least once would ensure that physiological conditions are maintained in the subsequent steps. This additional washing step aids in achieving a more physiological pH (about 7.2) in the keratin precipitate phase. [0062] In further embodiments, after covering the keratin precipitate phase with the liquid medium, the keratin solution is left standing for a period of time before the next step. For example, the keratin solution may be left standing for about 1 to 10 minutes, such as 1 minute, or 3 minutes, or 5 minutes, or 7 minutes, or 10 minutes. The keratin solution is left standing for a period of time before the next step so as to allow the liquid medium to diffuse into the keratin precipitate phase, thereby neutralizing the pH of the keratin precipitate phase.

[0063] After allowing the pH neutralization of the keratin precipitate phase, the supernatant phase may be removed. In various embodiments, the supernatant phase is removed from the container using a manual or pump-assisted pipette or syringe, or simply by decanting.

[0064] After the desired pH of the keration solution is achieved, living cells suspended in a liquid medium are added to the keratin precipitate phase and mixing both to form a homogeneous solution.

[0065] In various embodiments, the above discussion on the liquid medium used for the adjustment of the pH of the keratin solution also applies to the liquid medium used for the suspension of the living cells.

[0066] In various embodiments, the suspension of living cells is added to the keratin precipitate phase via a pipette or syringe.

[0067] In various embodiments, adding the living cells suspended in the liquid medium comprises adding a volume of the liquid medium about equivalent to the volume of supernatant removed. In certain embodiments, the desired number of cells is determined and suspended in the liquid medium.

[0068] In various embodiments, mixing the keratin precipitate phase and the cell suspension to form a homogeneous solution includes pipetting the keratin precipitate phase and the liquid medium phase. Other forms of mixing are also possible so long as a homogeneous solution is obtained. A homogeneous solution is said to be obtained when the boundary between the two phases, i.e. between the keratin precipitate phase and the liquid medium phase, disappears.

[0069] After obtaining the homogeneous solution, the homogeneous solution is then incubated to form a keratin-containing hydrogel encapsulating the living cells. In various embodiments, incubating the homogeneous solution includes incubating at a temperature range of about 25 to 40 °C. For example, the incubation temperature may be about 25 °C, or 30 °C, or 32 °C, or 35 °C, or 37 °C, or 40 °C. In an illustrative embodiment, the homogeneous solution is incubated at about 37 °C.

[0070] In certain embodiments, incubating the homogeneous solution includes incubating for about 1 minute to 24 hours. The keratin-containing hydrogel with the living cells are kept in cell culture conditions to maintain cell viability. In one illustrative embodiment where mammlian cells are used, 37°C at 5% C0₂ conditions are used. During this time, the keratin-containing hydrogels are able to re-estabilsh some cross-links that were destroyed during the mixing step. For example, the homogeneous solution may be incubated for about 1 minute, or 1 h, or 5 h, or 10 h, or 24 h. In an illustrative embodiment, the homogeneous solution is incubated for about lmin.

[0071] In one embodiment, the homogeneous solution is incubated at 37 °C and 5% co₂.

[0072] In a further embodiment, the living cells encapsulated keratin-containing hydrogel is cultured for a period of time, with media change carried out about every 24 h.

[0073] The process described herein is such that the cells of interest are subjected to only physiological conditions in order to maximize their viability and phenotypic profile. Existing literature on keratin hydrogels or freeze-dried sponges typically describe seeding cells directly onto the external surfaces of the matrix. Such strategies either do not achieve homogeneous cell distribution or subject cells to non- physiological conditions in the process, which may compromise their viability. This disclosure allows mixing of cells in suspension with a keratin hydrogel precursor solution under physiological conditions followed by the formation of the hydrogel in a controlled environment, thereby promoting uniform cell distribution, maximum viability and reproducible hydrogel micro-architecture.

[0074] In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of the following non- limiting examples.

Examples

[0075] Fig. 2 shows the evaluation of extracted human hair protein samples by SDS- PAGE and Western blotting. Left-hand side of the figure showscoomassie-stained gel showing presence of dominant double bands of sizes 40-60 kDA expected of basic and acidic keratins. Right-hand side of the figure showsimmunoblotting using antibody against hair cortex keratins (AE13) proved presence of basic and acidic keratin fractions.10 μg (for coomassie blue staining) or 1 μg (for Western blot) of keratin in solution was mixed witt^l LDS sample buffer and2μl sample reducing agent and made up to 20μ1 with deionized water. The samples were heated at 75°C for 10 min prior to gel electrophoresis in NuPAGENovex 4-12% Bis-Trisgels, at 120V for 90min. Gels were subsequently washed three times with deionized water and were either stained for 60 min with coomassie blue(SimplyBlueSafeStainsolution) or transferred onto nitrocellulose membranes using the iBlot Dry Blotting System at 20V for 6 min. Membranes were blocked with 5% dry milk in phosphate buffered saline containing 0.05% Tween-20 (PBST) for 30 min and then incubated with a mouse polyclonal primary antibody against total human hair keratins (1 : 1000 in blocking solution) for 60 min at room temperature. Thereafter, the membranes were washed with PBST and incubated with HRP-conjugated goat anti mouse secondary antibody (1 :4000 in blocking solution) for 30min at room temperature. Chemiluminescence was generated by adding the Super Signal West Pico chemiluminescent substrate.

[0076] Fig.3 shows A) mixing of L929 fibroblasts in human hair extracted keratin. Cells are homogenously suspended in physiological conditions; B) keratin hydrogel with encapsulated L929 fibroblasts in 6-well plate. Keratin hydrogel reforms in 1 minute, trapping viable cells within the gel matrix. L929 murine fibroblasts were maintained on tissue culture polystyrene (TCPS) in DMEM supplemented with 10% FBS, 2mM L-glutamine, ImM sodium pyruvate, O.lnM non-essential amino acids, lOOUnits/ml penicillin and lOOug/ml streptomycin. Keratin initial precipitation was done by mixing lOmg/ml extracted keratin solution with 1M PH3 citrate buffer and PBS buffer at volume ratio of 10:0.5:10. After 8 hours incubation at 37°C, keratin precipitate was washed with L929 cell culture medium for 3 times. L929 cells were harvested from a sub-confluent culture using 0.25% trypsin, counted using a haemocytometer and mixed with keratin precipitates at a density of 50,000 cells/cm³.

[0077] Fig.4 shows the distribution and proliferation of L929 fibroblasts within keratin hydrogels. Live cells were labeled green by Calcein AM and observed under a fluorescent microscope. At A) day 1 of culture, cells were evenly distributed within the hydrogels;by B) day 3 of culture, single cells have proliferated to small multi-cell colonies, indicating their proliferative capacity within the keratin hydrogels. Out-of- focus colonies could be observed, demonstrating the distribution of colonies throughout the depth of the hydrogels. Scale bars represent 100 um. Live cells were labeled green by Calcein AM and observed under a fluorescent microscope (CKX41, Olympus, Japan).

[0078] Fig.5 shows the relative proliferation of L929 fibroblasts in keratin hydrogels measured using PicoGreen DNA quantification assay. Data represent relative double- stranded DNA (dsDNA) levels over 6 days of culture, normalized to controls at day 1, presented as means ± standard deviation of quadruplicate samples. This result suggests that the cells proliferated normally, indicating that the keratin hydrogels were biocompatible. At days 1, 2, 4 and 6, L929 cells were lysed with 0.01% SDS. Cell proliferation was tracked by measuring the total amount of double stranded DNA in the lysate using the PicoGreen(invitrogen) assay (n=4).

[0079] Fig. 6 shows the formation of keratin floes (or precipitates) with increasing keratin concentration. The ratio of PBS:1 M pH 3 citrate buffer is 10:0.5 (v/v).

Brightfield images were obtained using an upright phase contrast light microscope (CKX41, Olympus, Japan).

[0080] Fig. 7 shows the effect of citrate buffer concentration on keratin hydrogel formation. At ratios of 10:0.5 and above, keratin solution reaches flocculation point. pH7.2 PBS buffer was mixed with 1M pH3 citrate buffer at the volume ratio of 10:0 (control), 10:0.3, 10:0.5, 10:1 and 10:2. Equal volumes of 10 mg/ml keratin solution were then added into the different buffers. Gel transparency was then measured using UV/vis spectroscopy to determine extent of gelling.

[0081] Fig. 8 shows that almost 96.7% of the keratin in the solution was flocculated under this condition. Only 3.89% remained inside supernatant. Keratin flocculation was done as described before. Protein concentration in the supernatant was measured with 660nm protein quantification kit (pierce). [0082] Fig. 9 shows temperature dependent crosslinking of keratin floes. Suspensions of keratin floes prepared with 10:0.5 PBS to citric buffer (v/v) were tested for their stability (transparency). Results show that at 37 °C, adding water has no effect on the stability of the floes. Adding PBS (increasing pH) destabilizes the floes resulting in increased transparency (low absorbance). However, with time, adding PBS gradually stops destabilizing the keratin floes. If the incubation was carried out at 4 °C, keratin floes will always destabilize the floes, suggesting that crosslinking is temperature dependent. At 2 hourly timepoints after induction of keratin gelling with 10:0.5 PBS to citric buffer (v/v), equal volumes of either water or PBS was added to the resulting opaque keratin floes at 4 °C or 37 °C. Stability of the resulting gels was tested by measuring light transparency as described before.

[0083] Fig. 10 shows microarchitecture of keratin gels made with different keratin concentrations: a) 25 mg/ml; b) 40 mg/ml; c) 50 mg/ml. The micro-architecture of keratin hydrogels was examined by scanning electron microscopy (SEM). Keratin hydrogels were frozen at -20 C for 8 hours and subsequently lyophilized for two days. The resulting sponges were gold-sputtered at 18mA for 10 sec and observed under a scanning electron microscope (JSM-6360, JEOL, USA) at an accelerating voltage of 5kV.

[0084] Fig. 11 shows the storage modulus measurement of keratin gels made with different keratin concentrations.Oscillatory rheology experiments were performed at 37°C on a rheometer (Anton Paar, Germany) with a 50mm diameter stainless steel parallel plate. Keratin hydrogel was formed from an initial volume of 6ml solution in a 50mm petri dish. After zeroing the gap height, the parallel plate was lowered until it touched the hydrogel surface. For frequency sweep, the storage modulus (G') was monitored as a function of angular frequency. [0085] Fig. 12 shows subcutaneous implantation of keratin hydrogels for acute toxicity assessment. Keratin hydrogels were implanted subcutaneously into 2 black 6 mice over 7 days (*). Masson Trichrome staining shows insignificant fibrotic encapsulation. No acute toxicity was observed.

[0086] Fig. 13 shows subcutaneous implantation of keratin hydrogels for acute toxicity assessment.H&E staining shows remodeling at gel/tissue interface after 7 days (*). Significant cell infiltration was evident. In Figs. 12 and 13,keratin hydrogels were subcutaneously implanted into 2 immune-competent wild type C57BL/6 mice. Up to 4 small incisions were created on the backs of the animals, from which subcutaneous pockets not larger than 1 cm each were created to house an implant (~5 x 5 x 3 mm). At day 7 post implantation, animals were sacrificed, implants were harvested and fixed in 4% paraformaldehyde for 48 hours at room temperature and embedded in paraffin using standard protocols. 5μτη serial sections were cut, mounted on glass slides and stained with Masson's Trichrome, or haematoxylin and eosin. Brightfield images were obtained using an upright phase contrast light microscope (CKX41, Olympus, Japan).

[0087] By "comprising" it is meant including, but not limited to, whatever follows the word "comprising". Thus, use of the term "comprising" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present.

[0088] By "consisting of is meant including, and limited to, whatever follows the phrase "consisting of. Thus, the phrase "consisting of indicates that the listed elements are required or mandatory, and that no other elements may be present.

[0089] The invention illustratively described herein may suitably be practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. shall be read expansively and without limitation. Additionally, the terms and expressions employed herein have been used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention claimed. Thus, it should be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the inventions embodied therein herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0090] By "about" in relation to a given numberical value, such as for temperature and period of time, it is meant to include numerical values within 10% of the specified value.

[0091] The invention has been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0092] Other embodiments are within the following claims and non- limiting examples. In addition, where features or aspects of the invention are described in terms of

Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group.

Claims

1. A method for encapsulating living cells in a keratin-containing hydrogel,

comprising:

(i) providing a solution of keratin;

(iv) removing the supernatant phase;

(v) adjusting the pH of the keratin solution by covering the keratin

(vi) optionally repeating steps (iv) and (v) until the desired pH is achieved;

(vii) adding living cells suspended in a liquid medium to the keratin

precipitate phase and mixing both to form a homogeneous solution; and

2. The method of claim 1 , wherein the pH buffer has a pH of about 3.

3. The method of claim 1 or 2, wherein the pH buffer is selected from the group consisting of citrate buffer, phosphate buffered saline (PBS), acetic acid, citric acid-disodium hydrogen phosphate [Na₂HP0₄], acetic acid-sodium acetate, and a mixture thereof.

4. The method of any one of claims 1-3, wherein step (ii) further comprises adding a liquid medium.

5. The method of any one of claims 1-4, wherein the liquid medium used in step(s) (ii), (v) and/or (vii) has a pH of about 7 to 7.5.

6. The method of claim 5, wherein the liquid medium has a pH of about 7.2.

7. The method of any one of claims 1-6, wherein step (ii) further comprises adding a liquid medium and the solution of keratin, the pH buffer, and the liquid medium are mixed in a ratio of about 10:1 :10 (v/v/v).

8. The method of any one of claims 1 -7, wherein incubating the keratin solution

comprises incubating for about 1 to 30 hours.

9. The method of claim 8, wherein incubating the keratin solution comprises

incubating for about 24 hours.

10. The method of claim 8 or 9, wherein incubating the keratin solution further

comprises incubating at about 37 °C.

1 1. The method of any one of claims 1-10, wherein the liquid medium used in any one of steps (ii), (v) and (vii) is a cell culture medium.

12. The method of any one of claims 1 -14, wherein incubating the homogeneous solution comprises incubating at about 37 °C and 5 % C0₂.

13. A living cells encapsulated keratin-containing hydrogel obtained from the method of any one of claims 1-12.

14. Use of the living cells encapsulated keratin-containing hydrogel for cell delivery, drug delivery, wound dressing, or tissue regeneration.